@article {pmid42450173,
year = {2026},
author = {Effah, SN and Barrera, SC and Urturi Ortiz, N and Dampier, W and Nonnemacher, MR and Wigdahl, B},
title = {CRISPR/Cas9-Based Genome Editing: Understanding Differences in DNA Repair Pathways, Profiles, and Outcomes.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450173},
issn = {1422-0067},
support = {MH110360/MH/NIMH NIH HHS/United States ; MH092177/MH/NIMH NIH HHS/United States ; MH079785/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *DNA Repair/genetics ; Animals ; Genetic Therapy/methods ; },
abstract = {Over a decade of advances in Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-based technologies have culminated in the first-ever FDA-approved CRISPR/Cas-based therapy. Aside from this approved therapy for sickle cell anemia, several CRISPR/Cas-based therapies are currently under development or testing for a range of chronic diseases, including viral diseases like human immunodeficiency virus type 1 (HIV-1) infection, genetic diseases like familial hypercholesterolemia, and cancer. The success of these therapies hinges on the effective delivery of CRISPR/Cas9 components to target regions, efficient Cas endonuclease editing, repair profiles generated, and their resulting outcomes. Here, we discuss the factors that influence the generation of CRISPR/Cas9-generated repair edits, the overall profiles, and outcome prediction(s), as well as the analytical tools that have been developed to date. Finally, how this technology has been used towards a functional HIV-1 cure is discussed.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*DNA Repair/genetics
Animals
Genetic Therapy/methods

@article {pmid42447097,
year = {2026},
author = {Zhang, Y and Xing, J and Zhang, H and Kong, J and Nang, SC and Zhang, M and Pan, Y and Zhai, Y and Yuan, L and Zhao, J and Wu, H},
title = {Disruption of rcnB modulates colistin susceptibility in Acinetobacter baumannii AB5075.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2697100},
pmid = {42447097},
issn = {2150-5608},
mesh = {*Colistin/pharmacology ; *Acinetobacter baumannii/drug effects/genetics/metabolism ; *Anti-Bacterial Agents/pharmacology ; *Bacterial Proteins/genetics/metabolism ; Microbial Sensitivity Tests ; Drug Resistance, Bacterial ; Drug Resistance, Multiple, Bacterial/genetics ; Oxidative Stress ; CRISPR-Cas Systems ; },
abstract = {Acinetobacter baumannii AB5075 is a clinically relevant multidrug-resistant (MDR) isolate that poses a major therapeutic challenge. Although colistin has been reinstated as a last-resort antibiotic against MDR Gram-negative infections, the rapid emergence of colistin resistance threatens its clinical utility. Here, we employed a CRISPR-Cas9-based genome editing system to generate an A. baumannii AB5075 ΔrcnB mutant and uncovered a previously underappreciated role of rcnB in modulating colistin susceptibility. Loss of rcnB markedly potentiated colistin-mediated killing through multiple associated changes, including compromised membrane integrity, impaired oxidative stress defenses, and reduced efflux pump activity. Transcriptomic profiling further revealed that rcnB deletion reshaped global stress-response networks, including suppression of fatty acid biosynthesis and reactive oxygen species (ROS)-detoxifying pathways, alongside altered metal ion and sulfur metabolism during colistin exposure. Collectively, our findings suggest that rcnB may contribute to colistin susceptibility of colistin resistance and provide mechanistic insights that may inform the development of targeted strategies to enhance colistin efficacy against MDR A. baumannii.},
}

*Colistin/pharmacology
*Acinetobacter baumannii/drug effects/genetics/metabolism
*Anti-Bacterial Agents/pharmacology
*Bacterial Proteins/genetics/metabolism
Microbial Sensitivity Tests
Drug Resistance, Bacterial
Drug Resistance, Multiple, Bacterial/genetics
Oxidative Stress
CRISPR-Cas Systems

@article {pmid42449928,
year = {2026},
author = {Poonooru, R and Park, KE and Schmelzle, A and Telugu, BP},
title = {Functional Inactivation of PAX4 Results in Disrupted Endocrine Pancreas Development and Neonatal Diabetes in Pigs.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42449928},
issn = {1422-0067},
support = {W81XWH-22-1-0017//Congressionally Directed Medical Research Programs/ ; },
mesh = {Animals ; *Paired Box Transcription Factors/genetics/metabolism ; *Islets of Langerhans/metabolism/pathology/growth & development ; Swine ; *Homeodomain Proteins/genetics/metabolism ; Animals, Newborn ; CRISPR-Cas Systems ; *Diabetes Mellitus/genetics/metabolism/pathology ; Humans ; },
abstract = {Variants in the human PAX4 gene are associated with both monogenic and complex forms of diabetes, yet their pathogenic effects remain difficult to define in models that accurately mimic human islet architecture and neonatal metabolic transitions. Here, we created a porcine PAX4 loss-of-function model using CRISPR/Cas9 cytidine deaminase base editing to introduce a premature stop codon in the PAX4 coding sequence. PAX4 knockout piglets developed severe hyperglycemia within 24 h of birth, followed by rapid postnatal clinical deterioration and uniform death by day 3. Biochemical analysis showed significant diabetic decompensation, including electrolyte imbalances, hyperosmolality, azotemia, dyslipidemia, and metabolic acidosis. Gross and histological examinations revealed notable pancreatic hypoplasia with preservation of exocrine tissue. Single-nucleus RNA sequencing and immunohistochemistry demonstrated an almost complete loss of insulin- and somatostatin-producing β- and δ-cells, respectively, with relative preservation of glucagon-expressing α-cells. Overall, these results establish PAX4 as a crucial factor in pancreatic endocrine development and postnatal glucose regulation in a large-animal model. This platform offers a human-relevant system for studying diabetes-associated PAX4 variants and for testing regenerative and gene-based therapies for insulin-deficient diabetes.},
}

Animals
*Paired Box Transcription Factors/genetics/metabolism
*Islets of Langerhans/metabolism/pathology/growth & development
Swine
*Homeodomain Proteins/genetics/metabolism
Animals, Newborn
CRISPR-Cas Systems
*Diabetes Mellitus/genetics/metabolism/pathology
Humans

@article {pmid42450131,
year = {2026},
author = {Samoń, M and Przyborowski, M},
title = {Wheat's Up with CRISPR-Cas-Current Advances, Obstacles and Perspectives.},
journal = {International journal of molecular sciences},
volume = {27},
number = {13},
pages = {},
pmid = {42450131},
issn = {1422-0067},
support = {Dotacja Celowa task 4.1//Ministry of Agriculture and Rural Development/ ; },
mesh = {*Triticum/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; Genome, Plant ; Genetic Engineering/methods ; },
abstract = {The emergence of CRISPR-Cas editing systems-comprising clustered regularly interspaced short palindromic repeats and associated Cas proteins-marked a breakthrough in genetic engineering, owing to the simplicity, efficiency, and adaptability of the method. Despite continuous improvements and the incorporation of innovative discoveries to develop reliable, fine-tuned tools, the effective application of CRISPR-Cas technology in cereals remains challenging. This review provides a technically oriented overview of CRISPR-Cas-mediated genome editing in wheat (Triticum aestivum L.), one of the world's fundamental crops. While focusing on established solutions and progressive methodological modifications, we also discuss pertinent topics, including plant genetic transformation, prospective innovations, and compliance considerations.},
}

*Triticum/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Plants, Genetically Modified/genetics
Genome, Plant
Genetic Engineering/methods

@article {pmid41168295,
year = {2026},
author = {Yeo, JH and Lee, S and Kim, S and Min, JG and Gopalappa, R and Oh, HC and Kim, HK and Nam, EJ and Kim, HH},
title = {High-throughput evaluation of in vitro CRISPR activities enables optimized large-scale multiplex enrichment of rare variants.},
journal = {Nature biomedical engineering},
volume = {10},
number = {7},
pages = {1410-1430},
pmid = {41168295},
issn = {2157-846X},
support = {RS-2022-NR070713, 2018R1A5A2025079, RS-2022-NR067326, RS-2022-NR067345, RS-2023-00260968//National Research Foundation of Korea (NRF)/ ; 2021R1I1A1A01047269//National Research Foundation of Korea (NRF)/ ; RS-2023-NR076625//National Research Foundation of Korea (NRF)/ ; 2024-22-0165//Yonsei University/ ; 22B-000-0101//Seoul National University Hospital (SNUH)/ ; RS-2024-00467177//Korea Drug Development Fund (KDDF)/ ; 1730158, 1540112, 1541349, 1826967, 2138811, 2112167, 2100237, 2120019, 1419152, 1743354, 2027170//National Science Foundation (NSF)/ ; 6-2019-0166//Yonsei University | Yonsei University College of Medicine (YUCM)/ ; },
mesh = {RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *High-Throughput Nucleotide Sequencing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Base Sequence ; Genetic Variation ; },
abstract = {Previous high-throughput evaluations of CRISPR activities for a large number of target and guide RNA sequences were based on measuring insertion-deletion frequencies rather than cleavage efficiencies. Here we develop two high-throughput in vitro methods, Cut-seq1 and Cut-seq2, to evaluate Cas9 cleavage efficiency for tens of thousands, or even hundreds of thousands, of guide RNA-target pairs. These methods reveal low correlations between in vitro cleavage efficiencies and insertion-deletion frequencies in cells, yet high concordances in protospacer adjacent motif compatibility. Using the resulting large datasets of in vitro cleavage efficiencies, we develop DeepCut, a set of deep learning models that can identify optimized single-guide RNAs that can selectively cleave specific sequences, even in the presence of similar noise sequences. Using these optimized single-guide RNAs, we develop a method, CLOVE-seq (which stands for cleavage for large-scale optimized variant enrichment sequencing), to enrich rare variants in a multiplexed manner by Cas9-mediated specific cleavage of noise or rare variant sequences. Our methods can enhance the understanding of CRISPR nuclease activities and could be used to detect a large number of rare variants in various biomedical contexts.},
}

RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems/genetics
Humans
*High-Throughput Nucleotide Sequencing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Base Sequence
Genetic Variation

@article {pmid41947506,
year = {2026},
author = {Park, JC and Song, Y and Choi, HW and Sung, J and Jin, H and Gwak, W and Yoo, K and Lee, S and Park, J and Kim, J and Jo, HJ and Koo, J and Jeong, Y and Lee, KH and Kee, SJ and Kim, H},
title = {Viral Infection-Inspired Autonomous Detection of Fusion-Competent Viruses for Screening and Environmental Surveillance.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {40},
pages = {e21241},
doi = {10.1002/adma.202521241},
pmid = {41947506},
issn = {1521-4095},
support = {2021R1A2C2013961//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; 2022R1C1C2007002//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; RS-2024-00345402//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; RS-2023-00209955//The National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT)/ ; Institutional 2E33111//Korea Institute of Science and Technology (KIST)/ ; RS-2024-00396818//Korea Institute of Planning and Evaluation for Technology in Food, Agricultural and Forestry (IPET) through High-Risk Animal Infectious Disease Control Technlogy Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA)/ ; 6634-332//The Korea Disease Control and Prevention Agency (KDCA)/ ; RS-2025-25409767//The Materials & Components Technology Development Program funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea)/ ; },
mesh = {*Environmental Monitoring/methods ; Humans ; RNA, Viral/analysis ; SARS-CoV-2/isolation & purification/genetics ; CRISPR-Cas Systems ; Virus Internalization ; Respiratory Syncytial Viruses/isolation & purification ; Influenza A virus/isolation & purification/genetics ; Liposomes/chemistry ; },
abstract = {The persistent burden of respiratory viruses requires rapid, simple, and robust screening and environmental surveillance technologies that enable widespread and frequent testing. Importantly, these technologies should be based on infectivity-relevant signals, as RNA detection alone has limited correlation with transmission risk. Here, we present a membrane fusion-mediated platform that autonomously detects viruses by recapitulating the native viral entry mechanism. Fusogenic vesicles selectively fuse with fusion-competent viral particles, triggering encapsulated CRISPR-Cas13a components to generate fluorescent signals upon recognition of the released viral RNA. Through an autonomous workflow and accelerated signal generation within a confined vesicle, our platform achieves one-step detection of viruses within 2 min. The assay robustly detects three major respiratory viruses, with analytical sensitivities down to 5 TCID50/mL for RSV and 50 TCID50/mL for SARS-CoV-2 and IAV. Clinical validation with 100 nasopharyngeal samples achieved 91.7% sensitivity. Remarkably, the sprayable format enables large-area surveillance of surface contamination-like luminol revealing hidden bloodstains, it makes invisible viral threats visible. This approach establishes an intuitive real-time detection platform, extending beyond clinical specimens to encompass environmental threats.},
}

*Environmental Monitoring/methods
Humans
RNA, Viral/analysis
SARS-CoV-2/isolation & purification/genetics
CRISPR-Cas Systems
Virus Internalization
Respiratory Syncytial Viruses/isolation & purification
Influenza A virus/isolation & purification/genetics
Liposomes/chemistry

@article {pmid42086608,
year = {2026},
author = {Cao, J and Liu, Z and Chen, X and Lan, Z and Liu, L and Zhai, Y and Wang, W and Xue, C and Cheng, H and Yao, Y and Cheng, T and Rao, S},
title = {Engineered dCas12f1-SAM enables robust transcriptional activation and gain-of-function screening in primary human cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42086608},
issn = {2041-1723},
support = {82370118, 82470240 and 82370117//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Transcriptional Activation/genetics ; T-Lymphocytes/metabolism/immunology ; *CRISPR-Cas Systems/genetics ; Transcription Factors/genetics/metabolism ; Hematopoietic Stem Cells/metabolism ; Interleukin-2/genetics/metabolism ; Kruppel-Like Transcription Factors/genetics/metabolism ; LIM-Homeodomain Proteins/genetics/metabolism ; Single-Cell Gene Expression Analysis ; Receptors, Chimeric Antigen/metabolism/genetics ; },
abstract = {Despite considerable powers, the application of CRISPR activation (CRISPRa) screens in primary human cells remains a formidable challenge. Here, we develop dCas12f1-SAM, a compact SAM-based transcriptional activation platform, that outperforms existing systems in both immortalized cell lines and primary human T cells and hematopoietic stem/progenitor cells (HSPCs). Using dCas12f1-SAM, we perform a pooled CRISPRa screen targeting 1559 human transcription factors (TFs) in primary human T cells and identify multiple positive regulators of IL-2 expression. We further implement a single-cell CRISPRa screen via our miCROP-seq construct, resolving how these genetic perturbations reshape T cell activation dynamics and drive functionally distinct cellular states. Among the top-ranking genes, we spotlight KLF12 and LHX5, whose overexpression significantly improves antigen-specific responses of chimeric antigen receptor T (CAR-T) cells. Collectively, these findings establish dCas12f1-SAM as a robust transcriptional activation tool, highlighting its potential to advance applications in cellular engineering and immunotherapy.},
}

Humans
*Transcriptional Activation/genetics
T-Lymphocytes/metabolism/immunology
*CRISPR-Cas Systems/genetics
Transcription Factors/genetics/metabolism
Hematopoietic Stem Cells/metabolism
Interleukin-2/genetics/metabolism
Kruppel-Like Transcription Factors/genetics/metabolism
LIM-Homeodomain Proteins/genetics/metabolism
Single-Cell Gene Expression Analysis
Receptors, Chimeric Antigen/metabolism/genetics

@article {pmid42103719,
year = {2026},
author = {Bourgeois, W and Rice, HE and Wenge, DV and Perner, F and Yue, H and Regalado, BD and Wan, G and Schroeder, JC and Sommerschield, A and Hatton, C and Singh, S and Singh, S and Bijpuria, S and McKeever, BM and Miller, WH and Safer, JF and Iqbal, S and Perry, JA and Fischer, ES and Doench, JG and McGeehan, GM and Cutler, JA and Armstrong, SA},
title = {CRISPR base editor screening identifies spectrum of MEN1 mutations impacting menin inhibitors in clinical trials.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42103719},
issn = {2041-1723},
support = {Chromatin Collaborative award//St. Jude Children's Research Hospital/ ; P50 CA206963/CA/NCI NIH HHS/United States ; R01 CA259273/CA/NCI NIH HHS/United States ; NIH 5 P30 CA06516//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; P01 CA066996/CA/NCI NIH HHS/United States ; CA206963//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 511811315//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 2021-EKEA.111//Else Kröner-Fresenius-Stiftung (Else Kroner-Fresenius Foundation)/ ; CA066996//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; CA259273//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; PE 3217/4-1, PN: 517204983//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Proto-Oncogene Proteins/genetics/antagonists & inhibitors/chemistry/metabolism ; Humans ; Histone-Lysine N-Methyltransferase/genetics/metabolism/antagonists & inhibitors ; *Mutation ; Nucleophosmin ; Myeloid-Lymphoid Leukemia Protein/genetics/metabolism ; Animals ; Drug Resistance, Neoplasm/genetics ; Nuclear Proteins/genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Mice ; },
abstract = {Menin inhibitors have entered clinical trials for histone lysine methyltransferase 2 A (KMT2A)-rearranged and nucleophosmin 1 (NPM1)-mutant acute leukemias and are demonstrating promising activity. CRISPR base editor screening previously predicted several MEN1 (menin) mutations that have arisen in patients receiving SNDX-5613 and confer resistance. The extent to which MEN1 mutations will impact each menin inhibitor is mostly unknown. Here we show that CRISPR base editor screens can be leveraged to profile the MEN1 mutations that may impact five different menin inhibitors in clinical trials. We identify shared (M327I/V/T, G331D) and inhibitor-specific (C334R, E368K/V, V372A) resistance mutations. Co-crystal structures of menin bound to each menin inhibitor suggest resistance mechanisms related to how each inhibitor engages the KMT2A binding pocket of menin. Orthogonal in vitro and in vivo MEN1 mutation generation under therapeutic pressure suggest the MEN1 mutations identified with CRISPR base editor screening are likely to arise and impact all menin inhibitors.},
}

*Proto-Oncogene Proteins/genetics/antagonists & inhibitors/chemistry/metabolism
Humans
Histone-Lysine N-Methyltransferase/genetics/metabolism/antagonists & inhibitors
*Mutation
Nucleophosmin
Myeloid-Lymphoid Leukemia Protein/genetics/metabolism
Animals
Drug Resistance, Neoplasm/genetics
Nuclear Proteins/genetics
CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Mice

@article {pmid42103787,
year = {2026},
author = {Cipria, D and Baccega, T and Rizzo, M and Quarato, P and Reschigna, A and El Khoury, R and Cappelluti, MA and Ammann, S and Poeta, VM and Conti, M and Valsoni, S and Spinelli, P and Merelli, I and Cathomen, T and Casucci, M and Lombardo, A},
title = {Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42103787},
issn = {2041-1723},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Cell Engineering/methods ; *Gene Editing/methods ; T-Lymphocytes/metabolism ; Epigenome Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epigenesis, Genetic ; Receptors, Antigen, T-Cell/genetics ; Transgenes ; Gene Silencing ; Receptors, Chimeric Antigen/genetics ; },
abstract = {The parallel disruption of multiple genes coupled with targeted transgene insertion offers a powerful strategy for more effective and precise cell engineering. However, such orthogonal editing involves the induction of multiple DNA breaks, raising safety concerns related to the risks of chromosomal translocations. Here, we present a polyfunctional CRISPR-Cas9-based strategy that enables both transgene insertion and epigenetic silencing at distinct genomic loci in a single treatment without inducing reciprocal chromosomal translocations. This is accomplished through an optimized all-in-one epigenome editor equipped with a catalytically active Cas9, whose endonuclease activity is selectively disabled at epigenetically silenced loci using truncated gRNAs. As a proof of concept, we demonstrate that this platform enables efficient multi-locus editing, including functional replacement of the endogenous TCR with a tumor-selective one, targeted insertion of a prototypic CAR with either a selectable marker or an immunomodulatory receptor into a TCR locus or a ubiquitously expressed gene, and durable, multiplexed epigenetic silencing of clinically relevant genes in primary human T cells. Polyfunctional editing establishes a versatile and safe framework for orthogonal editing, broadening the scope of genome and epigenome engineering in cancer immunotherapy and beyond.},
}

Humans
*CRISPR-Cas Systems/genetics
*Cell Engineering/methods
*Gene Editing/methods
T-Lymphocytes/metabolism
Epigenome Editing
RNA, Guide, CRISPR-Cas Systems/genetics
Epigenesis, Genetic
Receptors, Antigen, T-Cell/genetics
Transgenes
Gene Silencing
Receptors, Chimeric Antigen/genetics

@article {pmid42120395,
year = {2026},
author = {Morency, C and Rousseau, GM and Morneau, Z and Moineau, S},
title = {Phage satellites induced by virulent phages are mobilized by natural competence leading to phage resistance in a new host.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42120395},
issn = {2041-1723},
mesh = {*Streptococcus thermophilus/virology/genetics ; *Bacteriophages/genetics/pathogenicity/physiology ; CRISPR-Cas Systems ; Chromosomes, Bacterial/genetics ; Virulence ; Genome, Viral ; Prophages/genetics ; },
abstract = {A phage satellite (PS) typically resides within repeat regions (attL and attR sites) of a bacterial genome. Its genome ranges from 7 to 20-kb and includes genes encoding an integrase along with regulatory and DNA replication functions. However, it lacks genes associated with viral structural proteins. Streptococcus thermophilus (S.t.) is extensively used to produce yogurt and specialty cheeses. Intriguingly, the majority of S.t. strains harbor a PS while very few possess a complete prophage, suggesting that PSs may confer advantages to their hosts. In this study, we showed that PSs of S.t. can excise from the bacterial chromosome, at a very low rate, without any phage interaction. Furthermore, we found that they can also be induced by virulent phages. By leveraging CRISPR-Cas9, we selected S.t. cells devoid of any PS (delta-PS strain). Then, we mobilized a PS from one strain to a delta-PS strain, using only natural competence, bypassing the need for a helper phage. The resulting strain exhibited increased resistance to virulent phages. Through the isolation of phage mutants escaping the resistance phenotype, we pinpointed a specific phage protein responsible for the induction of a PS. Lastly, we demonstrated that a PS can be significantly induced by a virulent phage, which, in turn, greatly promotes its transfer and specific integration into new cells through natural competence. Our study introduces a novel natural approach to develop phage-resistant strains.},
}

*Streptococcus thermophilus/virology/genetics
*Bacteriophages/genetics/pathogenicity/physiology
CRISPR-Cas Systems
Chromosomes, Bacterial/genetics
Virulence
Genome, Viral
Prophages/genetics

@article {pmid42140952,
year = {2026},
author = {Wang, S and Hou, S and Luo, C and Zhang, H and Jin, Y and Zhang, R and Zhao, Y and Xiong, X and Guo, R and Wang, C and Bao, Y and Wen, L and Pan, D and Ye, Y and Zeng, Z and Gao, Z},
title = {Arid3b suppresses CD8 + T cell infiltration and function in microsatellite-stable colorectal cancer via Runx3.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42140952},
issn = {2041-1723},
support = {92478117//National Natural Science Foundation of China (National Science Foundation of China)/ ; 92374116//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32470664//National Natural Science Foundation of China (National Science Foundation of China)/ ; T2321001//National Natural Science Foundation of China (National Science Foundation of China)/ ; L248043//Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; },
mesh = {*Core Binding Factor Alpha 3 Subunit/genetics/metabolism/immunology ; Animals ; *Colorectal Neoplasms/genetics/immunology/pathology/metabolism ; *CD8-Positive T-Lymphocytes/immunology/metabolism ; Tumor Microenvironment/immunology/genetics ; Humans ; Mice ; *DNA-Binding Proteins/genetics/metabolism ; Cell Line, Tumor ; Lymphocytes, Tumor-Infiltrating/immunology ; CRISPR-Cas Systems ; Microsatellite Repeats ; *Transcription Factors/genetics/metabolism ; },
abstract = {Microsatellite-stable/proficient mismatch repair (MSS/pMMR) colorectal cancer (CRC) is characterized by a cold tumor microenvironment, with limited CD8[+] T cell infiltration and poor responsiveness to immune checkpoint inhibitors (ICIs). Here, using an in vivo CRISPR/Cas9 screen in a CMT93 cell-derived murine tumor model, we identify Arid3b as a key negative regulator of CD8[+] T cell infiltration and antitumor activity. Genetic ablation of Arid3b in CD8[+] T cells significantly enhances their intratumoral accumulation and promotes robust tumor control. Mechanistically, Arid3b deficiency upregulates Runx3, driving a tissue-resident memory-like phenotype and effector function. Notably, the benefits conferred by Arid3b deficiency are abrogated upon Runx3 deletion, indicating a RUNX3-dependent mechanism. Together, targeting ARID3B could offer a promising strategy to reshape the tumor microenvironment and sensitize MSS CRC to immunotherapy.},
}

*Core Binding Factor Alpha 3 Subunit/genetics/metabolism/immunology
Animals
*Colorectal Neoplasms/genetics/immunology/pathology/metabolism
*CD8-Positive T-Lymphocytes/immunology/metabolism
Tumor Microenvironment/immunology/genetics
Humans
Mice
*DNA-Binding Proteins/genetics/metabolism
Cell Line, Tumor
Lymphocytes, Tumor-Infiltrating/immunology
CRISPR-Cas Systems
Microsatellite Repeats
*Transcription Factors/genetics/metabolism

@article {pmid42264060,
year = {2026},
author = {Zaki, HF and Bishri, J and Abdul Muqtadir, M and Abu-Zaid, A},
title = {Correcting photoreceptor diseases at their source: CRISPR strategies for cone-rod dystrophy and achromatopsia.},
journal = {Experimental eye research},
volume = {270},
number = {},
pages = {111119},
doi = {10.1016/j.exer.2026.111119},
pmid = {42264060},
issn = {1096-0007},
mesh = {*Color Vision Defects/therapy/genetics ; Humans ; Animals ; *Genetic Therapy/methods ; *Cone-Rod Dystrophies/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Disease Models, Animal ; },
abstract = {BACKGROUND: Cone-rod dystrophy (CORD) and achromatopsia (ACHM) are inherited retinal dystrophies for which conventional adeno-associated virus (AAV) gene augmentation has important limitations, particularly in autosomal-dominant gain-of-function CORD and recessive ACHM. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) offers the potential for one-time, mutation-specific gene correction or allele ablation. This systematic review summarizes preclinical evidence on CRISPR/Cas9-based approaches for CORD and ACHM, focusing on editing efficiency, phenotypic rescue, and safety.

METHODS: This review followed PRISMA guidelines. PubMed, Google Scholar, and ScienceDirect were searched through June 2025 for original experimental studies using CRISPR/Cas9 in CORD or ACHM animal models or human-derived cell lines. Dual independent screening and data extraction were performed. Outcomes related to editing efficiency, structural or functional rescue, and safety were synthesized narratively.

RESULTS: Four studies were included: three targeting CORD and one targeting ACHM. In vivo studies used AAV-delivered SaCas9 to disrupt GUCY2D (or murine orthologs) in mouse and macaque photoreceptors, achieving approximately 8-45% on-target editing in mice and approximately 13% in macaques. Although ablation alone reduced retGC1 expression, it did not improve retinal function; however, a dual-AAV "ablate-and-replace" strategy preserved outer nuclear layer thickness for up to 24 weeks in CORD6 mice. In vitro, PROM1 correction in patient-derived iPSCs restored CD133 expression, and SpCas9-HiFi-mediated PDE6C correction in ACHM iPSCs achieved approximately 80% editing efficiency while preserving pluripotency and showing no detectable off-target effects. Safety data were limited, with immune responses assessed in only one primate study.

CONCLUSIONS: CRISPR/Cas9 shows promising preclinical efficacy for CORD and ACHM, particularly allele-specific ablate-and-replace strategies for CORD and precise HDR-based correction for ACHM. However, the available evidence remains limited, underscoring the need for expanded safety assessment, non-human primate studies, and standardized functional outcomes measures before clinical translation.},
}

*Color Vision Defects/therapy/genetics
Humans
Animals
*Genetic Therapy/methods
*Cone-Rod Dystrophies/therapy/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Disease Models, Animal

@article {pmid42284763,
year = {2026},
author = {Liu, L and Wang, H and Shi, L and Zhang, C and Zhang, L and Lv, L and Wang, Y},
title = {Development of super Vδ2 T cells for relapsed/refractory acute myeloid Leukemia via non-viral site-specific integration.},
journal = {International immunopharmacology},
volume = {185},
number = {},
pages = {116981},
doi = {10.1016/j.intimp.2026.116981},
pmid = {42284763},
issn = {1878-1705},
mesh = {Humans ; *Leukemia, Myeloid, Acute/therapy/immunology ; Animals ; *Immunotherapy, Adoptive/methods ; *Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism ; Receptors, Chimeric Antigen/genetics ; *T-Lymphocytes/immunology/transplantation ; NK Cell Lectin-Like Receptor Subfamily K/genetics/metabolism ; Mice ; Cell Line, Tumor ; CD3 Complex/genetics ; CRISPR-Cas Systems ; Cytotoxicity, Immunologic ; Mice, SCID ; },
abstract = {The efficacy of chimeric antigen receptor (CAR)-T cell therapy in relapsed/refractory acute myeloid leukemia (R/R AML) is limited by tumor heterogeneity, antigen evasion, and treatment-related toxicities. Gamma delta (γδ) T cells mediate antitumor activity independent of MHC by sensing stress-induced ligands. A prominent mechanism involves NKG2D ligand (NKG2DL) recognition, which is highly upregulated in malignancies but generally low or restricted expression in healthy tissues under homeostatic conditions. In human peripheral blood, the Vδ2 subset represents the predominant population. Vδ2 T cells transduced with the NKG2D-CD3ζ construct, which incorporates into the natural γδ TCR/CD3 complex, preserve innate phosphoantigen recognition while acquiring potent NKG2DL-directed cytotoxicity, enabling dual-pathway tumor recognition. These cells are termed "Super Vδ2 T cells." We successfully generated TRAC-specific integrated Super Vδ2 T cells using CRISPR/Cas9 technology, achieving 90-93% CAR[+] expression. In vitro assays demonstrated that the engineered "Super Vδ2 T cells" exhibited potent cytotoxic activity against multiple AML targets, including cell lines and primary R/R AML blasts, in contrast to their negligible toxicity on monocytes. In vivo, Super Vδ2 T cells demonstrated substantial tumor reduction without graft-versus-host disease (GvHD) reaction. Collectively, our data demonstrated that Super Vδ2 T cells represent a viable allogeneic therapy for AML.},
}

Humans
*Leukemia, Myeloid, Acute/therapy/immunology
Animals
*Immunotherapy, Adoptive/methods
*Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism
Receptors, Chimeric Antigen/genetics
*T-Lymphocytes/immunology/transplantation
NK Cell Lectin-Like Receptor Subfamily K/genetics/metabolism
Mice
Cell Line, Tumor
CD3 Complex/genetics
CRISPR-Cas Systems
Cytotoxicity, Immunologic
Mice, SCID

@article {pmid42439627,
year = {2026},
author = {Park, SH and Hong, J and Hwang, W and Kim, M and Yu, HJ and Bae, T and Lee, HK and Lee, JY and Lee, YC and Park, CK and Hur, JK},
title = {CRISPRi-Mediated Epigenetic Suppression of TERT Reduces Cell Growth in Non-Small-Cell Lung Cancer Cells.},
journal = {Cells},
volume = {15},
number = {13},
pages = {},
pmid = {42439627},
issn = {2073-4409},
support = {RS-2021-NR056589; RS-2023-00261114; RS-2025-02218918//Ministry of Science and ICT/ ; RS-2025-16063805//the Korea US Collaborative Research Fund/ ; 2023R1A6C101A009//the Ministry of Education/ ; RS-2023-00260529, RS-2026-25493418//National Research Foundation of Korea/ ; },
mesh = {Humans ; *Telomerase/genetics/metabolism ; *Carcinoma, Non-Small-Cell Lung/genetics/pathology ; *Lung Neoplasms/genetics/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; *Epigenesis, Genetic ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Neoplastic ; Cell Survival/genetics ; Epigenome Editing ; },
abstract = {TERT, the catalytic subunit of telomerase, is aberrantly activated in most cancers and represents an attractive therapeutic target. However, conventional TERT-targeting strategies, including chemical inhibitors and siRNA, are limited by several issues, such as insufficient efficacy and off-target effects. In this study, we investigated whether dCas9-KRAB-mediated CRISPR interference (CRISPRi) could overcome the limitations by transcriptional repression of TERT without DNA cleavage. We first assessed the efficacy of the dCas9-KRAB system by applying it to H1299 non-small-cell lung cancer cells and observed reduction in TERT expression up to approximately 80% and significant decreases in cell viability and growth. Transcriptome-wide analysis showed limited detectable changes in non-target-gene expression under the conditions tested. Together, the results suggest that dCas9-KRAB-mediated CRISPRi could serve as a proof-of-principle approach for targeted repression of TERT in cancer cells with limited detectable effects on non-target-gene expression.},
}

Humans
*Telomerase/genetics/metabolism
*Carcinoma, Non-Small-Cell Lung/genetics/pathology
*Lung Neoplasms/genetics/pathology
Cell Line, Tumor
Cell Proliferation/genetics
*Epigenesis, Genetic
*CRISPR-Cas Systems/genetics
Gene Expression Regulation, Neoplastic
Cell Survival/genetics
Epigenome Editing

@article {pmid42439976,
year = {2026},
author = {Mundada, AR and Badikol, AR and Mangu, K},
title = {CRISPR-Cas9-based therapies for Huntington's disease and Friedreich's ataxia: mechanisms, advances, and future perspectives.},
journal = {Neurogenetics},
volume = {27},
number = {1},
pages = {},
pmid = {42439976},
issn = {1364-6753},
mesh = {*Friedreich Ataxia/therapy/genetics ; Humans ; *Huntington Disease/therapy/genetics ; *CRISPR-Cas Systems ; *Genetic Therapy/methods/trends ; *Gene Editing/methods ; Animals ; Trinucleotide Repeat Expansion ; },
abstract = {Huntington's disease (HD) and Friedreich's ataxia (FRDA) are progressive inherited neurodegenerative disorders caused by trinucleotide repeat expansions but characterized by distinct pathogenic mechanisms. HD arises from a coding-region CAG expansion in the HTT gene that produces toxic gain-of-function effects of mutant huntingtin (mHTT), whereas FRDA results primarily from intronic GAA repeat expansion in FXN, leading to epigenetic repression and frataxin deficiency. The emergence of CRISPR-based genome engineering has created new opportunities to address these diseases at their genetic origin. This review examines current CRISPR therapeutic strategies for HD and FRDA, including allele-specific editing, transcriptional suppression, repeat excision, epigenetic reactivation, and emerging precision editing approaches such as base editing and prime editing. We compare the molecular rationale, preclinical outcomes, and translational limitations associated with each approach while highlighting how disease architecture influences therapeutic design. Although preclinical studies demonstrate promising restoration of cellular phenotypes and functional improvement, significant barriers remain. Efficient delivery to the central nervous system and cardiac tissue, control of editing duration, immune responses, off-target activity, and emerging concerns regarding on-target genomic instability continue to limit clinical translation. Recent advances in delivery engineering, non-viral systems, and programmable editing platforms suggest that future therapeutic success will depend on integrating disease-specific biology with increasingly precise and controllable genome engineering technologies. Ethical and regulatory concerns remain substantial, particularly regarding informed consent in the context of cognitive decline and the irreversibility of genomic modification.},
}

*Friedreich Ataxia/therapy/genetics
Humans
*Huntington Disease/therapy/genetics
*CRISPR-Cas Systems
*Genetic Therapy/methods/trends
*Gene Editing/methods
Animals
Trinucleotide Repeat Expansion

@article {pmid42441240,
year = {2026},
author = {Yu, W and Huang, X and Hu, Y and Chen, S},
title = {Beyond adaptive immunity: Functional diversity of the type III-A CRISPR-Cas system in Mycobacterium tuberculosis.},
journal = {Cell insight},
volume = {5},
number = {4},
pages = {100342},
pmid = {42441240},
issn = {2772-8927},
abstract = {CRISPR-Cas systems are best known as prokaryotic adaptive immune pathways that defend against invading genetic elements. Mycobacterium tuberculosis (Mtb) harbors a type III-A CRISPR-Cas system that is structurally conserved yet exhibits little evidence of ongoing spacer acquisition. Nevertheless, its interference machinery remains functional, and increasing evidence suggests that this system has evolved roles beyond canonical adaptive immunity. Accumulating studies indicate that this system is deeply integrated into cellular regulatory networks by governing stress responses, metabolic adaptation, and host-pathogen interactions. Mechanistically, the Mtb type III-A CRISPR-Cas system operates through transcription-dependent target recognition and cyclic oligoadenylate (cOA)-mediated signal amplification, in which the ancillary ribonuclease Csm6 serves as a key effector. Functionally, CRISPR-associated proteins influence antibiotic susceptibility, oxidative stress resistance and host immune responses, and may even act as secreted immunomodulatory factors. In this review, we summarize current understanding of the genomic organization, regulatory mechanisms, and non-canonical functions of the Mtb type III-A CRISPR-Cas system, with particular emphasis on its emerging roles in stress adaptation and host immune regulation.},
}

@article {pmid42442865,
year = {2026},
author = {Zheng, J and Zhang, W and Conrad, M},
title = {Ferroptosis induction via genetic approaches - CRISPR/Cas9-based disruption on key anti-ferroptotic genes.},
journal = {Methods in cell biology},
volume = {209},
number = {},
pages = {91-103},
doi = {10.1016/bs.mcb.2026.05.002},
pmid = {42442865},
issn = {0091-679X},
mesh = {*Ferroptosis/genetics/drug effects ; Humans ; *CRISPR-Cas Systems/genetics ; *Phospholipid Hydroperoxide Glutathione Peroxidase/genetics ; Amino Acid Transport System y+/genetics ; Cell Line, Tumor ; Gene Knockout Techniques/methods ; },
abstract = {Unlike apoptosis, necroptosis, or pyroptosis which are executed by dedicated proteins, ferroptosis is a distinct form of regulated cell death driven by lipid peroxidation downstream of metabolic dysfunction. In most physiological settings, the cyst(e)ine/glutathione/glutathione peroxidase 4 (GPX4) axis constitutes the central anti-ferroptotic machinery, and disruption of this axis is usually sufficient to trigger ferroptosis. For in vitro studies, commonly employed ferroptosis inducers include erastin, which blocks cystine uptake by targeting system xc[-], and (1S,3R)-RSL3, which inhibits GPX4 activity. However, both compounds exhibit off-target effects - erastin can activate voltage-dependent anion channels in mitochondria, whereas (1S,3R)-RSL3 affects other selenoproteins in addition to GPX4. Thus, genetic approaches to induce ferroptosis provide a valuable complement to chemical inducers by excluding off-target concerns. Here, we describe an efficient CRISPR/Cas9-based strategy to generate SLC7A11- and GPX4-knockout HT1080 cells. These knockout lines require routine culture in medium supplemented with β-mercaptoethanol or liproxstatin-1, while withdrawal of these supplements readily induces ferroptosis.},
}

*Ferroptosis/genetics/drug effects
Humans
*CRISPR-Cas Systems/genetics
*Phospholipid Hydroperoxide Glutathione Peroxidase/genetics
Amino Acid Transport System y+/genetics
Cell Line, Tumor
Gene Knockout Techniques/methods

@article {pmid42444979,
year = {2026},
author = {Yang, M and Song, Y and Wang, Z and Chao, K and Li, L and Zhang, X and Duan, X and Yu, C and Xue, R and Zhao, J},
title = {Delivery Systems for Therapeutic Genome Editing: Challenges, Innovations, and Future Perspectives.},
journal = {MedComm},
volume = {7},
number = {7},
pages = {e70791},
pmid = {42444979},
issn = {2688-2663},
abstract = {Therapeutic genome editing has advanced rapidly with the development of diverse programmable nucleases, from zinc-finger nucleases and transcription activator-like effector nucleases to clustered regularly interspaced short palindromic repeats (CRISPR)-based systems such as base and prime editors. Despite these breakthroughs, clinical translation remains constrained by the challenge of achieving safe, efficient, and tissue-specific delivery. Viral vectors, particularly adeno-associated viruses, have enabled durable editing in selected organs but are limited by their restricted cargo capacity, immunogenicity, and complex manufacturing. Nonviral platforms, most notably ionizable lipid nanoparticles, have demonstrated remarkable efficacy for hepatic targets, with clinical trials reporting up to 93% protein knockdown after a single dose. An expanding set of emerging modalities, including virus-mimicking nanosystems, cell-derived extracellular vesicles, cell-penetrating peptides, and intelligent-responsive multifunctional scaffolds, further enriches the delivery toolbox by supporting transient expression and programmable targeting across diverse editors and tissues. Parallel advances in high-throughput barcoded screening and machine learning are accelerating vector optimization, while rational chemical modification of payloads improves in vivo stability and specificity. This review provides a comprehensive overview of current and emerging delivery systems for genome editing, highlighting key innovations, unresolved challenges, and interdisciplinary strategies poised to unlock broader therapeutic potential.},
}

@article {pmid42445839,
year = {2026},
author = {Kruglova, NA and Borovikova, SE and Shepelev, MV},
title = {Production of virus-like particles with AsCas12a nuclease and CMV-driven crRNA for mammalian genome editing.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1735339},
pmid = {42445839},
issn = {2673-3439},
abstract = {CRISPR/Cas genome editing tools represent a promising technology for biomedicine with significant therapeutic potential for numerous human diseases. However, efficient delivery of these tools into primary cells, particularly in the form of ribonucleoprotein (RNP) complexes, remains a critical bottleneck that limits clinical translation. Virus-like particles (VLPs) derived from human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) have emerged as promising delivery vehicles for RNP complexes, yet their activity is limited by suboptimal nuclease and guide RNA packaging. Previously, we generated NanoMEDIC VLPs incorporating the AsCas12a nuclease with CMV-driven crRNA, which demonstrated substantially enhanced editing efficiency over SpCas9-VLPs with U6-driven gRNA. Here, we describe a detailed protocol for a small-scale production of AsCas12a-VLPs using three distinct transfection methods [cationic lipids, polyethyleneimine (PEI), and calcium-phosphate] and a large-scale production of VLPs using calcium-phosphate transfection. We show that both production scales yield comparable nuclease loading into VLPs and similar editing efficiencies, reaching up to 60% of CXCR4 knockout in Jurkat T cells.},
}

@article {pmid42120643,
year = {2026},
author = {Cui, H and Peng, J and Song, J and Yang, Y and Hao, X},
title = {An ultrasensitive CRISPR-strand displacement amplification biosensor achieves piRNA-54265 detection and imaging in colorectal cancer cells.},
journal = {Analytical and bioanalytical chemistry},
volume = {418},
number = {14},
pages = {4613-4621},
pmid = {42120643},
issn = {1618-2650},
support = {20252BAC250153//Natural Science Foundation of Jiangxi Province/ ; 22277047//National Natural Science Foundation of China/ ; 82160631//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Biosensing Techniques/methods ; *Colorectal Neoplasms/genetics/diagnosis ; *Piwi-Interacting RNA/analysis/genetics ; *CRISPR-Cas Systems ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Cell Line, Tumor ; *RNA, Small Interfering/analysis/genetics ; },
abstract = {PIWI-interacting RNAs (piRNAs) are well-recognized as promising diagnostic biomarkers for cancer, yet their quantitative detection remains a great challenge owing to their short sequences, low cellular abundance, high degradation susceptibility, and significant sequence homology among family members. Herein, we developed an ultrasensitive and highly specific biosensor for the detection of piRNA-54265-a colorectal cancer (CRC)-associated piRNA-by integrating strand displacement amplification (SDA) with the CRISPR/Cas12a system. After systematic optimization, the biosensor exhibited remarkably enhanced amplification efficiency and target specificity, achieving an ultra-low limit of detection (LOD) of 57.54 aM for piRNA-54265. Notably, this CRISPR-SDA platform enabled accurate discrimination of CRC cells from other cancer cells via high-fidelity intracellular imaging of piRNA-54265 and also realized reliable detection of the target in complex biological matrices with favorable recovery. Benefiting from its simple sequence design, user-friendly operation, and isothermal reaction conditions, the developed biosensor not only overcomes the inherent technical bottlenecks in piRNA detection but also shows great potential for applications in cellular imaging and early clinical diagnosis of CRC.},
}

Humans
*Biosensing Techniques/methods
*Colorectal Neoplasms/genetics/diagnosis
*Piwi-Interacting RNA/analysis/genetics
*CRISPR-Cas Systems
Limit of Detection
*Nucleic Acid Amplification Techniques/methods
Cell Line, Tumor
*RNA, Small Interfering/analysis/genetics

@article {pmid42130163,
year = {2026},
author = {Nomura, C and Kanzaki, H and Kanzaki, E and Shimizu, M and Oikawa, K and Utsushi, H and Ito, K and Sugimura, Y and Terauchi, R and Abe, A},
title = {Fine-tuning quantitative agronomic traits by manipulating gene copy number in rice.},
journal = {The New phytologist},
volume = {251},
number = {4},
pages = {1609-1616},
doi = {10.1111/nph.71258},
pmid = {42130163},
issn = {1469-8137},
support = {KAKENHI JP22K20584//Japan Society for the Promotion of Science/ ; KAKENHI JP23K26882//Japan Society for the Promotion of Science/ ; JPJ007097//Bio-oriented Technology Research Advancement Institution/ ; },
mesh = {*Oryza/genetics ; *Gene Dosage ; *Quantitative Trait, Heritable ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Genes, Plant ; Phenotype ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Although plant pan-genome studies have revealed extensive copy number variations, their phenotypic consequences remain poorly understood. Here, we manipulated the copy number of OsMADS18 in rice (Oryza sativa) cv 'Hitomebore' using the CRISPR/Cas9 system. We established rice lines harboring one to three tandem copies of OsMADS18, as identified by quantitative PCR and sequencing. The presence of one to three OsMADS18 tandem copies was reflected in stepwise increases in transcript levels and concomitant agronomic trait values. These results demonstrate that manipulating gene copy number can fine-tune important quantitative traits, providing a novel breeding strategy for crop improvement.},
}

*Oryza/genetics
*Gene Dosage
*Quantitative Trait, Heritable
Plants, Genetically Modified
Gene Expression Regulation, Plant
Genes, Plant
Phenotype
Plant Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics

@article {pmid42155537,
year = {2026},
author = {Albeladi, HA and Al-Zahrani, MH and Alghamdi, RA},
title = {Modulating claudin-2 with CRISPR-Cas9 to improve photodynamic therapy outcomes in colorectal cancer.},
journal = {Tissue & cell},
volume = {102},
number = {},
pages = {103586},
doi = {10.1016/j.tice.2026.103586},
pmid = {42155537},
issn = {1532-3072},
mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology/drug therapy/therapy ; *Photochemotherapy ; Apoptosis/drug effects/genetics ; *CRISPR-Cas Systems/genetics ; Reactive Oxygen Species/metabolism ; *Claudin-2/metabolism/genetics ; Cell Movement/drug effects/genetics ; Chlorophyllides ; HCT116 Cells ; Gene Expression Regulation, Neoplastic/drug effects ; Porphyrins/pharmacology ; Claudins ; },
abstract = {Claudin-2 (CLDN2) is a tight junction protein that is overexpressed in colorectal cancer (CRC) and is associated with chemoresistance. Photodynamic therapy (PDT) is an emerging treatment that utilizes a photosensitizer (in this case, chlorin e6 [Ce6]) and light to generate cytotoxic reactive oxygen species (ROS). This paper investigated the influence of the combination of Ce6-PDT and CRISPR-Cas9-mediated CLDN2 knockout (KO) on the relative metabolic activity of the CRC cell line. CRISPR-Cas9 was used to produce HCT116 cells with CLDN2 KO. Ce6 was placed on the cells, and the red laser (659 nm, 6 J/cm[2]) was used to illuminate the cells. The relative metabolic activity, migration, Apoptosis, cell cycle, and ROS generation, gene expression, protein expression were measured by MTT assay, wound healing assay, flow cytometry, DCFH-DA method, RT-PCR, western blot and bioinformatics, respectively. PDT significantly decreased the relative metabolic activity and/or migration, more in CLDN2KO cells (p < 0.0001) than in the WT. The CLDN2KO cells had a high level of Apoptosis (46.56 ± 2.05%), compared to the WT (26.03 ± 6.72%), the p = 0.0072. The production of ROS was also increased to 779.51 % in CLDN2KO cells, which is higher than the production in WT cells at 767.10 %. Upregulation of P53 and BAX following PDT was greatly enhanced, and BCL2 expression was significantly reduced as compared to wild-type groups. Coexistence of Ce6-PDT with CLDN2 KO enhances the Apoptosis of the CRC cell line. The Western blot results showed a decrease in ZO-1 and Occludin proteins after Ce6-PDT. Bioinformatics analysis demonstrated that increased CLDN2 expression in CRC, associated with multiple genes and implicated in various cellular pathways. Although the initial results are encouraging regarding the possibility of synergy, further studies are needed to determine its effectiveness and safety in clinical practice. The results of this research suggest the possibility of a therapeutic approach aimed at enhancing the effectiveness of CRC treatment through genetic regulation with the aid of PDT.},
}

Humans
*Colorectal Neoplasms/genetics/pathology/drug therapy/therapy
*Photochemotherapy
Apoptosis/drug effects/genetics
*CRISPR-Cas Systems/genetics
Reactive Oxygen Species/metabolism
*Claudin-2/metabolism/genetics
Cell Movement/drug effects/genetics
Chlorophyllides
HCT116 Cells
Gene Expression Regulation, Neoplastic/drug effects
Porphyrins/pharmacology
Claudins

@article {pmid42284917,
year = {2026},
author = {Zhao, D and Peng, W and Liu, Z and Zhou, Q and Wu, L and Zhou, Y and Ran, F},
title = {A sensitive detection of C-reactive protein based on the combination of CRISPR/Cas13a, MNPs and RNase H.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {280},
number = {},
pages = {117609},
doi = {10.1016/j.jpba.2026.117609},
pmid = {42284917},
issn = {1873-264X},
mesh = {*C-Reactive Protein/analysis ; Humans ; *Biosensing Techniques/methods ; *Ribonuclease H/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; *Magnetite Nanoparticles/chemistry ; },
abstract = {C-reactive protein (CRP) is a potential risk factor for disease. Here, developed a rapid and accurate fluorescence biosensor for detecting CRP, which contributes to early diagnosis and timely treatment of diseases. The CRP binds with the aptamer resulting in the probe 1 (P1) releasing from the complex of aptamer/P1/magnetic nanoparticles (MNPs). After magnetic separation, the free P1 hybridized with the RNA (P2) modified on the MNPs, leading to the P2 being multiple-turnover cut by ribonuclease H (RNase H). The formed free RNA can specifically bind with the crRNA and the tans-cleavage activity of CRISPR/Cas13a was triggered, leading to the RNA reporter containing a dye and quencher pair being cleaved and generating the fluorescence signal. This developed fluorescent biosensor takes full advantage of the synergy of aptamer, RNase H, MNPs and CRISPR/Cas13a. Here, the developed fluorescent biosensor exhibits excellent sensitivity and specificity towards the detection of CRP with a linear range from 10 pg/mL to 200 ng/mL. The detection limit is low down to 7.5 pg/mL. Additionally, this method successfully detected the CRP in human serum samples with satisfactory recoveries. Therefore, this developed biosensor will offer a valuable tool for the rapid diagnosis of CRP-related diseases.},
}

*C-Reactive Protein/analysis
Humans
*Biosensing Techniques/methods
*Ribonuclease H/chemistry/metabolism
*CRISPR-Cas Systems/genetics
Limit of Detection
Aptamers, Nucleotide/chemistry
*Magnetite Nanoparticles/chemistry

@article {pmid42341421,
year = {2026},
author = {Le, HT and Nghi, NB and My, VD and Vu, HA and Thanh, MC and Tri, BM and Niem, VVT and Phuong, HA},
title = {Functional characterization of PIK3CA E545A mutation in MCF-7 breast cancer cells reveals enhanced proliferation and resistance to Alpelisib.},
journal = {Biochemical and biophysical research communications},
volume = {829},
number = {},
pages = {154189},
doi = {10.1016/j.bbrc.2026.154189},
pmid = {42341421},
issn = {1090-2104},
mesh = {Humans ; *Class I Phosphatidylinositol 3-Kinases/genetics/antagonists & inhibitors/metabolism ; Cell Proliferation/drug effects/genetics ; *Breast Neoplasms/genetics/drug therapy/pathology ; Female ; *Drug Resistance, Neoplasm/genetics/drug effects ; MCF-7 Cells ; *Mutation ; *Thiazoles/pharmacology ; CRISPR-Cas Systems ; },
abstract = {PIK3CA mutations are central oncogenic drivers in hormone receptor-positive, HER2-negative breast cancer; however, the functional and therapeutic relevance of noncanonical variants remains incompletely defined. The E545A mutation, increasingly reported in specific patient populations, has not been systematically investigated. We generated an isogenic MCF-7 cell model harboring the PIK3CA E545A mutation using CRISPR/Cas9-mediated homology-directed repair to delineate its phenotypic and pharmacological consequences. E545A induced a robust gain-of-function phenotype, characterized by a mesenchymal-like morphological transition with reduced circularity and decreased cell size. This structural shift was accompanied by enhanced tumor cell fitness, including accelerated proliferation kinetics, increased metabolic activity, and significantly elevated clonogenic capacity compared with wild-type controls. Notably, growth trajectories showed sustained divergence between mutant and control cells across all time points, indicating a stable proliferative advantage. Importantly, E545A conferred diminished sensitivity to the PI3Kα inhibitor Alpelisib. Mutant cells retained migratory capacity under treatment and exhibited a pronounced, time-dependent increase in IC50, consistent with adaptive resistance. Collectively, these findings identify E545A as a functionally active and therapeutically consequential PIK3CA variant. Our study expands the current understanding of PIK3CA-driven oncogenic diversity beyond canonical hotspot mutations and underscores the need for variant-resolved stratification to improve the efficacy of PI3K-targeted therapies.},
}

Humans
*Class I Phosphatidylinositol 3-Kinases/genetics/antagonists & inhibitors/metabolism
Cell Proliferation/drug effects/genetics
*Breast Neoplasms/genetics/drug therapy/pathology
Female
*Drug Resistance, Neoplasm/genetics/drug effects
MCF-7 Cells
*Mutation
*Thiazoles/pharmacology
CRISPR-Cas Systems

@article {pmid42342973,
year = {2026},
author = {Duan, M and Meng, B and Zhou, L and Wu, L and Tong, X and Huang, D and Yin, H and Liu, ZJ and Zhang, Y},
title = {Structural basis of AtCas9 recognition of PAM mutants in underwound DNA topology.},
journal = {Nature structural & molecular biology},
volume = {33},
number = {7},
pages = {1062-1074},
pmid = {42342973},
issn = {1545-9985},
mesh = {Cryoelectron Microscopy ; *DNA/chemistry/metabolism/genetics ; Models, Molecular ; Mutation ; *Alicyclobacillus/enzymology/genetics ; Nucleic Acid Conformation ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism ; },
abstract = {The CRISPR-Cas9 system locates targets through guide RNA pairing and recognition of a protospacer-adjacent motif (PAM). Although PAM specificity is sequence-determined, DNA topology can relax PAM requirements and enable near-PAMless cleavage by the type II-C Alicyclobacillus tengchongensis Cas9 (AtCas9). However, the structural mechanism underlying this regulation remains unknown. Here we report cryogenic-electron microscopy (cryo-EM) structures of AtCas9 bound to B-form DNA or a 340 bp underwound minicircle DNA containing wild-type or mutant PAMs. Despite PAM sequences differences, all three underwound complexes adopt an almost identical architecture distinct from the B-form DNA-bound state. On B-form DNA, AtCas9 recognizes the PAM through base-specific hydrogen bonds and steric exclusion, conferring preference for N4CNNN and N4RNNA (R = A/G). By contrast, underwound DNA widens the PAM major groove and promotes sequence-independent backbone contacts, explaining the near-PAMless cleavage. These findings uncover a topology-dependent mechanism of PAM recognition and establish a cryo-EM platform using underwound minicircle DNA for structural studies under native-like topological states.},
}

Cryoelectron Microscopy
*DNA/chemistry/metabolism/genetics
Models, Molecular
Mutation
*Alicyclobacillus/enzymology/genetics
Nucleic Acid Conformation
CRISPR-Cas Systems
*CRISPR-Associated Proteins/chemistry/metabolism

@article {pmid42432276,
year = {2026},
author = {Tian, Y and Li, M and Liu, C and Liu, T and Zhang, X and Liu, Q},
title = {Simultaneous detection of multiple foodborne pathogens using a CRISPR/Cas12a-based pump-free microfluidic chip.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {42432276},
issn = {1618-2650},
abstract = {The development of microfluidic chips for nucleic acid detection provides efficient technical support for monitoring food safety. With the increasing maturity of CRISPR technology, it has the advantages of high specificity and high sensitivity in the detection of single or multiple nucleic acids. In this study, a microfluidic biosensor based on the CRISPR/Cas12a system was constructed using a pump-free microfluidic chip as the carrier, with a focus on the rapid, simultaneous detection of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Cronobacter sakazakii. For each target pathogenic bacterium, two replicate channels for each pathogen were established, along with corresponding negative and positive controls, which effectively ensured the reliability and repeatability of the detected results and successfully achieved the simultaneous high-sensitivity, high-specificity, and high-accuracy detection of multiple foodborne pathogens. The detection sensitivity of the sensor for S. aureus, E. coli O157:H7, L. monocytogenes, and C. sakazakii was as low as 10[3] CFU/mL, 10[3] CFU/mL, 10[2] CFU/mL, and 10[3] CFU/mL, respectively. This integrated CRISPR/Cas12a sensor chip has the advantages of the simultaneous efficient detection of multiple pathogens, parallel verification, and control settings, and the detection results can be visualized by fluorescence, indicating broad application prospects in the field of on-site rapid nucleic acid analysis.},
}

@article {pmid42434562,
year = {2026},
author = {Liang, Z and Li, Z and Li, C and Zhao, Y and Liu, J and Zhang, J},
title = {Advances in gene editing tools for four typical Gram-positive bacteria.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1882312},
pmid = {42434562},
issn = {1664-302X},
abstract = {Gram-positive bacteria serve as important chassis microorganisms in synthetic biology, industrial fermentation, and probiotic development. The rapid advancement of gene editing technologies has provided critical technical support for the iterative construction and functional validation of engineered strains. However, due to factors such as cell wall structure, differences in genetic backgrounds, and tool compatibility, the development and editing efficiency of gene editing systems for Gram-positive bacteria still face many challenges. This review focuses on four representative Gram-positive bacterial species-Lactobacillus plantarum, Lactococcus lactis, Bacillus subtilis, and Corynebacterium glutamicum-and traces the evolution and current state of their editing tools, from traditional homologous recombination to CRISPR-Cas9, base editors, and large-fragment integration tools. On this basis, we summarize the common challenges and corresponding strategies concerning host repair capacity, tool compatibility, and inherent limitations of editors in these four bacterial species, and propose recommendations for tool selection based on different application scenarios. This review aims to provide a technical reference for gene editing studies of the above-mentioned bacterial species. Although the conclusions cannot be directly extended to all Gram-positive bacteria, the common issues summarized here may inform the development of gene editing tools for other Gram-positive bacteria.},
}

@article {pmid42434939,
year = {2026},
author = {Wang, S and Hasan, R},
title = {CRISPR Biosensing for Environmental Monitoring: Workflow Design and Performance Benchmarking.},
journal = {Environmental science & technology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.est.6c02006},
pmid = {42434939},
issn = {1520-5851},
abstract = {CRISPR-based biosensing has rapidly emerged as a promising platform for environmental monitoring due to its high specificity, programmability, and compatibility with portable readouts. However, translation from biomedical diagnostics to environmental matrices remains challenging because of diverse sample types, complex inhibitors, and the breadth of biological and chemical targets. This Review provides a comprehensive analysis of CRISPR-based sensing technologies tailored for environmental contaminant detection, spanning both biological and chemical targets. We systematically evaluate published studies across target classes, Cas effectors, recognition mediators, sample matrices, pretreatment strategies, preamplification or signal-gain approaches, readout modalities, and reported performance metrics. To support practical implementation, we summarize a five-step experimental framework for environmental CRISPR sensing. We then propose a decision-guided design flowchart that links monitoring goals and matrix constraints to the selection of effectors, mediator-enabled transduction routes, pretreatment modules, amplification strategies, readouts, and validation controls. We further benchmark reported detection limits by normalizing units and comparing trends across preamplification-aided versus preamplification-free designs and by contextualizing performance against relevant regulatory or guideline thresholds when available. Across the literature, most studies rely on spiked-matrix validation, highlighting the need for broader nonspiked real environmental sample testing and more transparent reporting of sampling, pretreatment, and performance evaluation. Finally, we advocate standardized data reporting, including consistent units, workflow metadata, and matrix-matched validation, to enable cross-study comparison and accelerate the deployment of CRISPR-based sensors for real-world environmental monitoring.},
}

@article {pmid42435220,
year = {2026},
author = {Khan, MA and Durand, A and Skouri-Panet, F and Benzerara, K and Cassier-Chauvat, C and Chauvat, F and Ouchane, S},
title = {Targeted genome editing of the non-model cyanobacterium Cyanothece PCC 7425 via CRISPR/Cas12a.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00253-026-13959-y},
pmid = {42435220},
issn = {1432-0614},
support = {ANR-19-CE44-0017//Agence Nationale de la Recherche/ ; ANR-19-CE44-0017//Agence Nationale de la Recherche/ ; },
abstract = {Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of some cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece sp. PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a-mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of the ΔsodB mutant further confirmed its targeted deletion. Overall, CRISPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece sp. PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity, and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. KEY POINTS: • Rapid and efficient CRISPR/Cas12a editing established in Cyanothece sp. PCC 7425. • Fully segregated knockout mutants obtained after a single selection round. • Platform for exploring the biotechnological potential of Cyanothece sp. PCC 7425.},
}

@article {pmid42436385,
year = {2026},
author = {Vásquez-Herrera, L and Vallejos, OP and Acevedo-López, J and Campos-Gajardo, S and Piña-Iturbe, A and Tichy-Navarro, D and Seeram, D and Uhlemann, AC and González, PA and Kalergis, AM and Moreno-Switt, AI and Bueno, SM},
title = {Whole genome sequence-based comparative genomics reveals preliminary genomic features of Salmonella enterica subsp. enterica serovar Enteritidis phage type 1 and phage type 4 strains from EnteroBase.},
journal = {BMC microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12866-026-05329-5},
pmid = {42436385},
issn = {1471-2180},
support = {N° 21251772//ANID National Doctoral Fellowship/ ; N° 1240971//Fondo Nacional de Desarrollo Científico y Tecnológico/ ; N° 1231851//Fondo Nacional de Desarrollo Científico y Tecnológico/ ; N° 1231905//Fondo Nacional de Desarrollo Científico y Tecnológico/ ; ICN 2021_045//Millennium Institute of Immunology and Immunotherapy/ ; ECOS220027//ECOS-ANID/ ; },
abstract = {BACKGROUND: Whole Genome Sequencing (WGS) enables detailed characterization of circulating and emerging bacterial strains. Although tens of thousands of Salmonella genomes have been acquired over the years, analyses of the genomic differences between strains of different phage types are scarce.

RESULTS: We compared two Salmonella enterica subsp. enterica serovar Enteritidis (SEn) phage types, namely phage types 1 and 4 from available databases, using bioinformatic tools and nanopore sequencing of a Chilean PT1 strain. Comparisons between the two phage types show very low genomic divergence and high genomic sequence similarity. Single nucleotide polymorphism (SNP) searches identified SNPs specific to each phage type. Although a translocated region was identified in the Chilean PT1 strain analyzed in this study when compared to the genome of a PT4 strain, this was not present in the genomes of other PT1 strains, suggesting a local strain-specific rearrangement. Further analyses yielded no differences in the CRISPR-Cas locus, but a slight difference was observed in Gifsy-2 prophage detection and DNA modification systems between PT1 and PT4 strains.

CONCLUSIONS: Our findings provide insights into the genomic differences between SEn strains of two different phage types, serving as a basis for future genomic studies, yet further analyses with more diverse geographical locations collected over a longer time span are essential to validate these differences with the potential to establish molecular markers for strain identification and characterization in the context of epidemiological surveillance as a complement to WGS when this technique is not available.},
}

@article {pmid42437521,
year = {2026},
author = {Asemoloye, MD},
title = {Enhancing the Secretion Systems: Genetic Engineering of Super Bioagents for Effective Plant Disease Control.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70303},
pmid = {42437521},
issn = {1097-0290},
abstract = {The escalating threat of plant diseases to global agriculture and food security necessitates innovative and sustainable control strategies. Conventional biological control agents (BCAs), while environmentally friendly, often suffer environmental challenges and secretion of limited/poor antimicrobial compounds. Advances in CRISPR/Cas genome editing, protease engineering, and synthetic biology have enabled precise modifications that improve pathogen targeting and secretion efficiency. Interest should now be shifted on development of "Super Bioagents (SBs)" with enhanced secretion systems (SSs) for plant disease suppression against changing environmental factors. This will create sustainable ecofriendly alternative to chemical pesticides. This review explores a detailed overview of molecular mechanisms of microbial SSs and the potentials of SBs as a frontier in plant disease management. While there are still challenges in mass deployment of BCAs in sustainable agriculture, this review is guided by the hypothesis that rational, quantitative engineering of microbial SSs can transform conventional BCAs into integrated SBs. It synthesizes current advances within a systems‑level bioengineering framework linking secretion efficiency, regulation, and field performance. It further explores possible integration of SBs in plant-microbiome interactions to further enhance their adaptability and effectiveness. Finally, the review dives into recent breakthroughs, current challenges, and future directions for SBs development and application as next-generation plant disease control agents.},
}

@article {pmid42438474,
year = {2026},
author = {Ravendran, S and Fammé, S and Noer, MG and Skov, TW and Mikkelsen, NS and Lee Schneller, J and Dorset, SR and Wolff, JH and Møller, AMJ and Haslund, D and Revenfeld, ALS and Holm, M and Mogensen, TH and Møller, BK and Mikkelsen, JG and Bak, RO},
title = {AAV vector production in suspension cells using PEI transfection and sodium butyrate with orthogonal assessment of function and quality.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {3},
pages = {201787},
pmid = {42438474},
issn = {3117-387X},
abstract = {Adeno-associated virus (AAV) vectors are widely used in gene therapy, yet academic in-house production remains dominated by labor-intensive adherent cell workflows with limited scalability. Here, we describe an AAV vector production platform using suspension cells in orbital shaking Erlenmeyer flasks, based on polyethyleneimine (PEI) transfection and sodium butyrate supplementation. Following systematic evaluation of transfection conditions, this approach yields vectors with performance comparable to a commercial production kit. Vector quality was interrogated using orthogonal methodologies, including two-dimensional ddPCR, mass photometry, and nanopore sequencing, enabling comparative assessment of genome packaging, capsid composition, and vector heterogeneity. Functional validation was performed by in vitro transduction of K562 cells and primary human CD34+ hematopoietic stem and progenitor cells, as well as in vivo gene delivery to mouse liver and heart. Across assays, vectors produced using this protocol demonstrated comparable genome integrity and transgene expression. Comparative purification analysis revealed that iodixanol density gradient purification resulted in higher proportions of full capsids and reduced producer-cell-derived impurities relative to PEG 8000 precipitation. Together, this work establishes a scalable suspension-based AAV production workflow and demonstrates the value of orthogonal quality assessment combined with functional validation for robust vector benchmarking.},
}

@article {pmid42438737,
year = {2026},
author = {Faleiros, CA and Gonçalves, OS and Nunes, AT and Pires, CS and Poleti, MD and Fukumasu, H},
title = {Host breed and geography shape the antiviral defense landscape of the bovine rumen microbiome.},
journal = {ISME communications},
volume = {6},
number = {1},
pages = {ycag162},
pmid = {42438737},
issn = {2730-6151},
abstract = {The rumen microbiome represents a complex, phage-rich ecosystem where microbial survival depends on both metabolic cooperation and antiviral defense. However, global and breed-associated variations in rumen prokaryotic immune systems remain poorly understood. Here, we performed the most comprehensive profile to date of antiviral defense systems (DS) in the rumen, analyzing 6530 microbial genomes and metagenome-assembled genomes (MAGs) from diverse cattle breeds and geographic regions. In this global dataset, we identified >90 000 DS, the most abundant of which were restriction-modification, PDC-S01, deoxyribonucleic acid modification systems (DMS_other), AbiE and SoFic, with variations influenced by both host the lineage and geographic region. A more in-depth analysis was performed using two complementary antiviral annotation frameworks for Nellore cattle (Bos indicus) from Brazil. Data exhibited a remarkably enriched antiviral defense repertoire, with over 15 632 DS encoded across 547 high-quality MAGs. These systems were densely clustered in dominant rumen lineages, such as Prevotella, and positively correlated with prophage abundance, consistent with virus-host coevolution. Notably, we also detected viral contigs encoding both antiviral defense and anti-defense genes, underscoring the arms race between the phages and their microbial hosts. Metatranscriptomic data from North America and Oceania revealed high expression levels of toxin-antitoxin modules, clustered regularly interspaced short palindromic repeats components, and restriction enzymes, suggesting a basal level of antiviral activity. These findings reveal the rumen as an antiviral innovation hotspot, highlighting microbiome resilience with implications for ecology, adaptation, and phage-based interventions.},
}

@article {pmid42439519,
year = {2026},
author = {Iyer, MS and Hagström, E and Näslund, K and Andersson, SGE},
title = {Harnessing endogenous CRISPR-Cas9 for inducible genetic engineering of Apilactobacillus kunkeei.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0072826},
doi = {10.1128/aem.00728-26},
pmid = {42439519},
issn = {1098-5336},
abstract = {UNLABELLED: Despite substantial advances in bacterial genome engineering, functional genetic analysis remains challenging in many non-model bacterial species, particularly among host-associated gram-positive bacteria. The fructophilic species Apilactobacillus kunkeei has been investigated for more than two decades and is a dominant member of the honeybee microbiome, where it contributes to pathogen resistance and colony fitness. Nevertheless, the mechanistic investigation of this ecologically important species has remained limited despite its growing probiotic relevance. To enable functional genomics in this organism, we developed an inducible genome-engineering platform that leverages its endogenous Type II-A CRISPR-Cas9 system. The system uses a sakacin-responsive dual-plasmid initiator-effector design in which phage-derived recombineering genes and a single-guide RNA are coordinately expressed, while DNA cleavage is mediated by natively expressed Cas9. Using this approach, we achieved scarless deletion of individual genes, including targets as large as ~25 kb, gene replacement with a fluorescent reporter, C-terminal epitope tagging, and precise nucleotide substitutions, with editing efficiencies approaching 100%. Both plasmids can be readily cured following modification, allowing recovery of clean mutant genotypes. We further demonstrate that endogenous Cas9 can be repurposed for CRISPR interference using a single, self-contained plasmid to enable targeted transcriptional repression. Together, this work establishes a robust strategy for genetic manipulation of A. kunkeei and expands the toolkit available for harnessing endogenous CRISPR-Cas systems in genetically recalcitrant, non-model gram-positive bacteria.

IMPORTANCE: Many ecologically and industrially important bacteria remain genetically recalcitrant, limiting functional genomic studies. As research increasingly extends beyond traditional model organisms, these limitations are especially apparent in non-model gram-positive bacteria from host-associated or environmental niches. Here, we establish an inducible genome-editing framework exploiting the endogenous Cas9 system of Apilactobacillus kunkeei, a key member of the honeybee microbiota. This approach enables reliable scarless gene deletions, precise nucleotide changes, large-scale genome modifications, and programmable transcriptional repression. By enabling genetic manipulation in A. kunkeei, this work facilitates experimental studies of its roles in honeybee health, microbial interactions, and host-associated adaptation, and highlights the potential of endogenous CRISPR-Cas systems for expanding genetic access in non-model bacteria.},
}

@article {pmid42135407,
year = {2026},
author = {Bazick, HO and James, LM and Zylka, MJ},
title = {Nickase NmCas9 unsilences paternal Ube3a in a mouse model of Angelman syndrome without causing AAV vector integration.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42135407},
issn = {2045-2322},
support = {631904//Simons Foundation/ ; 1R01NS109304/NS/NINDS NIH HHS/United States ; 1R01NS109304/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Mice ; *Dependovirus/genetics ; *Angelman Syndrome/genetics/therapy/metabolism ; Genetic Vectors/genetics ; Disease Models, Animal ; Neurons/metabolism ; *Deoxyribonuclease I/metabolism/genetics ; Gene Editing/methods ; Male ; Virus Integration ; CRISPR-Cas Systems ; },
abstract = {Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of maternal UBE3A. In neurons, the paternal (pat)UBE3A allele is silenced by a long non-coding antisense transcript called Ube3a-ATS. Previous genome-editing approaches used active nucleases to unsilence patUbe3a by disrupting Ube3a-ATS. However, these methods create DNA double-strand breaks (DSBs) and promote integration of adeno-associated virus (AAV) vector genomes, both of which raise potential safety concerns. Here, we found that a nickase Neisseria meningitidis Cas9 variant (nNmCas9-D15A) disrupted Ube3a-ATS transcription when targeted to the non-template strand and unsilenced patUbe3a in cultured mouse neurons without generating DSBs or causing AAV integration. Intracerebroventricular delivery of AAV9-nNmCas9-D15A in AS model mice potently and durably reduced Ube3a-ATS and elevated Ube3a throughout the cerebral cortex and hippocampus for at least 6 months. Further, this vector restored UBE3A expression in ~ 87% of cortical neurons, which compares favorably to previously reported efficiencies with active Cas9, dead Cas9, and zinc finger nuclease vectors. These results demonstrate that nNmCas9 is a highly effective and potentially safer genome editor for the treatment of AS.},
}

Animals
*Ubiquitin-Protein Ligases/genetics/metabolism
Mice
*Dependovirus/genetics
*Angelman Syndrome/genetics/therapy/metabolism
Genetic Vectors/genetics
Disease Models, Animal
Neurons/metabolism
*Deoxyribonuclease I/metabolism/genetics
Gene Editing/methods
Male
Virus Integration
CRISPR-Cas Systems

@article {pmid42303099,
year = {2026},
author = {Zhou, Z and Dong, S and Li, S and Tan, X and Zhou, Z and Fan, L and Liang, J and Li, G},
title = {CRISPR/Cas12a and nanocomposite-based electrochemical/ colorimetric parallel dual-channel aptasensor for highly sensitive LDL detection.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {75},
number = {},
pages = {102981},
doi = {10.1016/j.nano.2026.102981},
pmid = {42303099},
issn = {1549-9642},
mesh = {*Nanocomposites/chemistry ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *Lipoproteins, LDL/analysis/blood ; Humans ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Colorimetry/methods ; Graphite/chemistry ; DNA, Single-Stranded/chemistry ; },
abstract = {Atherosclerotic cardiovascular disease (ASCVD) remains a leading global health threat, necessitating precise monitoring of low-density lipoprotein (LDL) as a key risk biomarker for assessing ASCVD risk. Herein, an electrochemical/colorimetric dual- channel aptasensor was developed by integrating nitrogen-doped reduced graphene oxide-Hemin-trimanganese tetroxide nanoparticles (NrGO-Hemin-Mn3O4 NPs) with the CRISPR/Cas12a system. The CRISPR/Cas12a system introduces a powerful signal amplification cascade: a single target binding event activates the trans-cleavage of numerous ssDNA probes, translating into a highly amplified electrical and optical response. The NrGO-Hemin-Mn3O4 NPs serves as a conductive redox probe and exhibits superior peroxidase-like activity through the synergistic effect between Hemin and Mn3O4. Mechanistically, surface-bound single-stranded DNA (ssDNA) initially induces steric hindrance, which obstructs electron transfer and suppresses the enzyme-mimicking performance of the NrGO-Hemin-Mn3O4 NPs. Upon the target LDL binding, the released activator DNA triggers the trans-cleavage activity of Cas12a to degrade the ssDNA, thereby restoring both the electroactivity and catalytic performance of the probe. Experimental results demonstrated that the dual-channel aptasensor achieved a wide linear range from 0.01 to 1000 nM with a detection limit of 0.01 nM, demonstrating that CRISPR integration is pivotal for achieving high sensitivity in complex biological matrices. This dual-channel strategy offers a sensitive, intuitive tool for early clinical screening of ASCVD diseases.},
}

*Nanocomposites/chemistry
*Aptamers, Nucleotide/chemistry
*Biosensing Techniques/methods
*Lipoproteins, LDL/analysis/blood
Humans
*Electrochemical Techniques/methods
*CRISPR-Cas Systems/genetics
*Colorimetry/methods
Graphite/chemistry
DNA, Single-Stranded/chemistry

@article {pmid42420735,
year = {2026},
author = {Rijal, S and Zhang, R and Tian, XJ},
title = {Harnessing CRISPRi Competition to Develop Multimodule Controllers for Resource-Aware Circuit Design.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {305-318},
pmid = {42420735},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Regulatory Networks ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; },
abstract = {Cellular resource limitations give rise to resource competition, undermining the modularity and predictability of engineered genetic circuits. In systems containing positive feedback, such competition can drive Winner-Takes-All (WTA) dynamics, resulting in severe imbalances in resource allocation across circuit modules. In this chapter, we present an experimental implementation of a Negatively Competitive Regulatory (NCR) controller based on CRISPR interference (CRISPRi) in dual self-activation (DSA) circuits. We describe a detailed workflow for chromosomal integration of a tunable dCas9 expression cassette, as well as the design of self-activation modules and module-specific guide RNAs that induce self-repression through competition for limiting dCas9. This architecture introduces effective negative feedback to the more active module while reallocating resources to the less active one, thereby promoting balanced module activity. Finally, we provide guidelines for quantitatively assessing the regulation of resource competition between DSA modules using the NCR strategy. Overall, these guidelines demonstrate how CRISPRi can be leveraged to implement NCR strategy in gene circuits, thereby enhancing circuit modularity and predictability.},
}

*CRISPR-Cas Systems
*Gene Regulatory Networks
RNA, Guide, CRISPR-Cas Systems/genetics
*Genetic Engineering/methods

@article {pmid42423795,
year = {2026},
author = {Ata, A and Topuz Ata, D},
title = {A comprehensive review of CRISPR-Cas9-mediated genome editing in Leishmania strains: methodologies, applications, challenges and future directions.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42423795},
issn = {1573-4978},
mesh = {*Leishmania/genetics ; *Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; Animals ; Genome, Protozoan ; Leishmaniasis/parasitology/genetics ; Humans ; },
abstract = {Genome editing employing CRISPR-Cas9 has rapidly transformed experimental research in Leishmania, providing opportunities to investigate the genetic factors responsible for parasite survival, response to drugs and pathogenic traits. This review provides a comprehensive synthesis of CRISPR-based systems implemented across Leishmania species, spanning Cas9-mediated gene deletion, precise genome editing, endogenous locus tagging and pooled screening strategies. Furthermore, we highlight the emergence of Cas variants and next-generation CRISPR systems which expand the range of targetable genomic regions, improve editing precision and reduce the need for generation of double-strand DNA breaks (DSBs). Particular emphasis is placed on conditional and inducible genome-editing platforms, cytosine base-editing technologies, and recently developed CRISPR-based approaches such as prime editing, CRISPR activation/interference and Cas12-associated implementations. This review also discusses the principal biological and technical constraints influencing CRISPR-based studies in Leishmania, including genome plasticity, multicopy gene families, required genes, guide RNA design limitations and off-target considerations. Notably, the review also addresses CRISPR-Cas implementations in sand-fly vector biology, drawing on a foundational study in Phlebotomus papatasi. Through systematic compilation of published studies into comparative tables, we evaluate the strengths, limitations, experimental utility, delivery strategies, experimental workflows and representative applications of major CRISPR platforms. Together, these advances highlight the transition of CRISPR-Cas systems from proof-of-concept tools to versatile platforms for functional genomics, target validation and translational research in Leishmania, while offering a consolidated guide for selecting suitable CRISPR-Cas technologies and underscoring important considerations for their continued development in leishmaniasis research.},
}

*Leishmania/genetics
*Gene Editing/methods/trends
*CRISPR-Cas Systems/genetics
Animals
Genome, Protozoan
Leishmaniasis/parasitology/genetics
Humans

@article {pmid42424815,
year = {2026},
author = {Tang, Q and Zhang, Y and Garza, DR and Ruan, C and Liu, B and Rocha, U and Shen, P and Wei, Y and Deng, Y and Zhang, J and Richnow, HH},
title = {Virus-mediated fate of antimicrobial resistance genes in livestock manure anaerobic digestion.},
journal = {Water research},
volume = {305},
number = {},
pages = {126401},
doi = {10.1016/j.watres.2026.126401},
pmid = {42424815},
issn = {1879-2448},
abstract = {Antimicrobial resistance (AMR) poses a critical global health challenge, with livestock manure acting as a significant environmental reservoir for antimicrobial resistance genes (ARGs). Anaerobic digestion (AD) is a pivotal process for mitigating ARG dissemination at the livestock-environment-human interface. This study aims to elucidate the global dynamics of ARGs in AD systems, focusing on virus-host interactions and arms race, to identify actionable strategies for AMR control. We analyzed 205 metagenomic (4.5 Tb) and 36 meta-transcriptomic (640 Gb) datasets, including 15 newly generated datasets, revealing that pig manure AD harbors the highest ARG abundance (0.668 ARGs/16S rRNA), while AD systems generally exhibit limited transcriptional activation of ARGs. We constructed a viral dataset for livestock manure AD (GVD_LMAD), comprising 59,316 DNA and 727 RNA viral operational taxonomic units (vOTUs). Virus-host interactions established by CRISPR-Cas spacer, tRNA and homology matches revealed 889 lytic infections of antimicrobial-resistant bacteria (ARB) compared to only 18 ARG transduction events. Further analysis showed that the relative abundance of vOTUs assigned to the reduction role (4.11% ± 3.19%) was substantially higher than that of reproduction (0.72% ± 0.64%) and transduction (0.19% ± 0.30%), demonstrating that, among viral processes, lysis outweighs transduction in contributing to ARG abundance reduction in AD. Furthermore, an antiviral defense system (ADS) catalogue (GADSC_LMAD), derived from 2760 high-quality metagenome-assembled genomes (MAGs) containing 39,307 ADS, with ADS prevalence in ARB (7.8 ± 6.0 per MAG), indicating an intensified virus-host arms race in AD that may shield ARB from phage lysis. The resulting CRISPR-Cas immune network with expressed spacers targets foreign ARG-carrying sequences (primarily plasmids and ICEs), suggesting a mechanism that restricts horizontal gene transfer (HGT) via conjugation and transformation, despite shielding ARB from phage lysis. Collectively, these findings highlight that viral communities significantly contribute to ARG reduction through phage lysis relative to transduction, while the ADS-mediated arms race, despite protecting ARB, constructs a biological firewall that potentially limits HGT of ARGs. This study provides novel insights into virus-host dynamics as a key mechanism for controlling ARG dissemination in AD systems.},
}

@article {pmid42424969,
year = {2026},
author = {Samad, MA and Ahmad, I and Jabir, NR and Rehan, M and Zaidi, SK and Al-Abbasi, F and Tabrez, S},
title = {Role of long non-coding RNAs in therapeutic resistance and clinical applications in cancer.},
journal = {European journal of medicinal chemistry},
volume = {317},
number = {},
pages = {119090},
doi = {10.1016/j.ejmech.2026.119090},
pmid = {42424969},
issn = {1768-3254},
abstract = {Cancer is one of the leading causes of mortality worldwide and is recognized as a complex, multifactorial disease with no clearly defined etiology for its onset and progression. Long non-coding RNAs (lncRNAs) are widely distributed across the human body and play varied roles in regulating cellular processes. In recent years, they have gained the attention of the scientific community as key regulators of cancer due to their diverse functional roles and complex regulatory mechanisms. Aberrant expression of lncRNAs contributes to tumor progression, functioning as oncogenes that modulate various pathways through different mechanisms. Early technologies could not study lncRNAs effectively and considered it as "junk" RNA. Studies using gene-expression analyses, functional experiments, and animal-based models have shown that dysregulated lncRNAs are implicated in the maintenance of cancer stem cells (CSCs) and in driving therapeutic resistance. Additionally, lncRNAs have shown promise as valuable biomarkers for cancer diagnosis, prognosis, predicting patient outcomes, and guiding treatment strategies. Moreover, therapeutic strategies targeting lncRNAs, such as antisense oligonucleotides (ASOs), RNA interference (RNAi), exosome-based delivery systems, nanomedicine, virus-mediated therapy, and CRISPR-Cas technologies, have opened new avenues for cancer treatment. This review highlights the diverse roles of lncRNAs in therapeutic resistance and emphasizes their clinical potential as diagnostic and prognostic tools and emerging therapeutic strategies.},
}

@article {pmid42425355,
year = {2026},
author = {V, MS and Chaudhary, N and Hasan, M and Kumar, A and Tripathi, MK},
title = {Filamentous fungi as microbial cell factories for lignocellulosic biomass valorization: A comprehensive review.},
journal = {International journal of biological macromolecules},
volume = {375},
number = {},
pages = {153415},
doi = {10.1016/j.ijbiomac.2026.153415},
pmid = {42425355},
issn = {1879-0003},
abstract = {The transition toward a sustainable bioeconomy requires efficient conversion of lignocellulosic biomass (LCB), the most abundant renewable biological macromolecular resource on Earth, into fuels, chemicals, and other high-value products. However, the complex architecture of cellulose, hemicellulose, and lignin imparts significant recalcitrance, limiting biomass deconstruction and industrial utilization. Although recent reviews have examined fungal biorefineries, lignocellulolytic enzymes, or fungal strain engineering separately, an integrated synthesis linking lignocellulosic biomass characteristics, fungal deconstruction mechanisms, hydrolysate utilization, and cell-factory engineering remains limited. This review presents an integrated framework for lignocellulosic biomass valorization using filamentous fungi as microbial cell factories. We examine biomass composition, recalcitrance, and pretreatment strategies, followed by the fungal macromolecular machinery responsible for biomass deconstruction, including cellulases, hemicellulases, lignin-active oxidoreductases, and auxiliary activity enzymes. Particular emphasis is placed on the regulatory networks and engineering strategies that govern fungal performance, including transcription factor engineering, promoter engineering, metabolic rewiring, heterologous pathway engineering, RNA interference, and CRISPR-Cas-based genome editing. The review further discusses the conversion of lignocellulose-derived hydrolysates into biofuels, organic acids, industrial enzymes, and other high-value compounds, together with emerging advances in co-culture fermentation, downstream processing, and integrated biorefinery design. Collectively, this review highlights how the integration of fungal enzymatic systems, strain engineering, and process-level innovations can overcome biomass recalcitrance and improve lignocellulosic bioconversion efficiency. These insights provide a framework for developing robust fungal platforms for the sustainable production of high-value bioproducts from renewable biomass.},
}

@article {pmid42426280,
year = {2026},
author = {Grigg, S and Shembrey, C and Fareh, M and Blombery, P and Corn, JE and Seymour, JF and Casan, JML},
title = {CRISPR in clinical oncology: translational advances from molecular diagnostics to therapeutics.},
journal = {Nature reviews. Clinical oncology},
volume = {},
number = {},
pages = {},
pmid = {42426280},
issn = {1759-4782},
abstract = {Cancer care is increasingly driven by molecular classification, yet many key oncogenic drivers remain undruggable, and intrinsic or acquired resistance to treatment frequently limits durable clinical benefit. CRISPR-Cas technologies provide a modular, programmable platform to interrogate and directly manipulate cancer biology via sequence-specific targeting of DNA or RNA and have advanced from experimental tools to the early stages of clinical translation. In this Review, we outline how CRISPR-enabled functional genomics approaches can reveal unexpected cancer dependencies and resistance mechanisms. We discuss emerging applications of CRISPR-based diagnostics in oncology that convert precise nucleic acid sequence recognition into rapid mutation detection. We also discuss applications of CRISPR in therapeutic strategies ranging from ex vivo immune cell engineering to nascent in vivo interventions that directly target tumour-related sequences such as fusion junctions or single-nucleotide variants. Finally, we highlight technological and regulatory challenges, including effective delivery of the editing machinery to cells in vivo, safety and platform-level regulatory frameworks, that will determine the clinical utility of CRISPR-based diagnostics and therapies in oncology.},
}

@article {pmid42427176,
year = {2026},
author = {Łakomy, W and Myślińska, M and Tarnawska, E and Rogóż, W and Kulig, K and Owczarzy, A and Maciążek-Jurczyk, M},
title = {Biotechnological strategies to combat antibiotic resistance.},
journal = {Polimery w medycynie},
volume = {56},
number = {1},
pages = {41-51},
doi = {10.17219/pim/218777},
pmid = {42427176},
issn = {0370-0747},
mesh = {Humans ; Antimicrobial Peptides/pharmacology ; Phage Therapy ; *Biotechnology ; CRISPR-Cas Systems ; Gene Editing ; *Drug Resistance, Microbial ; *Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology ; Nanoparticles ; Nanotechnology ; Animals ; },
abstract = {This article aims to present the current state of knowledge on four major biotechnological antimicrobial strategies and to evaluate their potential clinical applications in the context of increasing antibiotic resistance. Approaches such as phage therapy, CRISPR-Cas9 gene editing, nanoparticles, and antimicrobial peptides (AMPs) may significantly contribute to limiting the spread of resistance genes. Particular attention is given to advances in genetic engineering that enable precise targeting and elimination of resistance determinants, as well as to the therapeutic potential of the microbiome. A literature review of studies published between 2010 and 2025 was conducted using the following keywords: antimicrobial resistance, phage therapy, CRISPR-Cas9, AMPs, and nanotechnology. Both review articles and original studies, including preclinical and clinical data, were considered. Phage therapy demonstrates high efficacy against antibiotic-resistant pathogens, particularly in the form of phage cocktails and genetically engineered phages. Antimicrobial peptides exhibit broad-spectrum activity and can be structurally optimized to improve stability and selectivity. CRISPR-Cas9 systems enable targeted elimination of resistance genes or direct disruption of pathogen genomes, while nanotechnology facilitates drug delivery, biofilm penetration, and bactericidal activity, particularly through metal-based nanoparticles. Notably, all approaches show potential for synergistic use with conventional antibiotics. Biotechnological treatment strategies may become a key component in combating antibiotic resistance. However, their clinical implementation requires further research, comprehensive safety evaluation, regulatory development, and integration into medical practice. Advances in these areas could significantly reduce the global burden of infectious diseases.},
}

Humans
Antimicrobial Peptides/pharmacology
Phage Therapy
*Biotechnology
CRISPR-Cas Systems
Gene Editing
*Drug Resistance, Microbial
*Drug Resistance, Bacterial
Anti-Bacterial Agents/pharmacology
Nanoparticles
Nanotechnology
Animals

@article {pmid42428244,
year = {2026},
author = {Zhang, X and Shi, H and Yang, J and Du, L and Zhang, X and Li, X},
title = {The application of CRISPR gene-editing technology in influenza prevention and control.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1844919},
pmid = {42428244},
issn = {2673-3439},
abstract = {Influenza A virus (IAV) and influenza B virus (IBV) remain major global public health threats because of their rapid antigenic evolution and efficient human-to-human transmission. In contrast, influenza C virus (ICV) and influenza D virus (IDV) generally exhibit narrower host ranges and milder pathogenicity, yet their potential for interspecies transmission and zoonotic spillover still warrants attention. Conventional prevention strategies, such as inactivated and live-attenuated vaccines, suffer from prolonged development timelines and diminished efficacy against rapidly evolving viral strains. However, antiviral drugs are increasingly limited by the rapid emergence of drug-resistant variants. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene-editing technology has emerged as a promising platform for influenza prevention and control owing to its programmability and precise targeting capability. In this paper, we summarize recent advances in CRISPR-based strategies for influenza prevention and control. The RNA-targeting CRISPR-associated protein 13 (Cas13) system can recognize conserved viral RNA sequences and suppress replication across influenza subtypes, whereas the DNA-targeting CRISPR-associated protein 9 (Cas9) system can edit host susceptibility genes and thereby reduce cellular permissiveness to infection. In addition, lipid nanoparticle (LNP)-based delivery systems have become important tools for improving the in vivo delivery and expression of CRISPR components by enhancing targeting efficiency and reducing immunogenicity. CRISPR-based diagnostics, such as Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK), further expand the clinical utility of this technology by enabling rapid and sensitive detection of influenza viruses. Despite these advances, substantial challenges remain, including delivery inefficiency, off-target activity, long-term safety concerns, and the risk of viral escape. With continued technological refinement and careful translational development, CRISPR may become a versatile tool for influenza prevention, diagnosis, and therapy.},
}

@article {pmid42429865,
year = {2026},
author = {Preetam, S and Rath, P and Al-Enazi, NM and Sharaf, AAM and Jumah, JB and Govindarajan, RK and Goud, P and Thiruvengadam, M and Mathivanan, K},
title = {Engineering extracellular vesicle biogenesis for therapeutic gene delivery: emerging genetic programming strategies and translational prospects.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42429865},
issn = {1573-4978},
mesh = {Humans ; *Extracellular Vesicles/metabolism/genetics ; CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; Gene Editing/methods ; *Genetic Therapy/methods ; *Genetic Engineering/methods ; Animals ; },
abstract = {Extracellular vesicles (EVs) have emerged as promising biological nanocarriers for gene therapy due to their intrinsic ability to transport nucleic acids, proteins, and lipids between cells. Advances in EV biology have revealed complex regulatory mechanisms governing vesicle biogenesis, cargo sorting, secretion, and uptake, offering multiple opportunities for therapeutic engineering. Concurrently, modern genetic technologies, including the CRISPR-Cas9 genome editing system and synthetic biology tools, have enabled precise manipulation of EV composition and functionality. This review integrates current knowledge of EV biogenesis with emerging genetic engineering strategies to transform EVs into programmable gene delivery systems. We discuss recent advances in genetic tools for studying EV dynamics, methods for engineering EV cargo and targeting specificity, and the application of EV platforms for RNA and genome-editing therapies. Furthermore, key challenges related to vesicle heterogeneity, large-scale production, and clinical translation are examined. Finally, we highlight future perspectives on programmable EV therapeutics and their potential role in next-generation precision medicine.},
}

Humans
*Extracellular Vesicles/metabolism/genetics
CRISPR-Cas Systems/genetics
*Gene Transfer Techniques
Gene Editing/methods
*Genetic Therapy/methods
*Genetic Engineering/methods
Animals

@article {pmid42429888,
year = {2026},
author = {Thevendran, R and Maheswaran, S and Lee, SY},
title = {Development of attenuated and inactivated Dengue strains using advanced gene editing tools.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42429888},
issn = {1573-4978},
support = {FRGS/1/2023/SKK06/AIMST/03/2//Ministry of Higher Education, Malaysia/ ; },
mesh = {Humans ; *Dengue Virus/genetics/immunology/pathogenicity ; *Dengue Vaccines/immunology/genetics ; *Dengue/prevention & control/immunology/virology/genetics ; *Gene Editing/methods ; Vaccines, Attenuated/immunology/genetics ; Animals ; CRISPR-Cas Systems ; Vaccine Development/methods ; },
abstract = {Dengue fever remains a persistent viral threat, affecting millions of families every year, turning a simple mosquito bite into a potentially life-threatening emergency. This disease remains a constant burden on our global healthcare system, demanding innovative solutions to protect worldwide communities. While many researchers discuss general treatments, preventions and modern medical interventions, there is often a lack of focus on how current, cutting-edge molecular and genetic tools are employed to engineer dengue strains as vaccine candidates. Therefore, in this paper, we explore the recent genetic strategies, such as targeted virulent gene deletions, CRISPR-Cas inactivation, and viral codon deoptimization approaches used to attenuate or inactivate Dengue viruses specifically. Assays and techniques used in validating Dengue viral attenuation or inactivation are also discussed in detail, highlighting the importance of the balance between safety and immunogenicity for Dengue vaccine uses. The article also briefly elaborates the complex biological challenges and safety concerns that centre on Dengue vaccine developments. By bridging the gap between advanced genetics and public health, this review provides readers with a comprehensive understanding of how modern genetics is paving the way for the next generation of safe and effective Dengue vaccines.},
}

Humans
*Dengue Virus/genetics/immunology/pathogenicity
*Dengue Vaccines/immunology/genetics
*Dengue/prevention & control/immunology/virology/genetics
*Gene Editing/methods
Vaccines, Attenuated/immunology/genetics
Animals
CRISPR-Cas Systems
Vaccine Development/methods

@article {pmid42431872,
year = {2026},
author = {Kaya, NH and Abukhalaf, M and Fuentes, G and Taubenheim, J and Hentschel, U and Tholey, A and Fraune, S},
title = {c-JUN controls microbial colonization via selective phagocytosis in the sea anemone Nematostella.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42431872},
issn = {2041-1723},
support = {CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project Z3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project B1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC 1182, Project Z3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *Sea Anemones/microbiology/immunology/genetics/metabolism ; *Phagocytosis/immunology ; Immunity, Innate ; Vibrio/immunology ; *Proto-Oncogene Proteins c-jun/genetics/metabolism/immunology ; Microbiota/immunology ; CRISPR-Cas Systems ; Lysosomes/metabolism ; },
abstract = {Innate immunity is traditionally viewed as a broad defense system with limited specificity. However, increasing evidence suggests that innate immune cells can discriminate between distinct microbial partners. How such specificity arises in early-diverging animals remains unclear. Here, we identify in the sea anemone Nematostella vectensis a selective host innate immune mechanism mediated by nematosomes, motile multicellular bodies that differentially process bacterial cells. Nematosomes preferentially engulf non-native Vibrio isolates while showing reduced uptake of native host-associated strains. We identify the transcription factor cJUN as a key regulator of this process. CRISPR/Cas9-mediated knockout of cJUN reduces nematosome abundance, impairs lysosomal response, alters microbiome assembly, and increases susceptibility to bacterial infection. These results link immune gene function to microbial selectivity and demonstrate that even early-diverging animals exhibit sophisticated innate immunity mechanisms for microbiome regulation. Our findings support the idea that immune specificity can arise through repurposing deeply conserved pathways and may have deep evolutionary origin.},
}

Animals
*Sea Anemones/microbiology/immunology/genetics/metabolism
*Phagocytosis/immunology
Immunity, Innate
Vibrio/immunology
*Proto-Oncogene Proteins c-jun/genetics/metabolism/immunology
Microbiota/immunology
CRISPR-Cas Systems
Lysosomes/metabolism

@article {pmid42163455,
year = {2026},
author = {Xu, Z and Qiu, S and Tan, Y and Kuang, Y and Yang, C and Yan, F and Zhou, X and Zhou, H},
title = {Optimized tRNA processing and TREX2-SpCas9 fusion enable high-efficiency multiplex genome editing in plants.},
journal = {Plant communications},
volume = {7},
number = {7},
pages = {101921},
doi = {10.1016/j.xplc.2026.101921},
pmid = {42163455},
issn = {2590-3462},
mesh = {*Gene Editing/methods ; *Oryza/genetics ; *RNA, Transfer/genetics/metabolism ; CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Genome, Plant/genetics ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; },
abstract = {Multiplex genome editing is a powerful approach for dissecting gene networks and engineering complex traits in crops because it enables the simultaneous modification of multiple genomic loci. However, achieving high editing efficiency across multiple targets remains a significant challenge. To address this, we developed an optimized CRISPR system for rice that combines a monomeric TREX2-SpCas9 fusion with a novel array of tRNA-based gRNA processing elements. The TREX2-SpCas9 fusion significantly enhanced editing performance, resulting in higher editing efficiency, larger deletions, and increased mutation frequencies compared with wild-type SpCas9 and other exonuclease fusions. By systematically evaluating 38 endogenous rice tRNA genes, we identified 13 high-performing candidates, including tRNA[Leu-1] and tRNA[Pro-1], that outperformed the widely used tRNA[Gly] and tRNA[Met] elements, enabling highly efficient processing of multiplexed gRNA arrays. Incorporating these top-performing tRNAs into our system enabled simultaneous editing of up to 29 OsCPK genes in a single rice plant. Furthermore, we demonstrated the cross-species applicability of this platform in the dicot Nicotiana benthamiana using transient expression, where rice-derived tRNA elements facilitated high-efficiency editing. This optimized multiplex gene-editing system provides a robust, scalable platform for accelerating plant functional genomics and engineering complex agronomic traits.},
}

*Gene Editing/methods
*Oryza/genetics
*RNA, Transfer/genetics/metabolism
CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism
*Genome, Plant/genetics
Plants, Genetically Modified
Plant Proteins/genetics/metabolism

@article {pmid42240651,
year = {2026},
author = {Ma, SH and Yu, G and Park, S and Sung, H and Jeong, UJ and Kim, HH and Cho, J and Yoon, SS},
title = {Adapting prime editing with split prime editors in Escherichia coli and its application to Staphylococcus aureus genome editing.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {42240651},
issn = {1432-0614},
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *Methicillin-Resistant Staphylococcus aureus/genetics ; *Staphylococcus aureus/genetics ; *Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; Streptococcus pyogenes/genetics/enzymology ; },
abstract = {Prime editing is a precise and rapid genome-editing technique that modifies short DNA sequences using tailored guide RNAs. To implement this technique in bacteria, we used Prime Editor 2 (PE2) with the DeepPrime gRNA design tool and assessed its gene-editing efficiency in Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) cells. Our findings indicate that a split PE2, comprising a reverse transcriptase and two Cas9 nickase domains, exhibited gene-editing efficiency comparable to that of the intact PE2. The efficiency observed in E. coli was significantly affected by the target sites, edit type, and the presence of exonucleases. In MRSA, which serves as a model to evaluate the applicability in non-model bacterial species, Streptococcus pyogenes PE2 (SpPE2) exhibited superior performance relative to Staphylococcus aureus PE2 (SaPE2). Furthermore, the split SpPE2 lacking the reverse transcriptase successfully induced the intended mutation in MRSA. This study demonstrates the feasibility of prime editing within bacterial systems.},
}

*Escherichia coli/genetics
*Gene Editing/methods
*Methicillin-Resistant Staphylococcus aureus/genetics
*Staphylococcus aureus/genetics
*Genome, Bacterial
RNA, Guide, CRISPR-Cas Systems/genetics
Streptococcus pyogenes/genetics/enzymology

@article {pmid42310097,
year = {2026},
author = {Fajardo, AF and Gowda, CP and Johnson, E and Petroni, R and Tomar, VS and Liu, Z and Andres Blanco, M and Janssen, J and Elcheva, IA and Lanza, M and Fuchs, SY and Spiegelman, VS},
title = {In vivo CRISPR knockout screen identifies Polr1a as a key driver and a potential therapeutic target for melanoma metastasis.},
journal = {Oncogene},
volume = {45},
number = {29},
pages = {2978-2987},
pmid = {42310097},
issn = {1476-5594},
support = {CA243167//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; CA288849//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; CA304343//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {*Melanoma/genetics/pathology ; Humans ; Animals ; Cell Movement/genetics ; Mice ; Cell Line, Tumor ; *DNA-Directed RNA Polymerases/genetics/metabolism ; Neoplasm Metastasis ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Identification and characterization of novel mechanisms driving melanoma metastases and ways to target them are paramount for the development of effective treatment modalities. Here, we employed in vivo CRISPR knockout screening targeting the genes associated with poor prognosis to identify Polr1a as a potent driver of melanoma metastasis. High Polr1a levels correlate with increased metastasis and reduced survival in patients. Polr1a inhibition suppressed migration, invasion, and the ability of melanoma cells to colonize lungs. Ribo-seq analysis revealed that Polr1a is involved in regulating the non-canonical NF-κB pathway. Indeed, targeting Polr1a decreased levels of RelB and p52 and suppressed non-canonical NF-κB transcriptional activity; this suppression was responsible for the effects of Polr1a on melanoma cell migration. Accordingly, pharmacological inhibition of Polr1/Polr1a suppressed cell migration, tumor growth, and metastases. We discuss the potential utilization of Polr1 inhibitors for neoadjuvant treatment of melanoma.},
}

*Melanoma/genetics/pathology
Humans
Animals
Cell Movement/genetics
Mice
Cell Line, Tumor
*DNA-Directed RNA Polymerases/genetics/metabolism
Neoplasm Metastasis
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42366688,
year = {2026},
author = {Wu, J and Yan, J and Li, C and Liu, X and Zhao, K and Wu, T},
title = {CRISPR/Cas12a-Enhanced Cascade Amplification for Ultra-sensitive DNA Ligase Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {27},
pages = {20113-20121},
doi = {10.1021/acs.analchem.6c00404},
pmid = {42366688},
issn = {1520-6882},
mesh = {*DNA Ligases/analysis/metabolism ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism ; Limit of Detection ; Humans ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism ; },
abstract = {DNA ligases are essential enzymes for maintaining genomic integrity, serving as critical biomarkers for the early diagnosis of various malignancies. However, current detection paradigms are often hindered by laborious workflows, high costs associated with chemical modifications, and insufficient sensitivity for low-abundance targets. In this study, we developed an integrated, label-free detection system where DNA ligase serves as a molecular gatekeeper to initiate CRISPR/Cas12a activity. This strategy exploits the discovery that nicked activators exhibit significantly attenuated affinity for the Cas12a-crRNA ribonucleoprotein complex, whereas ligase-mediated repair restores backbone continuity to create a high-affinity intact activator. Upon this ligation-gated activation, the system triggers a subsequent circular DNA-mediated autocatalytic cascade, exponentially amplifying the initial enzymatic signal. Through this dual-stage amplification, we achieved an ultimate limit of detection (LOD) of 2.59 × 10[-6] U/mL. Notably, the platform can reach the analytical sensitivity of established methods in as little as 30 min (LOD of 6.12 × 10[-5] U/mL), significantly compressing the diagnostic time frame. The system demonstrates high selectivity against diverse physiological interferents and has been successfully validated for quantifying endogenous DNA ligase in MC38 tumor cell extracts. This innovative ligase-gated CRISPR cascade provides a modular and robust framework for rapid clinical diagnostics and advanced enzymology research.},
}

*DNA Ligases/analysis/metabolism
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism
Limit of Detection
Humans
*Endodeoxyribonucleases/metabolism
*Bacterial Proteins/metabolism

@article {pmid42381600,
year = {2026},
author = {Dong, P and Gao, Y and Zhao, W and Fan, L and Wang, Y},
title = {A real-time microfluidic surveillance system for multiplex detection of heavy metal contamination in wastewater.},
journal = {Lab on a chip},
volume = {26},
number = {14},
pages = {4229-4234},
doi = {10.1039/d6lc00331a},
pmid = {42381600},
issn = {1473-0189},
mesh = {*Wastewater/chemistry/analysis ; *Metals, Heavy/analysis ; *Water Pollutants, Chemical/analysis ; *Microfluidic Analytical Techniques/instrumentation ; *Lab-On-A-Chip Devices ; Limit of Detection ; CRISPR-Cas Systems ; },
abstract = {Water pollution, particularly from heavy metals, poses a critical threat to ecosystems and human health. This study integrates the CRISPR-Cas12a system with MOF-based bio-barcode technology to create a platform for the rapid, real-time and on-site detection of multiple heavy metal ions, demonstrating exceptional sensitivity and selectivity. The detection limits for Cu[2+], Pb[2+], and Hg[2+] are 0.26 nM, 0.06 nM, and 0.80 nM, respectively. Inductively coupled plasma-mass spectrometry analysis of real water samples confirmed the high accuracy and reliability of this method. Furthermore, a mobile phone-assisted portable device paired with a microfluidic chip facilitates real-time, rapid multi-channel metal ion detection in resource-limited settings.},
}

*Wastewater/chemistry/analysis
*Metals, Heavy/analysis
*Water Pollutants, Chemical/analysis
*Microfluidic Analytical Techniques/instrumentation
*Lab-On-A-Chip Devices
Limit of Detection
CRISPR-Cas Systems

@article {pmid42393911,
year = {2026},
author = {Zhu, L and Liao, L and Huang, Y and Nie, Z and Lei, C},
title = {Label-Free Electrochemical CRISPR Platform Gated by Allosteric Transcription Factors for Ultrasensitive Small-Molecule Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {27},
pages = {20617-20627},
doi = {10.1021/acs.analchem.6c02971},
pmid = {42393911},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Transcription Factors/metabolism/chemistry/genetics ; Allosteric Regulation ; *Tetracycline/analysis ; Animals ; Copper/analysis ; Milk/chemistry ; *CRISPR-Cas Systems ; G-Quadruplexes ; DNA Probes/chemistry ; Parabens/analysis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Limit of Detection ; },
abstract = {The highly sensitive analysis of small-molecule targets holds profound significance across diverse fields, ranging from clinical diagnosis and environmental monitoring to food safety. Herein, we developed a label-free electrochemical CRISPR platform gated by allosteric transcription factors (aTFs) for the ultrasensitive detection of various small molecules. In this system, the specific binding of target analytes to their cognate aTFs induces the release of programmable DNA adaptors, which subsequently trigger Cas12a to trans-cleave DNA probes anchored to the electrode surface. Consequently, the truncated DNA probes serve as initiators to form electroactive G-quadruplex/hemin complexes in situ via terminal deoxynucleotidyl transferase (TdT)-mediated elongation, generating a robust electrochemical response signal. Using TetR as a model aTF, this integrated electrochemical CRISPR biosensor achieved tetracycline detection with picomolar sensitivity. Furthermore, the versatility of this platform was demonstrated by extending its application to p-hydroxybenzoic acid and copper ions through the simple substitution of the aTF modules. The practical utility of the assay was further demonstrated by the robust detection of tetracycline in complex matrices such as milk. Ultimately, this study not only provides a novel strategy for constructing universal, label-free electrochemical CRISPR platforms but also paves the way for the sensitive detection of low-abundance non-nucleic acid targets.},
}

*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*Transcription Factors/metabolism/chemistry/genetics
Allosteric Regulation
*Tetracycline/analysis
Animals
Copper/analysis
Milk/chemistry
*CRISPR-Cas Systems
G-Quadruplexes
DNA Probes/chemistry
Parabens/analysis
*Clustered Regularly Interspaced Short Palindromic Repeats
Limit of Detection

@article {pmid42397942,
year = {2026},
author = {Tong, Z and Huang, Z and Liu, J and Su, J and Zhu, R and Xiong, R and Yang, Y and Xie, W and Xiao, R},
title = {Heterojunction-Enhanced Interfacial Evanescent-Tunable Fiber Optic Probe for Amplification-free CRISPR/Cas12a-Based Rapid and Ultrasensitive Detection of MPXV.},
journal = {Analytical chemistry},
volume = {98},
number = {27},
pages = {20429-20441},
doi = {10.1021/acs.analchem.6c02129},
pmid = {42397942},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Gold/chemistry ; *Fiber Optic Technology ; Zinc Oxide/chemistry ; *Optical Fibers ; Metal Nanoparticles/chemistry ; Surface Plasmon Resonance ; Limit of Detection ; },
abstract = {Conventional polymerase chain reaction (PCR)-based detection methods suffer from time-consuming procedures, reliance on specialized equipment, and difficulty in achieving early viral diagnosis. In this study, interferometric fiber-optic sensing is integrated with the CRISPR/Cas12a system for the first time. With sensitivity further enhanced by immobilizing ZnO@Au on the fiber surface, the platform enables rapid, amplification-free detection of monkeypox virus (MPXV) at the single-molecule level. Whispering-gallery modes (WGMs) excited in the fiber probe provide high sensitivity to ambient refractive-index changes, while the ZnO@Au layer induces localized surface plasmon resonance (LSPR) and coupled plasmon-waveguide resonance (CPWR) on the fiber surface. By controlling the AuNPs occupancy on ZnO, the LSPR and CPWR absorption peaks can be tuned to match the demodulation spectral band. Moreover, the ZnO-Au heterojunction further strengthens the LSPR, thereby improving the sensitivity of the fiber probe. The resulting sensing probe achieves amplification-free detection of plasmid targets from both MPXV subtypes down to 10° copies/μL, with the entire assay completed within 9 min. The detection capability was validated using real clinical MPXV samples, showing complete agreement with qPCR results. The strategy proposed in this work offers a feasible approach for early and rapid viral detection.},
}

*CRISPR-Cas Systems/genetics
Gold/chemistry
*Fiber Optic Technology
Zinc Oxide/chemistry
*Optical Fibers
Metal Nanoparticles/chemistry
Surface Plasmon Resonance
Limit of Detection

@article {pmid42420723,
year = {2026},
author = {Kim, G and Kim, HJ and Seo, SW},
title = {Construction of a Tl-CRISPRi Genetic Circuit in Bacteria for Translation-Level Gene Knockdown.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {47-57},
pmid = {42420723},
issn = {1940-6029},
mesh = {*Gene Knockdown Techniques/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Protein Biosynthesis ; *Bacteria/genetics ; Plasmids/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Regulatory Networks ; Genetic Engineering/methods ; },
abstract = {The Tl-CRISPRi system, which harnesses the specific RNA-binding activity of CRISPR-dCas13, has been recently developed for translation-level gene knockdown in bacteria. By introducing spacers complementary to the translation initiation region of the mRNA, dCas13 can be directed to block the ribosome and inhibit the translation of that mRNA. Here, we discuss how to construct the Tl-CRISPRi genetic circuit and implement this system for gene knockdown. This chapter describes how to design spacer sequences and install them into the guide RNA expression plasmid. Also, we describe how to mutate the handle of gRNA to achieve tunable knockdown of a target gene. By following the method described in this chapter, we anticipate that a precise and controllable knockdown of a target gene in bacterial cells can be performed in a programmable manner.},
}

*Gene Knockdown Techniques/methods
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
*Protein Biosynthesis
*Bacteria/genetics
Plasmids/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Regulatory Networks
Genetic Engineering/methods

@article {pmid42420724,
year = {2026},
author = {Santos-Moreno, J},
title = {Design of CRISPRi-Based Synthetic Gene Circuits in Bacteria.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {59-83},
pmid = {42420724},
issn = {1940-6029},
mesh = {*Gene Regulatory Networks ; *Synthetic Biology/methods ; *CRISPR-Cas Systems ; *Bacteria/genetics ; *Genes, Synthetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; Gene Expression Regulation, Bacterial ; Escherichia coli/genetics ; },
abstract = {Synthetic gene circuits are key elements of engineered biological systems that allow us to control and program cellular behavior. Yet, circuit design can be challenging to newcomers due to the numerous design choices and the abundance and variety of parameters that can influence circuit performance. While transcription factors have dominated the circuit construction toolbox for two decades, CRISPRi-based tools offer important benefits-especially for large circuits-but also require unique design considerations. Here I provide a detailed guide for designing CRISPRi circuits in bacteria, using the CRISPRlator, the first CRISPRi oscillator, as an example that illustrates the design process.},
}

*Gene Regulatory Networks
*Synthetic Biology/methods
*CRISPR-Cas Systems
*Bacteria/genetics
*Genes, Synthetic
*Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Engineering/methods
Gene Expression Regulation, Bacterial
Escherichia coli/genetics

@article {pmid42420725,
year = {2026},
author = {Park, D and Shin, W and Kang, H and Kim, J},
title = {Design of Conditional Guide RNAs for the Logical Regulation of Gene Expression.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {85-107},
pmid = {42420725},
issn = {1940-6029},
mesh = {Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Synthetic Biology/methods ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Bacterial ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The CRISPR interference (CRISPRi) is an RNA-guided regulator that silences gene expression by binding to its cognate DNA target, halting transcription in both prokaryotic and eukaryotic cells. Recent advances in RNA synthetic biology have endowed CRISPR guide RNAs (gRNAs) with conditional functionality: these so-called conditional guide RNAs (cgRNAs) fold into strong hairpins that block their activity until a specific trigger RNA is present. Upon introduction of the cognate trigger RNAs, the hairpin structure unfolds, allowing the activated cgRNA to direct transcriptional repression with large dynamic ranges, minimal crosstalk, expanded tunability, and logic-gated signal processing. Furthermore, cgRNAs can be integrated into endogenous gene circuits to achieve sophisticated and logical regulation of gene expression. This chapter describes the design of cgRNAs and provides detailed protocols for their in vivo characterization in E. coli.},
}

Escherichia coli/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
Synthetic Biology/methods
*CRISPR-Cas Systems
*Gene Expression Regulation, Bacterial
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42420731,
year = {2026},
author = {Pujar, A and Sharma, A and Kushwaha, M},
title = {Intercellular CRISPRi for Distributed Genetic Circuits.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {229-243},
pmid = {42420731},
issn = {1940-6029},
mesh = {*Gene Regulatory Networks ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacteria/genetics ; Synthetic Biology/methods ; Escherichia coli/genetics ; },
abstract = {Microbial communities and multicellular organisms employ diverse strategies for allocation of available resources, achieved through task distribution among specialized cells. Drawing inspiration from nature, several synthetic multicellular circuits have been recently constructed where a larger circuit is distributed into several cells in order to reduce the burden on individual cells. Here, we describe the implementation of multicellular logic-gate circuits in bacterial co-cultures that combine DNA messaging with CRISPRi regulation. Leveraging the easily programmable and information-dense DNA molecules, our system is composed of sender bacteria that transmit DNA messages encoding guide RNAs and receiver bacteria that receive them and express the guide RNAs to regulate transcription by CRISPR interference. We demonstrate several functional multicellular circuits representing digital logic gates that operate on timescales comparable to small molecule signaling: NOT, YES, AND, and AND-AND-NOT. The receiver cells process the inputs received to perform computations and generate a logical output.},
}

*Gene Regulatory Networks
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Bacteria/genetics
Synthetic Biology/methods
Escherichia coli/genetics

@article {pmid42340184,
year = {2026},
author = {Kawai-Harada, Y and You, S and Scarborough, T and Siraj, N and Yedla, J and Rennells, T and Walton, SP and Chan, C and Harada, M},
title = {Generation of Cellular Biofactories for the Scalable Production of Surface-Engineered Extracellular Vesicles via CRISPR Genome Editing.},
journal = {ACS biomaterials science & engineering},
volume = {12},
number = {7},
pages = {3821-3831},
doi = {10.1021/acsbiomaterials.6c00782},
pmid = {42340184},
issn = {2373-9878},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/metabolism/genetics/chemistry ; HEK293 Cells ; },
abstract = {Extracellular vesicles (EVs) are versatile biological nanoparticles with applications in therapeutics, diagnostics, and biotechnology. Current production methods relying on transient transfection or chemical conjugation suffer from high variability, limited scalability, and heterogeneous EV populations. Here, we present a synthetic-biology-based biomaterial manufacturing platform that uses CRISPR-Cas9 genome editing to generate stable HEK293T cell lines for continuous production of surface-functionalized EVs. A fusion construct encoding mCherry-C1C2 was site-specifically integrated into the AAVS1 safe-harbor locus, enabling consistent and heritable expression of EV membrane proteins without repeated transfection. Engineered cells produced EVs with uniform size (120-130 nm), preserved canonical markers (CD63 and ALIX), and enhanced surface-display efficiency compared with transiently transfected controls. These vesicles exhibited robust cellular uptake and maintained structural and functional stability for over 25 passages (∼3 months), confirming durable genome-encoded production. Overall, this platform eliminates batch-to-batch variability inherent to transient systems and provides a genetically defined route to biofunctional nanomaterial fabrication. This approach links genetic design to nanoscale surface functionality, establishing a versatile foundation for reproducible biomanufacturing of engineered EVs for biomaterial, therapeutic, and diagnostic applications.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Extracellular Vesicles/metabolism/genetics/chemistry
HEK293 Cells

@article {pmid42416053,
year = {2026},
author = {Xia, C and Lian, M and Ma, B and Yu, H and Zhang, R and Wen, L and Wang, X and Zhao, Y and Ouyang, Z and Ye, Y and Feng, X and Wu, H and Lai, L},
title = {Development of a BM7G(TKO/hCD46/hCD55/hTHBD/hEPCR) donor pig with endogenous promoter-driven transgenes for xenotransplantation.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1827497},
pmid = {42416053},
issn = {1664-3224},
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Promoter Regions, Genetic ; Animals, Genetically Modified ; Humans ; Swine ; Galactosyltransferases/genetics ; *Transgenes ; *Thrombomodulin/genetics ; *Membrane Cofactor Protein/genetics ; Graft Rejection/immunology/genetics/prevention & control ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Heterografts ; Mixed Function Oxygenases/genetics ; Graft Survival ; N-Acetylgalactosaminyltransferases ; },
abstract = {INTRODUCTION: Xenotransplantation holds promise for addressing the organ shortage crisis. Multi-genetic modification of pigs, such as knockout of three carbohydrate antigen-related genes and expression of immunoprotective proteins, can significantly improve xenograft survival. However, existing multi-gene modification strategies face challenges: transposon-based transgenic technology may lead to unstable expression, while exogenous promoters used in site-specific integration strategies are susceptible to epigenetic silencing, making it difficult to maintain long-term, stable expression levels. Therefore, developing a donor pig model capable of achieving stable and long-lasting multi-gene expression is a critical need in the field.

METHODS: CRISPR-Cas9 technology was used to knockout three major glycan antigen genes (GGTA1, CMAH, β4GalNT2) to eliminate hyperacute rejection. Subsequently, four human protective genes (hCD55, hCD46, hTHBD, hEPCR) were site-specifically integrated into the porcine Rosa26 safe-harbor locus. Their expression was driven by the porcine endogenous Rosa26 promoter and the THBD core promoter, respectively, to ensure long-term stable and tissue-specific expression. Furthermore, the selection marker gene was efficiently removed using the Cre/loxP system.

RESULTS: The three glycan antigens were completely absent at both cellular and tissue levels in BM7G genetically modified pigs. What's more, four protective proteins were stably expressed in vascular endothelial cells and major organs such as the heart, liver, and kidneys. Among them, hCD55 and hCD46 were widely expressed, while hTHBD and hEPCR were specifically expressed in the vascular region. In-vitro functional assays confirmed that BM7G porcine vascular endothelial cells significantly reduced the binding of human antibodies, effectively inhibited complement-dependent cytotoxicity, and decreased the formation of thrombin-antithrombin (TAT) complexes.

CONCLUSION: In summary, by combining the knockout of xenoantigens with the use of endogenous promoters to drive the expression of multiple human protective genes, we successfully constructed a seven-gene modified pig model with low immunogenicity and synergistic protective functions. This provides an important donor resource for preclinical research in xenotransplantation.},
}

Animals
*Transplantation, Heterologous/methods
*Promoter Regions, Genetic
Animals, Genetically Modified
Humans
Swine
Galactosyltransferases/genetics
*Transgenes
*Thrombomodulin/genetics
*Membrane Cofactor Protein/genetics
Graft Rejection/immunology/genetics/prevention & control
CRISPR-Cas Systems
Gene Knockout Techniques
Heterografts
Mixed Function Oxygenases/genetics
Graft Survival
N-Acetylgalactosaminyltransferases

@article {pmid42416071,
year = {2026},
author = {Akula, S and Wernersson, S and Hellman, L},
title = {Immunity: defense against infections essential for all living organisms.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1840774},
pmid = {42416071},
issn = {1664-3224},
mesh = {Animals ; Humans ; *Adaptive Immunity ; Immunity, Innate ; Host-Pathogen Interactions/immunology ; },
abstract = {All organisms need protection against infection. Bacteria are often primarily seen as infectious agents, but they also need protection against bacterial viruses, so-called bacteriophages. To this end, bacteria have developed very complex defense systems, including apoptosis-like mechanisms, restriction enzymes, and even adaptive-type mechanisms involving immunological memory of immune responses through a system called CRISPR-Cas. An earlier dominating view was that adaptive immunity in eukaryotes only exists in jawed vertebrates, as their immune system includes the classical and highly variable immunoglobulins (Igs) and T-cell receptors (TCR). However, other types of variable molecules, which may be involved in immunity, have also been identified in insects, snails, lancelets, plants, sea urchins, and jawless fishes. Interestingly, fishes without jaws, such as the hagfish and lamprey, have a very complex adaptive immunity built on lymphocyte-like cells and variable lymphocyte receptors (VLRs). Notably, the variability of these VLRs has been estimated to be in the same range as Igs and T-cell receptors. This illustrates that very diverse strategies have been used to create an adaptive immune system in different organisms, indicating potent convergent evolution. Vertebrate immunity includes both adaptive and non-adaptive components, which work closely together to form a very powerful immune system for defense against infections. In contrast to adaptive immunity, the majority of the non-adaptive innate defense mechanisms, such as pattern recognition receptors, antimicrobial peptides (AMPs), iron-binding proteins, the complement system, and lysozymes, can be traced back to early eukaryotes. Immunity of invertebrates seems to rely almost entirely on innate defense mechanisms, while the presence of complex adaptive mechanisms in invertebrates, such as the VLRs of jawless fishes and Igs and TCR of jawed vertebrates, is questionable. This review summarizes old and recent findings of importance for our understanding of how immunity became an integrated part of all living organisms, from bacteria to humans, and the very different strategies that different organisms use in the protection against infection.},
}

Animals
Humans
*Adaptive Immunity
Immunity, Innate
Host-Pathogen Interactions/immunology

@article {pmid42416743,
year = {2026},
author = {Raj D, D and Maurya, AK and Singh, J and Kumar G, M and Ramani, A and Khurana, AK and Purwar, S and Biswas, D},
title = {Off-Target activity as a Translational Barrier in Programmable Gene-Editing Strategies for Nontuberculous Mycobacteria: Narrative Review.},
journal = {Maedica},
volume = {21},
number = {2},
pages = {495-503},
pmid = {42416743},
issn = {1841-9038},
abstract = {OBJECTIVES: To review the clinical and translational implications of off-target activity associated with clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches in nontuberculous mycobacteria (NTM) and discuss current strategies aimed at specificity and safety.

MATERIALS AND METHODS: The relevant published literature on the application of CRISPR-Cas systems, including Cas9, Cas12a and CRISPR interference (CRISPRi), in NTM research was reviewed. Particular attention was given to off-target mechanisms, mycobacteria-specific genomic challenges, computational predictions, experimental detection methods, high-fidelity nucleases and delivery optimisation approaches.

RESULTS: Nontuberculous mycobacteria infections often require prolonged treatment and are frequently associated with relapse and rising antimicrobial resistance, particularly in Mycobacterium abscessus infections. CRISPR-based technologies provide advantages in precision diagnostics, functional genomics and therapeutic development; however, high guanine-cytosine (GC) content, repetitive PE/PPE gene families, mismatch tolerance and unique DNA repair mechanisms contribute considerably to off-target effects. Emerging high-fidelity nucleases, guide RNA optimisation, artificial intelligence (AI)-assisted prediction platforms and alternative editing systems demonstrate considerable potential for improving editing specificity and translational safety.

CONCLUSIONS: Advances in nuclease engineering, computational modelling, delivery systems, and genome-wide validation approaches may improve therapeutic precision and diagnostic reliability. Addressing these challenges through interdisciplinary innovation will be essential for the future clinical integration of CRISPR-based antimycobacterial strategies.},
}

@article {pmid42417038,
year = {2026},
author = {Abdullah, N and Lewis, J and Arumugam, P},
title = {Genome-wide CRISPR/Cas9 screening reveals lipid metabolism and inflammatory signalling as modulators of ganoderic acid DM cytotoxicity.},
journal = {Journal of genetics},
volume = {105},
number = {},
pages = {},
pmid = {42417038},
issn = {0973-7731},
mesh = {Humans ; *Triterpenes/pharmacology ; *Lipid Metabolism/drug effects/genetics ; Signal Transduction/drug effects ; *CRISPR-Cas Systems/genetics ; *Inflammation/genetics/metabolism ; Cell Line, Tumor ; Sterol Regulatory Element Binding Proteins/genetics/metabolism ; *Melanoma/genetics/drug therapy/pathology/metabolism ; NF-kappa B/genetics/metabolism ; Cholesterol/biosynthesis ; },
abstract = {Ganoderic acid DM (GA-DM), a triterpenoid derived from Ganoderma lucidum, exhibits anti-cancer and anti-diabetic activities, but the underlying mechanisms of action remain unclear. To identify genetic modulators of the GA-DM response, we conducted a genome-wide CRISPR/Cas9 knockout screen in human melanoma cells. The screen revealed key roles for genes regulating lipid metabolism and inflammatory signalling, particularly those involved in the SREBP (sterol regulatory element-binding protein) and NF-jB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathways, in the cellular response to GA-DM. While loss of genes involved in the regulation of cholesterol biosynthesis conferred resistance to GA-DM, disruption of genes involved in ubiquitin-mediated proteolysis and the Hippo pathway sensitised cells to GA-DM. Inflammatory genes enriched at later time points suggests that a delayed cellular response contributes to cytotoxicity. Our findings propose a mechanistic model wherein GA-DM perturbs lipid and inflammatory pathways to exert cytotoxic effects and highlight potential targets to enhance its therapeutic efficacy. This work demonstrates the utility of functional genomics in elucidating the mechanisms of action of natural products and guiding rational drug development.},
}

Humans
*Triterpenes/pharmacology
*Lipid Metabolism/drug effects/genetics
Signal Transduction/drug effects
*CRISPR-Cas Systems/genetics
*Inflammation/genetics/metabolism
Cell Line, Tumor
Sterol Regulatory Element Binding Proteins/genetics/metabolism
*Melanoma/genetics/drug therapy/pathology/metabolism
NF-kappa B/genetics/metabolism
Cholesterol/biosynthesis

@article {pmid42417866,
year = {2026},
author = {Tang, Y and Zhang, L and Wang, W and Yang, X},
title = {An integrated signal amplification strategy based on catalytic hairpin assembly and hybridization chain reaction for driving a CRISPR/Cas12a biosensor toward ultrasensitive detection of microRNAs.},
journal = {Mikrochimica acta},
volume = {193},
number = {8},
pages = {},
pmid = {42417866},
issn = {1436-5073},
mesh = {*MicroRNAs/blood/genetics/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Hybridization ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Humans ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; DNA Probes/chemistry/genetics ; },
abstract = {A novel biosensing platform is proposed that integrates catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) cascade isothermal amplification with the CRISPR/Cas12a system, enabling ultrasensitive detection of microRNAs (miRNAs) targets. Within this platform, two modules are integrated: a target recognition and signal amplification module constructed by the cascade of CHA and HCR, and a signal transduction module in which the CRISPR/Cas12a system acts in concert with DNA probes loaded onto gold nanoparticles (AuNPs). This design achieves cascaded amplification from target recognition to signal output, thereby conferring high signal gain. Experimental results demonstrate that the proposed biosensor had high sensitivity toward the target miRNA, with a detection limit as low as 37 fM. Moreover, it shows remarkable single-base discrimination capability, effectively distinguishing sequences with single-nucleotide mismatches. Notably, the sensor maintains stable and reliable performance in complex biological matrices, including serum samples and lysates from various tumor cells. This strategy effectively couples signal amplification with the CRISPR system, achieving both high sensitivity and specificity, making it a useful tool for miRNA detection and early cancer screening.},
}

*MicroRNAs/blood/genetics/analysis
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Nucleic Acid Hybridization
Metal Nanoparticles/chemistry
Gold/chemistry
Humans
Limit of Detection
*Nucleic Acid Amplification Techniques/methods
DNA Probes/chemistry/genetics

@article {pmid42420722,
year = {2026},
author = {Irvine, TCT and Bailey, AM and Gorochowski, TE},
title = {A Golden Gate-Compatible CRISPR-Associated Transposon Tool for Multiplexed Bacterial Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3041},
number = {},
pages = {33-45},
pmid = {42420722},
issn = {1940-6029},
mesh = {*Genome, Bacterial ; Escherichia coli/genetics ; *Gene Editing/methods ; *DNA Transposable Elements/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Synthetic Biology/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The insertion of large genetic circuits and metabolic pathways into bacterial genomes is becoming increasingly common within the field of synthetic biology due to the improved robustness and stability that come with genome integration. CRISPR-associated transposases (CASTs) enable RNA-guided DNA insertion without introducing double-stranded breaks and have been shown to function across diverse bacterial species. Here, we present an improved tool called pSPIN-GG and supporting protocols for simplified CAST-based genome engineering. The pSPIN-GG system includes Golden Gate-compatible promoter, guide, and cargo modules for simple assembly, a green fluorescent protein dropout cassette for rapid verification of guide replacement, and a set of tested sites within the Escherichia coli BL21 chromosome to enable gene dosing of genetic cargoes. These refinements support accelerated library construction, reduce assembly and screening burden, and expand the accessibility of CAST systems for multiplexed bacterial genome engineering.},
}

*Genome, Bacterial
Escherichia coli/genetics
*Gene Editing/methods
*DNA Transposable Elements/genetics
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Synthetic Biology/methods
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42415313,
year = {2026},
author = {Solanki, M and Yousuf, F and Srivastava, A and Vaikuntapu, PR and Molla, K and Neeraja, CN and Thakur, V and Barbadikar, KM and Sundaram, RM and Monhannath, G and Mangrauthia, SK},
title = {Powering Genome Editing in Rice by Harnessing Promising Gene Resources: A Comprehensive Roadmap.},
journal = {Physiologia plantarum},
volume = {178},
number = {4},
pages = {e71005},
doi = {10.1111/ppl.71005},
pmid = {42415313},
issn = {1399-3054},
support = {NASF/CRISPR-Cas-7003/2017-18//Indian Council of Agricultural Research/ ; NASF/BGAM-9021/2022-23//Indian Council of Agricultural Research/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; Plant Breeding/methods ; *Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {The imprecise breeding methods including recombination breeding, physical/chemical mutagenesis, and marker-assisted breeding have been extensively utilized for trait improvement of rice crop. Despite tremendous progress made through these breeding methods, the critical issues, such as linkage drag, unintended phenotype, and longer duration of time required to breed a cultivar, have been the major limitations. Among the new breeding technologies, genome editing (GE) has become the most promising approach because of its specificity, precision, and speed. Despite its transformative potential, genome editing continues to face several limitations in crop improvement. These include well-recognized policy challenges, such as biosafety regulations and intellectual property constraints, alongside technical barriers like inefficient tissue culture and transformation systems. Additionally, researchers remain constrained by the limited availability of precise gene information necessary for accurate targeted editing and effective trait enhancement. This review presents an analysis of genes that regulate abiotic and biotic stresses, yield, grain quality and nutrition, plant architecture, nutrient absorption and use efficiency, and other agronomically important traits of rice. The trait-wise probable target genes for genome editing have been discussed in detail. This review will serve as a ready reckoner for rice researchers and funding agencies.},
}

*Oryza/genetics
*Gene Editing/methods
Plant Breeding/methods
*Genome, Plant/genetics
Plants, Genetically Modified/genetics

@article {pmid41115768,
year = {2026},
author = {Jordan, AJ and Balmforth, C and Craig, N and Dhaun, N and Baker, A and Dweck, MR and Newby, DE},
title = {Ribonucleic acid and gene therapies in cardiovascular disease: clinical applications, delivery challenges and emerging precision tools.},
journal = {Heart (British Cardiac Society)},
volume = {112},
number = {15},
pages = {828-837},
doi = {10.1136/heartjnl-2024-325280},
pmid = {41115768},
issn = {1468-201X},
mesh = {Humans ; *Cardiovascular Diseases/therapy/genetics ; *Genetic Therapy/methods ; *RNA, Small Interfering/therapeutic use ; *Precision Medicine/methods ; Gene Therapy Agents ; Gene Editing/methods ; *RNAi Therapeutics/methods ; Gene Transfer Techniques ; CRISPR-Cas Systems ; },
abstract = {Cardiovascular diseases remain a leading cause of global mortality despite advancements in pharmacotherapies, with current treatments facing challenges related to efficacy, tolerability and patient adherence. In response, advanced therapies, such as RNA and gene therapies, have emerged as a promising alternative for addressing both acquired and monogenic cardiovascular conditions. This review explores the current landscape of RNA and gene therapies for cardiovascular disease, focusing on RNA-based therapeutics such as small-interfering RNAs (siRNAs), antisense oligonucleotides and clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9)-based gene editing systems. Recent European Medicines Agency and Food and Drug Administration-approved RNA therapies, including patisiran, vutrisiran and inclisiran, which employ lipid nanoparticle delivery systems, highlight the clinical potential of siRNAs for targeting hepatic molecular pathways. Emerging CRISPR-Cas9 technologies are poised to address genetic mutations at their source, offering permanent correction of pathogenic variants and the potential to treat a broad range of hereditary cardiovascular conditions. Together, these therapies represent a major leap forward in precision medicine, offering long-lasting therapeutic effects and improved patient care and adherence. However, many challenges remain, particularly in targeting such therapies to cardiac tissues and optimising delivery systems. This review discusses the current state of the art in cardiovascular RNA and gene therapies, including current evidence, delivery challenges and the current landscape of gene and RNA therapies in phase I clinical trials and beyond.},
}

Humans
*Cardiovascular Diseases/therapy/genetics
*Genetic Therapy/methods
*RNA, Small Interfering/therapeutic use
*Precision Medicine/methods
Gene Therapy Agents
Gene Editing/methods
*RNAi Therapeutics/methods
Gene Transfer Techniques
CRISPR-Cas Systems

@article {pmid42224782,
year = {2026},
author = {Dong, Z and Liu, Y and Wu, X and Ban, W and Zhao, L and Liu, X and Ding, J},
title = {Rapid multiplex detection of Echinococcus granulosus and Echinococcus multilocularis using a one-pot RPA-assisted CRISPR-Cas12a/Cas13a assay in a portable multi-tube device.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118857},
doi = {10.1016/j.bios.2026.118857},
pmid = {42224782},
issn = {1873-4235},
mesh = {Animals ; *Echinococcus multilocularis/isolation & purification/genetics ; *Echinococcosis/parasitology/diagnosis ; *Biosensing Techniques/instrumentation ; CRISPR-Cas Systems ; *Echinococcus granulosus/isolation & purification/genetics ; Dogs ; Rapid Diagnostic Tests ; DNA, Helminth/genetics/isolation & purification ; Limit of Detection ; Humans ; Equipment Design ; },
abstract = {Echinococcosis, caused by Echinococcus granulosus and Echinococcus multilocularis, remains a significant zoonotic threat, particularly in pastoral regions where rapid environmental surveillance is essential yet technically constrained. Here, we report a rapid and integrated one-pot recombinase polymerase amplification -assisted, orthogonal CRISPR-Cas12a/Cas13a platform for rapid and specific discrimination of these two species in environmental samples. Coupled with a simplified NaOH-based DNA extraction method, the assay enables a streamlined workflow completed within 60 min, achieving a detection limit of as low as 1 copy/μL without observable cross-reactivity. To facilitate point-of-care deployment, we further developed a low-cost, miniaturized handheld device capable of parallel analysis of up to eight samples with dual-target readout. The platform was validated using field samples, including canine feces, pasture grass, and vegetables, demonstrating complete agreement with quantitative PCR results, with 100% sensitivity and specificity. This integrated CRISPR-based biosensing system provides a robust and field-deployable solution for on-site echinococcosis surveillance and offers a scalable framework for multiplex environmental pathogen detection.},
}

Animals
*Echinococcus multilocularis/isolation & purification/genetics
*Echinococcosis/parasitology/diagnosis
*Biosensing Techniques/instrumentation
CRISPR-Cas Systems
*Echinococcus granulosus/isolation & purification/genetics
Dogs
Rapid Diagnostic Tests
DNA, Helminth/genetics/isolation & purification
Limit of Detection
Humans
Equipment Design

@article {pmid42247945,
year = {2026},
author = {Fan, Z and Yin, X and Ma, M and Du, B and Liu, Z and Xu, J and Liu, B and Tong, Z},
title = {Cas12a2-based multiplexed screen-printed electrode electrochemiluminescence biosensor for amplification-free SARS-CoV-2 detection in aerosols.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118885},
doi = {10.1016/j.bios.2026.118885},
pmid = {42247945},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation/methods ; *SARS-CoV-2/isolation & purification/genetics ; Humans ; Aerosols/analysis ; *COVID-19/diagnosis/virology ; Electrochemical Techniques/instrumentation/methods ; RNA, Viral/analysis/genetics ; Luminescent Measurements/instrumentation ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry ; Electrodes ; Limit of Detection ; *Endodeoxyribonucleases/chemistry ; Equipment Design ; Rapid Diagnostic Tests ; Bacterial Proteins ; },
abstract = {Airborne transmission of respiratory viruses poses a severe public health threat, urgently requiring portable and sensitive techniques for viral aerosol monitoring. CRISPR-Cas12 technology has brought extensive innovations to the field of nucleic acid detection. Among them, Cas12a2 exhibits unique RNA-triggered trans-cleavage activity, showing prominent advantages in the amplification-free detection of respiratory RNA viruses. Herein, we developed an amplification-free and electrode-modification-free electrochemiluminescence biosensing platform based on screen-printed electrodes by integrating the specific recognition capability of Cas12a2 and the synergistic activation effect of multiple crRNAs. The optimized Cas12a2-based system achieves ultrasensitive detection of SARS-CoV-2 RNA with a low limit of detection of 76 aM. Furthermore, we constructed a stable viral aerosol generation and collection device and successfully validated the practical capability of the proposed platform for SARS-CoV-2 aerosol detection. This rapid and portable detection strategy offers a promising alternative for on-site monitoring of airborne pathogens and further expands the application scope of CRISPR biosensing technology in viral detection.},
}

*Biosensing Techniques/instrumentation/methods
*SARS-CoV-2/isolation & purification/genetics
Humans
Aerosols/analysis
*COVID-19/diagnosis/virology
Electrochemical Techniques/instrumentation/methods
RNA, Viral/analysis/genetics
Luminescent Measurements/instrumentation
CRISPR-Cas Systems
*CRISPR-Associated Proteins/chemistry
Electrodes
Limit of Detection
*Endodeoxyribonucleases/chemistry
Equipment Design
Rapid Diagnostic Tests
Bacterial Proteins

@article {pmid42250350,
year = {2026},
author = {Wang, Q and Choi, S and Heo, W and Kim, MW and Park, S and Park, SJ and Shin, J and Hyun, KA and Kim, J and Lim, CS and Jung, HI},
title = {Electrochemical-sensor-assisted lab-in-a-cartridge (EC-LIC) for on-site detection of SARS-CoV-2 with a self-contained heating system.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118899},
doi = {10.1016/j.bios.2026.118899},
pmid = {42250350},
issn = {1873-4235},
mesh = {*SARS-CoV-2/isolation & purification/genetics ; Humans ; *COVID-19/diagnosis/virology ; *Biosensing Techniques/instrumentation ; *Electrochemical Techniques/instrumentation ; Rapid Diagnostic Tests ; Equipment Design ; Limit of Detection ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Heating/instrumentation ; COVID-19 Nucleic Acid Testing/instrumentation ; },
abstract = {Rapid and accurate detection of respiratory viruses is essential for controlling disease transmission and enabling effective public health responses, particularly in resource-limited settings. In this study, we present an electrochemical-sensor-assisted lab-in-a-cartridge (EC-LIC) platform for on-site detection of SARS-CoV-2 featuring a self-contained chemical heating system. The device incorporates rotational and gravity-driven fluid handling along with exothermic heating using calcium oxide and a flameless ration heater to generate controlled temperature gradients. Coupled with a CRISPR-Cas13a-based electrochemical sensor, the system enables direct detection of the SARS-CoV-2 N gene without nucleic acid amplification, achieving high sensitivity and specificity. Integrated with a handheld electrochemical reader, the EC-LIC operates as a fully automated sample-to-answer system, completing the assay within 40 min over a wide dynamic range from 1.0 × 10° to 1.0 × 10[5] fg/mL with a limit of detection as low as 1.21 × 10[-1] fg/mL. Clinical validation using samples from 102 individuals (60 positive and 42 negative) demonstrated a sensitivity of 98% and a specificity of 90%. These results establish the EC-LIC as a robust nucleic acid detection platform for rapid clinical screening and early epidemic response.},
}

*SARS-CoV-2/isolation & purification/genetics
Humans
*COVID-19/diagnosis/virology
*Biosensing Techniques/instrumentation
*Electrochemical Techniques/instrumentation
Rapid Diagnostic Tests
Equipment Design
Limit of Detection
CRISPR-Cas Systems
Sensitivity and Specificity
Heating/instrumentation
COVID-19 Nucleic Acid Testing/instrumentation

@article {pmid42269453,
year = {2026},
author = {Ren, K and Yu, C and Wu, L and Ma, J and Xu, X and Jia, L and Yang, Q},
title = {Construction of an amplification-free dual-mode sensor based on CRISPR/Cas12a-mediated and dual-mode integrated reporter FU for ultrasensitive detection of non-nucleic acid target deoxynivalenol.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118879},
doi = {10.1016/j.bios.2026.118879},
pmid = {42269453},
issn = {1873-4235},
mesh = {*Trichothecenes/analysis/isolation & purification/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Colorimetry/methods ; Gold/chemistry ; DNA, Single-Stranded/chemistry ; },
abstract = {Sensitive and accurate detection of deoxynivalenol (DON) is crucial for public health. The CRISPR/Cas12a system exhibits high specificity and efficiency in biosensing, but challenges remain in non-nucleic acid detection, such as reliance on multiple reporters for dual-mode signal output and low detection sensitivity without amplification. In this study, leveraging the magnetic and fluorescence quenching properties of Fe3O4/Au/PDA and the fluorescence/catalytic capabilities of UiO-66-NH2, we developed a multimodal integrated reporter (FU) as Fe3O4/Au/PDA-ssDNA-UiO-66-NH2, enabling dual-mode signal output via a single reporter. In the presence of DON, the DON-Ab-aDNA complex activates CRISPR/Cas12a, which then indiscriminately cleaves the single-stranded DNA in FU, releasing free UiO-66-NH2. Consequently, the fluorescence signal of UiO-66-NH2 is restored while it catalyzes TMB to produce a blue color reaction. The CRISPR/Cas12a-based fluorescence-colorimetric dual-mode biosensor (CrisprFU) achieved a colorimetric limit of detection (LOD) for DON of 2.15 × 10[-3] ng/mL (detection range: 2-100 ng/mL) and a fluorescence LOD of 7.96 × 10[-4] ng/mL (detection range: 0.5-40 ng/mL). Successful application in real samples demonstrated average recovery rates of 97.04%-104.4% for fluorescence detection and 96.4%-101.8% for colorimetric detection, confirming its practical potential. Furthermore, by replacing the recognition antibody, the CrisprFU system can be extended to detect other analytes.},
}

*Trichothecenes/analysis/isolation & purification/chemistry
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Limit of Detection
Colorimetry/methods
Gold/chemistry
DNA, Single-Stranded/chemistry

@article {pmid42269938,
year = {2026},
author = {Wei, E and Tang, Y and Lei, Y and Liu, S and Xu, B and Du, B and Wang, Y and Liao, Y and Wang, Y and Zhao, J},
title = {Development and application of a fast and efficient CRISPR/Cas12f -based genetic toolkit in Bacillus cereus GW-01.},
journal = {Journal of microbiological methods},
volume = {247},
number = {},
pages = {107584},
doi = {10.1016/j.mimet.2026.107584},
pmid = {42269938},
issn = {1872-8359},
mesh = {*Bacillus cereus/genetics/metabolism ; *CRISPR-Cas Systems ; Pyrethrins/metabolism ; *Gene Editing/methods ; Gene Knockout Techniques ; Bacterial Proteins/genetics ; Probiotics ; Gene Deletion ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Bacillus cereus GW-01, an efficient degrader of β-cypermethrin (β-CY), has a high safety profile and probiotic potential for regulating intestinal flora and fermented foods, which is difficult to genetically engineer for modification due to its restrictive modification system. This study successfully developed a CRISPR/enCas12f-based genome editing system, first selecting the plcR gene for proof-of-concept validation with 100% knockout efficiency. Subsequently, this system was utilized to delete the virulence gene nheABC in GW-01, yielding a safer probiotic strain. Compared with the wild-type strain GW-01, the probiotic-related indicators of the ΔnheABC mutant, including cell surface hydrophobicity, auto-aggregation ability and biofilm formation ability, were 80%, 90% and 2.9 (OD595), respectively. There were no significant differences in these indicators between the mutant and the wild type. Meanwhile, the ΔnheABC mutant still maintained a high β-cypermethrin degradation efficiency of 80% at the concentration of 30 μg/mL. This work facilitates functional genomic research and genetic modification of Bacillus cereus GW-01. The established CRISPR/enCas12f system enables targeted gene deletion to explore gene functions and phenotypic mechanisms, and paves the way for its development into safe probiotics and excellent microbial chassis.},
}

*Bacillus cereus/genetics/metabolism
*CRISPR-Cas Systems
Pyrethrins/metabolism
*Gene Editing/methods
Gene Knockout Techniques
Bacterial Proteins/genetics
Probiotics
Gene Deletion
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42308857,
year = {2026},
author = {Li, L and Guo, X and Yang, X and Qin, S and Wang, W},
title = {High-sensitivity and portable detection of oral pathogens based on CRISPR/Cas13a combined with exonuclease-assisted cycling amplification and lateral flow assay.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118935},
doi = {10.1016/j.bios.2026.118935},
pmid = {42308857},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Ribosomal, 16S/genetics/isolation & purification ; Rapid Diagnostic Tests ; *Bacteria/isolation & purification/genetics/pathogenicity ; Limit of Detection ; Exonucleases/chemistry ; *Mouth/microbiology ; },
abstract = {Infectious diseases caused by oral pathogens represent a significant threat to human health. Current diagnostic technologies for oral pathogens lack the characteristics of speed, sensitivity, and convenience, making it difficult to meet the needs of rapid testing in laboratories and on-site. Consequently, the development of novel high-sensitivity and high-specificity pathogen analysis methods and sensing systems is imperative. In this study, we established a high-throughput CRISPR/Cas13a method for identifying pathogenic bacteria 16S rRNA, which we combined with isothermal enzyme cycling amplification technology (CRIE) to improve sample detection resolution, sensitivity, and speed. Furthermore, based on the characteristics of dopamine catalyzed by G4/hemin to form polydopamine and combined with CRIE, we developed lateral flow assay (CRIEC) for simple, portable, and rapid detection of pathogenic bacteria. Preliminary experiments were performed to verify its analytical performance and application potential. The obtained data may lay a basic foundation for the subsequent research and clinical application in the field of oral pathogen detection.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*RNA, Ribosomal, 16S/genetics/isolation & purification
Rapid Diagnostic Tests
*Bacteria/isolation & purification/genetics/pathogenicity
Limit of Detection
Exonucleases/chemistry
*Mouth/microbiology

@article {pmid42323937,
year = {2026},
author = {Zhang, Y and Li, L and Li, Y and Zeng, Y and Liu, H and He, M},
title = {Functionalized carbon nanotube-assisted dual-mode CRISPR/Cas12a detection of hepatitis C virus via catalytic assembly circuit-driven Y-shaped dsDNA activators.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118946},
doi = {10.1016/j.bios.2026.118946},
pmid = {42323937},
issn = {1873-4235},
mesh = {*Nanotubes, Carbon/chemistry ; *Hepacivirus/isolation & purification/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Hepatitis C/virology/diagnosis ; *RNA, Viral/genetics/isolation & purification/analysis ; Colorimetry/methods ; Limit of Detection ; Hydrogen Peroxide/chemistry ; Catalysis ; DNA/chemistry ; Endodeoxyribonucleases/chemistry ; CRISPR-Associated Proteins/chemistry ; },
abstract = {Hepatitis C virus (HCV) is a major etiological agent of liver diseases and remains a serious global health threat. Herein, we report a dual-modal HCV biosensing platform for ultrasensitive HCV RNA detection by integrating near-infrared fluorescence and colorimetric readouts. In this system, the presence of HCV RNA initiates a catalytic assembly circuit (CAC) that forms a Y-shaped DNA structure, exposing two double-stranded DNA activators with complete protospacer adjacent motif (PAM) to trigger CRISPR/Cas12a nuclease activity. The combination of CRISPR/Cas12a-driven signal amplification and hemin-binding aptamer-functionalized single-walled carbon nanotubes (HeApt-SWCNTs) enables highly sensitive target quantification. Upon exposure to hydrogen peroxide (H2O2), ferric ion in hemin catalyzes a Fenton-like reaction, generating hydroxyl radicals (·OH) that quench SWCNT fluorescence and oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to its blue oxidized form (oxTMB). In the presence of target HCV RNA, CRISPR/Cas12a-mediated HeApt cleavage inhibits ·OH generation, resulting in SWCNT fluorescence recovery and suppresses TMB oxidation. Under optimal conditions, detection limits of 0.23 fM and 4.1 fM are achieved for the fluorescence and colorimetric modes, respectively. This integrated CAC-Cas12a-HeApt-SWCNTs (CCHS) biosensing strategy offers high specificity, dual-mode signal reliability, and broad potential for early diagnosis of HCV and other RNA viruses.},
}

*Nanotubes, Carbon/chemistry
*Hepacivirus/isolation & purification/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*Hepatitis C/virology/diagnosis
*RNA, Viral/genetics/isolation & purification/analysis
Colorimetry/methods
Limit of Detection
Hydrogen Peroxide/chemistry
Catalysis
DNA/chemistry
Endodeoxyribonucleases/chemistry
CRISPR-Associated Proteins/chemistry

@article {pmid42330664,
year = {2026},
author = {Zhao, J and Xu, H and Fei, S and Xu, C and Yin, W and Wei, Q and Lin, J and Liu, G and Feng, S and Gao, F and Wang, Y},
title = {Smartphone-integrated RPA-CRISPR/Cas12a detection system with microneedle sampling for early point-of-care diagnosis of potato late blight.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118943},
doi = {10.1016/j.bios.2026.118943},
pmid = {42330664},
issn = {1873-4235},
mesh = {*Solanum tuberosum/microbiology/parasitology ; *Plant Diseases/microbiology/parasitology ; Smartphone ; *Phytophthora infestans/isolation & purification/genetics/pathogenicity ; *Biosensing Techniques/instrumentation ; Point-of-Care Systems ; CRISPR-Cas Systems ; Rapid Diagnostic Tests ; Nucleic Acid Amplification Techniques/instrumentation ; Plant Leaves/microbiology ; Equipment Design ; Needles ; },
abstract = {Potato late blight, caused by the oomycete pathogen Phytophthora infestans (P. infestans), is one of the most devastating diseases threatening global potato production. Conventional plant disease detection methods rely on a labor-intensive and time-consuming workflow and require bulky and expensive benchtop equipment, limiting their in-field applications. Here, we report a portable RPA-CRISPR/Cas12a-based diagnostic platform integrated with a polyvinyl alcohol (PVA) microneedle (MN) patch, which allows rapid in-field sampling, and smartphone-based fluorescence acquisition and analysis to detect P. infestans in potato at the early stage. The PVA MN enables leaf sampling rapidly within 1 min, and yields efficient DNA extraction of 56.3 ± 4.2 ng/mg, which is ∼3-fold higher than the traditional CTAB method (18.1 ± 2.1 ng/mg). The RPA-CRISPR/Cas12a isothermal assay achieved specific detection of P. infestans with no cross-reactivity against closely-related species Phytophthora sojae or Phytophthora capsici. The smartphone-based point-of-care test (POCT) system demonstrates a detection limit of 4 pg/μL for P. infestans genomic DNA, which is comparable to that acquired with commercial laboratory equipment. The method enables early-stage diagnosis of potato late blight as early as Day 2 post-inoculation, with detection rates of 37.5% on Day 2 and 75% on Day 3, prior to the development of visible symptoms on leaves. This portable "sample-to-result" platform provides a promising strategy for rapid, field-deployable early diagnosis and surveillance of plant disease.},
}

*Solanum tuberosum/microbiology/parasitology
*Plant Diseases/microbiology/parasitology
Smartphone
*Phytophthora infestans/isolation & purification/genetics/pathogenicity
*Biosensing Techniques/instrumentation
Point-of-Care Systems
CRISPR-Cas Systems
Rapid Diagnostic Tests
Nucleic Acid Amplification Techniques/instrumentation
Plant Leaves/microbiology
Equipment Design
Needles

@article {pmid42330669,
year = {2026},
author = {Zheng, X and Li, H and Yao, S and Wang, X and Wang, J and Yin, C and Wang, J and Zhao, C},
title = {Multiple DNA cycle amplification-assisted one-pot isothermal Cas12a for ultrasensitive nucleic acid detection.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118953},
doi = {10.1016/j.bios.2026.118953},
pmid = {42330669},
issn = {1873-4235},
mesh = {*SARS-CoV-2/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; Gold/chemistry ; *COVID-19/diagnosis/virology ; Metal Nanoparticles/chemistry ; Limit of Detection ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Proteins ; Colorimetry ; *COVID-19 Nucleic Acid Testing/methods ; Endodeoxyribonucleases/chemistry ; DNA Probes/chemistry ; DNA ; Bacterial Proteins ; Coronavirus Nucleocapsid Proteins ; Phosphoproteins ; Spike Glycoprotein, Coronavirus ; },
abstract = {Rapid and ultrasensitive nucleic acid detection is essential for environmental monitoring and biomedical diagnostics. Here, we report a modular one-pot isothermal platform that integrates catalytic hairpin assembly (CHA), rolling circle amplification (RCA), and CRISPR-Cas12a to construct a self-reinforcing multilayer DNA circuit (CRC). In this system, Cas12a cis-cleavage generates short DNA fragments that recursively activate CHA and RCA, forming a self-sustained cascade amplification loop, while trans-cleavage enables real-time fluorescence signal readout. Using this one-pot platform, ultralow detection limits of 62 aM and 58 aM were achieved for the SARS-CoV-2 S and N genes, respectively, with a total assay time ranging from 20 to 120 min depending on the required sensitivity. Furthermore, functionalizing single-stranded DNA probes on gold nanoparticles (AuNPs) allowed the cleaved DNA to restore fluorescence of fluorophore-quencher reporters, and freeze-thaw-induced AuNP aggregation produced visible colorimetric changes and measurable photothermal signals, enabling trimodal readout without sophisticated instruments. The system demonstrated effective discrimination in controlled experiments, indicating its potential suitability for point-of-care applications. This integrated, one-pot, and scalable platform provides a versatile strategy for fast, sensitive, and multimodal nucleic acid detection applicable to diverse targets.},
}

*SARS-CoV-2/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Humans
Gold/chemistry
*COVID-19/diagnosis/virology
Metal Nanoparticles/chemistry
Limit of Detection
CRISPR-Cas Systems/genetics
CRISPR-Associated Proteins
Colorimetry
*COVID-19 Nucleic Acid Testing/methods
Endodeoxyribonucleases/chemistry
DNA Probes/chemistry
DNA
Bacterial Proteins
Coronavirus Nucleocapsid Proteins
Phosphoproteins
Spike Glycoprotein, Coronavirus

@article {pmid42333535,
year = {2026},
author = {Labun, K and Rio, O and Dahal-Koirala, S and Komisarczuk, AZ and Valen, E and Haapaniemi, E},
title = {SNIPSNP: precision design of CRISPR/Cas9 knock-in reagents for variant correction and disease modeling.},
journal = {Nucleic acids research},
volume = {54},
number = {W1},
pages = {W145-W153},
doi = {10.1093/nar/gkag409},
pmid = {42333535},
issn = {1362-4962},
support = {331912//Norges Forskningsråd/ ; 190290//Kreftforeningen/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *Gene Knock-In Techniques/methods ; *Software ; Recombinational DNA Repair ; Computational Biology/methods ; Alleles ; RNA, Guide, CRISPR-Cas Systems/genetics ; INDEL Mutation ; },
abstract = {We present SNIPSNP (crisprtools.org/snipsnp), a comprehensive bioinformatics pipeline for designing experiments for CRISPR-induced homology-directed repair (HDR). The tool addresses the critical challenge of Cas9 re-cleavage by simplifying the selection of "blocking" silent variants that are effective at inhibiting RNP binding upon donor-templated editing. SNIPSNP handles complex edits, including indels, and uses multi-objective optimization to balance editing efficiency with biological safety. From user-defined wild-type and desired HDR alleles, the pipeline identifies candidate guides, annotating them with integrated efficiency scores and genome-wide off-target assessments. Uniquely, SNIPSNP evaluates guide binding against the post-edit genome to determine whether the therapeutic variant alone disrupts repeated Cas9 recognition. When necessary, it introduces synonymous blocking variants, prioritizing PAM and seed regions to minimize re-cleavage probability and editing of the wild-type (WT) allele when editing heterozygous variants. All candidate modifications undergo safety profiling and prioritization of known benign variants from dbSNP. We experimentally validated SNIPSNP and benchmarked it on pathogenic inborn error of immunity variants in primary patient T-cells. Across loci, SNIPSNP-designed templates outperform standard "correction-only" strategies, demonstrating enhanced precision editing, and reduced re-cleavage, establishing SNIPSNP as a robust platform for genome editing and disease modeling.},
}

*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
*Gene Knock-In Techniques/methods
*Software
Recombinational DNA Repair
Computational Biology/methods
Alleles
RNA, Guide, CRISPR-Cas Systems/genetics
INDEL Mutation

@article {pmid42342132,
year = {2026},
author = {Eskandani, NA and Mirzaee, D and Ramezani Farani, M and Hatami, A and Hatami, M and Ghasemzaei, M and Ghoreishian, SM and Hwang, SK and Huh, YS},
title = {Light-controlled CRISPR-dCas9 epigenome editing: advanced drug-delivery strategies and oncology applications.},
journal = {Advanced drug delivery reviews},
volume = {236},
number = {},
pages = {115921},
doi = {10.1016/j.addr.2026.115921},
pmid = {42342132},
issn = {1872-8294},
mesh = {Humans ; *Epigenome Editing/methods ; *Neoplasms/genetics/therapy/drug therapy ; *Drug Delivery Systems ; Animals ; *CRISPR-Cas Systems/genetics ; Light ; Optogenetics/methods ; Epigenesis, Genetic ; },
abstract = {Cancer is increasingly recognized as a disease of the dysregulated epigenome; however, current epi-drugs are blunt, systemically toxic instruments. Catalytically dead CRISPR nucleases (dCas9) linked to chromatin effectors have now made it possible not only to write and erase epigenetic marks at specified loci without double-strand breaks but also to add an element of optogenetics, or reversible and light-encoded control over the timing and localization of the editors. In this review, the technological underpinnings of light-controlled CRISPR-dCas9 epigenome editing, which include architectures of dCas9 scaffold and guide, blue-to-near-infrared photoswitches, and high-gain epigenetic effector designs, are synthesized, and viral, non-viral, and stimuli-responsive delivery platforms, which have to be co-optimized with clinical light interfaces, are discussed. We then outline four functional routes by which opto-epigenome editors may be used therapeutically in cancer: tumor suppressor reactivation; oncogene and super-enhancer repression with metabolic rewiring; control of cancer stem cell differentiation; and immunomodulation of the tumor microenvironment. Lastly, a translational roadmap is defined in terms of preclinical model tiers, biomarker strategies, regulatory and manufacturing factors, and future directions, including NIR and bioluminescent actuation, implantable μLED devices, and AI-guided closed-loop illumination. Together, these aspects constitute design principles for advancing light-addressable epigenome editors toward first-in-human studies and for integrating them into combination regimens as a new class of precision cancer therapeutics.},
}

Humans
*Epigenome Editing/methods
*Neoplasms/genetics/therapy/drug therapy
*Drug Delivery Systems
Animals
*CRISPR-Cas Systems/genetics
Light
Optogenetics/methods
Epigenesis, Genetic

@article {pmid42379043,
year = {2026},
author = {Zheng, Y and Tian, X and Wang, J and Zhang, Y and Liu, T and Huang, J and Guo, Y and Liang, S and Wang, C},
title = {CRISPR/Cas12a-based dual-modal signal platform using MIL-101(Fe) for colorimetric and electron spin resonance detection of HPV-16 nucleic acid.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118976},
doi = {10.1016/j.bios.2026.118976},
pmid = {42379043},
issn = {1873-4235},
mesh = {*Human papillomavirus 16/isolation & purification/genetics ; Humans ; Colorimetry/methods ; *CRISPR-Cas Systems/genetics ; Electron Spin Resonance Spectroscopy/methods ; *Biosensing Techniques/methods ; *DNA, Viral/genetics/isolation & purification/analysis ; *Papillomavirus Infections/virology/diagnosis ; *Metal-Organic Frameworks/chemistry ; Iron/chemistry ; Limit of Detection ; },
abstract = {Human papillomavirus (HPV) infection is a leading cause of cervical cancer and other malignancies, underscoring the urgent need for accurate and rapid early diagnostic strategies. Herein, we report a dual-mode colorimetric and electron spin resonance (ESR) method for the qualitative detection of HPV-16, based on the integration of the CRISPR/Cas12a system with a metal-organic framework (MOF). A peroxidase-mimicking iron-based MOF, designated MIL-101(Fe), was conjugated to magnetic beads via a single-stranded DNA linker to serve as a signal probe. Upon recognition of the target nucleic acid, MIL-101(Fe) catalyzes the decomposition of hydrogen peroxide to generate hydroxyl radicals, which oxidize a chromogenic substrate to produce a visible color change. Meanwhile, the generated radicals are captured by a spin trap and detected by ESR spectroscopy. The assay enables sensitive and rapid detection of HPV-16, with clear discrimination even in mixtures containing both HPV-16 and HPV-18. Importantly, when evaluated with clinical specimens, the method achieved 100% sensitivity and specificity. Overall, this work provides a feasible and promising strategy for ultrasensitive nucleic acid detection and offers a new avenue for advancing CRISPR-based multimodal diagnostic platforms toward practical applications.},
}

*Human papillomavirus 16/isolation & purification/genetics
Humans
Colorimetry/methods
*CRISPR-Cas Systems/genetics
Electron Spin Resonance Spectroscopy/methods
*Biosensing Techniques/methods
*DNA, Viral/genetics/isolation & purification/analysis
*Papillomavirus Infections/virology/diagnosis
*Metal-Organic Frameworks/chemistry
Iron/chemistry
Limit of Detection

@article {pmid42402242,
year = {2026},
author = {Wang, F and He, C and Lin, Y and Zhou, X},
title = {One-Tube RPA-CRISPR-Cas13a assay with rational design for single-molecule detection of waterborne viruses in drinking water treatment.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118983},
doi = {10.1016/j.bios.2026.118983},
pmid = {42402242},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; *Drinking Water/virology ; *Norovirus/isolation & purification/genetics ; *Rotavirus/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Water Purification ; Limit of Detection ; Water Microbiology ; Humans ; Rapid Diagnostic Tests ; },
abstract = {The global rise in waterborne viral infections has created an urgent need for portable, highly efficient environmental virus detection technologies. CRISPR-based nucleic acid detection coupled with isothermal amplification (e.g., Recombinase Polymerase Amplification, RPA) shows great promise for field applications. However, most reported designs fail to achieve the single-molecule sensitivity, which significantly limits their practical applications. To bridge the gap, we proposed a rational design strategy for the RPA primer and the CRISPR-Cas13a crRNA, suggesting that sensitivity can be enhanced by simplifying the secondary structure of the crRNA spacer region. Subsequently, we established a portable, one-tube CRISPR-Cas13a bioassay to detect two major waterborne viruses, achieving ultrasensitive detection limits of 5/8 aM for norovirus and 2/3 aM for rotavirus within 40 min. Thereafter, seasonal sampling across different treatment stages of a drinking water treatment plant was conducted, and water samples were analyzed using the one-tube CRISPR-Cas13a bioassay in comparison with qPCR and dPCR, revealing a positive detection rate of 15.79% (6/38) for the one-tube CRISPR-Cas13a bioassay, 18.42% (7/38) for qPCR, and 15.79% (6/38) for dPCR. The assay's modular design allows for broad applicability to other pathogens by simply modifying the target nucleic acid sequence, offering high sensitivity and specificity. This innovation paves the way for deployable point-of-care testing and large-scale spatiotemporal virus monitoring.},
}

*Biosensing Techniques
*CRISPR-Cas Systems/genetics
*Drinking Water/virology
*Norovirus/isolation & purification/genetics
*Rotavirus/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
Water Purification
Limit of Detection
Water Microbiology
Humans
Rapid Diagnostic Tests

@article {pmid42411270,
year = {2026},
author = {Baldenweck, L and Berg, N and Djisalov, M and Efremov, V and Novakovic, Z and Auger, S and Vidic, J},
title = {Isothermal amplification techniques for rapid bacterial detection: alternatives to culturing and PCR-based methods.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d6ay00756b},
pmid = {42411270},
issn = {1759-9679},
abstract = {Rapid identification of bacteria and their virulence factors is essential for global public health. Isothermal amplification has become a cornerstone of point-of-care diagnostics, enabling genetic testing to be faster, simpler, and more accessible than culturing or polymerase chain reaction (PCR). This review examines recent advances in some of the most commonly used isothermal amplification methods for bacterial detection: SDA, LAMP, HDA, RPA, RCA, and NASBA. The integration of isothermal amplification with the CRISPR/Cas system or microfluidic devices is also highlighted as an advanced gene detection technology. We present various readout methods used to detect gene amplification products or processes, including colorimetric, fluorescent, electrochemical, and quartz microbalance techniques. These integrated approaches can detect very small amounts of bacterial DNA, in under an hour, providing rapid, sensitive, versatile, and portable tools for health control.},
}

@article {pmid42411453,
year = {2026},
author = {Ono, Y and Peterka, M and Love, M and Khan, A and Bowers, F and Bhandari, A and Gordon, E and Ball, JS and Hammond, C and Tyler, CR and Rees, S and Bohlooly-Y, M and Maresca, M and Scholpp, S},
title = {Optimised genome editing for precise DNA insertion and substitution using prime editors in zebrafish.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {42411453},
issn = {2050-084X},
support = {BB/X008401/1//UK Research and Innovation/ ; BB/X001458/1//UK Research and Innovation/ ; DA 8438235/WT_/Wellcome Trust/United Kingdom ; NC/X001407/1//National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)/ ; 29317//Versus Arthritis Senior fellowship/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Genome ; *Mutagenesis, Insertional/methods ; *DNA/genetics ; Deoxyribonuclease I/metabolism ; *Gene Editing/methods ; },
abstract = {CRISPR/Cas9-mediated genome editing has rapidly become a popular tool for studying gene functions and generating genetically modified organisms. However, using this system, stochastic integration of random insertions and deletions restricts precise genome manipulation. Advanced CRISPR/Cas9 technologies using Prime Editors (PEs), Cas9 proteins fused with reverse transcriptase, enable programmed integration of short DNA modifications into the genome. However, its application in precise genome editing in animal models is challenging. Here, we utilise a nickase- and a nuclease-based PE to perform programmed short DNA substitutions and insertions at various loci in the zebrafish genome. Whereas nickase-based PE2 mediated a higher ratio of precise prime edits to the total edits, nuclease-based PEn was more efficient for short DNA modifications, achieving up to 27.3% precise insertion. To further evaluate our approach, we inserted a nuclear localisation signal into a reporter transgene to incorporate longer fragments by prime editing. These gene modifications were transmitted to the next generation. We show that PE-mediated prime editing can efficiently manipulate genome information in zebrafish without using exogenous donor DNA.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems
*Genome
*Mutagenesis, Insertional/methods
*DNA/genetics
Deoxyribonuclease I/metabolism
*Gene Editing/methods

@article {pmid42412324,
year = {2026},
author = {Adiga, U and Vasishta, S and Adiga, S and Augustine, AJ},
title = {Targeting the Gut-Heart Axis in Atherosclerosis: Microbial Metabolites, Molecular Mechanisms, and Precision Therapeutics.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {42412324},
issn = {1867-1314},
abstract = {Despite advances in lipid-lowering and anti-inflammatory medications, atherosclerotic cardiovascular disease (ASCVD) continues to be the leading cause of morbidity and mortality worldwide. Recent studies have identified the gut microbiota as a key modulator of cardiovascular health via the gut-heart axis. This review investigates the molecular processes by which microbial metabolites affect atherogenesis. Proatherogenic substances like trimethylamine-N-oxide (TMAO), which are produced from dietary precursors through gut microbial and hepatic metabolism, aggravate foam cell production, platelet aggregation, and vascular inflammation. Short chain fatty acids (SCFAs), such as butyrate and propionate, have been shown to protect against atherosclerosis by activating G-protein-coupled receptors, regulating gene expression, and improving endothelial function. Additionally, secondary bile acids, tryptophan derivatives, and phenylacetylglutamine have emerged as important microbial metabolites involved in vascular disease. The review also summarizes various therapeutic strategies such as use of probiotics, prebiotics, postbiotics, precision microbiome editing (using bacteriophages and CRISPR-Cas systems), and fecal microbiota transplantation (FMT) for targeting gut-heart axis. Multi-omic systems combined with artificial intelligence can now detect disease-specific microbial signatures, improving risk stratification and paving the way for precision microbiome-based therapeutics. However, challenges such as determining causality, regulatory intricacies, and inter-individual variability in host-microbiome interactions remain. Despite these obstacles, the gut-heart axis provides a disruptive paradigm in preventive cardiology by emphasizing tailored microbiome therapies as a complement to traditional ASCVD care.},
}

@article {pmid42412819,
year = {2026},
author = {Tziony, I and Orenstein, Y},
title = {CROP: a feature-independent context-aware method for CRISPR-Cas9 frameshift prediction.},
journal = {Bioinformatics (Oxford, England)},
volume = {42},
number = {Supplement_1},
pages = {},
pmid = {42412819},
issn = {1367-4811},
support = {358/21//Israel Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *Frameshift Mutation ; *Software ; *Computational Biology/methods ; Prediction Algorithms ; },
abstract = {MOTIVATION: The CRISPR-Cas9 complex has revolutionized genome-editing technologies. By designing a 20 nt-long guide RNA, a Cas9 nuclease can be guided to cleave almost any genomic target site (followed by NGG). The cleavage induces double-stranded DNA breaks, which are then repaired by cellular pathways. Accurate CRISPR-Cas9 repair-outcome prediction is essential for designing guide RNAs with desired genomic effects, such as gene knockout. A central challenge is quantifying the rate of frameshifts, i.e. repair-outcomes that lead to a change in the local length that is not a multiple of three. Previous methods for frameshift-rate prediction were trained on only a few experimental or cellular contexts, mostly relied on manually defined microhomology features, and were limited by sparse features and class labels.

RESULTS: We developed CROP, a feature-independent context-aware repair-outcome prediction method. By aggregating specific repair outcomes as Δlength classes, CROP overcomes class sparsity. We designed CROP to work with variable input sequence lengths and output classes to utilize multiple datasets simultaneously. We benchmarked CROP against state-of-the-art repair-outcome prediction methods over 18 datasets, which we curated and standardized from various studies. Across all datasets, CROP outperformed all competing methods in frameshift-rate prediction. We performed cross-experiment and cross-cellular frameshift-rate predictions to investigate the generalizability of repair mechanisms. Finally, we show that CROP learned microhomology principles from raw sequences without explicit feature engineering, establishing an end-to-end architecture for CRISPR-Cas9 repair-outcome prediction that learns from multiple datasets.

CROP is available at https://github.com/OrensteinLab/CROP.},
}

*CRISPR-Cas Systems
*Frameshift Mutation
*Software
*Computational Biology/methods
Prediction Algorithms

@article {pmid42413622,
year = {2026},
author = {Yu, Q and Waheed, A and Hanioui, M and Ma, P and Wang, X and Liu, K and Li, X and Xue, Z and Zhang, G and Zhao, M},
title = {Engineering a tyrosine-auxotrophic Escherichia coli chassis for residue-specific in vivo DOPA incorporation into mussel foot protein mimics.},
journal = {Journal of biotechnology},
volume = {418},
number = {},
pages = {47-56},
doi = {10.1016/j.jbiotec.2026.07.003},
pmid = {42413622},
issn = {1873-4863},
abstract = {Mussel foot proteins (Mfps) achieve exceptional marine adhesion through post-translational conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA). Recombinant production, however, is limited by poor solubility, low yields, and insufficient DOPA incorporation. We generated a genetically stable tyrA-deficient Escherichia coli chassis using CRISPR/Cas, thereby abolishing endogenous tyrosine biosynthesis and enabling residue-specific in vivo incorporation of exogenously supplied DOPA into mussel foot protein (MFP) mimics through selective pressure incorporation (SPI). A two-stage cultivation strategy decoupled biomass accumulation from DOPA-dependent protein synthesis, yielding 18.01 mg L[-1] of purified FP1. Residue-specific DOPA incorporation was verified by the characteristic + 16 Da shift in the [M-H][-] ion, accompanied by the loss of the tyrosine signal following exogenous DOPA supplementation in M9 medium. FP3 was largely soluble (65%) in crude extracts, while FP5 expression remained minimal. Fusion to thioredoxin (TrxA) and magnetoreceptor protein (MagR) further enhanced FP3 solubility to 90% and 85%, respectively. Proteins expressed in minimal M9 medium displayed exceptional shear stability, with viscosity fluctuations limited to ±2.2%, reflecting preserved catechol chemistry and structural integrity. This integrated strategy overcomes recurring trade-offs between DOPA incorporation, solubility, and yield, providing a basis for the potentially scalable production of functional, catechol-rich Mfps. Collectively, these findings support the development of next-generation mussel-inspired adhesives and catechol-based biomaterials.},
}

@article {pmid42413656,
year = {2026},
author = {Zhang, J and Shi, X and Ding, Y and Zheng, J and Li, H and Zhao, H and Zhang, T and Xing, Q and Zhao, C and Yao, S and Wang, J},
title = {Dynamic bidirectional diffusion-controlled multi-enzyme system for one-pot viral detection.},
journal = {Journal of advanced research},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jare.2026.07.019},
pmid = {42413656},
issn = {2090-1224},
abstract = {INTRODUCTION: Integrating isothermal amplification with CRISPR-based detection in a single reaction vessel holds significant promise for rapid and sensitive point-of-care virus diagnostics. However, conventional one-pot methods often suffer from mutual inhibition between amplification and CRISPR-Cas reactions, which compromises detection performance and limits their practical application.

OBJECTIVES: This study aims to overcome the inhibitory interactions between isothermal amplification and CRISPR-based detection by developing an integrated reaction system that enables efficient multi-enzyme coordination within a single tube.

METHODS: We designed a dynamic bidirectional diffusion-controlled RPA/CRISPR-Cas12a multi-enzyme system based on a dual-phase separation strategy. In this system, glycerol was used to modulate viscosity and accelerate the RPA reaction, while sucrose was introduced to create a density gradient that enables spatial separation. This configuration effectively coordinates the activities of multiple enzymes within one reaction vessel. Furthermore, a 3D-printed nucleic acid extraction device was integrated to simplify sample preparation and enhance overall detection efficiency.

RESULTS: The developed system achieved single-copy sensitivity and completed detection within 30 min, exhibiting over 100-fold higher sensitivity than conventional one-pot assays. The method was further validated by detecting Norovirus in both clinical and food samples, confirming its robustness and accuracy.

CONCLUSION: The proposed dual-phase RPA/CRISPR-Cas12a system provides a simple, rapid, and highly sensitive platform for nucleic acid detection. Its operational simplicity, compatibility with low-resource settings, and potential for home-based diagnostics highlight its strong applicability for decentralized virus detection.},
}

@article {pmid41930411,
year = {2026},
author = {Tyagi, DS and Banoo, H and Jha, DK and Meena, V and Joon, R and Agrwal, K and Yadav, P and Kumar, A and Satbhai, SB and Long, T and Pandey, AK},
title = {CRISPR/Cas9 Editing of the Wheat Iron Sensor TaHRZ1 Confirms Its Conserved Role in Iron Homeostasis and Allocation in Grains.},
journal = {Plant, cell & environment},
volume = {49},
number = {8},
pages = {4975-4991},
doi = {10.1111/pce.70516},
pmid = {41930411},
issn = {1365-3040},
support = {//NABI-CORE/ ; },
mesh = {*Triticum/genetics/metabolism ; *Homeostasis ; *Iron/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems/genetics ; Phylogeny ; Plants, Genetically Modified ; *Seeds/metabolism/genetics ; Arabidopsis/genetics ; Amino Acid Sequence ; Edible Grain/metabolism/genetics ; },
abstract = {Plants rely on specialized sensing systems, including transcriptional regulators, to maintain iron (Fe) homeostasis. Among these, Hemerythrin RING Zinc finger (HRZ) proteins have emerged as key regulators of Fe homeostasis. In this study, six Triticum aestivum L. (wheat) HRZ homoeologs referred to as TaHRZ1 and TaHRZ2, were identified by BLAST searches using rice (Oryza sativa) HRZ sequences and mapped to chromosomes 1 and 3. These encode for proteins with conserved N-terminal Hemerythrin (HHE) domains and C-terminal CHY-RING and Zn-ribbon motifs. Phylogenetic analysis grouped these genes into distinct clades, while expression profiling revealed strong root-specific and Fe-responsive expression patterns, indicating roles in nutrient sensing. Functional conservation was demonstrated by complementation of the Arabidopsis thaliana bts-1 mutant, where both wheat genes restored normal Fe regulation. Full-length TaHRZ1 and TaHRZ2 interacted with members of wheat bHLH IVc transcription factors, while truncated versions lacking the RING domain did not, emphasizing their conserved role in protein interactions. CRISPR-Cas9 editing of the conserved HHE3 domain of TaHRZ1, coupled with devlopmental regulators GRF4-GIF1 chimeric protein, achieved 6.4%-8.8% regeneration efficiency in wheat. Elemental analysis indicated enhanced Fe loading in the grains of the edited lines, particularly in the scutellum, suggesting improved Fe partitioning compared to the non-edited plants. Additionally, qRT-PCR revealed upregulation of TaFIT and TaIRO3, and downregulation of IDEF1 in edited lines, supporting an important regulatory role for TaHRZ1 in Fe homeostasis signalling. These findings position TaHRZ1 as a valuable target for biofortification strategies to enhance Fe content in wheat grains.},
}

*Triticum/genetics/metabolism
*Homeostasis
*Iron/metabolism
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*CRISPR-Cas Systems/genetics
Phylogeny
Plants, Genetically Modified
*Seeds/metabolism/genetics
Arabidopsis/genetics
Amino Acid Sequence
Edible Grain/metabolism/genetics

@article {pmid42010781,
year = {2026},
author = {Yue, K and Liang, X and Wang, Y and Jiang, N and Hu, Y and Su, J and Lu, C and Gao, H and Gai, X and Guo, W},
title = {A field-deployable RPA-CRISPR/Cas12a dual-mode assay for rapid detection of Fusarium oxysporum in Nicotiana tabacum.},
journal = {Pest management science},
volume = {82},
number = {8},
pages = {7610-7619},
doi = {10.1002/ps.70825},
pmid = {42010781},
issn = {1526-4998},
support = {2024530000241006//Yunnan Academy of Tobacco Agricultural Sciences/ ; 202405AD350100//Yunnan Applied Fundamental Research Projects/ ; 110202401006(JY-06)//China National Tobacco Corporation (CNTC)/ ; },
mesh = {*Fusarium/isolation & purification/genetics ; *Nicotiana/microbiology ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {BACKGROUND: Fusarium oxysporum is a devastating soil-borne pathogen that causes severe economic losses in tobacco and other crops, necessitating rapid and accurate detection methods for effective disease management. Herein, we developed a dual-mode RPA-Cas12a platform incorporating both lateral flow dipstick (LFD) and fluorescence-based detection (FBD), targeting the CYP51C gene to enable rapid and equipment-facilitated detection of F. oxysporum under isothermal conditions.

RESULTS: The entire assay from sample to result can be completed within 68 min. The platform offers two complementary detection formats. The LFD mode provides visual qualitative results with a detection limit of 360 copies, making it ideally suited for rapid on-site screening. In contrast, the FBD mode achieves a detection limit of 3.6 copies, demonstrating 100-fold higher sensitivity and enabling precise quantitative analysis.

CONCLUSION: The assay successfully identified F. oxysporum in both artificially inoculated and field-collected tobacco samples, showing high concordance with fluorescence intensity. This work provides a sensitive, rapid, and practical diagnostic tool for the on-site detection and monitoring of tobacco root rot caused by F. oxysporum. © 2026 Society of Chemical Industry.},
}

*Fusarium/isolation & purification/genetics
*Nicotiana/microbiology
*CRISPR-Cas Systems
*Plant Diseases/microbiology
Rapid Diagnostic Tests
*Nucleic Acid Amplification Techniques/methods

@article {pmid42092617,
year = {2026},
author = {Richter, E and Klöhn, M and Nocke, MK and Friedrich, ME and Todt, D and Steinmann, E and Brüggemann, Y},
title = {Development of a CRISPR-Cas13-based antiviral strategy against hepatitis E virus.},
journal = {JHEP reports : innovation in hepatology},
volume = {8},
number = {7},
pages = {101885},
pmid = {42092617},
issn = {2589-5559},
mesh = {*Hepatitis E virus/genetics/drug effects ; Humans ; *CRISPR-Cas Systems ; Virus Replication/drug effects/genetics ; *Antiviral Agents/pharmacology ; *Hepatitis E/virology/therapy ; Genome, Viral ; },
abstract = {BACKGROUND & AIMS: Effective antiviral drugs remain unavailable for many clinically relevant pathogens, including the hepatitis E virus (HEV). This study aimed to evaluate the CRISPR/Cas13d system as a potential antiviral strategy against HEV.

METHODS: We developed a reporter assay to screen CRISPR RNAs (crRNAs) targeting conserved regions of the HEV genome and tested their antiviral activity in human hepatoma cells using a robust HEV cell culture model. HEV replication was assessed using a subgenomic replicon, infectious particle production was quantified by immunofluorescence and titration assays. A bioinformatic analysis was performed to identify a minimal set of crRNAs capable of broadly targeting circulating human pathogenic HEV strains.

RESULTS: A crRNA screen identified multiple functional crRNAs targeting HEV-3, with ORF1-targeting crRNAs significantly reducing viral capsid expression (p <0.01) and the number of HEV-infected cells (p <0.01). Cas13d-mediated targeting led to robust reduction of HEV replication and markedly lowered infectious virus production in vitro (p <0.001). Bioinformatic analysis revealed that just three distinct crRNAs could cover ∼94% of known HEV genomes with zero mismatches, while four crRNAs achieved complete coverage.

CONCLUSIONS: Our findings demonstrate that CRISPR/Cas13d can target HEV replication and viral progeny production in vitro. The identification of a minimal crRNA set capable of broadly targeting circulating HEV strains suggests that the CRISPR/Cas13d system may offer an antiviral strategy to address challenges related to viral evolution and treatment escape.

IMPACT AND IMPLICATIONS: This study establishes CRISPR/Cas13d as a proof-of-concept antiviral strategy against hepatitis E virus (HEV), demonstrating suppression of viral replication and particle production in vitro. By identifying a minimal set of broadly effective crRNAs, we provide a framework for targeting diverse HEV variants and buffering against viral evolution. These findings highlight the potential of CRISPR-based systems as innovative antiviral strategies.},
}

*Hepatitis E virus/genetics/drug effects
Humans
*CRISPR-Cas Systems
Virus Replication/drug effects/genetics
*Antiviral Agents/pharmacology
*Hepatitis E/virology/therapy
Genome, Viral

@article {pmid42115764,
year = {2026},
author = {Lv, G and Li, Y and Chen, J and Wu, Z and Wu, W and Wu, X and Lin, H and Wang, T},
title = {CRISPR/Cas9-mediated knockout of ZmHMA3 reveals its essential role in zinc homeostasis and high-zinc stress tolerance in maize.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42115764},
issn = {2045-2322},
support = {2023YFD1201105//National Key Research and Development Program of China/ ; },
mesh = {*Zea mays/genetics/metabolism/physiology ; *Zinc/metabolism/toxicity ; *Homeostasis ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; *Stress, Physiological/genetics ; Gene Expression Regulation, Plant ; Plant Leaves/metabolism/genetics ; Gene Knockout Techniques ; Plant Roots/metabolism/genetics ; *Adenosine Triphosphatases/genetics/metabolism ; Oxidative Stress ; Plants, Genetically Modified ; },
abstract = {Excessive Zn is toxic to maize (Zea mays L.). The heavy metal ATPase gene ZmHMA3 is associated with heavy metal transport, but its function in maize tolerance to high Zn stress has not been fully characterized. In this study, CRISPR/Cas9 technology was used to generate zmhma3 knockout mutants to investigate its function under high Zn stress. High Zn stress significantly induced the expression of ZmHMA3 in maize leaves and roots. Phenotypic analysis showed that, compared to the WT plants, the zmhma3 mutants exhibited significantly reduced tolerance to excessive Zn, manifested as severe growth inhibition, impaired root structure, decreased activity of key antioxidant enzymes (CAT, POD, SOD), and aggravated membrane damage. Furthermore, the mutants accumulated significantly higher levels of Zn in both roots and leaves, accompanied by disordered subcellular Zn distribution, indicating disrupted intracellular Zn homeostasis. Our results demonstrate that ZmHMA3 is a key positive regulator in maize's response to high Zn stress, likely by coordinating Zn compartmentalization and alleviating oxidative damage. This study provides new genetic and physiological insights into the molecular mechanisms of Zn stress tolerance in maize and offers a potential target for breeding new maize varieties tolerant with improved high-Zn-efficiency.},
}

*Zea mays/genetics/metabolism/physiology
*Zinc/metabolism/toxicity
*Homeostasis
*CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism
*Stress, Physiological/genetics
Gene Expression Regulation, Plant
Plant Leaves/metabolism/genetics
Gene Knockout Techniques
Plant Roots/metabolism/genetics
*Adenosine Triphosphatases/genetics/metabolism
Oxidative Stress
Plants, Genetically Modified

@article {pmid42302780,
year = {2026},
author = {Ueki, H and Tomita, Y and Duong, C and Mitake, H and Kiso, M and Furusawa, Y and Zhao, D and da Silva Lopes, TJ and Wu, L and Feng, H and Yamayoshi, S and Fukuyama, S and Yamashita, M and Ozawa, M and Ikawa, M and Yoshida, N and Watanabe, T and Kawaoka, Y},
title = {A CRISPR knockout mouse library for functional genomics in influenza research.},
journal = {Cell},
volume = {189},
number = {14},
pages = {4471-4488.e7},
doi = {10.1016/j.cell.2026.05.032},
pmid = {42302780},
issn = {1097-4172},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Mice ; *Influenza A virus/genetics ; *Orthomyxoviridae Infections/genetics/virology ; *Genomics/methods ; Mice, Knockout ; Cytoskeletal Proteins/genetics/metabolism ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; Guanine Nucleotide Exchange Factors/genetics/metabolism ; Humans ; RNA, Small Interfering/metabolism/genetics ; Host-Pathogen Interactions/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Library ; Mice, Inbred C57BL ; },
abstract = {Functional validation of host factors in whole-animal models is a major bottleneck in virology; it hinders the translation of data from in vitro studies into a deeper understanding of the viral life cycle and pathogenesis. To address this challenge, we developed a systematic in vivo screening platform for influenza A virus. This platform comprises a library of 84 CRISPR-Cas9-generated gene-modified mouse lines targeting host factors prioritized from the literature and in vitro small interfering RNA (siRNA) screening studies. Using this resource, we identified 17 host factors whose genetic ablation conferred resistance to influenza A virus infection. Further studies of two of these factors, Arhgef28 and Lasp1, revealed distinct protective mechanisms against influenza A virus. We offer this mouse library to the research community as a powerful platform for studying virus-host interactions in a physiologically relevant context.},
}

Animals
CRISPR-Cas Systems/genetics
Mice
*Influenza A virus/genetics
*Orthomyxoviridae Infections/genetics/virology
*Genomics/methods
Mice, Knockout
Cytoskeletal Proteins/genetics/metabolism
Adaptor Proteins, Signal Transducing/genetics/metabolism
Guanine Nucleotide Exchange Factors/genetics/metabolism
Humans
RNA, Small Interfering/metabolism/genetics
Host-Pathogen Interactions/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Library
Mice, Inbred C57BL

@article {pmid42409605,
year = {2026},
author = {Huang, YW and Hu, CC and Cho, YH and Tsai, CH and Lin, NS and Hsu, YH and Dinesh-Kumar, SP},
title = {Efficient CRISPR-Cas9 delivery and transgene-free multiplex genome editing in plants using cymbidium mosaic virus-derived vectors.},
journal = {The Plant journal : for cell and molecular biology},
volume = {127},
number = {1},
pages = {e71031},
pmid = {42409605},
issn = {1365-313X},
support = {NSTC 114-2313-B-005-052-MY2//National Science and Technology Council/ ; //Ministry of Education in Taiwan/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Nicotiana/genetics ; Genetic Vectors/genetics ; Plants, Genetically Modified/genetics ; Oxidoreductases/genetics ; Genome, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transgenes/genetics ; Potexvirus ; },
abstract = {Virus-induced genome editing (VIGE) has become a useful method by enabling transient delivery of gene-editing reagents; however, many viral systems face limitations in cargo size, host range, or reliance on transgenic Cas9-expressing plants. In this study, we developed a cymbidium mosaic virus (CymMV)-based VIGE platform that enables simultaneous expression of Streptococcus pyogenes Cas9 (SpCas9) and one or more guide RNAs (gRNAs) from a single viral RNA. In Nicotiana benthamiana, this system induced editing in the Phytoene desaturase (PDS) gene, with indel rates exceeding 50% within 6 days after inoculation, outperforming traditional delivery methods by about fivefold. Notably, over 80% of regenerated plants contained targeted mutations, and 82% of these were both transgene- and virus-free, including tetra-allelic knockouts directly in the M0 generation. Adding a Ruby-based visual counterselection marker enabled rapid, reliable identification of transgene-free, edited plants without antibiotic selection. When adapted to Phalaenopsis aphrodite orchids, the platform efficiently edited the PaPDS gene, achieving a 47% indel frequency at 20 days post-inoculation, with visible bleaching in leaf tissue from inoculated protocorm-like bodies. Additionally, expressing multiple gRNAs from a single CymMV replicon enabled multiplex editing in orchid tissues, demonstrating the system's versatility for complex, polyploid crops. Our findings broaden the use of VIGE in orchids and provide a reliable framework for precision plant breeding.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Nicotiana/genetics
Genetic Vectors/genetics
Plants, Genetically Modified/genetics
Oxidoreductases/genetics
Genome, Plant/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Transgenes/genetics
Potexvirus

@article {pmid42409731,
year = {2026},
author = {Shangguan, YT and Xie, LL and Liu, WB and Gu, RX and Xu, YX and Wang, M and Wang, JX},
title = {[Novel CD6-targeted CAR-T cell therapy for T-cell acute lymphoblastic leukemia: a safe and efficient strategy to prevent fratricide through gene editing].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {47},
number = {5},
pages = {433-441},
doi = {10.3760/cma.j.cn121090-20260130-00068},
pmid = {42409731},
issn = {0253-2727},
support = {82341213, 82570279, 82570278//National Natural Science Foundation of China/ ; 23JCYBJC01060//Tianjin Natural Science Foundation Project/ ; },
mesh = {Humans ; *Immunotherapy, Adoptive ; *Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; *Gene Editing ; Receptors, Chimeric Antigen ; *Antigens, CD ; *Antigens, Differentiation, T-Lymphocyte ; CRISPR-Cas Systems ; T-Lymphocytes ; Receptors, Antigen, T-Cell ; },
abstract = {Objective: To explore a novel strategy that addresses the dual challenges of fratricide and on-target off-tumor toxicity in current chimeric antigen receptor T-cell (CAR-T) therapy for T-cell acute lymphoblastic leukemia (T-ALL) and to develop a safe and efficacious anti-T-ALL CAR-T product by identifying a new target and compatible gene-editing approach. Methods: Public single-cell RNA sequencing (scRNA-seq) datasets were utilized to analyze bone marrow cells extracted from patients with T-ALL and healthy donors, evaluating the differential expression profiles of CD6 and CD7. In investigating the endogenous role of CD6 in CAR-T cells, the CRISPR/Cas9 RNP system was first employed in a CD19 CAR-T model to evaluate the impact of CD6 knockout on the phenotype and activation status of CAR-T cells. Subsequently, CD6-knockout, CD6-targeted CAR-T cells (6KO-6CAR) were constructed, and their functional activities were evaluated. Results: scRNA-seq analysis revealed that CD6 is broadly expressed in T-ALL. Compared with the traditional target CD7, which is also expressed in a subset of normal hematopoietic stem/progenitor cells and myeloid cells, CD6 exhibits a more restricted expression profile, exhibiting superior safety characteristics. Studies on the CD19 CAR-T model indicated that CD6 knockout enables CAR-T cells to maintain a superior functional state: their baseline activation level (CD25 expression) was reduced (P<0.05) while generating a higher proportion of TNF-α(+)IFN-γ(+) cells (P<0.05) upon antigen stimulation. The further constructed 6KO-6CAR cells exhibited potent specific activation (significantly upregulated CD107a expression level, all P<0.001) and cytotoxicity (all P<0.05) against multiple CD6(+) T-ALL cell lines (MOLT-4, CCRF-CEM, and Jurkat) in vitro. Conclusion: CD6 is a novel therapeutic target for T-ALL with high coverage and a favorable safety profile, and knocking out endogenous CD6 globally optimizes the intrinsic functional state of CAR-T cells. Constructing 6KO-6CAR based on the CRISPR/Cas9 technology addresses fratricide in CAR-T cells while enhancing their antitumor functionality, thereby providing a novel immunotherapy regimen with safety and clinical translational potential for relapsed/refractory T-ALL.},
}

Humans
*Immunotherapy, Adoptive
*Precursor T-Cell Lymphoblastic Leukemia-Lymphoma
*Gene Editing
Receptors, Chimeric Antigen
*Antigens, CD
*Antigens, Differentiation, T-Lymphocyte
CRISPR-Cas Systems
T-Lymphocytes
Receptors, Antigen, T-Cell

@article {pmid42409995,
year = {2026},
author = {Goudarzi, F and Salehipour, P and Modarressi, MH and Hosseini, M},
title = {A "turn-on" CRISPR-mediated method using enhanced fluorescent bimetallic DNA nanoclusters for EGFR mutation detection in non-small cell lung cancer.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-61115-3},
pmid = {42409995},
issn = {2045-2322},
support = {981234//University of Tehran/ ; },
abstract = {An affordable, precise detection of mutations is critical for guiding targeted cancer therapies and improving patient outcomes. Epidermal growth factor receptor (EGFR), a protein on the surface of cells that regulates growth and division, is frequently mutated in non-small cell lung cancer (NSCLC). Early identification of these mutations enables clinicians to select the most effective tyrosine kinase inhibitors, thereby enhancing treatment response and survival rates. Recent studies have focused on developing CRISPR-based detection strategies incorporating nanomaterials to achieve more accurate results. In this study, we present a CRISPR-based "turn-on" detection platform that leverages the cleavage of a novel enhanced bimetallic DNA nanocluster to measure EGFR exon 19 deletion in non-small cell lung cancer (NSCLC). The system is innovatively designed using guide RNAs (gRNAs) rationally derived from the normal EGFR gene, enabling the determination of exon 19 deletion through CRISPR-Cas activation in samples containing the normal and mutant. Upon recognition of the normal EGFR gene, the Cas12a enzyme induces cleavage of the Spermiform-designed Ag/Au DNA nanocluster and fluorescence quenching. At the same time, fluorescence signal retention depends on mutation frequency, with higher mutation frequencies resulting in greater or "turn-on" fluorescence signals. This approach achieves a detection limit (LOD) of approximately 0.35 nM, which is capable of detecting about 1.5% mutation, offering a cost-effective, label-free diagnostic tool and a promising strategy for future detection of deletion-related subtypes in PCR products by targeting normal sequences. The integration of bimetallic nanocluster-based reporters with CRISPR precision provides an emerging platform for next-generation molecular diagnostics targeting EGFR and other clinically relevant mutations.},
}

@article {pmid42410413,
year = {2026},
author = {Fan, Q and Stevanie, S and Frielingsdorf, S and Neubauer, P and Lenz, O and Gimpel, M},
title = {Genomically integrated orthogonal translation system in Escherichia coli enables production of functional modified [NiFe]-hydrogenases.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {42410413},
issn = {1475-2859},
mesh = {*Hydrogenase/genetics/metabolism/biosynthesis ; *Escherichia coli/genetics/metabolism ; Cupriavidus necator/genetics/enzymology ; CRISPR-Cas Systems ; *Protein Biosynthesis ; Plasmids/genetics ; Gene Editing ; },
abstract = {The functional diversification of O2-tolerant [NiFe]-hydrogenases using orthogonal translation systems (OTSs) offers a promising strategy for developing advanced biocatalysts and biohybrid energy platforms. However, plasmid-based OTSs frequently impose metabolic burdens and suffer from plasmid instability during fermentation, particularly when co-produced with complex metalloenzymes. To overcome these bioprocess limitations, we employed CRISPR/Cas9-mediated genome editing to integrate a psychrophilic pyrrolysyl-tRNA synthetase/tRNA pair into the Escherichia coli BL21 genome. The resulting strain provided a plasmid-free orthogonal translation background that supported amber suppression-mediated expression of the regulatory [NiFe]-hydrogenase (RH) of Cupriavidus necator. Using this genomically integrated OTS, we achieved the production of a full-length, catalytically active RH variant. Our results demonstrate that chromosomal OTS is compatible with the efficient production and maturation of complex metalloenzymes. The present work lays the groundwork for the bio-orthogonal engineering of hydrogenases and related hybrid biocatalysts.},
}

*Hydrogenase/genetics/metabolism/biosynthesis
*Escherichia coli/genetics/metabolism
Cupriavidus necator/genetics/enzymology
CRISPR-Cas Systems
*Protein Biosynthesis
Plasmids/genetics
Gene Editing

@article {pmid41419637,
year = {2026},
author = {Meng, X and Reis, N and Bassik, MC and Pașca, SP},
title = {CRISPR screens in human neural organoids and assembloids.},
journal = {Nature protocols},
volume = {21},
number = {7},
pages = {3127-3147},
pmid = {41419637},
issn = {1750-2799},
mesh = {Humans ; *Organoids/cytology/metabolism ; *CRISPR-Cas Systems ; Neurodevelopment ; *Neurons/cytology/metabolism ; Induced Pluripotent Stem Cells/cytology ; *Gene Editing/methods ; Cell Differentiation ; },
abstract = {Studying the molecular mechanisms underlying the assembly of the human nervous system remains a significant challenge. The ability to generate neural cells from pluripotent stem cells, combined with advanced genome-editing techniques, provides unprecedented opportunities to uncover the biology of human neurodevelopment and disease. Organoids and assembloids enable the in vitro modeling of previously inaccessible developmental processes, such as the specification and migration of human neurons, including the integration of cortical interneurons from the ventral into the dorsal forebrain. Here, we present a detailed protocol that combines pooled CRISPR-Cas9 screening with neural organoid and assembloid models and illustrate how it can be applied to map hundreds of disease genes onto cellular pathways and specific aspects of human neural development. Our protocol outlines key steps, from planning and optimizing genetic perturbations to designing readouts for neuronal generation and migration, conducting the screening and validating candidate genes. The screening experiments take ~3 months to complete and require expertise in stem cell culture and neural differentiation, genetic engineering of human induced pluripotent stem cell lines, fluorescence-activated cell sorting and next-generation sequencing and analyses. The integration of genetic screening and human cellular models constitutes a powerful platform for investigating the mechanisms of human brain development and disease, paving the way for the discovery of novel therapeutics.},
}

Humans
*Organoids/cytology/metabolism
*CRISPR-Cas Systems
Neurodevelopment
*Neurons/cytology/metabolism
Induced Pluripotent Stem Cells/cytology
*Gene Editing/methods
Cell Differentiation

@article {pmid41832622,
year = {2026},
author = {Yuan, C and Chen, F and Gao, X and Yusufu, A and Lu, D and Wu, X and Ni, L},
title = {UBE2M Identified by CRISPR Screening as a Key Regulator of Cisplatin-Induced Acute Kidney Injury via the p53 Pathway.},
journal = {Endocrine, metabolic & immune disorders drug targets},
volume = {26},
number = {},
pages = {e18715303410982},
pmid = {41832622},
issn = {2212-3873},
mesh = {*Cisplatin/toxicity ; *Acute Kidney Injury/chemically induced/genetics/metabolism/pathology ; Animals ; *Ubiquitin-Conjugating Enzymes/genetics/metabolism ; *Tumor Suppressor Protein p53/metabolism/genetics ; Signal Transduction/drug effects/physiology ; Humans ; *CRISPR-Cas Systems ; *Antineoplastic Agents/toxicity ; Mice ; Apoptosis/drug effects ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {INTRODUCTION: Acute kidney injury caused by cisplatin (Cis-AKI) is a major limitation in its clinical use, primarily due to the lack of effective therapeutic targets to mitigate nephrotoxicity. Although several molecular pathways are involved in Cis-AKI, identifying reliable and actionable therapeutic targets has been challenging. Through a CRISPR-based genome-wide screening approach, UBE2M was identified as a novel gene involved in cellular survival during cisplatin-induced stress. However, its expression, biological function, and underlying mechanism in Cis-AKI have not been thoroughly investigated. This study aims to identify key therapeutic targets for Cis- AKI and investigate the role of UBE2M in this condition.

METHODS: A CRISPR-Cas9 genome-wide screening approach was employed to identify key genes involved in cisplatin-induced renal tubular epithelial cell injury. UBE2M, identified as a critical survival factor, was further investigated using both gain- and loss-of-function strategies to explore its biological function and underlying regulatory mechanisms in the Cis-AKI model.

RESULTS: CRISPR screening identified UBE2M as a key regulator of cellular survival in Cis-AKI, and subsequent validation experiments confirmed its suppression in cisplatin-induced renal injury models. UBE2M overexpression alleviated apoptosis and renal injury by reducing p53 activation. In contrast, UBE2M knockdown exacerbated these effects, leading to increased apoptosis and renal injury.

DISCUSSION: This study reveals that UBE2M is a critical regulator of cisplatin-induced renal tubular epithelial cell injury. By regulating the p53-mediated apoptotic pathway, UBE2M protects against Cis-AKI.

CONCLUSION: UBE2M could serve as a novel therapeutic target for the prevention and treatment of cisplatin-induced nephrotoxicity.},
}

*Cisplatin/toxicity
*Acute Kidney Injury/chemically induced/genetics/metabolism/pathology
Animals
*Ubiquitin-Conjugating Enzymes/genetics/metabolism
*Tumor Suppressor Protein p53/metabolism/genetics
Signal Transduction/drug effects/physiology
Humans
*CRISPR-Cas Systems
*Antineoplastic Agents/toxicity
Mice
Apoptosis/drug effects
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42135732,
year = {2026},
author = {Xiao, WT and He, JY and Yang, D and Xun, Y},
title = {Construction of a novel signature based on CRISPR-Cas9 screening for prognostic prediction in breast cancer.},
journal = {BMC cancer},
volume = {26},
number = {1},
pages = {},
pmid = {42135732},
issn = {1471-2407},
support = {82304083//National Natural Science Foundation of China/ ; 2023JJ40584//Natural Science Foundation of Hunan Province/ ; },
mesh = {Humans ; *Breast Neoplasms/genetics/mortality/pathology ; Female ; Prognosis ; *CRISPR-Cas Systems ; *Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; Gene Expression Profiling ; Transcriptome ; },
abstract = {BACKGROUND: Breast cancer (BC) is a highly heterogeneous malignancy and remains the leading cause of cancer-related mortality among women worldwide. Although advances in molecular classification and targeted therapies have improved outcomes for certain subtypes, robust prognostic biomarkers applicable across clinical contexts are still lacking. The CRISPR-Cas9 system offers a powerful platform for identifying cancer cell vulnerabilities and may facilitate the development of clinically relevant prognostic models.

METHODS: We integrated genome-wide CRISPR-Cas9 screening data from the DepMap database with transcriptomic and clinical data from TCGA and GEO datasets to identify BC cell survival-dependent genes (CSDGs). CSDGs prognostic signature was constructed using univariate Cox regression, LASSO, and stepwise multivariate Cox regression analyses. The model was validated in internal and external cohorts. Functional enrichment analyses, including GO, KEGG, WGCNA, and GSEA, were performed to explore the biological mechanisms underlying the signature. Random forest analysis and functional experiments were conducted to investigate the role of key gene in CSDGs signature.

RESULTS: A total of 1,622 CSDGs were identified, and a nine-gene prognostic CSDGs signature (BRD4, CHORDC1, COPZ1, HNRNPC, NUP43, RAD1, RBBP8, TUBA1B, and VPS28) was developed. This signature effectively stratified patients into high- and low-risk groups with significantly different overall survival, and its robustness was confirmed across multiple internal and external cohorts. High-risk patients exhibited a significant association with multiple adverse clinical features. A nomogram that combined the risk score with clinical variables showed robust predictive performance, and its C-index surpassed those of individual predictors, underscoring the enhanced accuracy of the integrated model. Functional analyses revealed enrichment of oncogenic pathways (e.g., MYC targets, G2/M checkpoint, mTORC1 signaling) in high-risk patients, while low-risk patients exhibited immune and hormone response signatures. CHORDC1 was identified as the most critical gene in the model. Knockdown of CHORDC1 significantly inhibited proliferation, migration, and invasion of BC cells. Transcriptomic profiling further linked CHORDC1 to oncogenic pathways, including EMT, mTORC1 signaling, and TNF-α/NF-κB signaling activation.

CONCLUSION: We developed a CRISPR-Cas9 screening-based prognostic signature for BC that effectively stratifies patient risk and demonstrates robust predictive performance across cohorts. CHORDC1 was identified as a key oncogenic driver, promoting tumor progression via pathways such as EMT and mTORC1 signaling, highlighting its potential as a therapeutic target. These findings may contribute to the development of personalized prognostic tools and therapeutic strategies in BC.},
}

Humans
*Breast Neoplasms/genetics/mortality/pathology
Female
Prognosis
*CRISPR-Cas Systems
*Biomarkers, Tumor/genetics
Gene Expression Regulation, Neoplastic
Gene Expression Profiling
Transcriptome

@article {pmid42235446,
year = {2026},
author = {Olfati Sumar, M and Mohammadi, F and Khoshbin, Z and Abbasi Ghaeni, F and Abnous, K and Taghdisi, SM},
title = {A CRISPR-driven aptasensor for colorimetric monitoring of lead (II) ion assisted by rolling circle amplification process: Effective in controlling food and health safety.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {362},
number = {},
pages = {128167},
doi = {10.1016/j.saa.2026.128167},
pmid = {42235446},
issn = {1873-3557},
mesh = {*Colorimetry/methods ; *Lead/analysis ; *Aptamers, Nucleotide/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Food Safety/methods ; Food Contamination/analysis ; },
abstract = {Herein, an efficient colorimetric aptasensor has been introduced for highly sensitive measurement of lead (II) ions (Pb[2+]) by integrating the advantages of clustered regularly interspaced short palindromic repeats (CRISPR) system, the rolling circle amplification (RCA) process, and the catalytic activity of gold nanoparticles (AuNPs) for the first time. The presence of Pb[2+] inactivates the CRISPR system, making it unable to cleave the complementary sequence (CS) on the surface of ferrofluids (FFDs) and the formation of RCA product. By trapping AuNPs inside the RCA mass and subsequent magnetic separation of FFDs, the supernatant color remains yellow after adding 4-nitrophenol (4-NP). In the absence of Pb[2+], the supernatant color changes to colorless, due to the activation of CRISPR-Cas12a and the lack of large DNA structures. The colorimetric aptasensor can monitor Pb[2+] ions in the concentration ranges of 0.1 pM-20 nM and 20 nM-800 nM with a detection limit of 0.024 pM. It can also quantify Pb[2+] in the biological, cosmetic, and marine samples.},
}

*Colorimetry/methods
*Lead/analysis
*Aptamers, Nucleotide/chemistry
Gold/chemistry
Metal Nanoparticles/chemistry
*Biosensing Techniques/methods
Limit of Detection
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
*Food Safety/methods
Food Contamination/analysis

@article {pmid42284937,
year = {2026},
author = {Yang, Q and Cao, Y and Yuan, J and Lu, W and Guo, Y and Sun, X and Wang, X and Li, X and Li, J and Zhang, N and Cao, L and Gong, P},
title = {A rapid and specific strategy for detecting Orientobilharzia turkestanicum, a water-associated schistosome of veterinary and environmental concern.},
journal = {Water research},
volume = {303},
number = {},
pages = {126262},
doi = {10.1016/j.watres.2026.126262},
pmid = {42284937},
issn = {1879-2448},
mesh = {Animals ; Cattle ; *Schistosoma/isolation & purification ; *Water/parasitology ; CRISPR-Cas Systems ; Environmental Monitoring ; },
abstract = {Orientobilharzia turkestanicum (O. turkestanicum) is a water-associated schistosome parasite widely distributed in pastoral regions of Asia and Europe, where freshwater systems act as key interfaces linking livestock hosts, snail intermediate hosts, and environmental transmission pathways. Despite its strong environmental dependency, effective surveillance of O. turkestanicum in water-related settings remains limited by conventional diagnostic approaches that are time-consuming, equipment-dependent, and poorly suited for field-based monitoring. In this study, a rapid and sensitive detection assay based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (RPA-CRISPR/Cas12a) was developed for on-site identification of O. turkestanicum. The ITS1-5.8S region was selected as the molecular target to enable reliable species discrimination from closely related schistosomes, particularly Schistosoma japonicum (S. japonicum). The assay operates under isothermal conditions at 37 °C and allows result interpretation through visual fluorescence and lateral flow strip (LFS) readouts without the need for sophisticated instrumentation. The established RPA-CRISPR/Cas12a assay exhibited high analytical specificity, showing no cross-reactivity with a range of non-target parasites and bacterial species. Sensitivity evaluation using serially diluted standard plasmids demonstrated analytical limit of detection of 0.16 copies/μL by visual fluorescence and 160 copies/μL by LFS. Field applicability was validated using snail samples, cattle feces, liver, intestinal tissues and simulated infected water samples, yielding an overall concordance rate of 97.55 % compared with qPCR. Collectively, these results indicate that the proposed RPA-CRISPR/Cas12a assay provides a practical and field-deployable tool for livestock and water-associated transmission environment of O. turkestanicum and offers a useful framework for improving field surveillance and risk assessment of livestock schistosomiasis in endemic regions.},
}

Animals
Cattle
*Schistosoma/isolation & purification
*Water/parasitology
CRISPR-Cas Systems
Environmental Monitoring