@article {pmid42323652,
year = {2026},
author = {Liu, X and Wang, Y and Wong, JCY and Zhang, X and Wu, C and Chen, P and Yang, Y and Liu, P and Lau, CS and Cook, M and Li, PH},
title = {Modeling Hereditary Angioedema With Personalized Expanded Potential Stem Cell-Derived Hepatocytes: A CRISPR-Validated Platform for Mutation-Specific Mechanisms and Therapeutic Innovation.},
journal = {Allergy},
volume = {81},
number = {8},
pages = {2858-2873},
pmid = {42323652},
issn = {1398-9995},
support = {//the InnoHK initiative of the Innovation and Technology Commission of the Hong Kong Special Administrative Region Government/ ; },
mesh = {Humans ; *Hepatocytes/metabolism/cytology ; *Mutation ; *Angioedemas, Hereditary/genetics/therapy/metabolism ; Cell Differentiation ; *Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems ; Complement C1 Inhibitor Protein/genetics/metabolism ; Models, Biological ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Precision Medicine ; },
abstract = {Hereditary angioedema (HAE) with C1 esterase inhibitor (C1INH) deficiency is caused by pathogenic SERPING1 mutations that disrupt production of the plasma protease inhibitor C1INH. However, the molecular mechanisms and consequences of patient-specific mutations remain poorly understood due to the lack of physiologically relevant human models. Here, we established a personalized, isogenic, stem-cell-derived hepatocyte platform to investigate the underlying mutation-specific mechanisms of HAE. Specifically, peripheral blood mononuclear cell (PBMC)-expanded erythroblasts from four representative HAE-C1INH-Type1 patients containing distinct point, insertion, deletion, or large fragment SERPING1 mutations were reprogrammed into expanded potential stem cells (EPSCs) and further differentiated into hepatocyte-like cells (HLCs). These HLCs exhibited appropriate transcriptional transitions, mature hepatic features, and C1INH secretion comparable to that observed in human plasma. All patient-derived HLCs demonstrated impaired C1INH secretion with mutation-specific differences in both SERPING1 transcription and intracellular accumulation. Moreover, to verify that the mutations directly drive the phenotype, we performed CRISPR/Cas9-mediated genome repair, which restored SERPING1 mRNA expression and C1INH secretion. Conversely, identical patient mutations installed into healthy EPSCs showed the same transcriptional and secretory defects, confirming sufficiency. Collectively, we have established a robust human hepatocyte model that accurately recapitulates key hepatocyte-specific aspects of HAE pathophysiology and provides a scalable foundation for investigation of future precision therapies.},
}

Humans
*Hepatocytes/metabolism/cytology
*Mutation
*Angioedemas, Hereditary/genetics/therapy/metabolism
Cell Differentiation
*Stem Cells/cytology/metabolism
*CRISPR-Cas Systems
Complement C1 Inhibitor Protein/genetics/metabolism
Models, Biological
*Clustered Regularly Interspaced Short Palindromic Repeats
Precision Medicine

@article {pmid42389905,
year = {2026},
author = {Minaiyan, G and Aussel, C and Ammann, S and Cathomen, T},
title = {Genome Editing for Familial Hemophagocytic Lymphohistiocytosis: Design Principles, Challenges, and Translational Perspectives.},
journal = {Human gene therapy},
volume = {37},
number = {15-16},
pages = {702-712},
doi = {10.1177/10430342261465555},
pmid = {42389905},
issn = {1557-7422},
mesh = {Humans ; *Lymphohistiocytosis, Hemophagocytic/genetics/therapy ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; Translational Research, Biomedical ; Perforin/genetics ; Membrane Proteins/genetics ; Mutation ; CRISPR-Cas Systems ; Hematopoietic Stem Cell Transplantation ; },
abstract = {Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome caused by genetic defects in cytotoxic lymphocyte function. Current therapies can control disease activity, but transplantation of allogeneic hematopoietic stem and progenitor cells (HSPCs) remains the only curative option and is associated with substantial risks. These limitations have accelerated development of genome editing approaches enabling precise correction of disease-causing mutations in autologous cells. Familial HLH (FHL) represents a compelling target for genome editing, but successful and safe clinical translation has remained challenging. Preclinical studies demonstrate that targeted editing of key genes, such as PRF1 and UNC13D, can restore cytotoxic function in HSPCs and T cells. Translation to the clinic, however, depends on multiple factors, including the choice of target cell population, the level of functional correction required, and gene-specific constraints such as locus complexity and regulation of gene expression. In this review, we synthesize current progress in genome editing for FHL and highlight critical biological and technical barriers to clinical implementation. We propose a conceptual framework for designing genome editing strategies tailored to FHL, emphasizing the alignment of editing platform, gene architecture, and cellular context to enable effective and clinically translatable therapies.},
}

Humans
*Lymphohistiocytosis, Hemophagocytic/genetics/therapy
*Gene Editing/methods
*Genetic Therapy/methods
Animals
Translational Research, Biomedical
Perforin/genetics
Membrane Proteins/genetics
Mutation
CRISPR-Cas Systems
Hematopoietic Stem Cell Transplantation

@article {pmid42393087,
year = {2026},
author = {Weickert, P and Liu, Y and Strecker, J},
title = {Prokaryotic Schlafen proteins cleave tRNAs during type III CRISPR immunity.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42393087},
issn = {2041-1723},
support = {DP2HL185100//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; },
mesh = {*RNA, Transfer/metabolism/genetics ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Schlafen nucleases restrict viral infection in mammals by cleaving self RNAs, however, their function and mechanism in prokaryotic immunity is unknown. Here, we uncover CRISPR-associated Schlafen (Cash) proteins containing a Schlafen domain fused to Csx15, an uncharacterized member of Rossmann-like nucleotide-binding sensors. Cash is activated by cyclic tetra-adenylate (cA4) produced during type III CRISPR interference and induces cell toxicity by cleaving tRNAs, primarily in the T-loop. Cryo-electron microscopy structures of Chloroflexi bacterium Cash reveal an inactive dodecamer, the formation of a filament upon cA4 binding to align catalytic interfaces, and the molecular basis of substrate recognition and cleavage in a tRNA-bound complex. We identify numerous families of prokaryotic Schlafen proteins associated with diverse antiviral defense systems and characterized by unique sensor domains. This work highlights tRNA depletion by Schlafen nucleases as an evolutionary recurring antiviral strategy and reveals mechanistic differences between Cash and human Schlafen members.},
}

*RNA, Transfer/metabolism/genetics
*CRISPR-Cas Systems
*Bacterial Proteins/metabolism/genetics/chemistry
Cryoelectron Microscopy
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42424999,
year = {2026},
author = {Dong, J and Li, X and Gu, T and Zhang, Y and Deng, L and Luo, X and Hou, C and Huo, D},
title = {Compartmentalization-inspired dual-chamber CRISPR sensing coupled with single-atom electrocatalysis for crosstalk-free multiplex microRNA detection.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119016},
doi = {10.1016/j.bios.2026.119016},
pmid = {42424999},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation & purification/analysis/blood ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Electrochemical Techniques ; Nucleic Acid Amplification Techniques ; Catalysis ; Equipment Design ; },
abstract = {Compartmentalization is a hallmark of cells, enabling parallel biochemical processes to proceed with high fidelity and minimal interference. Drawing inspiration from this spatial isolation principle, we developed a compartmentalization-inspired dual-chamber sensing platform for crosstalk-minimized multiplex miRNA analysis. In each physically isolated compartment, the target miRNA directs padlock-probe ligation to form a circular template, followed by rolling-circle-extension-driven loop-mediated isothermal amplification (R-LAMP). The resulting amplicons specifically activate the corresponding CRISPR/Cas12a-crRNA complex, triggering trans-cleavage of a hairpin-DNA biogate that seals Fe-MOF nanocontainers. Gate opening releases distinct electroactive reporters (3,3',5,5'-tetramethylbenzidine, TMB; or methylene blue, MB) from their respective chambers. After the compartmentalized reactions finish, the two supernatants are combined and read out on a screen-printed electrode modified with a Co-N-C single-atom catalyst, producing two well-resolved DPV peaks for simultaneous quantification. The platform achieves femtomolar detection limits in simultaneous assays (0.87 fM for miRNA-21 and 0.72 fM for miRNA-155), a broad linear range (1 fM-100 pM), and high discrimination against non-cognate or mismatched sequences. Accurate recoveries in diluted human serum and consistent trends in cell lysates (validated by RT-qPCR) confirm practical applicability. By integrating bioinspired compartmentalization with CRISPR precision and single-atom electrocatalysis, this platform provides a generalizable route to multiplex nucleic acid diagnostics with enhanced fidelity and sensitivity.},
}

*MicroRNAs/genetics/isolation & purification/analysis/blood
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
Limit of Detection
Electrochemical Techniques
Nucleic Acid Amplification Techniques
Catalysis
Equipment Design

@article {pmid42431049,
year = {2026},
author = {Ji, S and Wang, B and Yan, Y and Zhang, C and Ren, H and Yang, J and Wei, X and Hou, W and Huang, M and Song, J and Wang, S and Qiu, L and Wang, H},
title = {TOPS-CRISPR: Thermally-regulated and oligonucleotide-mediated one-pot CRISPR-Cas12a assay for ultra-sensitive and rapid on-site diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {118998},
doi = {10.1016/j.bios.2026.118998},
pmid = {42431049},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Biosensing Techniques/methods ; Oligonucleotides/chemistry/genetics ; *Brucella/isolation & purification/genetics/pathogenicity ; *Brucellosis/diagnosis/microbiology ; Rapid Diagnostic Tests ; Temperature ; CRISPR-Associated Proteins/genetics ; Limit of Detection ; },
abstract = {CRISPR-Cas12a has emerged as a powerful tool in molecular diagnostics, owing to its robust signal amplification and compact crRNA design. However, its uncontrolled enzymatic activity often hampers application in streamlined one-pot assays. Although existing temporal or spatial regulation strategies can mitigate this issue, they typically introduce operational complexity or increased cost. Here, we designed a Thermally regulated, Oligonucleotide-mediated one-Pot System for CRISPR-Cas12a (TOPS-CRISPR), which employs a programmable inhibition strategy based on complementary RNA blockers with tunable length and binding sites, enabling efficient and reversible steric inhibition of the LbCas12a-crRNA ribonucleoprotein (RNP) complex, resolving the inherent contradiction between amplification and cleavage in one-pot assay. TOPS-CRISPR not only is operational simple and cost-effective but also achieves over 60-fold higher sensitivity than conventional one-pot platforms. We demonstrated the clinical applicability of TOPS-CRISPR by accurately detecting Brucella and Streptococcus in both spiked and clinical samples. Moreover, the system integrates seamlessly with rapid sample processing, lyophilized reagents, and miniaturized workflows, enabling field-deployable pathogen identification within 50 min.},
}

*CRISPR-Cas Systems/genetics
Humans
*Biosensing Techniques/methods
Oligonucleotides/chemistry/genetics
*Brucella/isolation & purification/genetics/pathogenicity
*Brucellosis/diagnosis/microbiology
Rapid Diagnostic Tests
Temperature
CRISPR-Associated Proteins/genetics
Limit of Detection

@article {pmid42447864,
year = {2026},
author = {Langley, J and Baudrier, L and Curry, J and Narta, K and Todesco, HM and Potts, K and Morrissy, S and Mahoney, DJ and Billon, P},
title = {Compound delivery of eVLPs enhances prime editing for targeted genome engineering and high-throughput screening.},
journal = {Cell genomics},
volume = {6},
number = {8},
pages = {101302},
doi = {10.1016/j.xgen.2026.101302},
pmid = {42447864},
issn = {2666-979X},
mesh = {Humans ; *Gene Editing/methods ; *High-Throughput Screening Assays/methods ; HEK293 Cells ; CRISPR-Cas Systems/genetics ; Lentivirus/genetics ; *Virion/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Engineered virus-like particles (eVLPs) enable transgene-free ribonucleoprotein delivery for genome editing, yet optimized strategies for high-throughput applications remain unexplored. Prime editing enables precise genomic modifications but suffers from limited efficiency. Here, we present PRIME-VLP (Progressive Repeated Infections for Maximized Editing via Virus-Like Particles), which exploits eVLP-mediated editing kinetics through multiple sequential sub-saturating transductions at optimal intervals. PRIME-VLP achieves 1.5- to 2.9-fold improvements in prime editing efficiency across diverse genomic targets and cell types without increasing off-target editing, compromising cellular viability or causing transcriptional perturbations. By decoupling pegRNA and editor delivery through pegRNA-free eVLPs, PRIME-VLP enables pooled prime editing screens and circumvents transgene silencing limitations. Using a 6,000-pegRNA library targeting TP53, PRIME-VLP achieved 2.8-fold higher editing and improved reproducibility compared to conventional lentiviral delivery, identifying TP53 loss-of-function variants conferring Nutlin-3 resistance. This work expands the versatility of eVLPs beyond their current in vivo therapeutic applications, demonstrating their promise for high-throughput functional genomics.},
}

Humans
*Gene Editing/methods
*High-Throughput Screening Assays/methods
HEK293 Cells
CRISPR-Cas Systems/genetics
Lentivirus/genetics
*Virion/genetics
Tumor Suppressor Protein p53/genetics

@article {pmid42470832,
year = {2026},
author = {Azhar, M and Malviya, R and Chandra, P and Sridhar, SB and Shareef, J and Wadhwa, T},
title = {Precision prime editing of TP53 mutations for functional tumor suppression in colorectal cancer.},
journal = {Biochemical and biophysical research communications},
volume = {831},
number = {},
pages = {154311},
doi = {10.1016/j.bbrc.2026.154311},
pmid = {42470832},
issn = {1090-2104},
mesh = {*Colorectal Neoplasms/genetics/therapy ; Humans ; *Mutation ; *Tumor Suppressor Protein p53/genetics ; CRISPR-Cas Systems ; Animals ; Genetic Therapy/methods ; Precision Medicine/methods ; *Gene Editing/methods ; },
abstract = {BACKGROUND: Colorectal cancer (CRC) is a major global health concern, with high mortality due to genetic heterogeneity and resistance to treatment. Tumor Protein p53 (TP53) mutations are also among the most important molecular changes that can disrupt genomic stability and facilitate tumor progression, so it is a critical target for precision-based interventions.

AIM: This review aims to discuss the future potential of prime editing as a new generation of genome engineering to identify precise approaches to correct TP53 mutations in colorectal cancer.

METHOD: A focused literature review was conducted on PubMed, Scopus, Web of Science, and Google Scholar for articles published between the years of 2010 and 2026. The keywords used in the search were CRC, TP53 mutation, prime editing, Prime Editing Guide RNA (pegRNA), CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9), and precision oncology. Studies were screened for experimental, mechanistic, and translational, and the focus was on mutation-specific editing, delivery platforms, organoid validation, clinically relevant barriers, etc. RESULTS/DISCUSSION: Prime editing is a programmable search-and-replace method that does not involve two single-stranded Deoxyribonucleic Acid (DNA) breaks, resulting in fewer Insertions/deletions (indels) and greater precision compared with traditional CRISPR-Cas9 approaches. Recent systems like Prime Editor Max (PEmax), PE5/PE5max, engineered pegRNAs, twin prime editors, PrimeDel, and PASTE have enhanced the efficiency, range, and flexibility. Hotspot and organoid studies suggest that variants of TP53, particularly R175H, R248Q/W, R273 H/C, and R282W, can be repaired. But cargo size, delivery specificity, tumor heterogeneity, varying cargo editing efficiency, cargo recognition by the immune system, and off-target risk are all barriers to clinical translation.

CONCLUSION: Precision oncology with prime editing has the potential to be a useful tool for CRC, though optimized delivery, thorough preclinical testing, and safety monitoring will be required for therapeutic adoption.

ORIGINALITY: This review combines TP53 hotspot biology, recent breakthroughs in prime editing technology, and CRC-specific translational challenges, and provides a step-by-step approach to its clinical application in a unique way.},
}

*Colorectal Neoplasms/genetics/therapy
Humans
*Mutation
*Tumor Suppressor Protein p53/genetics
CRISPR-Cas Systems
Animals
Genetic Therapy/methods
Precision Medicine/methods
*Gene Editing/methods

@article {pmid42472526,
year = {2026},
author = {Liu, Z and Wang, J and Yang, Z and Huang, H and Zhang, Z and Wu, T},
title = {High-specificity gene point mutation detection by PAM-free Cas12a system with double-stranded substrate positioning-unwinding.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119043},
doi = {10.1016/j.bios.2026.119043},
pmid = {42472526},
issn = {1873-4235},
mesh = {*Point Mutation/genetics ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA/genetics/chemistry ; *CRISPR-Associated Proteins/genetics ; DNA Mutational Analysis/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The CRISPR/Cas12a system holds great promise for nucleic acid detection, but its strict dependence on the protospacer adjacent motif (PAM) severely limits its application in gene point mutation analysis, with fewer than 2% of known mutation sites naturally harboring adjacent PAM sequences. Herein, we developed a PAM-free Cas12a system with double-stranded substrate positioning-unwinding (dsPU-Cas12a), wherein "bubble" structures formed by unpaired base pairs release partial single-stranded target strand as a toehold, and excess auxiliary strands induce local unwinding of double-stranded DNA to facilitate R-loop formation. After optimization, the dsPU-Cas12a system achieved an ultra-low limit of detection of 0.013% for gene point mutations, with excellent linearity over the mutation abundance range of 0-10%. Furthermore, it exhibited robust feasibility and accuracy in detecting the JAK2 V617F mutation in blood samples from patients with myeloproliferative neoplasms. This simple and universal strategy overcomes the sequence limitation of Cas12a, providing a high-performance tool for clinical gene point mutation detection.},
}

*Point Mutation/genetics
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*DNA/genetics/chemistry
*CRISPR-Associated Proteins/genetics
DNA Mutational Analysis/methods
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42474386,
year = {2026},
author = {Liao, J and Su, Y and Jiang, F},
title = {CRISPR-Cas12a assay for rapid and specific detection of Shigella flexneri 2a in clinical samples.},
journal = {Journal of clinical microbiology},
volume = {64},
number = {8},
pages = {e0161525},
pmid = {42474386},
issn = {1098-660X},
mesh = {Humans ; Sensitivity and Specificity ; *Shigella flexneri/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Dysentery, Bacillary/diagnosis/microbiology ; Feces/microbiology ; Rapid Diagnostic Tests ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases ; },
abstract = {Shigella flexneri 2a is the most common cause of shigellosis, a major public health concern in developing countries. Rapid and reliable diagnostic tools are critical for timely outbreak detection and management. Leveraging clustered regularly interspaced short palindromic repeats (CRISPR) technology, we developed a CRISPR-Cas12a-based assay for the rapid and specific detection of S. flexneri 2a and validated its performance using stool specimens from patients. Two guide RNAs targeting the gtrII and gtrX genes, unique markers of the S. flexneri 2a serotype, were designed to ensure specificity. Recombinase polymerase amplification (RPA) was coupled with Cas12a-mediated collateral cleavage for signal amplification, with detection by fluorescence or lateral flow. Analytical sensitivity, specificity, and clinical accuracy were compared with conventional PCR using purified DNA and 588 clinical stool specimens. The CRISPR-Cas12a assay achieved a detection limit of 10 copies/µL, comparable to PCR, and showed 100% analytical specificity without cross-reactivity to other bacteria. The isothermal reaction operated at room temperature and was completed within 1 h. Both readouts allowed visual interpretation without specialized equipment. Clinical validation of the CRISPR-Cas12a assay demonstrated a diagnostic sensitivity of 95% and specificity of 98%, comparable to PCR when evaluated using the same clinical specimens. This study provides two key advances: it establishes a CRISPR-Cas12a assay specifically targeting S. flexneri 2a, the predominant serotype, and validates it using a large clinical cohort. The assay's simplicity, speed, and high diagnostic accuracy make it a valuable tool for clinical diagnostics and field-based surveillance in resource-limited settings.IMPORTANCERapid and accessible diagnostics are essential for effective management of infectious diseases such as shigellosis. We developed a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based assay that specifically detects Shigella flexneri 2a, the predominant serotype responsible for the global disease burden. This assay integrates isothermal amplification with CRISPR-mediated detection to achieve low-copy detection (10 copies/µL) within 1 h, eliminating the need for complex instrumentation. Dual fluorescence and lateral-flow readouts enable flexible use in both clinical laboratories and low-resource settings. The method's simplicity, accuracy, and adaptability demonstrate the practical potential of CRISPR diagnostics for point-of-care applications. By enabling rapid, on-site identification of S. flexneri 2a, this approach can significantly improve clinical diagnosis and strengthen public health responses to enteric pathogen outbreaks.},
}

Humans
Sensitivity and Specificity
*Shigella flexneri/genetics/isolation & purification
*CRISPR-Cas Systems
*Dysentery, Bacillary/diagnosis/microbiology
Feces/microbiology
Rapid Diagnostic Tests
*Molecular Diagnostic Techniques/methods
Bacterial Proteins/genetics
CRISPR-Associated Proteins/genetics
Endodeoxyribonucleases

@article {pmid42485703,
year = {2026},
author = {Tan, Y and He, X and Shao, E and Chen, Q and Zhang, L and Deng, S},
title = {A one-tube autocatalytic transcription-driven CRISPR cascade for ultrasensitive mRNA detection.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119049},
doi = {10.1016/j.bios.2026.119049},
pmid = {42485703},
issn = {1873-4235},
mesh = {*RNA, Messenger/genetics/analysis/isolation & purification ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Transcription, Genetic ; *Biomarkers, Tumor/genetics/isolation & purification ; *Breast Neoplasms/genetics/diagnosis ; Nucleic Acid Amplification Techniques ; Limit of Detection ; },
abstract = {The rapid identification and precise quantification of cancer biomarkers are essential for the purposes of diagnosis, classification, and therapeutic intervention. Traditional molecular diagnostic methodologies, such as polymerase chain reaction (PCR), provide considerable sensitivity; however, they depend on exponential amplification and advanced instrumentation, thereby constraining their applicability for point-of-care testing. In this study, we developed a Transcription-driven CRISPR Cascade Amplification (TCCA) for one-tube detection of mRNA at the concentration of 0.6 copies/μL within just 30 min. The target-induced assembly of a three-way junction (TWJ) facilitates the generation of a split T7 promoter, which initiates transcription and activates Cas13a collateral cleavage. The activated Cas13a subsequently cleaves cascade probes and releases new trigger strands, forming a transcription-driven cascade amplification circuit. The assay enables multiplex detection of breast cancer-associated mRNA biomarkers (HBB, KRT17, and CD55) in cell lysates, demonstrating robust performance in intricate biological matrices. Furthermore, incorporation of a multiplex OR-gated logic design enables parallel target recognition, thereby enhancing detection reliability for rapid and accurate clinical diagnostics.},
}

*RNA, Messenger/genetics/analysis/isolation & purification
Humans
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Transcription, Genetic
*Biomarkers, Tumor/genetics/isolation & purification
*Breast Neoplasms/genetics/diagnosis
Nucleic Acid Amplification Techniques
Limit of Detection

@article {pmid42504876,
year = {2026},
author = {Wang, P and Sayed, S and Buchholz, F},
title = {Restoring Cancer Genomes: Functional Mutation Correction as a Platform for Precision Oncology.},
journal = {Human gene therapy},
volume = {37},
number = {15-16},
pages = {663-669},
doi = {10.1177/10430342261468973},
pmid = {42504876},
issn = {1557-7422},
mesh = {Humans ; *Neoplasms/genetics/therapy/diagnosis ; *Mutation ; *Precision Medicine/methods ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Genome, Human ; Animals ; },
abstract = {Cancer genome sequencing has uncovered an extensive landscape of somatic mutations. However, determining which of these alterations are biologically consequential and therapeutically actionable remains a central challenge in oncology. CRISPR-based genome editing now enables precise correction of oncogenic mutations within their endogenous genomic context. Recent work demonstrates that repairing cancer hotspot mutations restores conserved tumor-relevant transcriptional programs across diverse tumor types, revealing tumor-agnostic dependencies. Beyond therapeutic implications, this mutation-correction framework provides a scalable functional platform to stratify drivers, interrogate variants of uncertain significance, and refine precision diagnostics. This perspective discusses how programmable mutation correction advances (i) mechanistic cancer biology, (ii) personalized cancer diagnostics, and (iii) next-generation precision gene therapies. We propose that systematic reversal of cancer mutations represents a conceptual shift from observing mutational landscapes to actively testing their biological necessity.},
}

Humans
*Neoplasms/genetics/therapy/diagnosis
*Mutation
*Precision Medicine/methods
*Gene Editing/methods
CRISPR-Cas Systems
*Genetic Therapy/methods
*Genome, Human
Animals

@article {pmid42508243,
year = {2026},
author = {Wei, K and Heng, H and Wang, T and Liu, Z and Wang, K},
title = {Integration of dual-output TMSD and CRISPR/Cas12a for crosstalk-free electrochemiluminescence detection of aflD gene and AFB1.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119062},
doi = {10.1016/j.bios.2026.119062},
pmid = {42508243},
issn = {1873-4235},
mesh = {*Aflatoxin B1/analysis/genetics/isolation & purification ; *Biosensing Techniques/methods ; Luminescent Measurements/methods ; Electrochemical Techniques/methods ; CRISPR-Cas Systems/genetics ; Aptamers, Nucleotide/chemistry ; Humans ; Limit of Detection ; Food Contamination/analysis ; Metallocenes/chemistry ; Ferrous Compounds/chemistry ; },
abstract = {Aflatoxins are among the most toxic mycotoxins and pose a severe threat to food safety and human health. In addition to the direct monitoring of aflatoxin B1 (AFB1), simultaneous detection of its key biosynthesis gene, aflD, can effectively indicate the presence of toxin-producing strains, thereby enhancing the early screening and traceability of AFB1 contamination. Due to the significant functional differences among various biomarkers, performing multi-target analysis on a single detection interface remains challenging. Herein, we constructed a novel dual-target electrochemiluminescence (ECL) biosensor for the sequential and quantitative detection of the aflD gene and AFB1. This sensor innovatively integrates a dual-output toehold-mediated strand displacement (TMSD) and CRISPR/Cas12a trans-cleavage mechanisms to establish a dynamic "signal writing-erasing" regulation on a single ECL emitter. Specifically, aflD triggers the TMSD reaction, driving the enrichment of ferrocene (Fc)-labeled DNA at the electrode interface and quenching the ECL signal, corresponding to signal "write" (signal-off). Subsequently, AFB1 is converted via aptamer recognition into an activator DNA that initiates Cas12a trans-cleavage, leading to the removal of Fc-DNA from the interface and recovery of ECL emission, corresponding to signal "erase" (signal-on). This strategy enables cross-category detection and quantitative analysis of small-molecule toxins and nucleic acid biomarkers within a single luminescence system, effectively avoiding signal crosstalk while offering high sensitivity, high specificity, and high interfacial utilization efficiency. It provides a versatile new approach for the early warning and source tracing of contaminants in complex food matrices.},
}

*Aflatoxin B1/analysis/genetics/isolation & purification
*Biosensing Techniques/methods
Luminescent Measurements/methods
Electrochemical Techniques/methods
CRISPR-Cas Systems/genetics
Aptamers, Nucleotide/chemistry
Humans
Limit of Detection
Food Contamination/analysis
Metallocenes/chemistry
Ferrous Compounds/chemistry

@article {pmid42520408,
year = {2026},
author = {Kumar, J and Alok, A and Fox, J and Srivastava, A and Voytas, DF and Zhang, F and Kianian, SF},
title = {A novel genome editing strategy in plants using broad-host-range viral vectors derived from geminiviruses.},
journal = {Plant physiology},
volume = {201},
number = {4},
pages = {},
doi = {10.1093/plphys/kiag553},
pmid = {42520408},
issn = {1532-2548},
support = {//U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS) National Plant Disease Recovery System (NPDRS)/ ; },
mesh = {*Geminiviridae/genetics ; *Genetic Vectors/genetics ; *Genome, Plant/genetics ; *Host Specificity/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {The use of viral vectors offers a promising alternative to traditional transformation methods for creating gene-edited plants. In this study, we developed a novel plant genome editing system by delivering Cas9, Cas12f, and Cas12j nucleases along with their guide RNAs using a broad-host-range geminivirus, Wheat dwarf India virus (WDIV), in combination with Ageratum yellow leaf curl betasatellite (AYLCB). Cas9, Cas12f, and Cas12j nucleases were efficiently expressed along with corresponding guide RNAs under viral promoters. By leveraging tRNA spacers in place of external promoters and terminators, we significantly reduced the overall cargo size, streamlining vector design. Additionally, we compared the traditional AtU6-driven gRNA delivery with a novel spacer:gRNA:spacer format in Cas9-expressing lines and observed comparable editing efficiencies. The broad host range of WDIV and AYLCB, combined with the novel genome-editing platform, opens possibilities for editing across a wide range of plant species.},
}

*Geminiviridae/genetics
*Genetic Vectors/genetics
*Genome, Plant/genetics
*Host Specificity/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid42526210,
year = {2026},
author = {Zhao, J and He, J and Du, H and Dai, C and Ma, X and Kong, D and Yue, Y and Zhang, S and Liu, K and Liu, Y and Zhang, G and Wu, Y and Wei, D},
title = {Photo-switchable CRISPR electrochemical system enables de-interferential biosensing signal output.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119067},
doi = {10.1016/j.bios.2026.119067},
pmid = {42526210},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *Carcinoma, Non-Small-Cell Lung/genetics/diagnosis ; *Lung Neoplasms/genetics/diagnosis ; Mutation ; Limit of Detection ; Equipment Design ; Nucleic Acid Amplification Techniques ; Light ; },
abstract = {Most clustered regularly interspaced short palindromic repeats (CRISPR)-based electrochemical biosensors lack controllable switching function and are vulnerable to false signals induced by intrinsic amplification signal crosstalk. Thus, exploring strategies that mitigate intrinsic amplification crosstalk with a precise switch would be extremely useful for reliable and accurate bioanalysis. Herein, we develop a photo-switchable CRISPR/Cas12a electrochemical (PSCE) system by introducing a photocleavable (PC) linker and adopting a light-responsive strategy, which enables output of de-interference electrochemical signals and achieves highly sensitive and accurate detection of gene mutations for non-small cell lung cancer (NSCLC). The system decouples nucleic acid amplification (NAA) from signal transduction and produces unique photo-switchable response signals by light irradiation to activate Cas12a activity. This architectural design further separates signal readout from sample pretreatment, suppressing intrinsic amplification-derived signal crosstalk rather than global biological interference to deliver an ultralow limit of detection (LoD). The PSCE achieves 98.1% sensitivity, 92.7% specificity, and 98.9% overall accuracy when tested with 67 clinical samples. Moreover, the exploration of PSCE system in flexible wearable electronics and machine-learning analysis of clinical patient samples demonstrates significant application potential clinical diagnosis of mutation-associated diseases.},
}

*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Electrochemical Techniques/methods
*Carcinoma, Non-Small-Cell Lung/genetics/diagnosis
*Lung Neoplasms/genetics/diagnosis
Mutation
Limit of Detection
Equipment Design
Nucleic Acid Amplification Techniques
Light

@article {pmid42546382,
year = {2026},
author = {Qin, L and Tang, Z and Yu, Y and Liu, T and Zhang, M and Zhu, X and Hu, Q and Zhao, Z and Zhang, F and Tang, BZ and Cen, Y},
title = {DIRECTOR: DNA-guided spatially ordered assembly of split crRNA for activating CRISPR/Cas12a.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119075},
doi = {10.1016/j.bios.2026.119075},
pmid = {42546382},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/chemistry/genetics ; Molecular Dynamics Simulation ; *MicroRNAs/genetics/analysis ; *Bacterial Proteins/chemistry/genetics ; },
abstract = {Precise regulation of Cas12a activity is crucial for expanding its application in molecular diagnostics. However, existing split crRNA systems exhibit hardly any activation efficiency at low-abundance target and lack a well-defined regulated mechanism, representing a persistent bottleneck for practical application. This work proposes a DNA-guided spatially ordered assembly of split crRNA for activating CRISPR/Cas12a (DIRECTOR) strategy. This work combines artificial intelligence-driven AlphaFold3 structure prediction, computer-powered molecular dynamics simulations with fluorescence analysis to demonstrate that the 3' terminal extension of activator acts as a spatial director, utilizing DNA-guided spatially ordered assembly of split crRNA and stabilizing key Cas12a domains, thereby activating Cas12a. Conversely, the 5' terminal extension serves as a spatial misdirector, inhibiting Cas12a activation by destabilizing the protein structure and introducing the steric hindrance to shield the catalytic center. Furthermore, the structural and energy thresholds required for effective Cas12a activation were identified. Finally, utilizing the spatial director as an energy amplification element, DIRECTOR achieves a limit of detection as low as 42.1 fM for single-target miR-155 and dual-response detection of wide-scope nucleic acids. Owing to its direct activation strategy, DIRECTOR provides mechanistic insights for affordable and programmable CRISPR molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
*DNA/chemistry/genetics
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
*Biosensing Techniques/methods
*Endodeoxyribonucleases/chemistry/genetics
Molecular Dynamics Simulation
*MicroRNAs/genetics/analysis
*Bacterial Proteins/chemistry/genetics

@article {pmid42546384,
year = {2026},
author = {Li, L and Wei, H and Wei, M and Yang, X and Wang, T and Min, S and Wang, Y and Wang, X and Li, M and Wang, S and Rong, Z},
title = {An integrated centrifugal microfluidic CRISPR-based diagnostics platform for multiplexed point-of-care testing of respiratory pathogens.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119083},
doi = {10.1016/j.bios.2026.119083},
pmid = {42546384},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/instrumentation ; *Point-of-Care Testing ; Equipment Design ; *CRISPR-Cas Systems ; *Respiratory Tract Infections/diagnosis/microbiology/virology ; Lab-On-A-Chip Devices ; Centrifugation/instrumentation ; *Microfluidic Analytical Techniques/instrumentation ; Limit of Detection ; Rapid Diagnostic Tests ; Point-of-Care Systems ; },
abstract = {Rapid, accurate, and multiplexed point-of-care testing (POCT) of respiratory pathogens is critical for clinical triage and infection control. However, existing platforms frequently necessitate trade-offs between sensitivity, throughput, and operational complexity. To address this, we developed the iCARD (integrated Centrifugal Assay on a Rotating Disc) platform, a streamlined microfluidic molecular diagnostic system based on single-step CRISPR kinetics and centrifugal microfluidics. Comprising a polymethyl methacrylate disc pre-loaded with lyophilized CRISPR/Cas13a reagents and a self-developed portable fluorescence analyzer, the platform enables automated multiplexed detection following sample loading. Furthermore, a novel resin-based pretreatment method was engineered to efficiently purify and concentrate nucleic acids from throat swabs. The iCARD system facilitates the parallel screening of six respiratory pathogens across four independent samples within 40 min. Analytical validation confirmed single-copy sensitivity (limit of detection: 0.25-1.0 copies/μL) without cross-reactivity. Clinical validation using 94 retrospective patient throat swabs demonstrated exceptional diagnostic accuracy, achieving a sensitivity of 95.5% (64/67; 95% CI: 87.64%-98.47%) and a specificity of 100.0% (27/27; 95% CI: 87.51%-100.00%). These findings demonstrate that the iCARD platform serves as a robust, high-throughput, and accurate diagnostic tool for decentralized molecular screening and epidemiological surveillance.},
}

Humans
*Biosensing Techniques/instrumentation
*Point-of-Care Testing
Equipment Design
*CRISPR-Cas Systems
*Respiratory Tract Infections/diagnosis/microbiology/virology
Lab-On-A-Chip Devices
Centrifugation/instrumentation
*Microfluidic Analytical Techniques/instrumentation
Limit of Detection
Rapid Diagnostic Tests
Point-of-Care Systems

@article {pmid42557300,
year = {2026},
author = {Du, J and Luo, Z and Xie, D and Chen, Y and Yang, M and Li, Q and Wang, L and Han, L and Zhang, Y and Li, H and Lan, Z and Shi, H and Li, Y and Cheng, Q and Dong, F and Gao, Y and Yao, Y and Cheng, T and Wei, T and Rao, S},
title = {Engineered lipid nanoparticles for in vivo and durable editing of haematopoietic stem cells within humanized mice.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {42557300},
issn = {2157-846X},
support = {82370117//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82470240//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82370117//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {In vivo genetic engineering of haematopoietic stem and progenitor cells (HSPCs) holds the potential to revolutionize the treatment landscape for numerous diseases. However, despite its transformative potential, it remains hindered by the difficulty in efficiently and specifically targeting quiescent human HSCs while maintaining their long-term functionality. Here, after screening 15 lipid nanoparticles (LNPs), we report an LNP that efficiently delivers reporter mRNA to human HSPCs both in ex vivo and in vivo settings when conjugated with the anti-CD34 antibody (CD34/LNP[DP]). Using CRISPR/Cas editing cargos, CD34/LNP[DP] achieves high editing efficiency in human HSPCs ex vivo. Intrafemoral administration of CD34/LNP[DP] in humanized mice results in efficient editing of the erythroid-specific BCL11A enhancer within human HSPCs, enabling the sustained long-term reactivation of fetal haemoglobin (HbF) expression in erythroid cells. In a humanized neutropaenia model harbouring an ELANE mutation, intrafemoral administration of CD34/LNP[DP] achieves robust editing, targeting exon 2 of ELANE in human HSPCs, partially restoring neutrophil development impairment under long-term observation. Collectively, CD34-targeted delivery enables in vivo HSPC modification without perturbing haematopoiesis, underscoring its suitability for clinical translation.},
}

@article {pmid42558756,
year = {2026},
author = {Sharma, N and Thakur, K and Zinta, R and Shaunak, I and Batta, S and Saini, A and Sehgal, R and Bhagta, S and Singh, B and Thakur, AK},
title = {Transgene-free genome editing in potato, a clonally propagated crop - strategies and future prospects.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {32},
number = {8},
pages = {1747-1757},
pmid = {42558756},
issn = {0971-5894},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas)-based genome editing technology has come out as very precise and effective tool for targeted modification in the gene of interest and offers unprecedented potentials in crop improvement. However, in the present regulatory framework for commercialization of genome edited crops, in many countries including India, the edited lines must be transgene-free. In India, only site directed nuclease (SDN) I and SDN II category of genome edited events which are transgene-free are permitted for commercialization. Potato is a vegetatively propagated crop, having autotetraploid genome and is highly heterozygous in nature. Removal of the transgene from potato genome of edited lines through genetic segregation, either by crossing or selfing, is not the appropriate method as the elite background of the genome gets disturbed due to heterozygous nature of the crop. Every individual seed of potato, i.e. true potato seed (TPS) behaves like a different individual than the parental line and is unable to maintain the genetic identity. In this review article, we have discussed several strategies that can be enacted for generation of transgene-free genome edited lines in potato. This article will provide deeper insight and enhance understandings about the optimum use of CRISPR as non-GMO technology in the genetic enhancement of potato and to adopt the best strategies in editing this important tuberous, clonally propagated crop.},
}

@article {pmid42559426,
year = {2026},
author = {Li, A and Cao, C and Yang, C and Liu, Y},
title = {Programmable transcriptional condensates for enhanced CRISPR-based gene regulation.},
journal = {Theranostics},
volume = {16},
number = {14},
pages = {8215-8229},
pmid = {42559426},
issn = {1838-7640},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Gene Expression Regulation ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; HeLa Cells ; Transcription, Genetic ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {RATIONALE: Efficient gene activation or repression through programmable CRISPR-Cas9 has revolutionized molecular biology and drug development. Nonetheless, the currently available CRISPRa/i approaches are modestly potent and require multi-component delivery, which hampers the wide use of the technology in both research and therapy.

METHODS: We developed a modular CRISPR-condensate platform by appending a multivalent RNA nanostar to the 3' end of a single-guide RNA, producing a sgRNA-nanostar chimera that mediates phase separation at Cas9-bound genomic loci. The nanostar scaffold also contains MS2 stem-loops, which recruit MCP-tagged transcriptional effectors (VP64 for activation, KRAB for repression) to the condensate microenvironment at high local concentration. We examined condensate formation, genome targeting, and transcriptional output by using live-cell imaging, RT-qPCR, ChIP-seq, RNA-seq and CUT&Tag in HEK293T, HeLa, U-2 OS, MDA-MB-231, as well as human iPSC cell lines.

RESULTS: The CRISPR-condensate design resulted in up to 50-100-fold target-gene activation, compared with 5-10-fold activation by direct VP64 fusion, and 20-30-fold transcriptional repression, compared with 3-5-fold repression by direct KRAB fusion, with high target specificity (12 versus 28 non-target differentially expressed genes assessed by RNA-seq). Orthogonal kissing-loop (KL) pairings enabled independent condensate systems for simultaneous activation and repression of multiplexed targets. Janus condensates containing both activating and repressive domains enabled bidirectional regulation at a single locus. The system requires delivery of only three independently expressible components-dCas9-NLS, an sgRNA-nanostar chimera bearing MS2 stem-loops (MS2SLs), and an MCP-fused effector (VP64-MCP for activation or KRAB-MCP for repression)-and showed minimal innate immune response and high cell viability.

CONCLUSIONS: The CRISPR-condensate system merges the dramatically enhanced transcriptional efficacy with the reduced complexity of components, providing a modular system for fine-tuned gene expression regulation. This strategy makes biomolecular condensation a general principle for enhancing CRISPR gene regulation, opening up possibilities for functional genomics, cell engineering, and therapy development.},
}

Humans
*CRISPR-Cas Systems/genetics
HEK293 Cells
*Gene Expression Regulation
RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
HeLa Cells
Transcription, Genetic
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42559496,
year = {2026},
author = {Lee, HK and Lim, J and Shepherd, S and Nguyen, MTT and Akin, L and Bacon, A and Sidavi, Y and Shurgalin, M and Cobi, A and Nikolayenko, A and Fuflyigin, V and Bashir, R and Wang, X and Cunningham, BT},
title = {A Dual-Port, Smartphone-Linked, Pocket-Size Fluorimeter for Rapid Molecular Diagnostic Assays at Point of Care.},
journal = {IEEE sensors journal},
volume = {26},
number = {14},
pages = {20538-20555},
pmid = {42559496},
issn = {1530-437X},
support = {U01 AA029348/AA/NIAAA NIH HHS/United States ; },
abstract = {We present the design, testing, and demonstration of a portable, pocket-size, smartphone-linked fluorimeter called the "VPodDuo." The instrument is capable of reading the output of several fluorescence-generating biomolecular detection assays with sensitivity that is similar to larger and more expensive laboratory-based instruments. In this work, we focus on demonstrating the capability for readout of assays used to detect target nucleic acid sequences associated with infectious pathogens and cancer with incubation times of approximately 10 min. The VPodDuo features a dual-port configuration that allows simultaneous measurements of a negative experimental control (CTRL) in parallel with the test sample. We demonstrated compatibility with several assay protocols, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), recombinase polymerase amplification (RPA), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, and the target recycling amplification process (TRAP). Benchmarking against three commercially available fluorimeters showed comparable detection limits for clinically relevant nucleic acid sequences, including Zika virus (ZIKV, 10[4] copies/μL), methicillin-susceptible Staphylococcus aureus (MSSA, 10[2] copies/μL), human immunodeficiency virus (HIV, 6.83 pM), a lung cancer-associated circulating tumor DNA sequence [L858R point-mutated epidermal growth factor receptor (EGFR) gene, 29.2 pM], and a microRNA biomarker associated with lung cancer (miR-375-3p, 500 pM). We further validated the VPodDuo's performance under varying ambient temperatures through in-lab simulations and real-world outdoor testing using the TRAP assay for miR-375-3p detection, demonstrating cancer-associated biomarker detection at point-of-care (POC) settings. With its compact form-factor, low cost, portability, and real-time data transmission and analytical capabilities, the VPodDuo represents a promising solution for expanding access to rapid, on-site molecular diagnostics in diverse clinical and field settings.},
}

@article {pmid42560391,
year = {2026},
author = {Uc-Chuc, MA and Aguilar-Hernández, V and Jiménez-Ramírez, IA and Brito-Argaez, L and Loyola-Vargas, VM},
title = {In vitro CRISPR/Cas9-RNP cleavage of CcYUC1 in Coffea canephora.},
journal = {Planta},
volume = {264},
number = {3},
pages = {},
pmid = {42560391},
issn = {1432-2048},
support = {1515//Secretaría de Ciencia, Humanidades, Tecnología e Innovación (Secihti)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Coffea/genetics/enzymology/metabolism ; Indoleacetic Acids/metabolism ; *Plant Proteins/genetics/metabolism ; *Ribonucleoproteins/metabolism/genetics ; Base Sequence ; },
abstract = {An in vitro CRISPR/Cas9-RNP system efficiently cleaves CcYUC1 in Coffea canephora, establishing a foundation for DNA-free genome editing in coffee. Somatic embryogenesis depends on auxin biosynthesis and signaling; however, functional validation of candidate genes remains limited. In this study, we identified CcYUC1, a putative flavin monooxygenase gene associated with indole-3-acetic acid biosynthesis, during SE induction in Coffea canephora. CcYUC1 transcripts accumulated during the early stages of SE, suggesting a role in embryogenic induction. To establish a genome editing platform in coffee, we designed a CRISPR/Cas9 ribonucleoprotein (RNP) system targeting exon 4 of CcYUC1. In vitro cleavage assays confirmed specific and efficient digestion of the target, achieving over 80% cleavage under optimized Cas9/sgRNA conditions. These findings establish a proof-of-concept CRISPR/Cas9-RNP platform for DNA-free genome editing in coffee and provide a basis for future functional studies of auxin biosynthesis during somatic embryogenesis.},
}

*CRISPR-Cas Systems/genetics
*Coffea/genetics/enzymology/metabolism
Indoleacetic Acids/metabolism
*Plant Proteins/genetics/metabolism
*Ribonucleoproteins/metabolism/genetics
Base Sequence

@article {pmid42562481,
year = {2026},
author = {Tan, G and Qi, S and Hu, M and Wang, D and Lin, K and Wang, Y and Chen, S and Zhang, Q and Zhao, L},
title = {Understanding phage dynamics and their potential roles during soy sauce fermentation using metagenome-assembled genomes.},
journal = {Food research international (Ottawa, Ont.)},
volume = {241},
number = {},
pages = {119707},
doi = {10.1016/j.foodres.2026.119707},
pmid = {42562481},
issn = {1873-7145},
mesh = {*Fermentation ; *Bacteriophages/genetics/classification/physiology ; *Soy Foods/microbiology/virology ; *Metagenome ; Genome, Viral ; *Food Microbiology ; Metagenomics ; },
abstract = {The composition and functional roles of phages in fermented foods have been gaining increasing attention. However, their ecological functions and underlying mechanisms in high-salt soy sauce fermentation remain largely unexplored. In this study, we investigated phage communities, their potential functions, phage-host interactions, and host defense mechanisms in two different soy sauce fermentation processes (Cantonese-type process, CP; Japanese-type process, JP) using shotgun metagenomics. A total of 823 phage species (viral operational taxonomic units, vOTUs) were identified, with the majority exhibiting a temperate lifestyle (89.19%). The most abundant family was Straboviridae (CP, 9.95%-11.39%; JP, 12.04%-13.73%), followed by Salasmaviridae (CP, 6.92%-7.94%; JP, 5.70%-7.02%). Although the phage composition differed between the two processes, the number of vOTUs was positively correlated with prokaryotic species richness, total acidity, and amino acid nitrogen content, and negatively correlated with pH. A comparative genomic analysis revealed that 91 phages were associated with 26 bacterial genomes (metagenome-assembled genomes, MAGs), with Lactococcus petauri (MAG16) and Halomonas elongata (MAG51) hosting the most phage species. An analysis of host defense mechanisms showed that all 45 bacterial MAGs harbored CRISPR-Cas type I systems, and 95.6% encoded restriction-modification systems. Functional annotation using the KEGG and CAZymes databases indicated that the phages predominantly encoded genes related to cell growth, replication, and metabolism of amino acids, carbohydrates, and nucleotides, with more genes enriched in the JP than in the CP. Additionally, auxiliary metabolic genes (e.g., pfkA, ldh, adhP, ilvE, and arcA) were identified in 29 phages. These genes are potentially involved in metabolic pathways that may be linked to flavor compound production. Together, these findings provide novel insights into the ecological and potential functional roles of phages during soy sauce fermentation.},
}

*Fermentation
*Bacteriophages/genetics/classification/physiology
*Soy Foods/microbiology/virology
*Metagenome
Genome, Viral
*Food Microbiology
Metagenomics

@article {pmid42563405,
year = {2026},
author = {Adil, M and Gul, I and Lu, S and Bashir, S and Razzaq, S and Lu, H and Daud, M and Iqbal, Y and Tao, Y},
title = {Crop Resilience to Combined Drought and Salinity Stress in Drylands: From Soil Processes to Genomic Solutions.},
journal = {Plant, cell & environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70804},
pmid = {42563405},
issn = {1365-3040},
support = {41871079//National Natural Science Foundation of China/ ; SKLECRA2023//Chinese Research Academy of Environmental Sciences/ ; //Open Foundation of State Key Laboratory of Environmental Criteria and Risk Assessment/ ; },
abstract = {Drought and soil salinization increasingly co‑occur, threatening global food security, particularly in dryland farming systems where these stresses can interact additively, synergistically or antagonistically to reduce crop yields. This review critically distinguishes these interaction types and moves beyond single‑stress frameworks to provide a multiscale synthesis of plant responses, soil water-salt dynamics, root‑system plasticity, rhizosphere microbiome modulation, emerging breeding modelling and agronomic tools. This review provides a detailed overview of the multilevel damage from these co-occurring stresses, including osmotic shock, ionic toxicity and systemic oxidative damage, and examines plant strategies such as ion homoeostasis, osmotic adjustment and antioxidant responses. Beyond plant-centric views, the review explores epigenetic reprogramming for stress memory and the potential of rhizosphere microbiome engineering with plant growth-promoting rhizobacteria as a biological stress-alleviation strategy. It critically examines how high-throughput multiomics techniques, combined with innovative breeding methods, provide a transformative approach to decode complex tolerance traits and accelerate cultivar development. These techniques cover genomics, epigenomics, transcriptomics, proteomics and metabolomics, while the breeding methods include genomic selection and CRISPR-Cas gene editing. Despite progress, key challenges remain: validating combined stress resilience under field conditions, harnessing epigenetic inheritance and integrating microbial solutions into breeding pipelines. We propose a unified framework that merges mechanistic discovery, microbiome-assisted breeding, soil and water management and systems-level predictive modelling. This integrative approach is essential for developing climate-resilient crops to sustain dryland agriculture.},
}

@article {pmid42567578,
year = {2026},
author = {Wang, H and Bao, C and Liu, L and Li, F and He, Y and Liu, X and Yin, Y and Xu, S},
title = {CRISPR/Cas12a and CHA-based SERS platform for ultrasensitive nucleic acid detection.},
journal = {Analytica chimica acta},
volume = {1418},
number = {},
pages = {345840},
doi = {10.1016/j.aca.2026.345840},
pmid = {42567578},
issn = {1873-4324},
mesh = {*Spectrum Analysis, Raman/methods ; *CRISPR-Cas Systems/genetics ; Zea mays/genetics ; Silver/chemistry ; Limit of Detection ; DNA, Single-Stranded/analysis ; Metal Nanoparticles/chemistry ; },
abstract = {BACKGROUND: Highly sensitive nucleic acid detection is essential for analytical applications. Conventional methods often require complex pre-amplification procedures, limiting their practical utility in screening. Developing nucleic acid detection strategies with high sensitivity and selectivity, without target gene pre-amplification, remains a significant challenge.

RESULTS: This study integrates CRISPR/Cas12a recognition, catalytic hairpin assembly (CHA) amplification, and surface-enhanced Raman spectroscopy (SERS). CRISPR/Cas12a recognizes target nucleic acids and cleaves single-stranded DNA (ssDNA), thereby blocking the toehold-mediated strand displacement reaction (TSDR) and triggering CHA. Hairpin probe HP1 with C-Ag[+]-C structures bridges CHA, releasing Ag[+] through cyclic amplification. Ag[+] induces charge transfer and aggregation of AgNPs@4-ABT, generating strong SERS signals. The platform achieved femtomolar sensitivity and high selectivity in detecting pCaMV35S, with 96.4% accuracy in maize seeds and 100% in maize leaves.

SIGNIFICANCE: This strategy eliminates the need for pre-amplification of target genes by combining CRISPR's targeting feature, CHA, and ultrasensitive SERS detection. It demonstrates excellent performance in genetically modified organism screening, seed quality testing, and leaf sample analysis, providing a promising tool for food safety and agricultural regulation.},
}

*Spectrum Analysis, Raman/methods
*CRISPR-Cas Systems/genetics
Zea mays/genetics
Silver/chemistry
Limit of Detection
DNA, Single-Stranded/analysis
Metal Nanoparticles/chemistry

@article {pmid42568984,
year = {2026},
author = {Sahu, A and Kumar, A and Vaidya, A and Mishra, J and Mishra, S and Prajapti, SK},
title = {Decoding the silent conversations: targeting quorum sensing to disarm bacterial pathogens in the age of antimicrobial resistance.},
journal = {RSC medicinal chemistry},
volume = {},
number = {},
pages = {},
pmid = {42568984},
issn = {2632-8682},
abstract = {Antimicrobial resistance (AMR) has emerged as a global health challenge, imposing significant clinical and economic burdens worldwide. The widespread and often indiscriminate use of antibiotics has accelerated resistance, necessitating alternative therapeutic strategies to combat microbial pathogenicity. Quorum sensing, a cell density-dependent signalling system, represents a promising target in this aspect. This review examines the molecular framework of quorum sensing across diverse microbial communities, its signalling cascades, and its role in regulating biofilm formation, efflux pump modulation and horizontal gene transfer with the quorum signalling. It further discusses quorum-sensing inhibition strategies, including natural products, synthetic compounds, quorum-quenching enzymes, and antibody-mediated and vaccine-mediated approaches. Application of CRISPR/Cas, engineered probiotic strains and nanocarrier-mediated delivery systems for quorum signalling disruption has been addressed. A key strength of this review is that it is the first to combine the underlying molecular mechanisms of quorum sensing with future translational tools such as artificial intelligence, CRISPR, engineered probiotics and nanotechnology to develop next-generation anti-virulence solutions for drug-resistant infection. While the preclinical findings have several challenges specific to the specificity and pharmacokinetic properties, strategies to resolve these considerations have been discussed. Overall, quorum signalling as a target is a major paradigm shift that may offer sustainable antimicrobial therapy to fight the global antimicrobial resistance issue. This can be achieved through a multidisciplinary approach to optimise antimicrobial therapy beyond the traditional concept of "killing".},
}

@article {pmid42569449,
year = {2026},
author = {Choi, Y and Yang, J and Kim, J and Enkhtaivan, K and Son, J and Jang, J and Lee, HY and Choi, J},
title = {Exosome-Based Liquid Biopsy in Biliary Tract Cancer: Nanotechnology-Enabled Strategies and Future Perspectives.},
journal = {International journal of nanomedicine},
volume = {21},
number = {},
pages = {613686},
pmid = {42569449},
issn = {1178-2013},
mesh = {Humans ; *Exosomes/chemistry ; *Biliary Tract Neoplasms/pathology/diagnosis ; Liquid Biopsy/methods ; *Nanotechnology/methods ; Biomarkers, Tumor/analysis ; Nanomedicine ; Biosensing Techniques ; },
abstract = {Biliary tract cancer (BTC) remains a formidable clinical challenge owing to its asymptomatic early stages, anatomical complexity, and lack of reliable and noninvasive diagnostic tools. Although traditional tissue biopsy is often limited by invasiveness and sampling bias, liquid biopsy, particularly the analysis of tumor-derived exosomes, has emerged as a promising and clinically relevant alternative for early detection and longitudinal monitoring. Exosomes are specialized extracellular vesicles that sequester diverse molecular cargo including proteins, lipids, and nucleic acids, thereby reflecting the physiological state of their parental tumor cells. However, the clinical translation of exosomal biomarkers is often hindered by technical challenges in achieving high-purity isolation and ultrasensitive detection in complex biological matrices, such as blood and bile. To address these limitations, this review provides a comprehensive overview of recent advancements in nanotechnology-enabled platforms designed to overcome these challenges. First, we examined sophisticated nanostructured systems, such as immuno-magnetic nanoparticles and microfluidic nanoVelcro chips, for high-yield exosome enrichment. Subsequently, we investigated next-generation biosensing modalities with a focus on surface-enhanced Raman scattering for label-free molecular fingerprinting and CRISPR-Cas-integrated nanosensors for amplification-free nucleic acid detection. Significant emphasis has now been placed on the integration of artificial intelligence and deep learning algorithms, which have become indispensable for deciphering complex exosomal signatures to differentiate BTC from benign conditions such as cholangitis. Finally, we discuss the emerging clinical significance of bile-derived exosomes and remaining challenges in standardizing nanomedicine-based liquid biopsies for precision oncology. These integrated platforms could potentially redefine the BTC management paradigm by bridging the gap between advanced nanomaterials and clinical diagnostics.},
}

Humans
*Exosomes/chemistry
*Biliary Tract Neoplasms/pathology/diagnosis
Liquid Biopsy/methods
*Nanotechnology/methods
Biomarkers, Tumor/analysis
Nanomedicine
Biosensing Techniques

@article {pmid42296510,
year = {2026},
author = {Zhang, ZM and Xu, C and Zhu, Y and Wei, Y and Pei, Y and Zhang, H and Pang, J and Xie, ZJ and Xu, K and Wang, J},
title = {Integrated CRISPR-Cas12a-Based Biosensors with Subwavelength Grating Microring Resonators for Ultrasensitive Mutation-Specific Detection.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5674-5682},
doi = {10.1021/acssensors.6c00141},
pmid = {42296510},
issn = {2379-3694},
support = {62422503//National Natural Science Foundation of China/ ; U21A20454//National Natural Science Foundation of China/ ; 2022A0505030024//Natural Science Foundation of Guangdong Province/ ; 2022A1515110756//Natural Science Foundation of Guangdong Province/ ; 2022B1515020093//Natural Science Foundation of Guangdong Province/ ; 2022B1515120012//Natural Science Foundation of Guangdong Province/ ; GJHZ20220913143207014//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20220818102406013//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20220818102618040//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20241202130558075//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; RCYX20210609103707009//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; RCYX20221008092907027//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; 2022YFB3207200//National Key Research and Development Program of China/ ; 2022B1515020057//Science Fund for Distinguished Young Scholars of Guangdong Province/ ; },
mesh = {*Biosensing Techniques/methods/instrumentation ; *CRISPR-Cas Systems/genetics ; Mutation ; Gold/chemistry ; *SARS-CoV-2/genetics/isolation & purification ; Metal Nanoparticles/chemistry ; Humans ; *CRISPR-Associated Proteins ; DNA Probes/chemistry/genetics ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; },
abstract = {The rapid and precise detection of nucleic acids is critical for identifying viral mutations, yet it presents formidable difficulties for conventional diagnostics. While established techniques such as quantitative polymerase chain reaction and next-generation sequencing involve complex workflows, emerging on-chip integrated photonic biosensing techniques are often limited by inadequate specificity and sensitivity. Here, we introduce an integrated photonic biosensing platform that synergizes the programmable recognition of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, with the superior sensitivity of subwavelength grating microring resonators. The sensor surface is functionalized with single-stranded DNA probes conjugated to gold nanoparticles. Upon target recognition, activated Cas12a cleaves the probes, releasing the nanoparticles and generating a quantifiable resonance wavelength shift. In particular, the spectral response gets further amplified by a resonance-enhanced photothermal effect. The detection of SARS-CoV-2 variants enables discrimination between wild-type, Delta, and Omicron strains. The extracted detection limit of 0.7 fM represents a four-order-of-magnitude improvement over conventional fluorescence-based CRISPR assays. Our work establishes a generalizable platform for ultrasensitive, mutation-resolved molecular diagnostics on a CMOS-compatible photonic chip, paving the way for advanced point-of-care testing and genomic surveillance.},
}

*Biosensing Techniques/methods/instrumentation
*CRISPR-Cas Systems/genetics
Mutation
Gold/chemistry
*SARS-CoV-2/genetics/isolation & purification
Metal Nanoparticles/chemistry
Humans
*CRISPR-Associated Proteins
DNA Probes/chemistry/genetics
*Endodeoxyribonucleases/genetics
Bacterial Proteins

@article {pmid42391661,
year = {2026},
author = {Zheng, C and Nong, L and Luo, J and Liang, L and Wei, Y and Luo, Q and Zhang, K and Liao, X},
title = {An electrochemiluminescence biosensor based on the hairpin-mediated exponential amplification and CRISPR/Cas12a amplification for ultrasensitive detection of MMP-2.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {267},
number = {},
pages = {115948},
doi = {10.1016/j.colsurfb.2026.115948},
pmid = {42391661},
issn = {1873-4367},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Matrix Metalloproteinase 2/analysis ; *Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; Humans ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Electrodes ; *Nucleic Acid Amplification Techniques/methods ; Inverted Repeat Sequences ; Limit of Detection ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases ; },
abstract = {In this study, we report a novel electrochemiluminescence (ECL) biosensor for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), an important biomarker associated with tumor invasion and metastasis. The biosensor integrates hairpin-mediated exponential amplification with CRISPR/Cas12a-based trans-cleavage for dual-stage signal amplification. In this design, MMP-2 specifically cleaves a peptide sequence (GPLG↓VRGK) on the DNA hairpin probe (HP1), releasing an initiator peptide nucleic acid (PNA) that triggers hairpin-mediated exponential amplification reaction. The amplified DNA products then activate the Cas12a/gRNA complex, which induces collateral cleavage of ferrocene (Fc)-labeled probes immobilized on a DNA tetrahedron-modified PEI-Ti3C2Tx/Ru/AuNPs electrode, thereby generating a strong ECL response. The incorporation of the DNA tetrahedron nanostructure provides a well-defined three-dimensional framework that ensures ordered probe orientation, enhanced hybridization efficiency, and reduced steric hindrance on the electrode surface. This structural organization significantly improves electron transfer and signal stability compared with conventional planar immobilization. Under optimized conditions, the biosensor exhibited a broad linear range from 0.01 fM to 10 nM and an ultralow detection limit of 10 aM. It displayed high specificity against interfering proteins (thrombin, IgG, BSA, lysozyme), excellent stability, and satisfactory recoveries (96.9%-105.0%) in LO2 cell culture supernatants. Overall, this enzyme-responsive, DNA-tetrahedron-assisted, CRISPR-amplified ECL biosensor represents a robust and versatile platform for precise and rapid detection of protease activity, showing great promise for biomedical diagnostics and clinical biomarker monitoring.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Matrix Metalloproteinase 2/analysis
*Luminescent Measurements/methods
*Electrochemical Techniques/methods
Humans
Gold/chemistry
Metal Nanoparticles/chemistry
Electrodes
*Nucleic Acid Amplification Techniques/methods
Inverted Repeat Sequences
Limit of Detection
*Bacterial Proteins/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
Endodeoxyribonucleases

@article {pmid42399104,
year = {2026},
author = {Liu, Z and He, Y and Lin, X and Pal, T and Pfeilsticker, A and Liu, BM and Chen, J},
title = {Indiscriminate Trans-Cleavage Activity of CRISPR/SuCas12a2 Enables Sensitive Detection of SARS-CoV-2.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5397-5403},
pmid = {42399104},
issn = {2379-3694},
support = {R35 GM147069/GM/NIGMS NIH HHS/United States ; R35GM147069/GM/NIGMS NIH HHS/United States ; },
mesh = {*SARS-CoV-2/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *COVID-19/diagnosis/virology ; *CRISPR-Associated Proteins/metabolism/genetics ; RNA, Viral/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; *COVID-19 Nucleic Acid Testing/methods ; Endodeoxyribonucleases/metabolism/genetics ; Fluorescent Dyes/chemistry ; Rapid Diagnostic Tests ; },
abstract = {Sensitive detection of SARS‑CoV‑2 remains critical for controlling COVID‑19 outbreaks and guiding patient care. Although reverse transcription-polymerase chain reaction (RT‑PCR), the gold standard for detecting SARS-CoV-2, is highly sensitive, the need for specialized equipment and trained personnel limits its widespread application in low or middle-resource settings. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology could overcome these limitations by providing simplicity, low cost, and high specificity. However, current CRISPR-based diagnostics can simultaneously cleave the target and fluorescence probes, as they are the same nucleic acid type (ssDNA or ssRNA), thereby reducing detection sensitivity. Herein, we developed a novel CRISPR-based viral detection method using SuCas12a2 (Cas12a2 from Sulfuricurvum sp. PC08-66), which harnesses its unique broad trans-cleavage activity and offers flexibility in selecting fluorescence probes. Using the conserved SARS‑CoV‑2 envelope gene as the model analyte, the analytical performance of the CRISPR/SuCas12a2 system for viral detection was evaluated. The CRISPR/SuCas12a2 detection workflow achieved a detection limit of 5 × 103 copies/μL for SARS-CoV-2 viral RNA. When detecting nasopharyngeal swab samples from patients, the CRISPR/SuCas12a2 system showed preliminary agreement with RT-qPCR in a set of clinical samples. Our CRISPR/SuCas12a2 system provides a flexible detection platform with simplified probe selection and enhanced compatibility, offering new insights into future portable diagnostic applications and enhancing global public health surveillance.},
}

*SARS-CoV-2/genetics/isolation & purification
Humans
*CRISPR-Cas Systems
*COVID-19/diagnosis/virology
*CRISPR-Associated Proteins/metabolism/genetics
RNA, Viral/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins/genetics/metabolism
*COVID-19 Nucleic Acid Testing/methods
Endodeoxyribonucleases/metabolism/genetics
Fluorescent Dyes/chemistry
Rapid Diagnostic Tests

@article {pmid42402044,
year = {2026},
author = {Kim, T and Scheeres, EC and Fiebig, A and Olive, AJ and Crosson, S},
title = {A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells.},
journal = {Infection and immunity},
volume = {94},
number = {8},
pages = {e0011726},
doi = {10.1128/iai.00117-26},
pmid = {42402044},
issn = {1098-5522},
support = {R01AI177619/NH/NIH HHS/United States ; R35GM146795/NH/NIH HHS/United States ; },
mesh = {Humans ; *Macrophages/microbiology/metabolism ; *Brucellosis/genetics/microbiology ; *Host-Pathogen Interactions/genetics ; *CRISPR-Cas Systems ; *Brucella ; Brucella abortus ; Cell Line ; },
abstract = {Brucella spp. are widespread intracellular animal pathogens that cause brucellosis, a significant zoonosis. Despite the global impact of brucellosis on animal and human health, the host genes that support Brucella infection remain incompletely defined. To address this knowledge gap, we developed a flow cytometry-based infection assay with fluorescent Brucella and performed a genome-wide CRISPR-Cas9 loss-of-function screen in human macrophage-like cells. Disruption of >150 host genes significantly reduced intracellular B. abortus signal at 3 hours post-infection. In addition to recovering known host factors, the screen revealed previously unappreciated genes linked to endosomal trafficking, cytoskeletal remodeling, and lipid homeostasis. The screen was robust, as validation within these functional categories confirmed that the small GTPase RAB14, the Src-family kinase regulator CSK, and the phospholipid flippase subunit TMEM30A support the B. abortus and B. ovis infection process at a post-entry step. Gene set enrichment analysis further identified positive regulators of mTORC1 signaling as host factors. This result was validated by genetic disruption of LAMTOR2 and AKT1, and pharmacologic inhibition of AKT1. Together, these data indicate that the AKT-Ragulator-mTORC1 axis contributes to establishing a permissive intracellular niche. Finally, to assess whether these host requirements extend beyond Brucella, we examined infection by the unrelated intracellular pathogen Mycobacterium abscessus. CSK, AKT1, and LAMTOR2 were required for efficient M. abscessus infection, whereas RAB14 was dispensable. Together, these results define host genes that impact Brucella infection and distinguish shared versus pathogen-specific host dependencies exploited by intracellular bacteria.},
}

Humans
*Macrophages/microbiology/metabolism
*Brucellosis/genetics/microbiology
*Host-Pathogen Interactions/genetics
*CRISPR-Cas Systems
*Brucella
Brucella abortus
Cell Line

@article {pmid42412687,
year = {2026},
author = {Pan, Y and Yang, Z},
title = {Paper Microfluidic Platform Using Multiplexed Isothermal Amplification and CRISPR/Cas12a for Aquatic Pathogen Detection.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5893-5908},
doi = {10.1021/acssensors.6c00719},
pmid = {42412687},
issn = {2379-3694},
support = {RL-2022-041//Leverhulme Trust/ ; FF\1920\1\36//Royal Academy of Engineering/ ; NE/R013349/2//National Centre for Earth Observation/ ; NE/V010441/1//National Centre for Earth Observation/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods/instrumentation ; *Paper ; *CRISPR-Cas Systems/genetics ; *Water Microbiology ; *Bacteria/isolation & purification/genetics ; *Microfluidic Analytical Techniques/instrumentation/methods ; Rapid Diagnostic Tests ; Colorimetry ; Limit of Detection ; Molecular Diagnostic Techniques ; },
abstract = {The global health threat posed by microbial contamination of aquatic systems demands feasible pathogen monitoring solutions. However, current detection methods are limited by expensive instrumentation and specialized personnel, which hinders their application in point-of-care testing (POCT). Here, we presented an integrated paper microfluidic platform for spatially multiplexed detection of pathogenic bacteria, including Salmonella, E. coli, C. perfringens, B. cereus, V. parahaemolyticus, S. aureus, and L. monocytogenes, selected due to their epidemiological significance and regulatory relevance in environmental and food safety monitoring. LAMP, RAA-CRISPR, and RPA-CRISPR assays were housed within physically isolated reaction chambers on two-layer chips. An engineered horseradish peroxidase (HRP) cascade-coupled crRNA modification system with DNA-conjugated labels was designed for colorimetric detection. Operation was enabled by solar-powered and portable hardware for incubation and imaging, coupled with a web application for quantitative analysis. Exceptional analytical performance was demonstrated, achieving an LOD of 1 CFU/mL, a dynamic range of 1-107 CFU/mL, high reproducibility (CV <5%), low batch-to-batch variation (<6%), low cost (£2.5 per test), and scalable integration, with a sample-to-answer time of 60 min. Successful field validation in diverse aquatic environments confirmed its practical feasibility, consistent with gold standard PCR (R2 = 0.98). This platform offers a promising POCT solution for public health protection and epidemic monitoring, particularly in resource-limited settings.},
}

*Nucleic Acid Amplification Techniques/methods/instrumentation
*Paper
*CRISPR-Cas Systems/genetics
*Water Microbiology
*Bacteria/isolation & purification/genetics
*Microfluidic Analytical Techniques/instrumentation/methods
Rapid Diagnostic Tests
Colorimetry
Limit of Detection
Molecular Diagnostic Techniques

@article {pmid42425083,
year = {2026},
author = {Nair, U and Akauliya, M and Warner, JE and Parikh, S and Ronsard, L and Ssozi, M and Rays Wahba, L and Esposito, AG and Kelley, B and McCarthy, C and Weldon, SR and Wu, L and Lingwood, D and Batista, FD},
title = {CRISPR-mediated precise large fragment insertion in zygotes enables rapid generation of humanized immunoglobulin heavy-chain mice.},
journal = {Immunity},
volume = {59},
number = {8},
pages = {2334-2350.e8},
pmid = {42425083},
issn = {1097-4180},
support = {R01 AI153098/AI/NIAID NIH HHS/United States ; R01 AI155447/AI/NIAID NIH HHS/United States ; INV-085294/GATES/Gates Foundation/United States ; R01 AI195539/AI/NIAID NIH HHS/United States ; R01 AI168114/AI/NIAID NIH HHS/United States ; INV-046626/GATES/Gates Foundation/United States ; },
mesh = {Animals ; Humans ; Mice ; *Immunoglobulin Heavy Chains/genetics ; *CRISPR-Cas Systems/genetics ; *Zygote ; Chromosomes, Artificial, Bacterial/genetics ; B-Lymphocytes/immunology ; Immunoglobulin Variable Region/genetics ; Mice, Transgenic ; V(D)J Recombination ; Somatic Hypermutation, Immunoglobulin ; },
abstract = {Current CRISPR-Cas9 methods are restricted to small genomic edits. We developed a CRISPR-guided approach that enables direct insertion of large genomic sequences into mouse zygotes. We deleted the murine 2.4-Mb immunoglobulin heavy-chain (IgH) variable (VH) locus and then precisely inserted a bacterial artificial chromosome (BAC) containing a 155-kb human VH DNA fragment flanked by 20-kb homology arms. Full-length, single-copy BAC integration occurred without ectopic recombination. Human sequences were stably transmitted and expressed VH segments that recombined with endogenous mouse sequences, and mice exhibited normal B cell development. Upon immunization, human VH-expressing B cells underwent class-switch recombination and somatic hypermutation, secreting antigen-specific antibodies. We demonstrated modular IgH humanization by replacing endogenous mouse diversity and joining (DH-JH) segments with human sequences, producing V(D)J recombination and diverse antibodies. Unlike traditional methods requiring more than a year, this approach enables the generation and validation of mice carrying large genomic insertions within 8 weeks.},
}

Animals
Humans
Mice
*Immunoglobulin Heavy Chains/genetics
*CRISPR-Cas Systems/genetics
*Zygote
Chromosomes, Artificial, Bacterial/genetics
B-Lymphocytes/immunology
Immunoglobulin Variable Region/genetics
Mice, Transgenic
V(D)J Recombination
Somatic Hypermutation, Immunoglobulin

@article {pmid42433170,
year = {2026},
author = {Qiu, Z and Chen, J and Wu, J and Wang, J and Hu, Y and Feng, W and Zhao, J and Minamoto, T and Chen, X and Zhou, L and Liu, M and Wang, C and Lin, M and Hu, M and Zhou, X and Wang, J},
title = {Optical-Controlled One-Pot RPA-CRISPR Assay for Environmental DNA Detection of a Critically Endangered Species.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5553-5565},
doi = {10.1021/acssensors.5c04638},
pmid = {42433170},
issn = {2379-3694},
support = {ZJZX-06//Ministry of Agriculture and Rural Affairs of the People's Republic of China/ ; },
mesh = {*Endangered Species ; *DNA, Environmental/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Recombinases/metabolism ; Animals ; },
abstract = {Developing a rapid, sensitive, and field-deployable assay for on-site environmental DNA (eDNA) detection of endangered species is crucial, as current PCR-based assays are slow and expensive and rely on laboratory-based thermal cycling. Here, we adapted a previously reported optically controlled one-pot RPA-CRISPR-Cas12a (OORC) assay to detect eDNA of the critically endangered Bahaba taipingensis. The assay combines isothermal recombinase polymerase amplification (RPA) with the high specificity of a CRISPR-Cas12a trans-cleavage reaction in a single tube. The entire process is operated at a constant temperature, avoiding thermal cycling. Low-template replicate experiments conservatively redefined the OORC limit of detection as 6 copies/reaction. To support near-field application, we further integrated the assay with a portable handheld fluorescence detector capable of 365 nm photoactivation and fluorescence readout. This portable and highly sensitive workflow extends the potential of eDNA monitoring, offering a practical tool for the conservation of endangered species.},
}

*Endangered Species
*DNA, Environmental/analysis/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
Recombinases/metabolism
Animals

@article {pmid42441514,
year = {2026},
author = {Wang, X and Zhao, S and Jiang, J and Wang, H and Encarnación, C and Zheng, L and Zhang, L},
title = {5' Dual-Overhang Short PAM-less dsDNA as Switchable Activators of Cas12a trans-Cleavage for Amplification-Free miRNA Detection.},
journal = {ACS sensors},
volume = {11},
number = {7},
pages = {5753-5764},
doi = {10.1021/acssensors.6c00524},
pmid = {42441514},
issn = {2379-3694},
support = {24DX2800100//Science and Technology Innovation Plan Of Shanghai Science and Technology Commission/ ; },
mesh = {*MicroRNAs/blood/analysis/genetics ; Humans ; *DNA/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry ; CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; },
abstract = {Precise and programmable regulation of CRISPR-Cas12a activity is essential for advancing controllable nucleic acid diagnostics, yet the structural determinants governing Cas12a activation by short PAM-less double-stranded DNA (dsDNA) remain largely unexplored. This study systematically investigates the effects of the terminal architectures of short PAM-less dsDNA on Cas12a trans-cleavage activity. By profiling a series of dsDNA constructs bearing distinct 5'/3' overhang configurations, a 5' dual-overhang motif was identified as a highly effective structural inhibitor that suppresses Cas12a activation. Kinetic fluorescence assays combined with computational structural modeling indicated that this inhibition arises from steric constraints imposed by the 5' terminal architecture. Leveraging this structure-guided regulatory mechanism, an amplification-free CRISPR-Cas12a assay was developed for the direct detection of oncogenic microRNAs miR-155 and miR-21, achieving femtomolar sensitivity without reverse transcription. The assay was further evaluated in human serum samples spiked with target miRNAs, supporting its proof-of-concept performance in a more complex matrix. Collectively, these findings highlight the potential of terminally engineered PAM-less dsDNA as a structural handle for programming Cas12a activity and provide useful insight for the design of CRISPR-based biosensing strategies.},
}

*MicroRNAs/blood/analysis/genetics
Humans
*DNA/chemistry/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/chemistry
CRISPR-Cas Systems
*Bacterial Proteins/metabolism/chemistry
*Endodeoxyribonucleases/metabolism/chemistry

@article {pmid42462715,
year = {2026},
author = {Zhu, Y and Wang, Q and Cao, Y and Hou, L and Yang, L and Cheng, C and Ye, S and Li, W and Zhang, J},
title = {Single cell CRISPR screen identifies antagonism between Nsd1-H3K36me2 and Ezh2-H3K27me3 orchestrates pluripotency transition.},
journal = {Stem cell reports},
volume = {21},
number = {8},
pages = {103016},
doi = {10.1016/j.stemcr.2026.103016},
pmid = {42462715},
issn = {2213-6711},
mesh = {Animals ; *Enhancer of Zeste Homolog 2 Protein/metabolism/genetics ; Mice ; *Histones/metabolism/genetics ; *Histone-Lysine N-Methyltransferase/metabolism/genetics ; Mouse Embryonic Stem Cells/metabolism/cytology ; Epigenesis, Genetic ; DNA Methylation ; CRISPR-Cas Systems ; *Single-Cell Analysis/methods ; DNA Methyltransferase 3A ; DNA (Cytosine-5-)-Methyltransferase 1/genetics/metabolism ; Cell Differentiation/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; *Intracellular Signaling Peptides and Proteins/metabolism/genetics ; },
abstract = {The transcriptional and epigenetic landscape imposes constraints on the self-renewal capacity and lineage specification potential of both naive and primed mouse embryonic stem cells (mESCs). CRISPR/Cas9-based functional screening coupled with single-cell RNA-seq (CROP-seq) establishes relationships between gRNA-mediated knockout genotype and transcriptome phenotype, providing a powerful tool to dissect gene regulatory networks. Here, we employed CROP-seq to investigate the epigenetic regulation governing the pluripotency network in mESCs. This highly sensitive method identified key genes essential for the acquisition and exit from pluripotency, and revealed a novel role for H3K36me2 in modulating DNA methylation through regulating the expression of Dnmt1 and Dnmt3a. Specifically, loss of Nsd1-mediated H3K36me2 delayed naive state exit, whereas Ezh2 deficiency accelerated primed entry. Collectively, our findings identify an epigenetic regulatory network critical for determining mESCs' pluripotent state transitions.},
}

Animals
*Enhancer of Zeste Homolog 2 Protein/metabolism/genetics
Mice
*Histones/metabolism/genetics
*Histone-Lysine N-Methyltransferase/metabolism/genetics
Mouse Embryonic Stem Cells/metabolism/cytology
Epigenesis, Genetic
DNA Methylation
CRISPR-Cas Systems
*Single-Cell Analysis/methods
DNA Methyltransferase 3A
DNA (Cytosine-5-)-Methyltransferase 1/genetics/metabolism
Cell Differentiation/genetics
*Pluripotent Stem Cells/metabolism/cytology
DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism
*Intracellular Signaling Peptides and Proteins/metabolism/genetics

@article {pmid42503780,
year = {2026},
author = {Shi, JY and Wu, SL and Tan, Y and Jiang, HX and Liu, SQ and Gan, XQ and Sun, WB and Li, MY and Li, KD and Tang, AN and Zhu, LN and Cai, QL and Kong, DM},
title = {DNA Nanowire-Assisted CRISPR/Cas12a Triple Cascade Amplification for Sensitive Detection of Myeloperoxidase Activity.},
journal = {Analytical chemistry},
volume = {98},
number = {31},
pages = {22931-22942},
doi = {10.1021/acs.analchem.6c02223},
pmid = {42503780},
issn = {1520-6882},
support = {22293030//National Natural Science Foundation of China/ ; 22474063//National Natural Science Foundation of China/ ; 72474154//National Natural Science Foundation of China/ ; 82570985//National Natural Science Foundation of China/ ; 25JCLZJC00250//Tianjin Municipal Science and Technology Program/ ; 25JCLZJC00640//Tianjin Municipal Science and Technology Program/ ; 23JCJQJC00080//Science Fund for Distinguished Young Scholars of Tianjin Municipality/ ; },
mesh = {*Peroxidase/metabolism/blood ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry/metabolism ; *Nanowires/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Myeloperoxidase (MPO) is an inflammation-associated heme enzyme implicated in cardiovascular oxidative stress, but sensitive activity-based detection in complex clinical samples remains challenging. Herein, we report a CRISPR/Cas12a-based triple-cascade amplification platform for rapid and sensitive MPO activity detection. The core sensing element consists of DNA nanowires containing multiple Cas12a activator strands, tethered to magnetic beads via biotin-labeled and phosphorothioate-modified linkers. In the chloride-containing assay system, MPO-catalyzed generation of HOCl oxidatively cleaves these linkers, releasing the nanowires and activating Cas12a, which, in turn, cleaves fluorescent reporters. This design integrates three amplification stages (MPO catalysis, multiactivator release, and Cas12a trans-cleavage), achieving ultrasensitive detection without additional nucleic acid amplification. Under the optimized chloride-containing conditions, the assay achieved a detection limit of 10.20 pg/mL for MPO. A preliminary pilot analysis using human serum samples from acute coronary syndrome patients and healthy individuals showed different signal distributions, supporting the feasibility of applying the platform to complex serum matrices, although contributions from eosinophil peroxidase/HOBr-mediated probe activation cannot be excluded. The platform is readily adaptable to lateral flow assays and portable fluorescence readouts, offering versatile formats for point-of-care-compatible analysis. This work provides a sensitive CRISPR/Cas12a-based strategy for MPO activity-related hypohalous oxidant assessment under defined assay conditions and demonstrates its preliminary applicability in complex serum matrices.},
}

*Peroxidase/metabolism/blood
Humans
*CRISPR-Cas Systems/genetics
*DNA/chemistry/metabolism
*Nanowires/chemistry
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Biosensing Techniques/methods
*CRISPR-Associated Proteins/metabolism
*Endodeoxyribonucleases/metabolism
*Bacterial Proteins/metabolism/genetics

@article {pmid42550765,
year = {2026},
author = {Torrance, R and Orf, K and White, N and Matti, C and McKenna, AJ and Cosentino, A and Casirati, G and Albuquerque, AS and Thrasher, AJ and Genovese, P and Booth, CA and Fox, TA and Burns, SO and Morris, EC},
title = {Functional restoration of immune defects in STAT1 gain-of-function disease following stem cell gene editing.},
journal = {Blood},
volume = {},
number = {},
pages = {},
doi = {10.1182/blood.2025032242},
pmid = {42550765},
issn = {1528-0020},
abstract = {Germline gain-of-function (GOF) mutations in the signal transducer and activator of transcription 1 (STAT1) gene cause a dominantly inherited inborn error of immunity (IEI) characterized by chronic mucocutaneous candidiasis, autoimmunity, severe opportunistic infections and an increased risk of malignancy. Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is curative but is associated with increased risk of morbidity and mortality in STAT1 GOF patients compared to other IEI. To develop a curative, autologous alternative to HSCT, we evaluated gene editing strategies in STAT1 GOF model cell lines, primary T cells, and patient-derived HSCs. Universal and mutation-specific strategies using CRISPR/Cas-mediated homology-directed repair (HDR) were limited by low efficacy (<25%), poor viability, and a lack of allele-specificity. In contrast, adenine base editing corrected the recurrent and highly pathogenic p.T385M mutation with upwards of 90% efficiency in patient T cells and HSCs without significant unintended on- or off-target genomic aberrations. Gene editing functionally restored total STAT1 expression (p<0.0217), STAT1 phosphorylation (p<0.0056), interferon-stimulated gene expression (OAS1; p=0.0005) and improved IL-17 production (p<0.0001). Edited HSCs retained multilineage differentiation capacity and sustained engraftment with persistence of the corrected allele at 16 weeks in humanized immunodeficient mice. These data demonstrate efficient and precise correction of STAT1 GOF mutations by base editing, with maintenance of the correction through long-term engraftment in vivo. This represents the first application of gene editing to correct a dominant gain-of-function mutation causing immunodeficiency, with potential applicability to other genetic disorders associated with heterozygous and gain-of-function mutations.},
}

@article {pmid42551559,
year = {2026},
author = {Khosrojerdi, M and Hashemi, SA and Besharati, R and Haghbin, A and Azimian, A},
title = {CRISPR-Cas systems as precision antimicrobials: Reversing the tide of antimicrobial resistance.},
journal = {Virus research},
volume = {371},
number = {},
pages = {199782},
doi = {10.1016/j.virusres.2026.199782},
pmid = {42551559},
issn = {1872-7492},
abstract = {Antimicrobial resistance (AMR) has escalated into a global health crisis, with resistant pathogens causing over 1.2 million direct deaths annually and threatening to render modern medicine unsustainable. This review provides a comprehensive and updated synthesis of CRISPR-Cas-based antimicrobial strategies with a unique focus on: (i) critical comparison with conventional antibiotics and emerging alternatives; (ii) quantitative evaluation of delivery platforms; (iii) novel strategies including AI-optimized guide design and the ATTACK-CreTA system; (iv) comprehensive analysis of ecological risks; and (v) technology readiness level assessments for clinical translation. The CRISPR-Cas system, originally discovered as a bacterial adaptive immune mechanism, has been repurposed as a programmable precision tool to combat AMR by selectively targeting and eliminating resistance genes. We systematically evaluate the mechanistic diversity of Cas effectors, from DNA-cleaving Cas9 and Cas3 to RNA-targeting Cas13, and their application in reversing resistance phenotypes in WHO priority pathogens. We critically assess emerging delivery platforms, including engineered bacteriophages, conjugative plasmids, nanoparticles, and outer membrane vesicles, quantitatively comparing their delivery efficiency, payload capacity, and biosafety profiles. Novel strategies such as CRISPR interference (CRISPRi) for gene silencing without genomic cleavage, the ATTACK-CreTA system for enhanced bactericidal activity, and AI-driven optimization of guide RNA design are examined with appropriate caveats. We comprehensively address clinical translation challenges including immunogenicity, pharmacokinetics/pharmacodynamics, manufacturing scalability, regulatory pathways, and bacterial resistance mechanisms including anti-CRISPR proteins. No CRISPR-based antimicrobial has yet received regulatory approval, and we critically evaluate the gap between proof-of-concept and clinical utility. A detailed roadmap for clinical development is proposed. By integrating recent advances in Cas protein engineering, delivery technologies, and diagnostic applications, this review positions CRISPR-Cas systems as next-generation precision therapeutics capable of both treating resistant infections and curtailing the spread of AMR across clinical and environmental settings.},
}

@article {pmid42554845,
year = {2026},
author = {Zubair, A and Hemal, MAKP and Ahmed, A and Ahmad, F and Waheed, Y and Afghan, N},
title = {CRISPR/Cas system as a novel therapeutic strategy to combat multi-drug-resistant bacteria.},
journal = {Archives of microbiology},
volume = {208},
number = {11},
pages = {},
pmid = {42554845},
issn = {1432-072X},
mesh = {*Drug Resistance, Multiple, Bacterial/genetics ; *CRISPR-Cas Systems ; *Bacteria/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; Humans ; *Bacterial Infections/therapy/microbiology ; Gene Editing/methods ; },
abstract = {Antimicrobial resistance (AMR) has diminished the effectiveness of present antibiotics, posing a huge threat to global community health and economic stability. This study investigates the CRISPR-Cas framework's potential as a cutting-edge tactic to fight antimicrobial resistance. Current applications, limitations, and prospective future uses are analyzed. CRISPR antimicrobial strategies, which bring together the latest developments in gene-targeting strategies, engineered delivery platforms, and translational applications to fight multidrug-resistant pathogens. CRISPR technology is different from traditional antimicrobial treatments that target general antimicrobial resistance genes, instead allowing targets to be eliminated specifically by sequence, while retaining beneficial microbial communities, which has the potential to be a transformative precision antimicrobial treatment. Nevertheless, there is still a need for optimization of delivery systems, specificity of targets, biosafety, and regulations to ensure successful clinical translation, especially given their amazing advances. Recent research confirms that CRISPR-based mechanisms also affect different bacterial species, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, playing a key function in averting the emergence of resistance genes in these bacteria. Changes to CRISPR loci affect how resistance genes are targeted in ESKAPE pathogens, and CRISPR-Cas9 successfully lowers resistance by focusing on genes like tetM and ermB. A promising application of CRISPR-Cas systems in combating antimicrobial resistance (AMR) is the precise targeting of plasmid-borne mcr-1 resistance genes and other mobile genetic elements that facilitate the dissemination of colistin resistance. But the efficiency of CRISPR-Cas is diminished in some bacterial strains due to variations in their CRISPR loci. Enhancing transformation approaches and minimizing off-target impacts are critical challenges to confirm the precision and safety of CRISPR-based mechanisms in therapeutic applications. Advances in these areas are likely to continue to enable the development of next-generation CRISPR therapeutics for the effective management of multidrug-resistant bacterial infections.},
}

*Drug Resistance, Multiple, Bacterial/genetics
*CRISPR-Cas Systems
*Bacteria/genetics/drug effects
Anti-Bacterial Agents/pharmacology
Humans
*Bacterial Infections/therapy/microbiology
Gene Editing/methods

@article {pmid42556039,
year = {2026},
author = {Li, H and Du, H and Xu, R and Xue, H and Liu, Y and Cao, Y and Guo, Z and Pan, J and Liu, Y},
title = {Point-of-care detection for respiratory diseases: From samples and biomarkers to principles and applications.},
journal = {Talanta},
volume = {312},
number = {Pt A},
pages = {130377},
doi = {10.1016/j.talanta.2026.130377},
pmid = {42556039},
issn = {1873-3573},
abstract = {Early screening can significantly reduce the severe morbidity and mortality of respiratory diseases and alleviate the burden on public healthcare systems. Point-of-care (POC) devices refer to portable instruments that meet the REASSURED criteria and can be conveniently used near the patient without professional laboratory conditions. POC devices played an important role in patient initiated early diagnosis during the COVID-19 pandemic, demonstrating great potential. From a macro-to-micro perspective, this review first comprehensively introduces clinically relevant sample types, including blood, respiratory tract and oral samples, and exhaled breath, along with the clinical significance of corresponding biomarkers (nucleic acids, proteins, gaseous molecules, extracellular vesicles, circulating tumor cells, and pathogen particles) and pre-processing methods. Subsequently, it summarizes the test principles and promising bioreceptors including base pairing-based systems (PCR, isothermal amplification, CRISPR/Cas), antibodies and antibody mimetics, and other affinity-based recognition elements, and innovatively presents portable integration platforms of biosensors from the perspective of bioreceptor compatibility. It then evaluates the application performance of transducers and corresponding optical or electrochemical portable detection devices, including SERS, e-nose, nanopore sensors, and portable GC-MS. Finally, the latest applications of computer technology and artificial intelligence tools in POC detection for respiratory diseases, spanning device design, bioreceptor screening, biomarker discovery, and diagnostic data processing, are presented by functional category. This review aims to provide a reference for the research and application of multiplex biomarker/sample/disease POC testing in respiratory diseases, and to offer perspectives on future directions for technological innovation, intelligentization, and commercial translation.},
}

@article {pmid42556347,
year = {2026},
author = {Zhou, Z and Saffarian-Deemyad, I and Shi, H and Weiss, T and Ur-Rehman, MM and Vohra, K and Skopintsev, P and Yoon, PH and Trinidad, MI and Langeberg, CJ and Kamalu, M and Amerasekera, J and Zhou, Y and Doherty, EE and Aris, KDP and Al-Sayyad, N and Thornton, BW and Weissman, RF and Wasko, KM and Esain-Garcia, I and DeTurk, EC and Savage, DF and Jacobsen, SE and Bryant, Z and Doudna, JA},
title = {Stepwise DNA-unwinding gates TnpB genome-editing activity.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2026.07.015},
pmid = {42556347},
issn = {1097-4164},
abstract = {TnpB is a compact RNA-guided endonuclease and an evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited, and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through a discrete, long-lived, partially unwound intermediate state before reaching a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, H4W-L304F-V305R (Ymu1-WFR), stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in plants. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.},
}

@article {pmid42557080,
year = {2026},
author = {Hu, YW and Zhang, Y and Ren, Y and Qin, H and Zhao, H},
title = {[Advances in phage therapy for pneumonia caused by Klebsiella pneumoniae].},
journal = {Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases},
volume = {49},
number = {8},
pages = {902-907},
doi = {10.3760/cma.j.cn112147-20251012-00632},
pmid = {42557080},
issn = {1001-0939},
support = {202304021301063//Shanxi Province scientific and technological achievements transformation guidance project/ ; 2023-190//Research Project Supported by Shanxi Scholarship Council of China/ ; },
mesh = {*Klebsiella pneumoniae/virology ; Humans ; *Phage Therapy/methods ; *Klebsiella Infections/therapy/microbiology ; *Pneumonia, Bacterial/therapy/microbiology ; Bacteriophages ; Animals ; Anti-Bacterial Agents/therapeutic use ; Drug Resistance, Multiple, Bacterial ; },
abstract = {Klebsiella pneumoniae (KP) has emerged as a formidable nosocomial pathogen in the era of antimicrobial resistance, with mortality from pneumonia caused by carbapenem-resistant strains exceeding 50%. Phage therapy has re-emerged as a promising alternative or adjunctive strategy for managing refractory KP infections. This review consolidates the current preclinical and clinical evidence base, outlines the molecular mechanisms of phage-host interactions, and appraises evolving therapeutic approaches. Preclinical investigations in murine pneumonia models have consistently demonstrated that intranasal or nebulization phage administration markedly reduces pulmonary bacterial burden, attenuates inflammatory lung injury, and improves survival, often exhibiting synergistic effects when combined with conventional antibiotics. Clinical case reports and small compassionate-use series have further provided preliminary yet compelling evidence supporting the safety and therapeutic promise of personalized phage formulations in critically ill patients with multidrug-resistant KP pneumonia who have exhausted standard treatment options. Mechanistically, phage tropism is mediated through the specific recognition of bacterial surface receptors-principally capsular polysaccharide and, to a lesser extent, lipopolysaccharide-by phage-encoded receptor-binding proteins, culminating in bacterial lysis. In response, KP has evolved a multilayered defensive arsenal encompassing receptor modification to impede adsorption, nucleic acid interference systems (e.g., CRISPR-Cas and restriction-modification), and abortive infection mechanisms that curtail phage propagation at the population level. To surmount the inherent limitations of narrow host range and the inevitable emergence of phage-resistant mutants, a suite of optimization strategies is under active refinement, including rationally designed phage cocktails, genetically engineered phages with extended tropism, artificial intelligence-assisted host-range prediction, and innovative delivery platforms such as hydrogel encapsulation to enhance pulmonary bioavailability. Despite ongoing challenges in mechanistic complexity, manufacturing standardization, and regulatory uncertainty, current initiatives- such as the establishment of geographically diverse phage libraries, real-time surveillance of phage resistance, and the development of phage-derived enzyme products-hold promise for establishing precision phage therapy as a viable and sustainable component of the antimicrobial stewardship armamentarium.},
}

*Klebsiella pneumoniae/virology
Humans
*Phage Therapy/methods
*Klebsiella Infections/therapy/microbiology
*Pneumonia, Bacterial/therapy/microbiology
Bacteriophages
Animals
Anti-Bacterial Agents/therapeutic use
Drug Resistance, Multiple, Bacterial

@article {pmid42557140,
year = {2026},
author = {Zhou, X and Wang, L and Peng, X and Liu, M and Li, Y and Gui, F and Zhou, L and Deng, Z},
title = {A light-activated one-pot ERA/CRISPR-Cas12a assay for cost-effective dual-mode detection of HBV DNA.},
journal = {Talanta},
volume = {},
number = {},
pages = {130329},
doi = {10.1016/j.talanta.2026.130329},
pmid = {42557140},
issn = {1873-3573},
abstract = {Light-activatable CRISPR-Cas systems offer an effective strategy to overcome the kinetic incompatibility in one-pot nucleic acid assays; however, their practical application remains limited by high reagent cost, the lack of compatible reaction buffers, and reliance on instrument-dependent readout. In this work, we developed a cost-effective light-controlled one-pot ERA/CRISPR-Cas12a platform for rapid hepatitis B virus (HBV) DNA detection. NPOM-caged crRNA was employed to temporarily suppress Cas12a activity during amplification and to trigger target-dependent trans-cleavage by a brief 30 s UV irradiation. By replacing RPA with ERA, the per-reaction reagent cost of the one-pot assay was reduced from approximately US$6.0 to US$2.0, corresponding to a reduction of approximately 66.7%, while maintaining comparable analytical performance. In addition, a PEG-free unified buffer was established through systematic optimization, enabling efficient integration of ERA and Cas12a reactions in a sealed single-tube format. The proposed platform provided dual-mode readout through fluorescence and lateral flow assay (LFA), with a limit of detection of 1 copy μL[-1] and a total assay time of 30 min. Clinical evaluation using 79 serum samples showed a sensitivity of 100% for fluorescence readout and 96.3% for LFA, with excellent agreement with qPCR (Cohen's κ = 0.94 - 1.00). Owing to its low cost, high sensitivity, and operational simplicity, this platform represents a promising tool for rapid HBV molecular diagnosis in both centralized laboratories and resource-limited settings.},
}

@article {pmid42133510,
year = {2026},
author = {Cimolato, C and Petrelli, S and Favaro, D and Frusteri Chiacchiera, A and Del Favero, S and Schenato, L and Pasotti, L and Bellato, M},
title = {Modeling of Conjugative- and Phage-Mediated CRISPR-Based System Against Antimicrobial Resistant Bacteria.},
journal = {IEEE transactions on bio-medical engineering},
volume = {73},
number = {8},
pages = {2961-2971},
doi = {10.1109/TBME.2026.3693402},
pmid = {42133510},
issn = {1558-2531},
mesh = {*Bacteriophages/genetics ; *Drug Resistance, Bacterial/genetics ; *CRISPR-Cas Systems/genetics ; *Models, Biological ; Computer Simulation ; *Bacteria/genetics/virology/drug effects ; *Phage Therapy/methods ; },
abstract = {OBJECTIVE: Antimicrobial resistance (AMR) poses a significant threat to global health by diminishing the effectiveness of conventional antibiotics. This study aims to assess, using a systems biology approach, a potential synthetic biology-based strategy that employs engineered conjugative probiotic bacteria and bacteriophages to combat AMR, examining the implications of implementing targeted gene silencing as an alternative to direct bacterial killing.

METHODS: A comprehensive mathematical model was developed to describe the dynamics of the delivery systems (engineered conjugative probiotic bacteria and engineered phages) and the antimicrobial actuators being studied (genome cutting via CRISPR systems and AMR-gene silencing through CRISPR interference), also compared to traditional phage therapy (selection of phages capable of killing pathogens through bacterial-specific viral infection). The target population includes antibiotic-resistant bacteria competing with other probiotic bacteria for colonizing the host environment. The model explicitly incorporates parameters for mutations that affect actuator functionality and simulates their impact on overall therapeutic performance.

RESULTS: Simulations show how variations in actuator efficiency and emergence of new mutations in target pathogens affect the long-term suppression of resistance genes. Including mutational effects provides insights into system robustness and guides optimal therapeutic design choices.

CONCLUSION: The proposed modeling framework effectively captures key biological and mechanistic aspects of engineered therapies, enabling the prediction and optimization of each intervention against resistant pathogens. It highlights engineered phages and CRISPR interference as the most promising candidates for the design of new engineered biological therapeutics.

SIGNIFICANCE: This study establishes a quantitative foundation for rational design and dosage optimization in engineered phage- and bacterial-based therapies, advancing the use of synthetic biology methods to fight antimicrobial resistance.},
}

*Bacteriophages/genetics
*Drug Resistance, Bacterial/genetics
*CRISPR-Cas Systems/genetics
*Models, Biological
Computer Simulation
*Bacteria/genetics/virology/drug effects
*Phage Therapy/methods

@article {pmid42405454,
year = {2026},
author = {Chen, Z and Wu, H and Chu, LT and Lin, J and Zhang, Y and Lin, X and Zeng, T},
title = {Aptamer-based CRISPR-Cas12a fluorescent biosensors for serum biomarker detection.},
journal = {The Analyst},
volume = {151},
number = {16},
pages = {4516-4534},
doi = {10.1039/d6an00123h},
pmid = {42405454},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/metabolism/genetics ; *CRISPR-Cas Systems ; Humans ; *Biomarkers/blood ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *Bacterial Proteins/genetics/chemistry/metabolism ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; Fluorescent Dyes/chemistry ; },
abstract = {The CRISPR-Cas12a system enables sensitive nucleic acid detection due to its programmability, trans-cleavage activity, and biocompatibility. To expand its applications beyond nucleic acid analysis, aptamers have emerged as ideal recognition elements owing to their high specificity, design flexibility, ease of modification and low cost. The integration of Cas12a with aptamers enables the conversion of target-binding signals into nucleic acid recognition signals, thereby combining molecular recognition with signal amplification for the detection of non-nucleic acid targets. This review provides a concise overview of the working mechanism and features of the Cas12a system, with particular emphasis on recent advances in Cas12a-aptamer-based fluorescent biosensors for serum biomarker detection. The advantages and limitations, current challenges, and future prospects are also discussed.},
}

*Biosensing Techniques/methods
*Aptamers, Nucleotide/chemistry/metabolism/genetics
*CRISPR-Cas Systems
Humans
*Biomarkers/blood
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
*Bacterial Proteins/genetics/chemistry/metabolism
*Endodeoxyribonucleases/genetics/metabolism/chemistry
Fluorescent Dyes/chemistry

@article {pmid42547606,
year = {2026},
author = {Akkoul, N and Kumar, T and Sharma, S and Sharma, V and Raja, V and Malik, TG and Wan Mohd Jaafar, WS and Gupta, AK and Hakeem, KR},
title = {CRISPR-Cas-based detection of Mycobacterium tuberculosis: current advances and translational bottlenecks.},
journal = {Protoplasma},
volume = {},
number = {},
pages = {},
pmid = {42547606},
issn = {1615-6102},
abstract = {Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health challenge due to persistent diagnostic gaps. CRISPR-Cas-based diagnostics have emerged as highly sensitive and programmable platforms for nucleic acid detection, enabling rapid identification of Mtb targets, including drug-resistance-associated mutations. These systems integrate isothermal amplification, diverse Cas effectors, and multiple signal readout strategies to achieve high analytical performance. This review provides a comparative analysis of clinically evaluated CRISPR-based TB diagnostic platforms, highlighting substantial variability in assay design, performance, and translational readiness. While many platforms demonstrate strong analytical sensitivity, their implementation remains constrained by workflow complexity and limited integration into true point-of-care formats. This highlights that successful clinical translation of CRISPR-based TB diagnostics is determined more by real-world adaptability than by analytical performance alone. The current review presents a comparative analysis of CRISPR-based diagnostic platforms for tuberculosis, evaluating the variability in assay design, analytical and clinical performance, and translational readiness across currently available systems.},
}

@article {pmid42548009,
year = {2026},
author = {Chen, SY and Yang, LH and Liang, ZQ and Li, JD and Li, SD and Deng, YL and Chen, GQ and Ling, JW and Zhou, SS and Chen, G and He, RQ},
title = {CRISPR Screen Reveals Pathways and Factors Driving Tyrosine Kinase Inhibitor Resistance in Hepatocellular Carcinoma.},
journal = {Cancer medicine},
volume = {15},
number = {8},
pages = {e72028},
pmid = {42548009},
issn = {2045-7634},
support = {2025GXNSFAA069130//Joint Project on Regional High-Incidence Diseases Research of Guangxi Natural Science Foundation/ ; NSFC82460783//The National Natural Science Foundation of China/ ; //Creative Research Development Grant from the First Affiliated Hospital of Guangxi Medical University/ ; S202410598060X//The China Undergraduate Innovation and Entrepreneurship Training Program/ ; YCBZ2025127//The Innovation Project of Guangxi Graduate Education/ ; },
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics/drug therapy/pathology/metabolism ; *Liver Neoplasms/genetics/drug therapy/pathology/metabolism ; *Drug Resistance, Neoplasm/genetics ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Signal Transduction ; },
abstract = {Tyrosine kinase inhibitor (TKI) resistance severely limits clinical outcomes in hepatocellular carcinoma (HCC), highlighting the urgent need to elucidate its underlying molecular mechanisms. In this study, an unbiased genome-wide CRISPR/Cas9 screening identified novel key factors related to the therapeutic responsiveness of TKI in HCC. By integrating data from 20 datasets encompassing 322 samples, a comprehensive TKI therapeutic response landscape for HCC was constructed. GO and Reactome enrichment analyses revealed that dysregulated RNA splicing, ubiquitination, endocytosis/exocytosis, and cell cycle pathways modulate TKI sensitivity, with close links to antitumor immunity. This study identified GPATCH4, CCT3, C19orf53, UACA, PPM1M, and LIN37 as key genes mediating TKI resistance in HCC. These six genes were found to be highly expressed in HCC and significantly associated with HCC patient prognosis. Drug sensitivity assays identified a significant association between their expression and responsiveness to TKI agents. In-house quantitative real-time PCR validated their differential expression levels in normal hepatocytes, parental HCC cells, and TKI-resistant HCC sublines. ssGSEA, TIMER2, and ESTIMATE analysis revealed that their expression modulates HCC immune infiltration. Bibliometric analysis revealed a growing focus on immunotherapy-based combination regimens to overcome TKI resistance. Ferroptosis, epithelial-mesenchymal transition and hypoxia were new research directions, which were closely related to the pathways investigated in this study. In conclusion, this study identified RNA splicing, ubiquitination, endocytosis/exocytosis, and cell cycle pathways, as well as GPATCH4, CCT3, C19orf53, UACA, PPM1M, and LIN37, as novel directions and targets for TKI-immunotherapy combination strategies, providing new insights for overcoming TKI resistance in HCC.},
}

Humans
*Carcinoma, Hepatocellular/genetics/drug therapy/pathology/metabolism
*Liver Neoplasms/genetics/drug therapy/pathology/metabolism
*Drug Resistance, Neoplasm/genetics
*Protein Kinase Inhibitors/pharmacology/therapeutic use
Gene Expression Regulation, Neoplastic
*CRISPR-Cas Systems
Cell Line, Tumor
Signal Transduction

@article {pmid42549139,
year = {2026},
author = {Pytlik, D and Gerovac, M and Bischler, T and Schlosser, A and Vogel, J and Schoen, C},
title = {The CRISPR/Cas-associated scaRNA modulates efeUOB expression and stress responses in Neisseria meningitidis.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqag027},
pmid = {42549139},
issn = {2633-6693},
abstract = {Neisseria meningitidis is a human-adapted commensal pathogen that must continuously balance nutrient acquisition with stress tolerance. Here, we identify a type II-C CRISPR/Cas-associated small RNA (scaRNA) as a posttranscriptional regulator of the efeUOB operon and oxidative stress responses. Using in vitro RNA binding and structure probing assays, we show that the scaRNA interacts with the 5' untranslated region of efeO mRNA, leading to reduced translation of this component of the ferrous iron transporter EfeUOB. Consistent with this, efeO translational fusions demonstrate repression by the scaRNA, whereas a ΔscaRNA mutant shows increased reporter expression. We further show that meningococcal Cas9 (Nme1Cas9) is able to cleave scaRNA in vitro, but in vivo phenotypes are primarily scaRNA-dependent, indicating that Nme1Cas9 contributes, at most, indirectly to this regulation. In line with this observation, comparative proteomics revealed overlapping but distinct roles of scaRNA and Nme1Cas9 in oxidative stress adaptation, energy metabolism, and ion transport. While steady-state protein abundances did not capture all scaRNA-dependent effects, functional assays confirmed that scaRNA inactivation reduces survival under oxidative stress. Together, our results identify scaRNA-mediated repression of efeO as a novel posttranscriptional mechanism that contributes to stress adaptation in meningococci. These findings expand the functional repertoire of CRISPR-associated elements and suggest a role for small RNA-based regulation in iron-related stress adaptation in a major human pathogen.},
}

@article {pmid42549572,
year = {2026},
author = {Iwe, IA and Liu, FX and Corsano, A and da Silva, SJR and Doucet, J and Singh, S and Lamothe, G and Zayani, R and Nguyen, J and Matthews, Q and Vigar, JRJ and Bayat, P and Simchi, M and Bozovicar, K and Charania, M and Panfilov, S and Kelly, P and Cai, R and Hubbard, BP and Li, X and Mazzulli, T and Tremblay, JP and Zhao, Y and Green, AA and Li, Z and Yao, S and Pardee, K},
title = {RAPID: evaluation of Cas12a protospacer nicking and chimeric reporters for PAM-independent RNA and DNA diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42549572},
issn = {1362-4962},
support = {//Precision Medicine Initiative/ ; PRMUHT2024-001//Canadian Institute of Health Research/ ; 202410MFE-531769-419793//Canadian Institute of Health Research/ ; //Ontario Graduate Scholarship/ ; 201610FDN-375469//CIHR Foundation/ ; 950-231075//Canada Research Chairs Program/ ; 950-233107//Canada Research Chairs Program/ ; RGPIN-2016-06352//NSERC Discovery/ ; N66001-23-2-4042//Defense Advanced Research Projects Agency/ ; PJT-189974//CIHR/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *RNA/analysis/genetics ; *DNA/analysis/genetics ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Genes, Reporter ; },
abstract = {CRISPR-Cas nucleases have revolutionized diagnostics and biotechnology by providing programmable specificity. Here, we extend the understanding of Cas12a biology with a screen that, unexpectedly, finds that Cas12a trans-cleavage activity can be modulated by nicks in the protospacer in a position-dependent manner. Wanting to explore the impact of non-conventional trans-cleavage substrates, we subsequently find that non-specific Cas12a cleavage can be significantly reduced with RNA and chimeric (mixed RNA/DNA) reporter sequences. Exploiting these features and building on emerging protospacer adjacent motif (PAM)-independent Cas12a diagnostics that use engineered DNA activators and split-guide architectures, we introduce RAPID (RNA/DNA Advanced chimeric, PAM-independent, Integrated Nicking, Diagnostics), a nick-tuned, PAM-duplex-mediated platform for PAM-independent RNA and DNA detection. By strategically introducing a nick within the spacer region, RAPID expands Cas12a detection to include target RNAs, which can be ligated in situ to create a hybrid protospacer-target with trans-cleavage activity matching conventional Cas12a. We then apply RAPID to detect single-point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems. In combination with RT-LAMP, RAPID is used for PAM-independent RNA detection in clinical samples, achieving sensitivity down to ∼1 aM and 100% concordance with RT-qPCR for samples with Ct ≤ 33.},
}

*CRISPR-Associated Proteins/metabolism/genetics
*RNA/analysis/genetics
*DNA/analysis/genetics
*CRISPR-Cas Systems
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
Genes, Reporter

@article {pmid42549575,
year = {2026},
author = {Li, Y and Han, P and Yuan, R and Bai, L and Qing, M},
title = {Cas-regulation-targeting chimera enables selective and tunable control of CRISPR/Cas12a.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42549575},
issn = {1362-4962},
support = {82072378//National Natural Science Foundation of China/ ; 22504009//National Natural Science Foundation of China/ ; CSTB2025NSCQ-LZX0126//Natural Science Foundation of Chongqing/ ; CSTB2025NSCQ-GPX0375//Natural Science Foundation of Chongqing/ ; CQYC202005015//Chongqing Talents: Exceptional Young Talents Project, China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Selective and tunable regulation of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a activity enables on-demand control, yet current strategies remain hindered by nonspecific regulation and limited tunability. Inspired by proximity effect, we present a Cas-regulation-targeting chimera (CasTAC) strategy that employs CRISPR RNA (crRNA) as a proximity mediator to carry phosphorothioate regulators to interfere with catalytic or recognition domains of Cas12a and consequently suppress its activity. This crRNA-induced proximity approach can effectively eliminate nonspecific interaction between phosphorothioate regulators and proteins within complex multi-enzyme systems, thereby enabling selective control over CRISPR/Cas12a activity. Furthermore, CRISPR/Cas12a activity can be finely tuned to different inhibitory levels by varying the number of phosphorothioate regulators. The CasTAC strategy also improves nuclease resistance and single-nucleotide discrimination, offering potential advances in the sensitivity of molecular diagnostics and the accuracy of gene editing. Notably, the CasTAC balances the kinetics of nucleic acid amplification and CRISPR cleavage, facilitating efficient product accumulation and resolving compatibility issues in one-pot assays. As a proof of concept, we develop a one-pot, one-step recombinase polymerase amplification-CasTAC assay that achieves over 1000-fold higher detection sensitivity than the conventional one-pot recombinase polymerase amplification-CRISPR/Cas12a assay. The CasTAC strategy provides a versatile framework for fine-tuning Cas activity and advances CRISPR technology toward refined and context-adaptable functionality.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics

@article {pmid42549577,
year = {2026},
author = {Winter, E and Emiliani, F and Cook, A and Abderrahim, A and McKenna, A},
title = {BASELINE: a CRISPR base editing platform for mammalian-scale single-cell lineage tracing.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42549577},
issn = {1362-4962},
support = {DP2GM149750/NH/NIH HHS/United States ; //V Foundation/ ; //Pew Biomedical Scholars/ ; },
mesh = {*Cell Lineage/genetics ; *Single-Cell Analysis/methods ; Animals ; *CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; Pancreatic Neoplasms/genetics/pathology ; },
abstract = {A cell's fate is shaped by its inherited state, or lineage, and the ever-shifting context of its environment. CRISPR-based recording technologies are a promising solution for mapping the lineage of a developing system; however, challenges remain regarding single-cell recovery, engineering complexity, and scale. Here, we introduce BASELINE, which uses base editing to generate high-resolution lineage trees in conjunction with single-cell profiling. BASELINE uses the Cas12a adenine base editor to irreversibly edit nucleotides across target arrays built from 50 synthetic target sites, which are integrated multiple times into a cell's genome. We demonstrate that BASELINE accumulates lineage-specific marks over a wide range of biologically relevant intervals, recording more than 4300 bits of information in a model of pancreatic cancer, a 50-fold increase over existing technologies. Single-cell sequencing reveals high-fidelity capture of these recorders, averaging 29 cell divisions captured per lineage, within the estimated range of mammalian development. We expect BASELINE to apply to a wide range of lineage-tracing projects in development and disease, especially those in which cellular engineering makes small, more distributed systems challenging.},
}

*Cell Lineage/genetics
*Single-Cell Analysis/methods
Animals
*CRISPR-Cas Systems
Humans
*Gene Editing/methods
Pancreatic Neoplasms/genetics/pathology

@article {pmid42549821,
year = {2026},
author = {Jafari, A and Manzari-Tavakoli, A and Manzari Tavakoli, M and Hashemian, SM and Jamaati, H and Akhtari, M and Tabarsi, P and Omrani, M},
title = {Theranostic innovation in infectious lung diseases: integrating biotechnology and nanotechnology for precision medicine.},
journal = {Expert review of molecular diagnostics},
volume = {},
number = {},
pages = {},
doi = {10.1080/14737159.2026.2714060},
pmid = {42549821},
issn = {1744-8352},
abstract = {INTRODUCTION: Introduction: Infectious lung diseases, including pneumonia, tuberculosis (TB), COVID-19, influenza, and emerging fungal infections, are major causes of illness and death worldwide. Traditional methods have serious limitations such as diagnostic delays, antimicrobial resistance, and non-targeted therapy. Theranostics offers a transformative precision medicine paradigm for pulmonary infections.

AREAS COVERED: This review looks closely at how biotechnology and nanotechnology synergistically advance theranostic strategies for infectious lung diseases. We explore biotechnological tools including CRISPR-Cas systems, non-coding RNAs (ncRNAs), and monoclonal antibodies (mAbs) for detecting specific pathogens and intervening directly. We also discuss nanotechnological platforms such as nanosensors, surface-enhanced Raman spectroscopy (SERS), and various nanocarriers (lipid nanoparticles, polymeric nanoparticles, liposomes, metallic nanoparticles, mesoporous silica nanoparticles, and biomimetic systems) for drug, gene, and vaccine delivery with better targeting, controlled release, and imaging capabilities. Integrated case studies across major diseases, including COVID-19, influenza, TB, pneumonia, COPD, and idiopathic pulmonary fibrosis, demonstrate effective theranostic applications. We also address associated challenges like safety, manufacturing, regulatory hurdles, and economic feasibility.

EXPERT OPINION: The combination of biotechnology and nanotechnology represents a paradigm shift toward personalized pulmonary medicine. Future success needs to develop smart, multi-stimuli-responsive nanoplatforms, integrating artificial intelligence for predictive modeling and treatment optimization, and establishing closed-loop theranostic systems that connect real-time diagnostics with adaptive therapies. Key priorities include standardized preclinical models, clear regulations for combination products, and health economic analyses demonstrating cost-effectiveness. Interdisciplinary collaboration among material scientists, molecular biologists, clinicians, and regulatory specialists will be essential to translate these promising platforms from bench to bedside.},
}

@article {pmid42550282,
year = {2026},
author = {Yang, L and Ji, X and Li, Z and Duan, F and Jia, Q and Zhang, S and Hu, B and Zhang, Z},
title = {Ultrasensitive electrochemiluminescence determination of Salmonella based on CRISPR/Cas12a integrated with bimetallic semiconductive metal-organic frameworks.},
journal = {Mikrochimica acta},
volume = {193},
number = {9},
pages = {},
pmid = {42550282},
issn = {1436-5073},
support = {252300420760//Natural Science Foundation of Henan Province/ ; 252300421534//Natural Science Foundation of Henan Province/ ; 254200510025//Zhongyuan Sci-Tech Innovation Leading Talents/ ; 25IRTSTHN003//Program for Innovative Research Team (in Science and Technology) in University of Henan Province/ ; },
mesh = {*Biosensing Techniques/methods ; *Luminescent Measurements/methods ; *Salmonella/isolation & purification/chemistry ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *Metal-Organic Frameworks/chemistry ; Limit of Detection ; DNA, Single-Stranded/chemistry ; Semiconductors ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; Bacterial Proteins ; },
abstract = {An ultrasensitive electrochemiluminescence (ECL) biosensor was established by combining CRISPR/Cas12a technique and semiconductive bimetallic-organic framework (scMOF) [[CuxNi3-x(HITP)2] (HITP = 2,3,6,7,10,11-hexaiminotriphenylene)]] emitter and employed to detect Salmonella using the allosteric probe as the recognition component. Given that CuxNi3-x(HITP)2 has demonstrated large specific surface area, both in-plane and out-of-plane charge transfer ability, narrowed band gap, and enhanced separation of holes and electrons, it can be simultaneously employed as the superior ECL emitter and bioplatform for anchoring single-strand DNA (ssDNA), thus improving the detection sensitivity toward Salmonella. The CRISPR/Cas12a-based system can specifically recognize the target sequence of Salmonella and activate the nuclease activity of Cas12a, and the activated Cas12a possesses trans-cleavage ability toward ssDNA. The CuxNi3-x(HITP)2 emitter is then released, resulting in the decline of the ECL response. The developed CuxNi3-x(HITP)2-CRISPR/Cas12a-based ECL biosensor exhibits the ultralow detection limit of 0.25 CFU mL[- 1] in the linear range from 1.0 CFU mL[- 1] to 10[6] CFU mL[- 1], significantly lower than those of reported ones. Furthermore, the developed biosensor exhibits outstanding overall biosensing properties with high selectivity, favorable reproducibility and stability, together with promising practical applicability for the determination of Salmonella in a variety of foodstuffs.},
}

*Biosensing Techniques/methods
*Luminescent Measurements/methods
*Salmonella/isolation & purification/chemistry
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
*Metal-Organic Frameworks/chemistry
Limit of Detection
DNA, Single-Stranded/chemistry
Semiconductors
*Endodeoxyribonucleases/chemistry/genetics/metabolism
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
Bacterial Proteins

@article {pmid42050729,
year = {2026},
author = {Sofianos, G and Petmezas, A and Samaras, A and Karaoglanidis, G},
title = {Resistance of Botrytis cinerea to anilinopyrimidine fungicides: A novel ARMS-PCR method for the detection of Bcpos5 mutations and characterization of resistance using CRISPR/Cas9 editing.},
journal = {Pest management science},
volume = {82},
number = {9},
pages = {8189-8198},
pmid = {42050729},
issn = {1526-4998},
support = {//General Secretariat for Research and Technology/ ; },
mesh = {*Botrytis/drug effects/genetics ; *Fungicides, Industrial/pharmacology ; *Drug Resistance, Fungal/genetics ; *Pyrimidines/pharmacology ; *Polymerase Chain Reaction/methods ; Mutation ; *CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {BACKGROUND: Anilinopyrimidine (AP) fungicides have been widely used against Botrytis cinerea, yet their resistance mechanisms have only recently been clarified. Resistance is primarily linked to mutations G408V, L412V, and L412F in the Bcpos5 gene, whose encoded protein is localized to the mitochondria. In this study, we developed a detection method and tested the fitness of L412F/V mutants obtained by using the CRISPR/Cas9 editing technique.

RESULTS: For rapid and cost-effective mutation identification, a TETRA-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) was developed to rapidly detect the nucleotide alterations that lead to L412F and L412V mutations, producing a 702 bp band in all isolates, with additional 470 bp (F) or 252 bp (V) fragments. Isolates harboring only the 702 bp band were further digested with MlyI to confirm the mutation leading to the amino acid substitution G408V mutation (467 bp + 235 bp). Results were validated by Sanger sequencing. Application of the assay to 170 isolates from strawberry and tomato revealed mutation frequencies of 70.2% (L412F), 8.3% (L412V), and 4.7% (G408V) within the resistant fraction of the population (n = 82 resistant isolates). Furthermore, sequencing analysis revealed also a low frequency of the E407K mutation in Bcmdl1, along with evidence of additional, yet undefined, resistance mechanisms. To further characterize the mutations, the B. cinerea reference strain B05.10 was transformed with L412F and L412V alleles via CRISPR/Cas9 and homologous recombination. The resulting mutants displayed resistance to cyprodinil, and potential fitness costs were assessed in both field-derived and CRISPR/Cas9-generated isolates through measurements of mycelial growth and sporulation in vitro, and pathogenicity in planta. The L412F and L412V transformants did not differ significantly from the parental B05.10 strain in any of the evaluated fitness parameters.

CONCLUSION: Overall, the developed ARMS PCR offers a fast, cost-effective tool for resistance monitoring aiming to identify the most common mutations conferring resistance to APs, while CRISPR/Cas9 provides an efficient approach for functional validation of resistance mutations in B. cinerea. Using this approach, we confirmed that L412F and L412V mutations in Bcpos5 confer resistance to APs, while are not associated with fitness cost. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.},
}

*Botrytis/drug effects/genetics
*Fungicides, Industrial/pharmacology
*Drug Resistance, Fungal/genetics
*Pyrimidines/pharmacology
*Polymerase Chain Reaction/methods
Mutation
*CRISPR-Cas Systems
*Fungal Proteins/genetics/metabolism
Gene Editing

@article {pmid42546506,
year = {2026},
author = {Pal, P and Anand, U and Saha, SC and Sundaramurthy, S and Okeke, ES and Kumar, M and Radha, and Bontempi, E and Albertini, E and Dey, A and Di Maria, F},
title = {Retraction notice to "Novel CRISPR/Cas technology in the realm of algal bloom biomonitoring: Recent trends and future perspectives" [Environ. Res 231 (2023) 115989].},
journal = {Environmental research},
volume = {306},
number = {Pt 4},
pages = {125335},
doi = {10.1016/j.envres.2026.125335},
pmid = {42546506},
issn = {1096-0953},
}

@article {pmid42547416,
year = {2026},
author = {Zhuang, Q and Wang, F and Zhang, H and Zhang, Q and Gao, Y and Feng, Z and Gao, H and Sun, S and Lin, S and Li, S and Zhao, Q and Huang, X and Li, Q and Sheng, W and Huang, G},
title = {A cardiac-related, promoter-proximal, regulatory element shapes chromatin and JAG1 transcription.},
journal = {Life science alliance},
volume = {9},
number = {10},
pages = {},
pmid = {42547416},
issn = {2575-1077},
mesh = {*Jagged-1 Protein/genetics/metabolism ; *Chromatin/genetics/metabolism ; *Promoter Regions, Genetic/genetics ; Animals ; Transcription, Genetic ; Mice ; Humans ; Enhancer Elements, Genetic ; Gene Expression Regulation ; Apoptosis/genetics ; Cell Movement/genetics ; Cell Proliferation/genetics ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {The Jagged1 (JAG1) gene is essential for cardiac development, yet its tissue-specific transcriptional regulation remains poorly understood. In this study we used an integrative screening approach to identify 19 candidate enhancers within the ±100 kb region flanking the JAG1 locus, among which R7 exhibited the highest activity in dual-luciferase assays. CRISPR/Cas9-mediated deletion of R7 in AC16 cells significantly reduced JAG1 expression, decreased proliferative and migratory capacities, and increased apoptosis. Mechanistically, R7 deletion altered local chromatin contacts and reduced accessibility at CTCF-bound regions near the JAG1 promoter, accompanied by decreased H3K27ac, H3K4me3, RNA polymerase II, and SRF occupancy. These findings identify R7 as a cardiac-associated promoter-proximal regulatory element with enhancer-like activity that contributes to local chromatin organization and transcriptional activity at the JAG1 locus.},
}

*Jagged-1 Protein/genetics/metabolism
*Chromatin/genetics/metabolism
*Promoter Regions, Genetic/genetics
Animals
Transcription, Genetic
Mice
Humans
Enhancer Elements, Genetic
Gene Expression Regulation
Apoptosis/genetics
Cell Movement/genetics
Cell Proliferation/genetics
CRISPR-Cas Systems
Cell Line

@article {pmid42379397,
year = {2026},
author = {Shao, D and Wen, X and Luo, Q and Liu, X and Li, L and Men, S},
title = {Combined T-DNA and CRISPR/Cas9 mutagenesis reveals redundant developmental roles of the Arabidopsis BAG family.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {371},
number = {},
pages = {113305},
doi = {10.1016/j.plantsci.2026.113305},
pmid = {42379397},
issn = {1873-2259},
mesh = {*Arabidopsis/genetics/growth & development/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; DNA, Bacterial/genetics ; Gene Expression Regulation, Plant ; Plant Growth Regulators/metabolism ; Indoleacetic Acids/metabolism ; *Molecular Chaperones/genetics/metabolism ; Germination/genetics ; },
abstract = {BAG (Bcl-2-associated athanogene) genes encode evolutionarily conserved co-chaperones that participate in proteostasis regulation, stress responses, and programmed cell death. However, their collective functions during plant development remain poorly understood. Promoter cis-element analysis revealed multiple hormone-responsive elements in promoters of Arabidopsis thaliana (Arabidopsis) BAG genes, suggesting potential involvement of BAG genes in phytohormone-mediated developmental regulation. To investigate this, we generated a bag-septuple (bag-s) mutant in which all seven Arabidopsis BAG genes were knocked out using a combination of T-DNA insertion alleles and CRISPR/Cas9-mediated mutagenesis. Phenotypic characterization revealed pleiotropic defects, including delayed seed germination, increased seed coat mucilage accumulation, reduced primary root elongation, decreased rosette diameter and plant height, and delayed leaf senescence. Consistent with the delayed leaf senescence phenotype, expression of senescence-associated genes and senescence-promoting transcription factors was downregulated in the bag-s mutant. RT-qPCR analyses further showed that genes involved in auxin biosynthesis and auxin signaling were downregulated in the bag-s mutant. Furthermore, exogenous IAA partially rescued the root elongation defect of the bag-s mutant, supporting a functional association between BAG genes and auxin-dependent root growth. Collectively, these findings indicate that BAG genes redundantly regulate seed germination, vegetative growth, auxin-related root development, and leaf senescence, providing a genetic framework for further dissecting BAG-mediated coordination of proteostasis, hormone signaling, and plant development.},
}

*Arabidopsis/genetics/growth & development/metabolism
*Arabidopsis Proteins/genetics/metabolism
*CRISPR-Cas Systems
DNA, Bacterial/genetics
Gene Expression Regulation, Plant
Plant Growth Regulators/metabolism
Indoleacetic Acids/metabolism
*Molecular Chaperones/genetics/metabolism
Germination/genetics

@article {pmid42391902,
year = {2026},
author = {Liao, W and Li, S and Wu, S and Yang, X and Liu, B and Ling, L and Zeng, Q and Zhang, J},
title = {Sensitive detection of prostate cancer antigen 3 (PCA3) in urine based upon CRISPR/Cas12a and gold nanorods (AuNRs).},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {363},
number = {Pt 2},
pages = {128309},
doi = {10.1016/j.saa.2026.128309},
pmid = {42391902},
issn = {1873-3557},
mesh = {Humans ; Male ; *Gold/chemistry ; *Nanotubes/chemistry/ultrastructure ; *Antigens, Neoplasm/urine ; *Prostatic Neoplasms/urine/diagnosis ; *CRISPR-Cas Systems ; Limit of Detection ; Spectrometry, Fluorescence ; *CRISPR-Associated Proteins/metabolism ; Biosensing Techniques/methods ; Biomarkers, Tumor/urine ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism ; },
abstract = {Prostate Cancer Antigen 3, a type of long non-coding RNA, exhibits outstanding specificity as a biomarker for the diagnosis of prostate cancer, offering a highly effective diagnostic indicator, whereas the currently used prostate specific antigen exhibits low specificity, leading to reduced accuracy in prostate cancer diagnosis. Herein, we designed a novel fluorescent sensing platform for targeted detection of PCA3, which integrates the non-specific trans-cleavage activity of the CRISPR/Cas12a with the remarkable fluorescence quenching effect of Gold Nanorods. The Cas12a recognizes and binds to specific sequences of PCA3, thereby activating nonspecific cleavage activity, which cleaves fluorescent reporter probes adsorbed on AuNRs, thus leading to the recovery of fluorescence signals and enabling sensitive detection. The proposed fluorescent sensor exhibits excellent accuracy and convenience for the detection of PCA3 in urine, and a detection limit as low as 1.65 pM was obtained. This sensing system has achieved effective detection of clinical samples of prostate cancer and is expected to provide significant assistance in the screening and therapeutic feedback of prostate cancer in clinical diagnosis.},
}

Humans
Male
*Gold/chemistry
*Nanotubes/chemistry/ultrastructure
*Antigens, Neoplasm/urine
*Prostatic Neoplasms/urine/diagnosis
*CRISPR-Cas Systems
Limit of Detection
Spectrometry, Fluorescence
*CRISPR-Associated Proteins/metabolism
Biosensing Techniques/methods
Biomarkers, Tumor/urine
*Bacterial Proteins/metabolism/chemistry
*Endodeoxyribonucleases/metabolism

@article {pmid42437612,
year = {2026},
author = {Li, X and Xin, C and Guo, J and Zheng, Y and Huang, F and Li, X and Guo, H and Xu, Y and Shao, W},
title = {Multiplex CRISPR-Cas9 editing of starch branching enzyme II and vacuolar invertase simultaneously enhances resistant starch content and cold-induced sweetening resistance in Solanum chacoense.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {371},
number = {},
pages = {113319},
doi = {10.1016/j.plantsci.2026.113319},
pmid = {42437612},
issn = {1873-2259},
mesh = {*beta-Fructofuranosidase/genetics/metabolism ; *Starch/metabolism ; *1,4-alpha-Glucan Branching Enzyme/genetics/metabolism ; *Solanum/genetics/enzymology/metabolism ; *CRISPR-Cas Systems ; Cold Temperature ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {Potato processing suffers from a high glycemic index due to amylopectin-rich starch and from undesirable color and acrylamide formation during frying, mainly caused by cold-induced sweetening (CIS). To address both issues simultaneously, we used CRISPR-Cas9 to knock out two key genes in diploid Solanum chacoense: ScSBE II (starch branching enzyme II), which controls amylopectin biosynthesis, and ScVInv (vacuolar invertase), a central regulator of CIS. Knockout of ScSBEⅡ increased tuber fresh weight-based absolute amylose content by ∼5-fold versus wild type. Amylose proportion in total starch elevated from 22% to 54%, while amylopectin abundance declined 1.5-fold, substantially optimizing the amylose/amylopectin mass ratio of tuber starch. These lines also showed a 4-fold reduction in rapidly digestible starch (RDS), 3.5‑fold and 1.2‑fold increases in slowly digestible starch (SDS) and resistant starch (RS), respectively, and markedly improved pasting properties. Enzyme assays confirmed a 2.5‑fold reduction in ScSBE II activity. In wild‑type (WT) tubers, cold storage (4 °C, 7 d) increased ScVInv activity ∼5‑fold (to 48 μg·min[-1]·g[-1]) and reducing sugars 6‑fold (from 11 to 68 mg·g[-1]). Notably, ScVInv single‑knockout and ScSBE II/ScVInv double‑knockout lines produced chips with lighter color and much lower acrylamide than WT or ScSBE II single‑knockout lines. This dual‑gene editing strategy creates novel potato germplasm with enhanced resistant starch (health benefit) and superior processing quality (safer, visually appealing fried products).},
}

*beta-Fructofuranosidase/genetics/metabolism
*Starch/metabolism
*1,4-alpha-Glucan Branching Enzyme/genetics/metabolism
*Solanum/genetics/enzymology/metabolism
*CRISPR-Cas Systems
Cold Temperature
*Gene Editing
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified

@article {pmid42543873,
year = {2026},
author = {Wang, J and Peng, Q},
title = {Innovations, Applications, and Future Trends in Veterinary Diagnostic Technologies.},
journal = {Transboundary and emerging diseases},
volume = {2026},
number = {1},
pages = {e6973879},
pmid = {42543873},
issn = {1865-1682},
support = {32470195//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Veterinary Medicine/trends/methods ; *Animal Diseases/diagnosis ; High-Throughput Nucleotide Sequencing/veterinary ; },
abstract = {Veterinary diagnostics is undergoing a significant transformation driven by technological advancements, extending its scope from the traditional confirmation of specific pathogens to the continuous, dynamic surveillance of animal population's health. This paradigm shift has the potential to enable more timely disease control, precise intervention, and enhanced public health security. Traditional clinical and laboratory diagnostic methods, such as microbial culture, serological assays, and nucleic acid-based polymerase chain reaction, form the cornerstone of the current diagnostic framework and are widely applied based on varying detection needs and practical environments. Nonetheless, the field is experiencing profound innovation. Firstly, novel detection technologies are emerging, such as digital PCR (dPCR), CRISPR-Cas-based molecular diagnostic tools, next-generation sequencing (NGS), and metagenomic sequencing. These technologies have not only achieved breakthroughs in sensitivity and specificity but, more importantly, enable the unbiased discovery of novel pathogens. Secondly, the deep integration of artificial intelligence (AI) and big data is reshaping the diagnostic pipeline. By consolidating and analyzing multimodal information streams from imaging, genomics, wearable devices, and production data, AI algorithms can provide objective, quantitative decision support, facilitating a transition from post-symptomatic diagnosis towards predictive and preventive health management. This scoping review systematically summarizes both mainstream and emerging veterinary diagnostic technologies, elaborates and discusses their advantages and limitations as well as future developmental directions, while highlighting that the combined application of multiple methods represents an optimal diagnostic strategy.},
}

Animals
*Veterinary Medicine/trends/methods
*Animal Diseases/diagnosis
High-Throughput Nucleotide Sequencing/veterinary

@article {pmid42545527,
year = {2026},
author = {Hu, Y and Fang, F and Cui, Z and Chen, S},
title = {A Reproducible Electroporation Strategy for CRISPR-Cas9 RNP and mRNA Delivery in Fish Embryos.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {28},
number = {4},
pages = {},
pmid = {42545527},
issn = {1436-2236},
support = {2023TD20//Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; 2023ZD0405503//STI 2030-Major projects/ ; 2022YFD2400101//the National Key R&D Program of China/ ; 32230107//National Natural Science Foundation of China/ ; 2023ZLYS02//the Key Research and Development Project of Shandong Province/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Electroporation/methods ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; *RNA, Messenger/genetics/metabolism ; Embryo, Nonmammalian/metabolism ; *Gene Editing/methods ; Gene Knockout Techniques ; Green Fluorescent Proteins/genetics ; Polyglutamic Acid ; Chorion/metabolism ; Zebrafish Proteins/genetics ; },
abstract = {This study presents a streamlined electroporation-based method for efficient macromolecular delivery into zebrafish embryos. We first characterized the physical barrier posed by the chorion using TEM (Transmission Electron Microscope) and established that its removal is prerequisite for effective delivery. A systematic optimization of electroporation parameters for dechorionated embryos identified optimal conditions (e.g., poring pulse: 25 V, 20 ms; transfer pulse: 5 V, 50 ms) that ensured high embryo survival and robust eGFP mRNA transfection. Applying this protocol, we achieved targeted gene knockout by electroporation-mediated delivery of Cas9 ribonucleoproteins (RNPs) against multiple loci. Targeting the tyr locus resulted in a phenotypic albinism rate of (38.6 ± 3.30)%. Furthermore, we incorporated polyglutamic acid (PGA) to modify the RNP complexes (target tyr), which inhibited aggregation and enhanced editing efficiency to (44.45 ± 1.41)%, outperforming a commercial. electroporation system, while targeting the pigmentation-related gene slc24a5 yielded an albinism or hypopigmentation rate of (38.33 ± 2.62)%. In addition, targeting the development-associated gene ddx19b produced developmental defect phenotypes in (38.33 ± 1.88)% of embryos. The successful introduction of indels at the target site was confirmed by sequencing. Our work establishes a highly effective electroporation strategy, augmented by nanotechnology, for the delivery of mRNA and RNP complexes, enabling high-efficiency protein expression and gene editing in zebrafish embryos, with broad potential applications in aquatic biotechnology.},
}

Animals
*Zebrafish/genetics/embryology
*Electroporation/methods
*Ribonucleoproteins/genetics/metabolism
*CRISPR-Cas Systems
*RNA, Messenger/genetics/metabolism
Embryo, Nonmammalian/metabolism
*Gene Editing/methods
Gene Knockout Techniques
Green Fluorescent Proteins/genetics
Polyglutamic Acid
Chorion/metabolism
Zebrafish Proteins/genetics

@article {pmid42545751,
year = {2026},
author = {Haneef, S and Zhou, YJ and Bai, F},
title = {Transcriptional regulation: Efficient genetic engineering tools for non-conventional yeasts.},
journal = {FEMS yeast research},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsyr/foag032},
pmid = {42545751},
issn = {1567-1364},
abstract = {Non-conventional yeasts are recognized as valuable hosts for producing biofuels, pharmaceuticals, and other high-value chemicals, owing to their diverse physiological traits, ability to utilize various substrates, and greater tolerance to environmental stresses compared to conventional model yeast Saccharomyces cerevisiae. To fully optimizing metabolic flux toward desired products, effective genetic engineering tools enabling precise modulation of gene expression and coordinated control of metabolic pathways are essential. In this context, we discussed classical transcriptional regulation tools like promoters, and transcription factors, alongside innovations in synthetic biology that allow metabolic engineering in non-conventional yeasts to produce higher biofuels and other useful products, promoting the development of sustainable resources, and assisting the development of innovative bio-products. It also discussed innovative programmable technologies, such as CRISPR/Cas-mediated transcriptional activation and repression, as well as dynamic regulatory systems that can fine-tune metabolic routes and balance cellular resources. Strategies for promoter engineering, transcription factor manipulation for transcriptional regulation, and metabolic rewiring were highlight as methods to boost pathway efficiency and yields. This review concluded with current challenges and future directions, focusing on integrating synthetic biology and systems biology to create robust, controllable transcriptional frameworks for next-generation yeast cell factories.},
}

@article {pmid42545979,
year = {2026},
author = {Patel, MA and Singh, M and Sinha, H and Vo, PQN and Chin, AB and Ellouzi, A and West, M and Hirukawa, A},
title = {A Digital Microfluidic Electroporation Platform for Low-input CRISPR Genome Editing and mRNA Transfection In Suspension T Cells and 3D Cell Models.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {233},
pages = {},
doi = {10.3791/70573},
pmid = {42545979},
issn = {1940-087X},
mesh = {Humans ; *Electroporation/methods/instrumentation ; *Transfection/methods/instrumentation ; HEK293 Cells ; *RNA, Messenger/genetics/administration & dosage ; *T-Lymphocytes/physiology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Microfluidic Analytical Techniques/methods/instrumentation ; },
abstract = {Digital microfluidic (DMF) electroporation enables precise, low-volume genetic manipulation of mammalian cells while minimizing cellular input by up to 100x and preserving viability. This study presents a high-throughput DMF-based transfection workflow for CRISPR-mediated knockout of the TRAC locus in primary human suspension T cells and for mRNA transfection of three-dimensional HEK293T spheroids. Using spatially deposited CRISPR guide RNAs and on- cartridge ribonucleoprotein (RNP) assembly, efficient TRAC locus disruption was achieved in both CD4[+] and CD8[+] T-cell populations using only 10,000 cells per condition, with post-editing viabilities exceeding 85%. Biophysical characterization using flow-induced and Taylor dispersion analyses revealed that polymer additives stabilize Cas9-sgRNA complexes under electroporation buffer conditions, supporting reproducible editing at sub microliter volumes. The workflow was further adapted for 3D applications by delivering EGFP mRNA into intact HEK293T spheroids, resulting in robust and spatially uniform fluorescence without impairing spheroid growth or morphology. Together, these results demonstrate that DMF electroporation enables efficient genome editing and mRNA delivery across both suspension immune cells and multicellular spheroids. This platform provides a scalable and low-input solution for applications in CAR-T cell therapy, functional genomics, and advanced 3D cellular models.},
}

Humans
*Electroporation/methods/instrumentation
*Transfection/methods/instrumentation
HEK293 Cells
*RNA, Messenger/genetics/administration & dosage
*T-Lymphocytes/physiology
*Gene Editing/methods
*CRISPR-Cas Systems
*Microfluidic Analytical Techniques/methods/instrumentation

@article {pmid42263565,
year = {2026},
author = {Wang, Q and Wang, Y and Jia, T and Hu, X and Chen, F},
title = {Evaluation of the performance of reverse transcription-recombinase polymerase amplification (RT-RPA) coupled with CRISPR/Cas12a and microfluidics for one-step detection of common HCV genotypes.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {3},
pages = {117482},
doi = {10.1016/j.diagmicrobio.2026.117482},
pmid = {42263565},
issn = {1879-0070},
mesh = {Humans ; *Hepacivirus/genetics/isolation & purification/classification ; Genotype ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Hepatitis C/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *Microfluidics/methods ; Rapid Diagnostic Tests ; *Genotyping Techniques/methods ; },
abstract = {BACKGROUND: Hepatitis C virus (HCV) genotyping is critical for guiding therapy, yet current methods are technically demanding and time-consuming.

AIM: To develop and evaluate a one-step, integrated assay combining reverse transcription-recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a detection on a microfluidic platform for rapid and accurate HCV genotyping.

METHODS: The assay was validated using 186 clinical samples genotyped by Sanger sequencing. The microfluidic chip enabled sequential RT-RPA amplification and CRISPR/Cas12a detection via centrifugal fluid transfer, with real-time fluorescence monitoring.

RESULTS: The assay demonstrated high concordance with Sanger sequencing (overall accuracy >98%), with sensitivities of 100% for genotypes 1b and 6a, and >97% for 2a and 3a. The limit of detection was 1 IU/mL (5 copies/mL)across major genotypes, with no cross-reactivity against other viruses.

CONCLUSION: The integrated RT-RPA-CRISPR/Cas12a-microfluidics platform offers a rapid, sensitive, and specific one-step assay for HCV genotyping, suitable for point-of-care applications in resource-limited settings.},
}

Humans
*Hepacivirus/genetics/isolation & purification/classification
Genotype
*CRISPR-Cas Systems
Sensitivity and Specificity
*Hepatitis C/diagnosis/virology
*Nucleic Acid Amplification Techniques/methods
*Microfluidics/methods
Rapid Diagnostic Tests
*Genotyping Techniques/methods

@article {pmid42289138,
year = {2026},
author = {Sata, TN and Sah, AK and Ismail, M and Sharma, G and Doloi, R and Nayak, B and Shalimar, and Venugopal, SK},
title = {Development of RPA and nested-RPA based CRISPR/Cas13a diagnostic platform for the identification of HBV DNA and HCV RNA in Indian patient cohort.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {3},
pages = {117514},
doi = {10.1016/j.diagmicrobio.2026.117514},
pmid = {42289138},
issn = {1879-0070},
mesh = {Humans ; *Hepatitis B virus/genetics/isolation & purification ; *Hepacivirus/genetics/isolation & purification ; India ; *Hepatitis B/diagnosis/virology ; *Hepatitis C/diagnosis/virology ; *DNA, Viral/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Viral/genetics ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *CRISPR-Cas Systems ; Recombinases/metabolism ; Cohort Studies ; },
abstract = {BACKGROUND: Among the Indian population, hepatitis B virus (HBV) is one of the major burdens and the hepatitis C virus (HCV) chronically infects around 1% Indian population. CRISPR-based detection platforms have shown to be a novel low-cost technology with high sensitivity and specificity. In the presence of target nucleic acids, Cas13a molecule is activated to trans-cleave the fluorophore quencher (FQ)-labeled ssRNA reporter, and illuminate detectable fluorescent signals.

METHODS: Leptotrichia wadei (Lwa) cas13a was expressed and purified. Recombinase Polymerase Amplification (RPA) was implemented to produce T7 RNA polymerase appended amplicons of conserved regions of HBV and HCV at 37°C and 42°C respectively. Corresponding crRNAs have been designed against amplified regions and produced using In-Vitro Transcription (IVT). With T7 RNA polymerase, the RPA-amplified HBV and HCV templates are transcribed into ssRNAs, which are further used in detection assay containing expressed Cas protein, crRNA, and fluorescent probes. This detection was performed in the microplate reader in kinetic format.

RESULTS: LwaCas13a was expressed and was purified using the strep tag. Conserved regions among Indian HBV and HCV genotypes are selected as targets of detection. RPA and Nested-RPA was performed using primers against conjunct region of HBV polymerase and surface antigen and RNA-dependent RNA polymerase (RdRp) region of HCV. The detection assay was performed from 45 HBV and 30 HCV human samples, out of which it could differentiate positive and healthy samples.

CONCLUSION: This approach can be a better alternative to be used in rural India and at the same time with high sensitivity, for a rapid detection of HBV and HCV, which could be used as a novel a low-cost diagnostics platform for identification of HBV DNA and HCV RNA.},
}

Humans
*Hepatitis B virus/genetics/isolation & purification
*Hepacivirus/genetics/isolation & purification
India
*Hepatitis B/diagnosis/virology
*Hepatitis C/diagnosis/virology
*DNA, Viral/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*RNA, Viral/genetics
Sensitivity and Specificity
Rapid Diagnostic Tests
*CRISPR-Cas Systems
Recombinases/metabolism
Cohort Studies

@article {pmid42328982,
year = {2026},
author = {Mao, Y and Fei, X and Yang, X and Hu, C and Zhu, C and Wang, J and Ge, Y and Ye, C and Yang, S and Cheng, P and Li, Y and He, X and Hu, Z and Qi, Y},
title = {CRISPR-Cas12a-based fluorescent and visual assays for universal detection and clade discrimination of mpox virus.},
journal = {Microbiology spectrum},
volume = {14},
number = {8},
pages = {e0010526},
pmid = {42328982},
issn = {2165-0497},
support = {2025YQZL01//Huadong Research Institute for Medicine and Biotechniques/ ; SKLADCPKFKT202512//State Key Laboratory for Animal Disease Control and Prevention/ ; },
mesh = {Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Orthopoxvirus/genetics/isolation & purification/classification ; Fluorescence ; *Molecular Diagnostic Techniques/methods ; CRISPR-Associated Proteins/genetics ; Polymerase Chain Reaction/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The global mpox outbreak has highlighted critical gaps in diagnostic capabilities, particularly the need for methods that can distinguish between the high-fatality Clade I and more transmissible Clade II of the mpox virus (MPXV). Current PCR-based approaches remain reliant on laboratory infrastructure, limiting their use in resource-limited settings. There is an urgent need for a versatile diagnostic platform that can provide both accurate clade discrimination and flexible deployment across diverse healthcare environments. We developed a dual-mode detection platform by integrating recombinase-aided amplification with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a technology, creating two distinct assays: a universal assay targeting OPG034, and a Clade I-discriminatory assay targeting OPG033. The platform achieved detection sensitivities of 1 copy/reaction for both fluorescence and visual readouts with OPG034, and 10 copies (fluorescence) and 1 copy/reaction (visual) with OPG033. Both assays demonstrated high specificity, successfully distinguishing MPXV from related orthopoxviruses and common viruses. Clinical validation using 23 Clade II samples and 10 healthy controls showed that, relative to quantitative PCR (qPCR) (cycle threshold ≤37), the OPG034 fluorescence assay detected all 13 qPCR-positive samples and 2 additional positives (100% sensitivity, 80.0% specificity), while the visual assay detected 12 of 13 positives (92.3% sensitivity, 100% specificity). For Clade I-specific detection, both OPG033 fluorescence and visual assays showed 100% specificity in Clade II samples (23/23). While the clade-discriminatory capability was established through sequence-specific design, further evaluation with authentic Clade I clinical specimens is warranted to confirm typing performance. The dual-mode design provides flexibility for both laboratory and field use, advancing mpox surveillance and outbreak response.IMPORTANCEMpox is a significant zoonosis. Accurate discrimination between its highly lethal Clade I and more transmissible Clade II is critical for clinical management and outbreak control, yet current methods primarily enable only general detection. To address this, we identified novel genetic markers (OPG034 for universal detection and OPG033 for Clade I specificity) and developed a dual-mode detection platform integrating Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a with recombinase-aided amplification. Its key advantage is providing two result readouts: a sensitive fluorescence mode for laboratories and an instrument-free visual colorimetric mode for field use. The demonstrated excellent performance on clinical samples confirms that this platform meets the precision requirements of clinical laboratories while remaining suitable for resource-limited settings like field clinics. Thus, it offers a flexible and practical tool for enhancing mpox surveillance and control globally, particularly in regions with constrained medical resources.},
}

Humans
Sensitivity and Specificity
*CRISPR-Cas Systems
*Orthopoxvirus/genetics/isolation & purification/classification
Fluorescence
*Molecular Diagnostic Techniques/methods
CRISPR-Associated Proteins/genetics
Polymerase Chain Reaction/methods
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42372673,
year = {2026},
author = {Yang, H and Li, X and Su, Y and Guo, J and Wang, T and Wang, Z and Fu, H and Chen, Z and Xie, Z and Li, D},
title = {Enhanced stability of RPA-CRISPR-Cas12a system for respiratory pathogen detection using Trehalose-Carboxymethyl Chitosan Lyoprotectant.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {3},
pages = {117523},
doi = {10.1016/j.diagmicrobio.2026.117523},
pmid = {42372673},
issn = {1879-0070},
mesh = {Humans ; Sensitivity and Specificity ; *Chitosan/analogs & derivatives/chemistry ; *CRISPR-Cas Systems ; Rapid Diagnostic Tests ; *Trehalose/chemistry ; *Respiratory Tract Infections/diagnosis/virology/microbiology ; SARS-CoV-2/genetics/isolation & purification ; Neisseria meningitidis/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; Streptococcus pneumoniae/genetics/isolation & purification ; Freeze Drying ; },
abstract = {PURPOSE: Acute respiratory infections caused by bacterial and viral pathogens pose a major global health burden. The recombinase polymerase amplification (RPA)-CRISPR-Cas12a system offers a promising point-of-care testing (POCT) platform, but its field deployment is limited by the instability of lyophilized reagents. This study aims to develop a composite lyoprotectant to enhance the stability of the RPA-CRISPR-Cas12a system for respiratory pathogen detection.

MATERIALS AND METHODS: A composite lyoprotectant composed of trehalose and carboxymethyl chitosan (Tre-CMC) was formulated at various mass ratios. The optimal ratio was identified by evaluating matrix microstructure, enzyme activity retention, and primer-dimer suppression. The lyophilized system was tested for sensitivity, specificity, and long-term stability against six respiratory pathogens (H1N1, IBV, Neisseria meningitidis, SARS-CoV-2, Streptococcus pneumoniae, and human adenovirus) using real-time fluorescence and gel electrophoresis.

RESULTS: This study establishes a promising proof-of-concept framework for an integrated, lyophilized RPA-CRISPR-Cas12a diagnostic system. The optimal Tre: CMC ratio (2:1) produced a porous, non‑hygroscopic matrix that preserved reagent integrity. The lyophilized system achieved a detection limit of 10 copies/reaction within 30 min for all six pathogens, with 100% specificity. After six months of storage at 4°C, RPA enzyme activity remained above 89%, and Cas12a-crRNA complex functionality was fully retained. Tre-CMC significantly reduced primer-dimer formation and nonspecific background fluorescence. While clinical evaluation using 21 nasopharyngeal swab samples demonstrated 100% concordance with RT-qPCR-preliminarily supporting the system's potential diagnostic accuracy-we acknowledge that this initial methodological framework requires broader and more rigorous downstream validation with larger clinical cohorts to formally establish its robust diagnostic performance.

CONCLUSION: The Tre-CMC composite lyoprotectant effectively stabilizes the RPA-CRISPR-Cas12a system, enabling cold-chain-independent storage and reliable POCT for respiratory pathogens in resource-limited settings.},
}

Humans
Sensitivity and Specificity
*Chitosan/analogs & derivatives/chemistry
*CRISPR-Cas Systems
Rapid Diagnostic Tests
*Trehalose/chemistry
*Respiratory Tract Infections/diagnosis/virology/microbiology
SARS-CoV-2/genetics/isolation & purification
Neisseria meningitidis/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
Streptococcus pneumoniae/genetics/isolation & purification
Freeze Drying

@article {pmid42539204,
year = {2026},
author = {Saxena, AG and Ramey, GD and Capra, JA and Conklin, BR and Macklin, BL},
title = {EXCAVATE-HT: A Bioinformatic Pipeline to Identify Targetable Genomic Variants for Allele-Specific Editing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42539204},
issn = {2692-8205},
abstract = {Allele-specific CRISPR/Cas editing is a powerful tool with great potential for treating genetic diseases and for uncovering the effects of allelic diversity. By targeting commonly inherited single nucleotide polymorphisms (SNPs), a small number of gRNAs can treat many more individuals than targeting rare disease mutations. However, current tools for identifying common targetable variants and generating CRISPR guide RNAs (gRNA) have fundamental conceptual and technical limitations. Here, we introduce EXCAVATE-HT (EXtracting Common Allelic VAriants for Targeted Editing in High-Throughput) a bioinformatic tool that mines population variant data to generate CRISPR libraries targeting genomic loci for allele-specific editing. Users define their loci of interest, Cas species, and SNP frequency, then EXCAVATE-HT outputs an annotated list of allele-specific gRNAs. EXCAVATE-HT can also generate libraries of gRNA pairs to enable excision. We illustrate the use of EXCAVATE-HT to design and characterize multiple gRNA libraries for allele-specific targeting of the disease gene, Cone-Rod Homeobox (CRX). EXCAVATE-HT revealed multiple excisions that could treat >30-fold more patients than targeting a single CRX disease mutation.},
}

@article {pmid42542233,
year = {2026},
author = {Liu, Y and Feng, L and Li, J and Zhang, R},
title = {CRISPR-based live-cell DNA imaging: Technologies, biological insights and future perspectives.},
journal = {Biotechnology advances},
volume = {92},
number = {},
pages = {108999},
doi = {10.1016/j.biotechadv.2026.108999},
pmid = {42542233},
issn = {1873-1899},
abstract = {Live-cell DNA imaging provides a direct view of genome behavior in real-time and has advanced rapidly with the development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system. Here, we review progress in live-cell DNA imaging from two major directions: non-repetitive loci visualization and multicolor imaging. We also highlight biological insights enabled by these technologies, covering DNA replication, damage and repair, chromatin organization and interactions, epigenetic regulation, extrachromosomal DNA, and viral genome dynamics. We then discuss future trends in live-cell DNA imaging and its potential impact on both biotechnology and biomedical research.},
}

@article {pmid41639251,
year = {2026},
author = {Khan, A and Herring, G and Zhu, JY and Petterson, M and Lister, R},
title = {Designing and testing CRISPRi-based synthetic gene circuits in plants.},
journal = {Nature protocols},
volume = {21},
number = {8},
pages = {3528-3551},
pmid = {41639251},
issn = {1750-2799},
support = {DP240103385//Department of Education and Training | Australian Research Council (ARC)/ ; CE230100015//Department of Education and Training | Australian Research Council (ARC)/ ; GNT2035042//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Protoplasts/metabolism ; Arabidopsis/genetics ; *Gene Regulatory Networks ; *Genes, Synthetic ; *CRISPR-Cas Systems ; Triticum/genetics ; *Plants/genetics ; Brassica napus/genetics ; Bryopsida/genetics ; },
abstract = {Synthetic gene circuits are powerful tools for precisely programming gene expression and introducing novel cellular functions. However, their development and application in plants has lagged behind other systems, due mainly to the limited availability of modular genetic parts. We recently developed a CRISPR interference (CRISPRi)-based synthetic gene circuit system for programming gene expression in plants. Using a robust and high-throughput protoplast-based dual luciferase assay, we demonstrated the development, testing and functionality of these circuits in various plant species. Here we detail the key design principles and considerations for building and testing programmable and reversible CRISPRi-based gene circuits in plants. We also provide detailed procedures for isolating protoplasts from multiple plant species, including Arabidopsis thaliana, Brassica napus, Triticum aestivum and Physcomitrium patens. Furthermore, we provide step-by-step instructions for the 96-well plate-based protoplast transfection assay for testing genetic parts and synthetic circuits, using a dual luciferase assay. The detailed descriptions of these developed systems will enhance the efficiency and reproducibility of the construction, testing, and implementation of synthetic gene circuits in a variety of plant species. This protocol enables the design and testing of CRISPRi-based gene circuits in plants within ~4 weeks.},
}

Protoplasts/metabolism
Arabidopsis/genetics
*Gene Regulatory Networks
*Genes, Synthetic
*CRISPR-Cas Systems
Triticum/genetics
*Plants/genetics
Brassica napus/genetics
Bryopsida/genetics

@article {pmid41933972,
year = {2026},
author = {Li, Z and Wang, X and Liu, J and Janssen, JM and Hoeben, RC and Gonçalves, MAFV},
title = {Selector adeno-associated viral vectors facilitate on-target precise genome editing and purge off-target chromosomal insertions.},
journal = {Trends in biotechnology},
volume = {44},
number = {8},
pages = {2422-2445},
doi = {10.1016/j.tibtech.2026.01.006},
pmid = {41933972},
issn = {1879-3096},
mesh = {*Dependovirus/genetics ; *Gene Editing/methods ; *Genetic Vectors/genetics ; Humans ; CRISPR-Cas Systems ; },
abstract = {Adeno-associated viral (AAV) vectors are commonly used for genome editing owing to the proclivity with which their single-stranded genomes serve as homologous recombination (donor) substrates during programmable nuclease-assisted gene targeting. However, the highly recombinogenic nature of AAV genomes also facilitates their nonhomologous end joining at off-target chromosomal breaks ('capture') created by said nucleases, mutagens, or DNA metabolic processes. Moreover, AAV donor constructs can equally yield imprecise on-target edits resulting from end-joining recombination pathways. Here, we demonstrate that endowing AAV vectors with exogenous marker-free selectable sequences permits enrichment for cells precisely coedited at endogenous target and ATP1A1 alleles. These selector AAV vectors install ATP1A1 polymorphisms conferring resistance to the small molecule ouabain, yielding high frequencies of on-target and precisely edited cell populations. Crucially, we further report that selector AAV vectors achieve a thorough removal of heterogeneous off-target DNA species resulting from conventional AAV-based genome editing procedures.},
}

*Dependovirus/genetics
*Gene Editing/methods
*Genetic Vectors/genetics
Humans
CRISPR-Cas Systems

@article {pmid42035261,
year = {2026},
author = {Tan, K and Del Bosque Siller, D and Xiong, AY and Wang, AXA and McCallister, TX and Mummadi, S and St John, LA and Lee, TK and Carrillo, AN and Renshaw, DG and Zhou, RH and Lim, CKW and He, J and Fields, CJ and Hayden, MR and Gaj, T},
title = {Treatment of Huntington's disease with a pan-HTT-targeting CRISPR nuclease.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {8},
pages = {4635-4655},
pmid = {42035261},
issn = {1525-0024},
support = {R01 GM141296/GM/NIGMS NIH HHS/United States ; R01 NS123556/NS/NINDS NIH HHS/United States ; U01 NS122102/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Huntington Disease/therapy/genetics/metabolism/pathology ; *Huntingtin Protein/genetics/metabolism ; Mice ; Disease Models, Animal ; *CRISPR-Cas Systems ; Humans ; Dependovirus/genetics ; *Genetic Therapy/methods ; Genetic Vectors/genetics/administration & dosage ; Gene Editing ; Trinucleotide Repeat Expansion ; Gene Targeting ; Neurons/metabolism ; },
abstract = {Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene, which leads to a mutant protein that destroys neurons in the brain. Despite intense effort, there remains no approved disease-modifying therapy for HD. Here, we develop a pan-HTT-targeting CRISPR-Cas9 system that, when delivered to the striatum of R6/2 and YAC128 mice by adeno-associated virus serotype 5 (AAV5), lowered mutant HTT mRNA and protein by 55%-80% via its induction of frameshift-inducing insertion or deletion (indel) mutations in HTT exon 1. Cas9 targeting improved motor coordination and locomotor activity, decreased anxiety-like deficits, reduced clasping and weight loss, limited striatal atrophy, and decreased the formation of intranuclear inclusions immunoreactive for the mutant HTT protein. In Hu21/21 mice, which carry the wild-type human HTT gene in lieu of the mouse ortholog, Cas9 lowered the HTT protein by 44% but induced no measurable behavioral deficits and had no adverse effect on neuronal viability, though its targeting was associated with neuroinflammation. Altogether, our results demonstrate the ability of a newly developed pan-HTT-targeting Cas9 system to affect HD-related phenotypes across models and provide insights into its tolerability.},
}

Animals
*Huntington Disease/therapy/genetics/metabolism/pathology
*Huntingtin Protein/genetics/metabolism
Mice
Disease Models, Animal
*CRISPR-Cas Systems
Humans
Dependovirus/genetics
*Genetic Therapy/methods
Genetic Vectors/genetics/administration & dosage
Gene Editing
Trinucleotide Repeat Expansion
Gene Targeting
Neurons/metabolism

@article {pmid42231480,
year = {2026},
author = {Xu, L and Liang, H and Bai, S and Xu, R and Liu, B and Xu, X and Xu, X and Liu, M},
title = {Protein arginine methyltransferase 5 is essential for virulence in Toxoplasma gondii.},
journal = {Parasites & vectors},
volume = {19},
number = {1},
pages = {},
pmid = {42231480},
issn = {1756-3305},
support = {2021A1515011707//Guangdong Provincial Natural Science Foundation/ ; },
mesh = {*Toxoplasma/pathogenicity/genetics/enzymology ; *Protein-Arginine N-Methyltransferases/genetics/metabolism ; Animals ; Virulence ; Mice ; Mice, Inbred BALB C ; Arginine/analogs & derivatives/metabolism ; *Protozoan Proteins/genetics/metabolism ; Female ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Protein arginine methyltransferase 5 (PRMT5) is a key enzyme responsible for catalyzing symmetric dimethylarginine (SDMA) modifications and plays crucial roles in epigenetic regulation, transcription, and cell cycle progression in eukaryotes. Although our previous study determined the expression and cellular localization of PRMT5 in tachyzoites and bradyzoites, and confirmed its type II PRMT activity, its functional significance in Toxoplasma gondii remains entirely uncharacterized.

METHODS: This study aimed to explore the biological functions of PRMT5 in T. gondii. The prmt5 gene was disrupted in the type I RH strain using the clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 system. The biological roles of PRMT5 were evaluated via multiple functional assays, including plaque formation, intracellular proliferation, host cell invasion, virulence, and tachyzoite to bradyzoite conversion assays. RNA sequencing was further performed to profile transcriptomic alterations induced by prmt5 disruption.

RESULTS: Phenotypic characterization revealed that the ∆prmt5 strain exhibited reduced symmetric dimethylarginine (SDMA) levels as well as severe defects in plaque formation, invasion, intracellular replication, and bradyzoite differentiation. Accordingly, the virulence of the ∆prmt5 strain was dramatically attenuated, as all infected BALB/c mice survived over a 10-day period, in stark contrast to the 100% mortality observed in the wild-type control group within 10 days. RNA-sequencing analysis uncovered the molecular basis for these phenotypes, demonstrating that prmt5 disruption leads to global transcriptional dysregulation. Specifically, we identified a significant downregulation of genes associated with motor protein function and fatty acid metabolism pathways.

CONCLUSIONS: Our research has demonstrated that PRMT5 plays a critical role in the proliferation, survival, pathogenicity, and regulation of gene expression in Toxoplasma gondii.},
}

*Toxoplasma/pathogenicity/genetics/enzymology
*Protein-Arginine N-Methyltransferases/genetics/metabolism
Animals
Virulence
Mice
Mice, Inbred BALB C
Arginine/analogs & derivatives/metabolism
*Protozoan Proteins/genetics/metabolism
Female
CRISPR-Cas Systems

@article {pmid42259916,
year = {2026},
author = {Zeng, J and Cheng, Z and Chen, H and Wang, Z and Thompson, J and Crosby, KT and Han, H and Singhal, A and Ngo, W and Xia, C and Rosas-Rivera, D and Zhang, Z and Kang, MH and Mao, Y and Diolaiti, ME and Lee, GC and Diffley, JFX and Song, Y and Qiu, L and Krah, NM and Murthy, N and Jackson, RN and Liu, Y and Ashworth, A and Doudna, JA},
title = {Targeting cancer-specific mutations with RNA-triggered chromatin shredding.},
journal = {Nature},
volume = {656},
number = {8126},
pages = {199-206},
pmid = {42259916},
issn = {1476-4687},
mesh = {Humans ; *Chromatin/metabolism/genetics ; *Mutation/genetics ; *Neoplasms/genetics/pathology/therapy ; CRISPR-Cas Systems/genetics ; DNA Damage ; Animals ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Genetic mutations that drive cancer often occur in tumour-suppressor proteins such as the p53 transcription factor, which is altered in 40-50% of cases[1,2]. However, current therapies often fail to target these mutations because the mutant proteins typically lack defined drug-binding pockets and restoring their endogenous function has proven challenging. Here we program Cas12a2, an RNA-guided CRISPR nuclease with trans-nucleolytic cleavage activity[3,4], to kill cancer cells selectively by targeting cancer-specific transcripts. This approach limited cell growth by inducing trans shredding of chromatin and triggering DNA-damage responses and cell death. In contrast to existing methods, RNA-guided Cas12a2 senses cellular RNA signatures, enabling precise targeting of undruggable mutations. Transcript-activated chromatin shredding provides an innovative approach to precision disease treatments for undruggable targets.},
}

Humans
*Chromatin/metabolism/genetics
*Mutation/genetics
*Neoplasms/genetics/pathology/therapy
CRISPR-Cas Systems/genetics
DNA Damage
Animals
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid42331809,
year = {2026},
author = {Zhou, C and Dong, C and Zhao, W and Liang, FS},
title = {Hierarchical interplay between H3K27ac and H3K4me3 in transcriptional regulation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42331809},
issn = {2041-1723},
support = {R01GM143256//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Histones/metabolism/genetics ; *Transcription, Genetic ; RNA Polymerase II/metabolism ; Epigenome Editing ; Humans ; Promoter Regions, Genetic ; *Gene Expression Regulation ; CRISPR-Cas Systems ; Chromatin/metabolism ; RNA Stability ; Animals ; },
abstract = {H3K27ac and H3K4me3 are enriched at transcriptional start sites and have been implicated in transcription. However, how these marks concertedly regulate transcription is not fully understood. Here, we develop a dual chemically inducible CRISPR/dCas9-based epigenome editing system that enables independent, temporal and transcription stage-specific modulation of H3K27ac and H3K4me3 at a specific gene locus. Stage-specific removal of H3K4me3 impairs RNA polymerase II recruitment, increases promoter-proximal pausing, reduces productive elongation, and accelerates mRNA decay via increased m[6]A deposition. Losing both H3K27ac and H3K4me3 rapidly abolishes transcriptional activity, while preserving H3K4me3 without H3K27ac can partially sustain transcription. These findings reveal a functional hierarchy and interdependence between H3K27ac and H3K4me3 in different transcription stages at the tested gene loci. This versatile tool will contribute to the functional dissection of the temporal dynamics of chromatin modifications in gene regulation.},
}

*Histones/metabolism/genetics
*Transcription, Genetic
RNA Polymerase II/metabolism
Epigenome Editing
Humans
Promoter Regions, Genetic
*Gene Expression Regulation
CRISPR-Cas Systems
Chromatin/metabolism
RNA Stability
Animals

@article {pmid42358199,
year = {2026},
author = {Gijsbertsen, M and Duarte, FM and Fuentes Manjón, A and Maduro, T and Kassem, M and van der Oost, J and Mathijssen, IMJ and van Leeuwen, JPTM and van de Peppel, J},
title = {Evaluation of Prime Editing Efficiency in Human Immortalized MSC-TERT Cells with Osteogenic Potential for Modeling FGFR2-Linked Craniosynostosis.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {191-206},
doi = {10.1177/25731599261457618},
pmid = {42358199},
issn = {2573-1602},
mesh = {Humans ; *Mesenchymal Stem Cells/metabolism/cytology ; *Receptor, Fibroblast Growth Factor, Type 2/genetics ; *Osteogenesis/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; HEK293 Cells ; *Craniosynostoses/genetics ; Telomerase/genetics/metabolism ; Mutation ; Cell Line ; },
abstract = {Craniosynostosis is a rare congenital bone condition where skull sutures fuse prematurely and is linked to mutations in over 60 genes. Generating mutation-specific in vitro models allows investigation of craniosynostosis-associated mutations without the need for patient-derived material or transgenic gene expression. Here, we developed a human in vitro disease model with the CRISPR-Cas9 prime editing variant, using an immortalized TERT-immortalized mesenchymal bone marrow-derived stem (MSC-TERT) cell line with osteogenic potential. MSC-TERT cells showed a higher resistance to prime editing, compared with HEK293FT cells. Addition of dnMLH1 and epegRNAs resulted in higher editing efficiencies in HEK293FT cells, but not in MSC-TERT cells. Prime editing efficiency varied between targeted loci and was found to be more efficient in nonadherent cells compared with adherent cells. Prime editing continued over 4 days in an isolated nonadherent HEK293FT culture. Our results present a foundation on the use of prime editing to establish FGFR2 mutation-specific in vitro models and their application in MSC-TERT cells.},
}

Humans
*Mesenchymal Stem Cells/metabolism/cytology
*Receptor, Fibroblast Growth Factor, Type 2/genetics
*Osteogenesis/genetics
*Gene Editing/methods
CRISPR-Cas Systems
HEK293 Cells
*Craniosynostoses/genetics
Telomerase/genetics/metabolism
Mutation
Cell Line

@article {pmid42413621,
year = {2026},
author = {Nan, Y and Yan, S and Zhang, L and Yang, F and Qiao, M and Xin, Y},
title = {Markerless large DNA integration in Lactococcus lactis through the coupling of homologous single-crossover and Cre/loxP system.},
journal = {Journal of biotechnology},
volume = {418},
number = {},
pages = {60-68},
doi = {10.1016/j.jbiotec.2026.07.004},
pmid = {42413621},
issn = {1873-4863},
mesh = {*Lactococcus lactis/genetics ; Plasmids/genetics ; *Integrases/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Lactococcus lactis is widely used in food fermentation and has great potential as a microbial cell factory for producing high-value compounds. Currently, heterologous gene expression in this host mainly relies on plasmid-based systems, which suffer from segregational instability without antibiotic selection. To address this problem, several genome integration tools have been developed, yet most are limited to single gene insertions or leave selection markers. In this study, we developed an efficient and markerless platform for integration single genes and large DNA fragments combining a temperature-sensitive plasmid for homologous single-crossover and the Cre/loxP system for plasmid backbone excision. First, using CRISPR/Cas9 assisted ssDNA recombineering, we introduced a loxP site into the ribB gene of L. lactis NZ9000/RecT, creating the chassis strain L. lactis loxP. We then constructed a donor plasmid, pG15AribB-Up-Cat-loxP, which carries a homology arm of ribB, the chloramphenicol resistance gene cat, and a second loxP site. This donor plasmid was integrated into the chromosome next to the existing loxP site through a single-crossover event. Subsequent expression of Cre recombinase then removed the plasmid backbone, leaving only the target gene cat at the insertion site. After 99 generations without chloramphenicol selection, the integrant L. lactis IMT maintained nearly 100% genetic stability. Using this method, we next successfully markerless integrated the ∼4.8 kb crtEBI cluster, and the seven gene tagatose-6-phosphate pathway (∼7.0 kb). This work provides an efficient platform for markerless and stable large DNA integration in L. lactis for constructing microbial cell factory to produce high-value compounds.},
}

*Lactococcus lactis/genetics
Plasmids/genetics
*Integrases/genetics/metabolism
CRISPR-Cas Systems

@article {pmid42456866,
year = {2026},
author = {Qu, ZL and Liu, T and Qin, TK and Yue, HM and Huang, L and Ruan, R and Wu, CS and Ye, H and Li, CJ},
title = {Establishment of a novel brain cell line from grass carp (Ctenopharyngodon idella) with high transfection efficiency and CRISPR/Cas9-mediated genome editing capacity.},
journal = {Fish & shellfish immunology},
volume = {177},
number = {},
pages = {111597},
doi = {10.1016/j.fsi.2026.111597},
pmid = {42456866},
issn = {1095-9947},
mesh = {Animals ; *Carps/genetics/immunology ; Cell Line ; Transfection/veterinary ; *CRISPR-Cas Systems ; *Brain/cytology ; *Gene Editing/veterinary ; Reoviridae/physiology ; Fish Diseases/immunology/virology ; Reoviridae Infections/veterinary/immunology ; },
abstract = {Fish cell lines serve as valuable tools in aquaculture research, particularly in immunology, pathology, and toxicology. In this study, we successfully established a novel cell line derived from brain tissue of grass carp, designated CIB. This cell line has been subcultured over 100 times and exhibits a fibroblast-like morphology. Chromosomal analysis revealed that the diploid chromosome number of CIB cells is 2n = 48, while sequencing of the 18S rRNA gene confirmed the cell line's origin. Notably, CIB cells demonstrated a transfection efficiency of 62.4% with pEGFP-N3, highlighting their potential for studies involving exogenous gene expression. Furthermore, CIB cells were susceptible to grass carp reovirus genotype I (GCRV-I), as evidenced by cytopathic effects (CPE), increased synthesis of viral proteins, and accumulation of viral particles within the cells. Both viral infection and poly(I:C) stimulation significantly increased the expression of intracellular interferon-related signaling molecules. Additionally, electroporation of a gRNA-Cas9 ribonucleoprotein (RNP) complex into CIB cells achieved the first successful large-fragment gene knockout in cultured grass carp cells and generated a homozygous clonal line, thereby enhancing the antiviral response. In summary, this novel cell line represents a significant advancement for studying gene functions, host-virus interactions, and genetic engineering in teleost fish.},
}

Animals
*Carps/genetics/immunology
Cell Line
Transfection/veterinary
*CRISPR-Cas Systems
*Brain/cytology
*Gene Editing/veterinary
Reoviridae/physiology
Fish Diseases/immunology/virology
Reoviridae Infections/veterinary/immunology

@article {pmid42460742,
year = {2026},
author = {Lv, B and Chen, Y and Zhou, R and Liu, R and Li, D},
title = {DNAzyme-mediated synergistic activation of CRISPR/Cas12a for Cd[2+] and Pb[2+] biosensing.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {61},
pages = {15252-15256},
doi = {10.1039/d6cc04053e},
pmid = {42460742},
issn = {1364-548X},
mesh = {*Lead/analysis ; *DNA, Catalytic/metabolism/chemistry ; *Cadmium/analysis ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {This work reports an isothermal, one-pot assay for Cd[2+] and Pb[2+] based on DNAzyme-triggered synergistic activation of CRISPR/Cas12a.},
}

*Lead/analysis
*DNA, Catalytic/metabolism/chemistry
*Cadmium/analysis
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/metabolism

@article {pmid42479891,
year = {2026},
author = {Feng, H and Wang, Y and Zhao, J and Fan, H and Wu, T and Qu, J and Pan, S and Wang, J and Han, Z and Liang, A and Xu, T and Chen, C and Hu, C and Hu, T},
title = {Topology-Gated λ Exonuclease Enables Amplification-Free Signal Boosting.},
journal = {Journal of the American Chemical Society},
volume = {148},
number = {30},
pages = {32782-32792},
doi = {10.1021/jacs.6c11250},
pmid = {42479891},
issn = {1520-5126},
support = {23ZR1450300//Natural Science Foundation of Shanghai Municipality/ ; 23-0178-0002//National University of Singapore/ ; A-8002032-00//Ministry of Education - Singapore/ ; 22304061//National Natural Science Foundation of China/ ; 22304157//National Natural Science Foundation of China/ ; LQ23H200005//Natural Science Foundation of Zhejiang Province/ ; ZR2023MB019//Natural Science Foundation of Shandong Province/ ; 24-0994-P0001//National Centre for Infectious Disease/ ; },
mesh = {*Exodeoxyribonucleases/metabolism/chemistry ; CRISPR-Cas Systems ; *RNA, Viral/analysis ; *Exonucleases/metabolism/chemistry ; Humans ; *Bacteriophage lambda/enzymology ; MicroRNAs/analysis ; Viral Proteins ; },
abstract = {Amplification-free detection remains a fundamental challenge in CRISPR-based RNA diagnostics. Here, we identify a previously unrecognized topological property of λ exonuclease, whereby duplex substrates bearing 5' phosphates at both termini undergo a self-sustained cyclic cleavage-reforming process. This topology-gated behavior enables signal renewal without external amplification. Through systematic biochemical and structural analyses, we elucidate the underlying mechanism and establish λ exonuclease as a topology-driven signal amplifier. Then, we design a topology-gated dumbbell probe that sequesters 5' phosphates within dual RNA hairpin loops. Upon target recognition, CRISPR/Cas13 specifically cleaves the loops, exposing the hidden phosphates and thereby activating the λ exonuclease-mediated cyclic reaction. The resulting cascade, termed Topo-CRISPR (Topology-gated λ exonuclease enables CRISPR amplification-free), achieves attomolar sensitivity within 25 min without preamplification. Applied to clinical samples, Topo-CRISPR enables robust and specific detection of enterovirus RNA, miR-21, and ciR1445, demonstrating performance comparable to RT-qPCR. We further extend the Topo-CRISPR to non-nucleic-acid targets via aptamer-mediated conformational gating. This work uncovers a previously overlooked enzymatic topology, positioning λ exonuclease as a cyclic signal transducer and offering a general framework for ultrasensitive molecular sensing.},
}

*Exodeoxyribonucleases/metabolism/chemistry
CRISPR-Cas Systems
*RNA, Viral/analysis
*Exonucleases/metabolism/chemistry
Humans
*Bacteriophage lambda/enzymology
MicroRNAs/analysis
Viral Proteins

@article {pmid42487445,
year = {2026},
author = {Collantes, JC and Xu, K and Ruiz-Urigüen, M and Samaniego-Villacis, AC and Stombaugh, J and Jin, S},
title = {Development and Characterization of RNA Aptamer-Mediated Modular Base Editors Containing Staphylococcus aureus Cas9 Derivatives and Novel Deaminase Orthologs.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {207-222},
doi = {10.1177/25731599261467606},
pmid = {42487445},
issn = {2573-1602},
mesh = {*Staphylococcus aureus/genetics/enzymology ; *Gene Editing/methods ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; Animals ; *Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cytidine Deaminase/genetics ; APOBEC-1 Deaminase/genetics ; HEK293 Cells ; Rats ; },
abstract = {Base editing enables precise genome modifications without introducing DNA double-strand breaks. Using Streptococcus pyogenes Cas9 as a prototype, we previously developed a modular base editing platform in which the deaminase is recruited by an RNA aptamer engineered into the gRNA, thereby separating sequence recognition from base modification. Here, we expanded this modular base editor toolbox by engineering Staphylococcus aureus Cas9 (SaCas9) in combination with various vertebrate effectors derived from activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic subunit 1 (APOBEC1) orthologs, from bat, lizard, human, and rat. Moreover, we adopted the SaCas9 variants with different protospacer adjacent motif requirements. These base editors generally showed high editing efficiency with low on-target indel formation and low-to-undetectable off-target activities. Quantitative and qualitative differences in editing occur among the base editors when applied to diverse loci, allowing sequence-specific optimization. Together, our study demonstrates the effectiveness of the SaCas9 modular base editors, the robustness of the platform's modularity, and its feasibility for convenient screening of target-specific base editors.},
}

*Staphylococcus aureus/genetics/enzymology
*Gene Editing/methods
Humans
*CRISPR-Associated Protein 9/genetics/metabolism
Animals
*Aptamers, Nucleotide/genetics
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Cytidine Deaminase/genetics
APOBEC-1 Deaminase/genetics
HEK293 Cells
Rats

@article {pmid42488965,
year = {2026},
author = {Shepard, A and Minones-Moyano, E and Mork, C and Pyhtila, B and Barrangou, R},
title = {The Diversifying Distribution Trends of Maturing CRISPR Technologies by Addgene.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {184-190},
doi = {10.1177/25731599261470096},
pmid = {42488965},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Plasmids/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {Since the advent of Cas9-based CRISPR technologies in 2012, there has been a remarkable growth in genome editing research, literature, applications, and translational impact. Much of this research has been fueled by the global dissemination of CRISPR plasmids through nonprofit distribution by Addgene, as both a repository and distributor of enabling biological material. Recently, key milestones have been reached, with over 20,000 plasmids deposited by over 1,000 labs, being distributed over 300,000 times globally. The driving trends reflect multidimensional diversification in terms of effectors (Cas9, Cas12 and beyond), editing modalities (base editing, prime editing, epigenetic modification, CRISPRi/a), and deployment across phylogenetic groups (mammalian, bacterial, plant, yeast, insects, and more). Noteworthy, guide RNA and HDR templates account for the bulk of deposits, while cloning backbones are the most requested, and lentiviral plasmids comprise the majority of expression material. The data reflect a continued diversification of the CRISPR-based toolbox, robust interest in genome editing applications across the tree of life, maturation in terms of adoption, and rising relative distribution beyond the USA and China, with Addgene continuing to play a critical role in access to equitable and disruptive technologies.},
}

*CRISPR-Cas Systems
*Gene Editing/methods/trends
*Plasmids/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Animals
RNA, Guide, CRISPR-Cas Systems/genetics
Humans

@article {pmid42525182,
year = {2026},
author = {Fang, G and Zheng, S and Miao, J and Zeng, Y and Peng, Y and Zhai, Y and Guo, H and Wang, J and Dong, Y and Tian, X and Liu, M and Liu, Y and Li, X and Wang, M and Zhao, X and Du, J and Wang, Y and Dong, J},
title = {The ApoE-Null Golden Hamster: A Novel Model of Atherosclerosis.},
journal = {Cardiovascular toxicology},
volume = {26},
number = {8},
pages = {},
pmid = {42525182},
issn = {1559-0259},
mesh = {Animals ; *Atherosclerosis/pathology/genetics/metabolism/blood ; Disease Models, Animal ; *Apolipoproteins E/genetics/deficiency ; Mesocricetus ; Diet, High-Fat ; Plaque, Atherosclerotic ; *Aortic Diseases/pathology/genetics/metabolism ; Cholesterol, Dietary ; CRISPR-Cas Systems ; *Aorta/pathology/metabolism ; Male ; Phenotype ; Genetic Predisposition to Disease ; Lipids/blood ; Liver/pathology/metabolism ; Hyperlipidemias/genetics/pathology ; Animals, Genetically Modified ; Fibrosis ; },
abstract = {Atherosclerosis is a chronic, progressive arterial disease characterized by the deposition of lipids on the inner arterial walls, leading to plaque formation and serious cardiovascular events. Traditional mouse models of atherosclerosis require prolonged dietary induction to exhibit arterial lesions due to significant differences in lipid metabolism compared to humans. In contrast, Golden hamsters share a lipid metabolic profile more closely aligned with humans. In this study, we utilized CRISPR/Cas9 to generate ApoE knockout (ApoE[-/-]) hamsters using, which spontaneously developed atherosclerotic lesions in the arterial wall after 8 weeks on a standard chow diet. When fed on a high-cholesterol/high-fat diet, they exhibited even more severe aortic atherosclerosis, fatty liver, and liver fibrosis. Our findings demonstrated that the ApoE[-/-] hamster model is highly valuable tool for translational research, offering significant potential for studying hyperlipidemia and atherosclerosis in a context more relevant to human physiology.},
}

Animals
*Atherosclerosis/pathology/genetics/metabolism/blood
Disease Models, Animal
*Apolipoproteins E/genetics/deficiency
Mesocricetus
Diet, High-Fat
Plaque, Atherosclerotic
*Aortic Diseases/pathology/genetics/metabolism
Cholesterol, Dietary
CRISPR-Cas Systems
*Aorta/pathology/metabolism
Male
Phenotype
Genetic Predisposition to Disease
Lipids/blood
Liver/pathology/metabolism
Hyperlipidemias/genetics/pathology
Animals, Genetically Modified
Fibrosis

@article {pmid42525385,
year = {2026},
author = {Kim, GD and Gu, D and Park, M and Wook Chi, S},
title = {abCRISPR: deep learning-based design of abasic gRNA sequences for specific CRISPR-Cas genome editing.},
journal = {Bioinformatics (Oxford, England)},
volume = {},
number = {},
pages = {},
doi = {10.1093/bioinformatics/btag568},
pmid = {42525385},
issn = {1367-4811},
abstract = {SUMMARY: CRISPR-Cas9 has become a widely used tool for genome editing. However, its off-target cleavage caused by partial sequence matches with guide RNAs (gRNAs) remains a critical limitation. Recently, abasic gRNAs (ØXØ) have been developed to enhance target specificity, but their effects vary depending on the positional sequence context. Here, we present abCRISPR, a deep neural network (DNN) framework for the rational design of ØXØ sequences with minimized off-target activity. abCRISPR leverages informative few-shot training with paired datasets of abasic and unmodified gRNAs, using high-quality random mismatch target libraries, exhaustively sequenced for mismatched off-target substrates (n = 97,583) in in vitro CRISPR-Cas9 cleavage experiments. Predicted off-target activities for both abasic and unmodified gRNAs showed strong correlation with experimental data (r ≥ 0.95, 10-fold cross-validation). Notably, these comprehensive training sets provide robust ground-truth negatives, enabling accurate and sensitive prediction of off-targets. For unmodified gRNAs, abCRISPR (AUC = 0.98) was validated to outperform existing deep learning-based methods (AUC = 0.45-0.68). When applied to the human genome, abCRISPR generated ØXØ sequences, covering 58,875,004 potent CRISPR-targetable sites with improved target specificity. Together, this work provides a comprehensive bioinformatics resource for safe and precise CRISPR-Cas9 genome editing.

The source code for abCRISPR and training data are available at https://doi.org/10.5281/zenodo.20398246. abCRISPR results for the human genome are available at http://clip.korea.ac.kr/abCRISPR/.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}

@article {pmid42528148,
year = {2026},
author = {du Plessis, J and Omar, A},
title = {Patient-Derived Organoid-Based CRISPR Screens in Cancer Research: Applications, Advances, and Challenges.},
journal = {Cancer medicine},
volume = {15},
number = {8},
pages = {e72112},
pmid = {42528148},
issn = {2045-7634},
mesh = {Humans ; *Organoids/metabolism ; *Neoplasms/genetics/pathology ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genomics/methods ; Animals ; Precision Medicine/methods ; },
abstract = {Patient-derived organoids (PDOs) have emerged as physiologically relevant cancer models that preserve key genetic, histological, and functional features of the tumors from which they are derived. In parallel, CRISPR-based perturbation technologies have transformed functional genomics by enabling scalable interrogation of gene function. Their integration provides a powerful framework for identifying cancer dependencies, modeling oncogenic evolution, and investigating mechanisms of drug response and resistance in patient-relevant settings. This review examines how CRISPR knockout, CRISPR interference/activation, and precision editing approaches have been applied in PDO systems to uncover context-specific vulnerabilities, reconstruct mutational trajectories, and study tumor heterogeneity. We further compare pooled and arrayed screening formats and discuss what is uniquely enabled by performing CRISPR screens in organoids rather than conventional 2D models. Particular emphasis is placed on the technical and analytical constraints of organoid-based screening, including variable editing efficiency, clonal bottlenecks, biological heterogeneity, and limited scalability. We argue that the major value of organoid-based CRISPR screening lies in its ability to identify functionally actionable cancer vulnerabilities in a patient-contextualized model, while also introducing methodological challenges that must be addressed for robust clinical translation.},
}

Humans
*Organoids/metabolism
*Neoplasms/genetics/pathology
*CRISPR-Cas Systems
Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
Genomics/methods
Animals
Precision Medicine/methods

@article {pmid42529880,
year = {2026},
author = {Zhang, W and Hang, Y and Zhan, S and Song, W and Li, B and Chen, N and Lv, M},
title = {Emerging point-of-care technologies for bacterial pathogen detection.},
journal = {Journal of Zhejiang University. Science. B},
volume = {27},
number = {7},
pages = {677-697},
pmid = {42529880},
issn = {1862-1783},
support = {32371439 and 22404026//the National Natural Science Foundation of China/ ; 24ZR1455600 and 24ZR1455900//the Natural Science Foundation of Shanghai/ ; },
mesh = {Humans ; *Point-of-Care Systems ; *Bacteria/isolation & purification/genetics ; *Point-of-Care Testing ; *Bacterial Infections/diagnosis/microbiology ; Rapid Diagnostic Tests ; Immunoassay ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; },
abstract = {Bacterial infections remain a significant threat to public health worldwide, driving an urgent need for rapid, accurate, and field-deployable diagnostic techniques. Point-of-care testing (POCT) has emerged as a transformative strategy, providing timely detection, operational simplicity, and portability. Recent studies have aimed at enhancing sensitivity, specificity, multiplexing capability, and automation through the integration of molecular diagnostics with microfluidics and lab-on-chip technologies, alongside the development of low-cost, portable devices equipped with smartphone-based readout and cloud connectivity for real-time surveillance in resource-limited settings. Nonetheless, evidence-based frameworks for selecting optimal detection targets-such as genomic sequences, conserved protein epitopes, or viable whole cells-and matching them to appropriate POCT modalities remain notably underrepresented in the literature. This review systematically summarizes recent advances in POCT strategies for bacterial detection, categorized according to three major types of detection targets, including cellular phenotypic characteristics, surface antigens, and nucleic acids. We discuss the principles, advantages, limitations, and representative applications of key POCT platforms, which include microscopy-based visualization, immunoassays, isothermal amplification, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems, and microfluidic biosensors. Critical challenges, such as sample pretreatment, detection sensitivity, and operational simplicity, have been partially addressed through recent innovations. Finally, we outline the main future research directions focused on the development of integrated, automated, and intelligent POCT systems for clinical deployment.},
}

Humans
*Point-of-Care Systems
*Bacteria/isolation & purification/genetics
*Point-of-Care Testing
*Bacterial Infections/diagnosis/microbiology
Rapid Diagnostic Tests
Immunoassay
Nucleic Acid Amplification Techniques
CRISPR-Cas Systems

@article {pmid42530034,
year = {2026},
author = {Sharma, N and Tanwar, D and Grewal, U and Keerthi, MM and Ahlawat, YK and Pandey, N and Makvana, CG},
title = {Pectinase-Based Bioprocesses for Circular Bioeconomy and Sustainable Industrial Development.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70312},
pmid = {42530034},
issn = {1097-0290},
abstract = {Pectinases play a vital role in the degradation of pectic part of the plant cell wall and are considered in the group of hydrolytic enzymes. In the present scenario, demand of economically feasible and environment friendly techniques and approaches has significantly led the research on the microbial production, standardization of process parameters and improvements of pectinases. The development of pectinase-based bioprocesses has been found to promote sustainable industrial practices by reducing the use of chemicals, energy, and waste, thereby supporting eco-friendly production systems. The unique aspect of this review is that it explains traditional methods of pectinase production alongside novel methods, which include solid-state and submerged fermentation, recombinant DNA techniques, heterologous gene expression, protein engineering, CRISPR/Cas genome editing technologies, enzyme immobilization, and nanobiotechnological methods, which improve the production, stability, and performance of pectinases. It further demonstrates the increasing importance of the use of pectinases in sustainable manufacturing through decreasing the use of chemicals and energy, as well as reducing waste production from industry. It illustrates that innovations in microbial strain engineering, process optimization, and utilization of low-cost substrates have significantly increased the economic viability and efficiency of producing pectinases. Yet, some issues associated with commercialization, standardization of production processes, recovery, and stability of enzymes still exist. Overall, this review paper gives an overview of the recent advancements and limitations of next-generation pectinases.},
}

@article {pmid42531075,
year = {2026},
author = {Zhou, R and Zhan, Y and Sun, Y and Wang, R and Wang, T and Liu, Z and Shan, Z and Li, X and Zhang, S and Sun, N and Zhang, H and Yuan, Z and Yang, J},
title = {Molecular basis of single-mismatch-induced nuclease-to-nickase conversion in TIGR-TasH.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42531075},
issn = {1362-4962},
support = {2024ZD037//Scientific Research Program of Tianjin Municipal Education Commission/ ; TJYXZDXK-009A//Tianjin Key Medical Discipline/ ; TJYXZDXK-3-004B//Tianjin Key Medical Discipline/ ; 2024ZD037//Scientific Research Program of Tianjin Municipal Education Commission/ ; },
mesh = {Cryoelectron Microscopy ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *Base Pair Mismatch ; *Deoxyribonuclease I/metabolism/genetics/chemistry ; Protein Domains ; Models, Molecular ; },
abstract = {Tandem interspaced guide RNA (TIGR)-Tas systems are a distinct class of RNA-guided double-stranded DNA nucleases that employ dual-spacer guide RNAs (tigRNAs) for PAM-independent target recognition. A single mismatch between the tigRNA and target DNA can convert Salicola phage CGphi29 (Sp)TasH from a double-strand nuclease into a nickase in a position-dependent manner, but the molecular basis underlying this functional switch remains unknown. Here, we combined biochemical analyses and cryo-electron microscopy to investigate tigRNA maturation and mismatched target recognition by the Nop domain of SpTasH. We show that the Nop domain is required for pre-tigRNA processing and stabilizes the mature tigRNA through extensive interactions, thereby establishing a cleavage-competent ribonucleoprotein complex. Structural analyses of SpTasH complexes bound to substrates containing single mismatches reveal that a mismatch at the 5'-most position of spacer A is readily accommodated through Nop domain-mediated stabilization of the spacer-target heteroduplex. In contrast, a mismatch proximal to the cleavage site destabilizes the heteroduplex, preventing recruitment of the corresponding HNH domain, thereby converting the complex into a nickase. Together, these findings establish the structural basis for position-dependent mismatch recognition and reveal how Nop domain-mediated tigRNA-target stabilization enables differential responses to mismatches, providing a foundation for engineering TIGR-Tas systems for genome-editing applications.},
}

Cryoelectron Microscopy
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry
*Base Pair Mismatch
*Deoxyribonuclease I/metabolism/genetics/chemistry
Protein Domains
Models, Molecular

@article {pmid42532990,
year = {2026},
author = {Liu, Y and Zhao, H and Cao, X and Jiang, S and Fu, J and Xue, J and Li, C and Xu, Z and Li, M and Du, W},
title = {Rapid and quantitative measurement of bacteriophage infectivity via fully automated droplet digital PCR.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42532990},
issn = {2041-1723},
support = {XDB0810000//CAS | State Key Laboratory of Microbial Resources (State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences)/ ; 22374016//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370090//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Bacteriophages/genetics/physiology/pathogenicity ; *Polymerase Chain Reaction/methods ; RNA, Ribosomal, 16S/genetics ; DNA, Viral/genetics ; Automation ; Bacteria/virology/genetics ; Phage Therapy/methods ; },
abstract = {The clinical translation of phage therapy for multidrug-resistant infections is constrained by the lack of rapid, standardized therapeutic phage selection. Here, we introduce digital phage susceptibility testing (dPhaST), an automated droplet digital PCR workflow that quantifies phage-induced DNA release as a molecular signature of lysis. By targeting conserved 16S rRNA regions, dPhaST measures lytic activity across diverse bacterial pathogens within 3 h. Across 122 phage-host combinations involving 19 bacterial strains from six species, dPhaST shows 95.9% concordance with spot tests while resolving weak and heterogeneous lytic activities that are not readily distinguished phenotypically. It remains robust during the early infection window despite phage-encoded nuclease activity and tolerates phage cross-contamination better than spot tests. The method captures defense-mediated interactions involving CRISPR-Cas and Sir2-HerA systems. In this work, we show that automated digital quantification enables rapid and mechanistically informative profiling of early phage lytic efficacy across Gram-positive and Gram-negative pathogens.},
}

*Bacteriophages/genetics/physiology/pathogenicity
*Polymerase Chain Reaction/methods
RNA, Ribosomal, 16S/genetics
DNA, Viral/genetics
Automation
Bacteria/virology/genetics
Phage Therapy/methods

@article {pmid42533766,
year = {2026},
author = {Kong, W and Fu, L and Li, Y and Pan, W and Li, N and Tang, B},
title = {Amplification-free CRISPR/Cas biosensors for point-of-care nucleic acid detection: recent advances and future perspectives.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d6cc02994a},
pmid = {42533766},
issn = {1364-548X},
abstract = {Nucleic acid biomarkers are critical targets for early diagnosis of disease, public health surveillance and environmental safety. However, their low abundance and the complexity of the sample matrix pose strict requirements for the high sensitivity and portability of detection technologies. Although traditional clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems exhibit advantages such as high specificity, operational simplicity, and compatibility with mild reaction conditions, their reliance on pre-amplification steps elevates the risk of false-positive results and hinders their broader application. To overcome these limitations, amplification-free CRISPR/Cas technologies have emerged and undergone extensive development. These approaches enable highly sensitive nucleic acid detection without the need for pre-amplification and are more amenable to integration with portable devices, thereby offering promising avenues for point-of-care testing (POCT). This review systematically examines the fundamental principles and design strategies underlying amplification-free CRISPR/Cas biosensors and summarizes recent advances in detection platforms based on autocatalytic signal enhancement, nanomaterial-coupled amplification, and integrated high-sensitivity readout systems, while also outlining their practical applications in POCT settings. Furthermore, the key technical challenges and future development directions of amplification-free CRISPR technologies are discussed based on current advances in the field. These insights and perspectives aim to provide a systematic reference for further research and to facilitate the expanded application of amplification-free CRISPR/Cas systems in POCT.},
}

@article {pmid42534996,
year = {2026},
author = {Zhu, L and Huang, J and Xie, C},
title = {AI-enhanced framework for optimizing CRISPR-Cas gene editing in crop biotechnology addressing regulatory challenges and opportunities in global agricultural practices.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1770472},
pmid = {42534996},
issn = {1664-462X},
abstract = {INTRODUCTION: The integration of CRISPR Cas genome editing with artificial intelligence (AI) offers significant potential for crop biotechnology by supporting more precise and adaptive strategies for trait improvement under complex agricultural and regulatory conditions. However, the global governance of gene edited crops remains highly heterogeneous, creating major challenges for the development of frameworks that can jointly support optimization, uncertainty management, and regulatory alignment. Conventional approaches often lack the ability to account for evolving regulatory requirements and multi source uncertainties in a unified manner.

METHODS: In this paper, we introduce the Adaptive Regulatory Optimizer (ARO), an AI enhanced framework designed to support CRISPR Cas genome editing in crop biotechnology under biologically, regulatorily, and contextually constrained conditions. The ARO consists of three interconnected modules: the Manifold Constrained Gene Editor, the Agent Driven Regulatory Planner, and the Uncertainty Propagation Filter. Together, these modules embed editing decisions within biologically feasible manifolds, incorporate jurisdiction aware regulatory planning, and model interacting uncertainties associated with gene editing and deployment contexts. The The framework combines constrained optimization refinement, probabilistic uncertainty modeling, and adaptive regulatory planning to provide a structured basis for compliance aware and context sensitive decision support.

RESULTS AND DISCUSSION: Experimental results on the evaluated datasets indicate that the ARO achieves improved performance on the selected metrics relative to the compared methods, while its architecture is explicitly designed to integrate regulatory constraints into the optimization process. These findings suggest that the proposed framework provides a promising foundation for supporting more transparent, adaptive, and analytically grounded decision making in CRISPR Cas applications for crop biotechnology.},
}

@article {pmid42537394,
year = {2026},
author = {Zemmouchi, M and El-Fermawi, A and Benagdi, A and Baaziz, S and Hiram, R},
title = {Translational consistency of gene therapy strategies targeting inflammation in atrial fibrillation's management.},
journal = {Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie},
volume = {202},
number = {},
pages = {119824},
doi = {10.1016/j.biopha.2026.119824},
pmid = {42537394},
issn = {1950-6007},
abstract = {Atrial fibrillation (AF) is the most common cardiac rhythm disorder. AF risk factors include pathological ageing, hypertension, obesity, diabetes, and cardiac conditions such as myocardial infarction. Chronic inflammation is a major pathophysiological profile commonly observed in AF and its risk factors. Clinical and preclinical studies have suggested that increased expression of proinflammatory biomarkers such as the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, interleukin (IL)-1β, or IL6 is associated with the development and maintenance of cardiac arrhythmias including AF. Current anti-arrhythmic and anti-inflammatory treatments are non-optimal in AF management. In parallel, mounting evidence suggests that new biotechnologies including gene therapy approaches, might help to target specific genes to prevent or promote their expression and their associated protein activity. Applied to cardiac arrhythmias, gene therapy might help to restore normal functions of ion channels, optimal calcium (Ca[2 +])-handling machinery, functional gap junctions, and efficient inflammatory signaling, known to be altered in AF. With an emphasis on the importance of gene therapy strategies targeting inflammation, this narrative review aims to: i) highlight the rationale and clinical relevance of gene therapy as an innovative strategy for the management of AF; ii) evaluate current knowledge regarding gene therapy vectors and delivery platforms applicable to cardiology and AF; iii) review emerging molecular targets explored in the context of AF gene therapy; and iv) identify promising gene-based therapeutic candidates, with a particular focus on inflammation-related pathways in AF.},
}

@article {pmid42538871,
year = {2026},
author = {Wang, X and Trypsteen, W and Anckaert, J and de Bony, E and Mestdagh, P},
title = {An Optimized Workflow for In Vitro Transcription of Single Guide RNAs Minimizes Innate Immune Activation.},
journal = {The CRISPR journal},
volume = {9},
number = {4},
pages = {223-232},
doi = {10.1177/25731599261467974},
pmid = {42538871},
issn = {2573-1602},
mesh = {*Immunity, Innate/genetics ; *Transcription, Genetic ; *RNA, Guide, CRISPR-Cas Systems/genetics/immunology ; Humans ; CRISPR-Cas Systems/genetics ; RNA, Double-Stranded/genetics ; Workflow ; Gene Knockdown Techniques ; Sodium Chloride ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; },
abstract = {CRISPR interference (CRISPRi) often uses single guide RNAs (sgRNAs) generated by in vitro transcription (IVT); however, IVT-derived RNAs can trigger innate immune responses that confound functional analyses. Here, we evaluate innate immune activation induced by IVT sgRNAs in a CRISPRi setting and show that enzymatic removal of the 5'-triphosphate group alone is insufficient to consistently eliminate this response. We therefore assessed modifications of IVT reaction conditions and found that supplementation with sodium chloride or urea further attenuated immune activation. Based on immune suppression, sgRNA yield, and knockdown efficiency, 0.15 M NaCl was selected for the optimized IVT condition. This condition showed a lower double-stranded RNA (dsRNA) concentration, providing direct support for reduced dsRNA by-products as a contributor to diminished immune activation. By integrating NaCl-supplemented IVT with phosphatase treatment, we establish an optimized and scalable workflow that minimizes innate immune responses while preserving sgRNA-mediated target knockdown efficiency in stable CRISPRi cells.},
}

*Immunity, Innate/genetics
*Transcription, Genetic
*RNA, Guide, CRISPR-Cas Systems/genetics/immunology
Humans
CRISPR-Cas Systems/genetics
RNA, Double-Stranded/genetics
Workflow
Gene Knockdown Techniques
Sodium Chloride
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells

@article {pmid40476386,
year = {2026},
author = {Su, S and Xu, Z and Suo, J and Zhou, Y and Zhang, X and Peng, X and Chen, M and Li, F},
title = {Function analysis of flightin gene in the global tortricid fruit borer Grapholita molesta using CRISPR/Cas9.},
journal = {Insect science},
volume = {33},
number = {4},
pages = {1357-1368},
doi = {10.1111/1744-7917.70079},
pmid = {40476386},
issn = {1744-7917},
support = {//the Coordinated Research Project (CRP) of the International Atomic Energy Agency/ ; //the National Key R&D Program of China/ ; //the Science and Technology Partnership Program of Ministry of Science and Technology of China/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Moths/genetics/growth & development/physiology/metabolism ; *Insect Proteins/genetics/metabolism ; Flight, Animal ; Larva/growth & development/genetics/physiology ; Female ; Pupa/growth & development/genetics ; Gene Knockout Techniques ; Male ; },
abstract = {Many tortricid moths are significant fruit borers characterized by limited flight capacity. However, some individuals within a population of tortricid species exhibit extended flight capabilities, facilitating gene flow between orchards and enabling host switching. To date, research on the proteins involved in flight among fruit borers is limited. Flightin is recognized as a flight muscle protein, yet its function remains unexplored in lepidopteran insects. In this study, quantitative polymerase chain reaction analysis revealed that the flightin gene is expressed at various developmental stages and tissues of Grapholita molesta, with the highest expression in adults and the thorax. Using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9) gene editing technology, we successfully generated a homozygous flightin gene knockout strain of G. molesta. The knockout of the flightin gene resulted in contraction of indirect flight muscle fibers, irregularities in the Z-disc of the flight muscles, and a significant elongation of sarcomere length. Additionally, cumulative flight distance and flight time were significantly reduced. The larval period and preoviposition period were significantly prolonged, while larval weight, pupal weight, longevity, and fecundity were all significantly decreased. The results indicate that the flightin gene not only plays an important role in the flight capacity of G. molesta, but also has an effect on the growth and development, and reproduction of the insect, suggesting that flightin may be a potential target for pest management of G. molesta. This is the first investigation into the function of the flightin gene using CRISPR/Cas9.},
}

Animals
CRISPR-Cas Systems
*Moths/genetics/growth & development/physiology/metabolism
*Insect Proteins/genetics/metabolism
Flight, Animal
Larva/growth & development/genetics/physiology
Female
Pupa/growth & development/genetics
Gene Knockout Techniques
Male

@article {pmid41159566,
year = {2026},
author = {Yen, PS and Verkuijl, SANR and Capriotti, P and Del Corsano, G and Yee, CKG and Hoermann, A and Inghilterra, MG and Aramburu-Gonzalez, I and Khan, MA and Vlachou, D and Christophides, GK and Windbichler, N},
title = {The nanosd integral gene drive enables population modification of the malaria vector Anopheles gambiae.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf246},
pmid = {41159566},
issn = {2160-1836},
support = {OPP1158151//Bill and Melinda Gates Foundation/ ; INV-058071//Bill and Melinda Gates Foundation/ ; },
mesh = {Animals ; *Anopheles/genetics/parasitology ; Female ; *Mosquito Vectors/genetics/parasitology ; *Malaria/transmission/parasitology ; Male ; *Gene Drive Technology ; CRISPR-Cas Systems ; Fertility/genetics ; *Insect Proteins/genetics ; },
abstract = {The modification of mosquito populations at scale through CRISPR-Cas9-mediated homing gene drives is a promising route for malaria vector control. Integral gene drives (IGDs) are designed to utilize the regulatory sequences of endogenous genes to reduce the size of the modification required for nuclease and effector expression. In this study, we describe the creation and characterization of the nanosd integral gene drive, which targets and is inserted into the nanos gene of the malaria vector Anopheles gambiae, and show that it achieves high rates of gene drive (98.4% in females, 99.5% in males). We find that homozygous nanosd females but not males show impaired fecundity and exhibit variable degrees of ovary underdevelopment. Transcriptomic analysis of ovaries points to decreased transcript levels of the nanos gene when harboring Cas9 and changes to other fertility-related genes. As a minimal genetic modification, nanosd does not induce widespread transcriptomic perturbations that would affect vector competence, and we show that its susceptibility to Plasmodium spp. and O'nyong nyong virus infection remains similar to wild-type mosquitoes. Importantly, we find that nanosd propagates efficiently in caged mosquito populations and is maintained as a source of Cas9 after the emergence of drive-resistant alleles, whilst also mobilizing a nonautonomous antiparasitic effector modification. The nanosd gene drive shows promise as a genetic tool for malaria vector control via population modification, and we outline steps towards its further optimization.},
}

Animals
*Anopheles/genetics/parasitology
Female
*Mosquito Vectors/genetics/parasitology
*Malaria/transmission/parasitology
Male
*Gene Drive Technology
CRISPR-Cas Systems
Fertility/genetics
*Insect Proteins/genetics

@article {pmid41706533,
year = {2026},
author = {Overton, MS and Guy, SE and Chen, X and Martsul, A and Carolino, K and Akbari, OS and Meyer, JR and Kryazhimskiy, S},
title = {Upper bound on the mutational burden imposed by a CRISPR-Cas9 gene-drive element.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf315},
pmid = {41706533},
issn = {2160-1836},
support = {HR0011-17-2-0047//DARPA/ ; R35GM153242/GF/NIH HHS/United States ; 5T32GM133351-02//Pathways in Biological Sciences NIH T32 program/ ; //Tata Institute for Genetics and Society BS/MS Fellowship/ ; R35GM153242/GM/NIGMS NIH HHS/United States ; },
mesh = {Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Mutation ; Loss of Heterozygosity ; Mutation Rate ; },
abstract = {Homing-based CRISPR-Cas9 gene drives (CCGDs) are powerful tools for genetic control of wild populations, with applications from disease eradication to species conservation. However, Cas9 alone and in a complex with guide RNA can cause double-stranded DNA breaks at off-target sites, which could increase the mutational load and lead to unintended loss-of-heterozygosity (LOH) events. These undesired effects raise potential concerns about the long-term evolutionary safety of CCGDs, but the magnitude of these effects is unknown. To measure how the presence of a CCGD or a Cas9 alone in the genome affects the rates of LOH events and de novo mutations, we carried out a mutation accumulation experiment in yeast Saccharomyces cerevisiae. We found no detectable effects on the genome-wide rates of mutations or LOH events. Our power calculations suggest that CCGD or Cas9 affect these rates by less than 30%, which is much less than natural variation for these traits in yeast. A more detailed examination shows that CCGD or Cas9 may alter the lengths and genomic distributions of LOH events, but the statistical support for these effects is weak. Thus, our results demonstrate that CCGDs impose at most a weak additional mutational burden in the yeast model. Although mutagenic effects of gene drives need to be further evaluated in other systems, our results add credence to the proposition that the evolutionary risks posed by well-designed gene drives may be acceptable.},
}

Saccharomyces cerevisiae/genetics
*CRISPR-Cas Systems
*Mutation
Loss of Heterozygosity
Mutation Rate

@article {pmid41774761,
year = {2026},
author = {Harvey-Samuel, T and Kaur, R and Leftwich, PT and Feng, X and Gantz, V and Alphey, L},
title = {Sequence mismatch between gene-drive and target-site flanking regions significantly impairs homing efficiency in Culex quinquefasciatus.},
journal = {Genetics},
volume = {233},
number = {4},
pages = {},
doi = {10.1093/genetics/iyag054},
pmid = {41774761},
issn = {1943-2631},
support = {BBS/E/I/00007033//UK Biotechnology and Biological Sciences Research Council/ ; BBS/E/I/00007038//UK Biotechnology and Biological Sciences Research Council/ ; BBS/E/I/00007039//UK Biotechnology and Biological Sciences Research Council/ ; },
mesh = {Animals ; *Culex/genetics/virology ; *Gene Drive Technology/methods ; CRISPR-Cas Systems ; Transgenes ; Mosquito Vectors/genetics ; },
abstract = {CRISPR/Cas9-based homing gene-drives (homing-drives) hold enormous potential as control tools for mosquito disease-vectors. These genomically encoded technologies spread themselves through target populations by creating double-stranded DNA breaks on homologous chromosomes, into which the homing-drives are copied ("homed"). Homing is dependent on sequence homology between the genomic regions flanking the transgene insertion and the break site. Homing efficiency (ie copying rate) substantially impacts the power of these systems: less efficient homing-drives spread slower, have fewer applications, and are more resistance-prone. Understanding what influences homing-drive efficiency is therefore vital to the successful use of these technologies. Here we report a novel mechanism by which a homing-drive's efficiency can be significantly impaired by natural sequence variation within a population into which it is spreading. Using a kmo-targeting "split" homing-drive in the West Nile virus mosquito Culex quinquefasciatus, we found that target-site heterology (sequence mismatch between the genomic regions flanking the target cut-site and the homing-drive transgene) of less than 10% reduced homing efficiency by up to 54%. While substantial research effort has been dedicated to increasing homing-drive efficiency through optimization of within-construct components, our results highlight that the real-world efficacy of these systems may in part depend on variation beyond these controllable factors.},
}

Animals
*Culex/genetics/virology
*Gene Drive Technology/methods
CRISPR-Cas Systems
Transgenes
Mosquito Vectors/genetics

@article {pmid42083788,
year = {2026},
author = {Surender, S and Haeusser, LA and Kuhlburger, L and Tsiami, F and Dogan, NO and Maise, L and Nahnsen, S and Beck, S and Merk, DJ and Tabatabai, G},
title = {Molecular modulators of cyclin-dependent kinase 4/6 inhibitor response in experimental glioma identified through genome-wide CRISPR-Cas9 screening.},
journal = {Neuro-oncology},
volume = {28},
number = {8},
pages = {1904-1920},
doi = {10.1093/neuonc/noag093},
pmid = {42083788},
issn = {1523-5866},
support = {2019_Kolleg_14//Else Kröner Fresenius Stiftung/ ; },
mesh = {Humans ; *Cyclin-Dependent Kinase 4/antagonists & inhibitors/genetics ; *Cyclin-Dependent Kinase 6/antagonists & inhibitors/genetics ; Animals ; *Glioma/drug therapy/genetics/pathology ; Mice ; *Protein Kinase Inhibitors/pharmacology ; *CRISPR-Cas Systems ; *Brain Neoplasms/drug therapy/genetics/pathology ; Cell Proliferation ; Checkpoint Kinase 1/genetics/antagonists & inhibitors ; Apoptosis ; Oncogene Proteins/genetics ; Cell Line, Tumor ; Cyclin E ; },
abstract = {BACKGROUND: Glioblastoma harbors frequent alterations in the retinoblastoma pathway, providing a genetic rationale for therapeutic targeting with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. The NOA-20 trial did not reveal a progression-free survival benefit of CDK4/6 inhibition plus radiation therapy in newly diagnosed, O6-methylguanine DNA methyltransferase (MGMT)-unmethylated glioblastoma. In fact, CDK4/6 inhibitor monotherapy has not demonstrated efficacy in solid tumors. We aimed at discovering response modulators to CDK4/6 inhibition, paving the way for rational combination therapies.

METHODS: We conducted genome-wide CRISPR-Cas9 screens in human glioma cell lines and stem-like cells (LN229, LN18, LNZ308, T98G, and GS-9) under CDK4/6 inhibition, employing knockout (Brunello library) and activation strategies (Calabrese library), followed by genetic and pharmacological validation of selected candidate genes in vitro and ex vivo (primary cultures) as well as the investigation of 1 functionally instructed combination therapy in vivo.

RESULTS: Loss of AMBRA1 and gain of function of CCNE1 reduced sensitivity to CDK4/6 inhibition in glioma cells, whereas disruption of checkpoint kinase 1 (CHEK1) or FAM122A resulted in synthetic lethality in combination with CDK4/6 inhibition. AMBRA1-deficient glioma cells exhibited increased sensitivity to CHK1 inhibition, revealing a context-specific vulnerability. Combined inhibition of CHK1 and CDK4/6 led to synergistic antiglioma activity in vitro, ex vivo, and in vivo.

CONCLUSIONS: Our data identify AMBRA1, CCNE1, CHEK1, and FAM122A as potential molecular modifiers of CDK4/6 inhibition response in experimental glioma and provide a biological rationale for combinatorial targeting with CDK4/6 inhibition in glioblastoma.},
}

Humans
*Cyclin-Dependent Kinase 4/antagonists & inhibitors/genetics
*Cyclin-Dependent Kinase 6/antagonists & inhibitors/genetics
Animals
*Glioma/drug therapy/genetics/pathology
Mice
*Protein Kinase Inhibitors/pharmacology
*CRISPR-Cas Systems
*Brain Neoplasms/drug therapy/genetics/pathology
Cell Proliferation
Checkpoint Kinase 1/genetics/antagonists & inhibitors
Apoptosis
Oncogene Proteins/genetics
Cell Line, Tumor
Cyclin E

@article {pmid42192133,
year = {2026},
author = {Elena, M and Giuliana, N and Giuseppina, R and Valeria, L and Geppino, F and Viola, C and Maria, V and Tiziana, A and Dario, A and Alessandra, P},
title = {Unveiling a novel role for p19Arf (alternative reading frame) in mESC differentiation toward the pancreatic lineage.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42192133},
issn = {2045-2322},
support = {2022R74KBC//MUR- Ministero Università Ricerca-PRIN 2022/ ; },
mesh = {Animals ; *Cell Differentiation/genetics ; Mice ; *Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; Endoderm/cytology/metabolism ; *Mouse Embryonic Stem Cells/cytology/metabolism ; *Pancreas/cytology/metabolism ; *Cell Lineage/genetics ; Gene Expression Profiling ; CRISPR-Cas Systems ; Mutation ; },
abstract = {The tumor suppressor ARF (p14 in human, p19 in mouse), has traditionally been characterized by its pivotal role in tumor surveillance. However, its involvement in an expanding range of cellular processes reveals that its functions are broader and more complex than initially appreciated. Here, we uncover a previously unrecognized role of p19ARF in endodermal differentiation, specifically in pancreatic lineage specification using an in vitro differentiation model of mouse embryonic stem cells (mESCs). Using CRISPR/Cas9-mediated mutagenesis, we show that mESCs with mutations in p19Arf are unable to efficiently differentiate towards pancreatic endoderm. Transcriptomic profiling reveals substantial alterations in gene networks associated not only with lineage commitment but also with cytoskeletal organization and cell morphology. These changes correlate with a disruption in stem cell architecture and suggest the persistence of pluripotency feature when only one copy of functional p19Arf is present. Taken together, our findings highlight a role for p19Arf in modulating endodermal differentiation while maintaining the essential cellular properties of stem cells, thus expanding its relevance beyond tumor suppression.},
}

Animals
*Cell Differentiation/genetics
Mice
*Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism
Endoderm/cytology/metabolism
*Mouse Embryonic Stem Cells/cytology/metabolism
*Pancreas/cytology/metabolism
*Cell Lineage/genetics
Gene Expression Profiling
CRISPR-Cas Systems
Mutation

@article {pmid42210018,
year = {2026},
author = {Malaiwong, N and Malaiwong, P and Kim, C and O'Donnell, M},
title = {FLInt 2.0: robust and customizable single-shot integration in C. elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkag138},
pmid = {42210018},
issn = {2160-1836},
support = {DP2 GM154014/GM/NIGMS NIH HHS/United States ; DP2 GM154014/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Transgenes ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Luminescent Proteins/genetics ; Gene Transfer Techniques ; },
abstract = {Transgenesis in Caenorhabditis elegans has revolutionized biological research by enabling the precise control of expression of both endogenous and exogenous genes. FLInt (Fluorescent Landmark Interference) was developed to integrate transgenes via CRISPR-Cas9 using visible changes in existing fluorescent protein expression strains. While the original FLInt method (FLInt 1.0) enabled a simple visual readout of potential transgene integration, the process was prone to false positives, leading to burdensome screening efforts. Here, we present an alternative FLInt strategy, FLInt 2.0, that reduces false positives by targeted CRISPR-Cas9 cutting of fluorescent protein landing sites in a manner which largely retains fluorescence in nonintegrative repair events but eliminates expression upon transgene integration. We demonstrate that this targeted approach maintains effective integration while significantly decreasing the proportion of false positives. Molecular and transmission analyses confirm that nonfluorescent F2 animals more reliably represent stably integrated multicopy transgenic lines. We show that integration efficiency and array transmission are influenced by DNA structure and composition, with linear DNA substrates promoting more robust array formation and insertion. We further show that multicopy transgene lines can be tailored to desired expression levels using a simple subsequent Cas9 targeting approach, reducing labor-intensive screening and increasing experimental throughput. Our strategy provides a robust, visually guided refinement of FLInt, offering a generalizable framework for improving site-specific transgene integration in C. elegans.},
}

Animals
*Caenorhabditis elegans/genetics
*Transgenes
CRISPR-Cas Systems
Animals, Genetically Modified
Luminescent Proteins/genetics
Gene Transfer Techniques