@article {pmid41718579,
year = {2026},
author = {Shih, J and Yokomi, RK and Hajeri, S and Roy, A and Wei, W},
title = {Field-Compatible Detection of Spiroplasma citri Associated with Citrus Stubborn Disease Using CRISPR-Cas12a and Crude Sample Preparation.},
journal = {Plant disease},
volume = {110},
number = {9},
pages = {3694-3702},
doi = {10.1094/PDIS-08-25-1699-RE},
pmid = {41718579},
issn = {0191-2917},
mesh = {*Citrus/microbiology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology ; *Spiroplasma citri/genetics/isolation & purification ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins/genetics ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; Endodeoxyribonucleases ; },
abstract = {Citrus stubborn disease (CSD), caused by Spiroplasma citri, presents a significant risk to citrus production, resulting in considerable yield losses when infections remain undetected. Accurate and timely diagnosis is crucial for effective disease management. However, existing nucleic acid-based methods, such as PCR and quantitative PCR (qPCR), require laboratory equipment and are not easily applicable in the field. This study developed a CRISPR-Cas12a-based DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) assay for the rapid, sensitive, and specific detection of S. citri, targeting the spiralin gene. An optimized recombinase polymerase amplification (RPA) primer pair and CRISPR-RNA (crRNA) were utilized for sequence-specific activation of Cas12a, enabling cleavage of fluorescent and lateral flow-compatible reporters. The assay demonstrated a detection limit of 1 attomolar (aM) (around 1.8 genome copies) using a fluorescence plate reader and 10 aM using blue-light visualization and a lateral flow assay (LFA). Specificity testing revealed discrimination against other phytopathogenic spiroplasmas, including S. kunkelii and S. melliferum. Validation in the plate-reader format with DNA extracted from symptomatic citrus samples showed 100% consistency with qPCR results. A 10-min NaOH-Tris crude extraction protocol was also assessed, facilitating straightforward and equipment-free sample preparation. Relative to DETECTR assays with kit-extracted samples, crude extracts preserved full diagnostic sensitivity in fluorescence assays and achieved 70% accuracy in LFA and visual formats in a subset of the same samples. These findings establish a dependable, portable, and highly sensitive diagnostic approach for S. citri, providing a practical tool for on-site detection and enhanced management of citrus stubborn disease.},
}

*Citrus/microbiology
*CRISPR-Cas Systems/genetics
*Plant Diseases/microbiology
*Spiroplasma citri/genetics/isolation & purification
CRISPR-Associated Proteins/genetics
Bacterial Proteins/genetics
Rapid Diagnostic Tests
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods
Endodeoxyribonucleases

@article {pmid41933454,
year = {2026},
author = {Ying, XY and Tang, DL and Sun, YM and Liu, MQ and Feng, XY and Wang, FY and Lu, C and Luo, ZD},
title = {Soil In Situ Enrichment Coupled with RPA-CRISPR/Cas12b for Rapid and Visual On-Site Detection of Fusarium oxysporum in Strawberry.},
journal = {Plant disease},
volume = {110},
number = {9},
pages = {4013-4021},
doi = {10.1094/PDIS-01-26-0110-RE},
pmid = {41933454},
issn = {0191-2917},
mesh = {*Fragaria/microbiology ; *Fusarium/isolation & purification/genetics ; *Soil Microbiology ; *Plant Diseases/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Soil/chemistry ; },
abstract = {Fusarium oxysporum is a representative soilborne fungal pathogen that causes strawberry wilt, a disease characterized by an extremely high mortality rate that poses a severe threat to the sustainable development of the global strawberry industry. However, traditional detection methods are often time-consuming and dependent on specialized laboratory equipment, while existing soil nucleic acid extraction protocols are highly susceptible to interference from inhibitors in complex matrices, leading to low detection efficiency or high false-negative rates. To address these limitations, this study developed a novel on-site detection platform based on in situ biological enrichment and purification-free nucleic acid release. A specialized enrichment rod targeting F. oxysporum was developed to leverage the tropic growth characteristics of the pathogen, achieving physical separation from the soil matrix and effectively eliminating interference from complex soil inhibitors such as humic acids. The enriched pathogens release nucleic acids via a rapid lysis buffer, which are then neutralized and used directly as templates for recombinase polymerase amplification (RPA)-CRISPR/Cas12b isothermal detection, enabling visual field identification through lateral flow strips. This method requires no specialized instrumentation, achieving a detection limit of 8.5 CFU/g and a sensitivity of 70 copies. After completing 48 h of in situ enrichment, the entire process from rod retrieval to detection completion requires only 40 min (with hands-on operation time <10 min). By effectively circumventing soil inhibitor interference and simplifying complex nucleic acid extraction into a rapid, integrated protocol, this platform provides a critical technical solution for the on-site monitoring and precise control of soilborne pathogens.},
}

*Fragaria/microbiology
*Fusarium/isolation & purification/genetics
*Soil Microbiology
*Plant Diseases/microbiology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Soil/chemistry

@article {pmid42628204,
year = {2026},
author = {Tanveer, A and Khan, SH and Atif, RM and Azhar, MT and Rehman, HM and Rana, IA and Yang, SH},
title = {Blocking the conversion of β-carotenoids into xanthophylls through HYb gene editing via CRISPR/Cas9 confers enhanced heat stress tolerance in Nicotiana tabacum.},
journal = {Plant physiology and biochemistry : PPB},
volume = {238},
number = {},
pages = {111638},
doi = {10.1016/j.plaphy.2026.111638},
pmid = {42628204},
issn = {1873-2690},
mesh = {*Nicotiana/genetics/metabolism/physiology ; *Xanthophylls/metabolism ; *Mixed Function Oxygenases/genetics/metabolism ; *beta Carotene/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; *Heat-Shock Response/genetics ; Chlorophyll/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Plants produce protective metabolites to withstand stress, and β-carotenoids act as crucial antioxidants. β-Carotene is converted into xanthophylls by β-carotene hydroxylase (BCH). In this study, the BCH gene in Nicotiana tabacum was targeted using CRISPR/Cas9 genome editing to redirect metabolic flux toward β-carotene accumulation and evaluate its effects on heat tolerance. Following Agrobacterium-mediated transformation with a BCH-specific gRNA, three mutant plants were obtained; two carrying frameshift mutations (P1 and P3) and one with mis-sense mutations (P2). A significant increase in chlorophyll contents was observed in the edited lines compared with wild-type plants. Interestingly, the frameshift mutants exhibited substantial increase in total chlorophyll (71% and 31% in P3 and P1 respectively) as compared to mis-sense mutant P2 (18%). Further, biochemical profiling using HPLC showed that the edited plants accumulated 3.74-fold increase in β-carotene than wild-type plants. Under heat stress (42 °C for 96 h), the edited plants exhibited enhanced thermotolerance by remaining greener and delayed wilting, whereas wild-type plants developed necrosis rapidly under heat stress. This enhanced thermotolerance might has resulted from the increased expression of ε-cyclase (an upstream key enzyme in β-carotene biosynthesis) as revealed by expression profiling of edited plants. In contrast, the expression of downstream β-cryptoxanthin and zeaxanthin biosynthetic genes was reduced, which seems consistent with mutation of BCH. DAB staining further confirmed reduced ROS accumulation in edited plants as compare to wild type supporting the protective role of β-carotene. In summary, the downregulation of BCH in N. tabacum boosted β-carotene levels and chlorophyll retention, enhancing heat stress tolerance. These findings demonstrate the potential of targeting carotenoid biosynthesis to improve crop resilience.},
}

*Nicotiana/genetics/metabolism/physiology
*Xanthophylls/metabolism
*Mixed Function Oxygenases/genetics/metabolism
*beta Carotene/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
*Heat-Shock Response/genetics
Chlorophyll/metabolism
Gene Expression Regulation, Plant

@article {pmid42679775,
year = {2026},
author = {Li, S and Liao, Y and Wang, X and Chen, G and Liu, Y and Pan, S and Zhen, T and Li, X and Zhu, F and Qin, R},
title = {Double-end blocker and split input mediated CRISPR-Cas12a system for modular single-base mutation detection with low abundance.},
journal = {Biosensors & bioelectronics},
volume = {314},
number = {},
pages = {119176},
doi = {10.1016/j.bios.2026.119176},
pmid = {42679775},
issn = {1873-4235},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Pancreatic Neoplasms/genetics/diagnosis ; *Proto-Oncogene Proteins p21(ras)/genetics ; DNA Mutational Analysis/methods ; Mutation ; Limit of Detection ; },
abstract = {Traditional CRISPR-Cas12a mutation detection systems are limited by poor single-base specificity, target-specific crRNA redesign, and insufficient sensitivity for low-abundance mutations, restricting their clinical liquid biopsy applications. Herein, we developed a crRNA-universal, sensitive and specific CRISPR-Cas12a detection platform, termed DESIC (double-end blocker and split-input mediated CRISPR-Cas12a system), for single-base mutation detection. The DESIC system adopts two key structural designs: double-end blocker (DEB) and duplicated split-input (SIN). The DEB spatially isolates crRNA recognition and target-binding regions, enabling universal detection of various mutation sites without crRNA redesign. The SIN strategy amplifies thermodynamic differences from single-base mismatches, greatly improving single-nucleotide discrimination. We targeted four prevalent pancreatic cancer KRAS mutations (G12D, G12R, G12V, Q61H) and optimized the system to achieve optimal discrimination. The optimized DESIC system exhibited ultra-low limits of detection down to 0.01% mutant allele fraction with reliable linear quantitative performance. Clinical validation using 15 pairs of pancreatic cancer tissue and peripheral blood samples confirmed that DESIC results were highly consistent with gold-standard NGS data. With a flexible modular design, this low-cost, easy-operated platform can be readily extended to multiple tumor mutations, holding great potential for tumor liquid biopsy and early molecular diagnosis.},
}

Humans
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Pancreatic Neoplasms/genetics/diagnosis
*Proto-Oncogene Proteins p21(ras)/genetics
DNA Mutational Analysis/methods
Mutation
Limit of Detection

@article {pmid42696818,
year = {2026},
author = {Zhang, S and Xie, S and Wang, J and Zhang, X and Cai, Y and Lai, X and Min, N and Qu, J and Peng, B and Li, T and Zhang, J and Yang, D and Shi, X and Xia, N and Ge, S},
title = {Mosaic switch for PAM-free and one-pot CRISPR/Cas12a detection.},
journal = {Biosensors & bioelectronics},
volume = {314},
number = {},
pages = {119177},
doi = {10.1016/j.bios.2026.119177},
pmid = {42696818},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *DNA/genetics/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics/chemistry ; DNA, Single-Stranded/chemistry/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas12a combined with nucleic acid amplification enables highly specific and sensitive detection. However, its broader deployment is constrained by protospacer adjacent motif (PAM) dependence, multistep workflows, and limited reagent practicality. Here, we identify a PAM-independent Cas12a activator, termed mosaic DNA, which exhibits structural features intermediate between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Building on this finding, we develop a PAM-independent mosaic switch triggered by a solid-liquid phase transition, thereby addressing these limitations within a single platform. Distinct from previous reports, our experiments show that this activator is generated prior to the digestion of dsDNA into ssDNA by the lambda exonuclease, forming the basis of lambda exonuclease-driven Cas12a activation, a process we refer to as the mosaic switch. Mosaic switch can detect arbitrary dsDNA with sensitivity comparable to that of PAM-containing dsDNA, and maintain single-nucleotide discrimination. Lyophilizing mosaic switch reagents and encapsulating them in paraffin improve stability and usability, which also enables straightforward one-pot integration with recombinase polymerase amplification (RPA) via a solid-liquid phase transition. Applied directly to 58 extraction-free mpox clinical samples, this platform showed complete concordance (100%) with quantitative PCR. This CRISPR/Cas12a platform maintains analytical performance while broadening the range of targets, simplifying the workflow, and enhancing reagent practicality, showing great potential for clinical deployment.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*DNA/genetics/chemistry
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/genetics/chemistry
DNA, Single-Stranded/chemistry/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42759108,
year = {2026},
author = {Tahira, M and Su, W and Maqsood, FK and Ikram, M and Javaid, MH and Rao, MJ and Zhang, J},
title = {CRISPR/Cas9 engineering: Insights into tomato fruit development, stress resilience via brassinosteroid pathways.},
journal = {Plant physiology and biochemistry : PPB},
volume = {238},
number = {},
pages = {111740},
doi = {10.1016/j.plaphy.2026.111740},
pmid = {42759108},
issn = {1873-2690},
mesh = {*Solanum lycopersicum/genetics/metabolism/growth & development ; *Brassinosteroids/metabolism ; *Fruit/growth & development/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Signal Transduction ; *Stress, Physiological ; Plant Growth Regulators/metabolism ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Tomato (Solanum lycopersicum), a cornerstone of global agriculture and nutrition, has undergone decades of breeding focused on yield and stress resilience. Yet, consumer-driven traits such as fruit uniformity, flavor complexity, and nutritional value remain suboptimal. The emerging convergence of hormonal biology and precision genome editing presents a transformative approach to addressing this gap. Brassinosteroids (BRs), a class of steroidal phytohormones, act as central regulators of cell expansion, tissue patterning, and developmental plasticity. Their signaling cascade, initiated by perception at the BRI1-BAK1 receptor complex and transduced via BES1/BZR1 transcriptional modules, intersects with networks controlling fruit set, morphology, ripening, and stress adaptation. Gene editing through CRISPR/Cas9 technology now enables targeted dissection and manipulation of these BR-regulated nodes with unprecedented accuracy. CRISPR/Cas9 studies have directly characterized BR signaling regulators such as SlBZR1, SlBIN2, and SlBES1, while complementary genetic and transgenic studies have provided functional evidence for other BR-related components, including SlBRI1 and the BR-biosynthetic gene SlDWF4, further supporting the roles of BR signaling in tomato fruit development, ripening, and carotenoid accumulation. Beyond improvement of traits, CRISPR/Cas9 offers the potential to tune BR pathways and their crosstalk with auxin and ethylene, providing a systems-level framework for engineering climate-resilient and nutritionally superior cultivars. This review integrates mechanistic insights into BR signaling with cutting-edge CRISPR/Cas9 applications, positioning tomato as a model for reprogramming fruit development and as a paradigm for next-generation crop improvement.},
}

*Solanum lycopersicum/genetics/metabolism/growth & development
*Brassinosteroids/metabolism
*Fruit/growth & development/metabolism/genetics
*CRISPR-Cas Systems/genetics
Signal Transduction
*Stress, Physiological
Plant Growth Regulators/metabolism
Plants, Genetically Modified
Plant Proteins/genetics/metabolism
Gene Editing

@article {pmid42786150,
year = {2026},
author = {Ramasamy, M and Li, G and Guo, L and Fang, H and Eid, A and Mandlik, R and Cheng, Y and Niedz, RP and Culver, JN and Coleman, GD and Liu, Z and Mandadi, KK and Qi, Y},
title = {Boosting genome editing in perennial plants by CRISPR-Combo mediated morphogenic gene activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42786150},
issn = {2041-1723},
support = {IOS-2132693//National Science Foundation (NSF)/ ; IOS-2224203//National Science Foundation (NSF)/ ; 2020-70029- 33161//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2021-70029-36056//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2025-70029-44033//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; },
mesh = {*Gene Editing/methods ; Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Solanum tuberosum/genetics ; Fragaria/genetics/growth & development ; *Genome, Plant ; Populus/genetics ; Plant Roots/genetics ; Transcriptional Activation ; Plant Shoots/genetics/growth & development ; Plant Proteins/genetics/metabolism ; Genes, Plant ; },
abstract = {A major bottleneck in genome editing of many perennial plants is their recalcitrance to transformation and regeneration. To boost genome editing in such perennial crops, transcriptional reprogramming of morphogenic genes is introduced by CRISPR-Combo, a versatile system for simultaneous genome editing and transcriptional activation in plant cells. In potato, we screen 17 morphogenic genes and identify 4 genes (WOX11/12, ARF5, ABI3-1, and ABI3-2) that promote regeneration of genome-edited hairy roots, and 3 of the 4 genes are also found to boost shoot regeneration by Agrobacterium-mediated stable transformation. Similarly, screening of 10 morphogenic genes in citrus leads to the identification of 5 genes (BBM3, FUS3, IPT1, SERK1, and STM) that enhance plant regeneration upon activation. In wild strawberry, we demonstrate that simultaneous activation of Baby Boom genes (BBM1 and BBM2) or of GRF3 and GIF1 reduces the generation time of genome-edited plants by over one month. Moreover, in poplar, we show that simultaneous activation of WUS and WOX11 synergistically promotes plant regeneration without exogenous plant hormones, which leads to a protocol of generating genome-edited poplar shoots in less than one month. Collectively, this study provides efficient strategies for boosting genome editing in four perennial crops.},
}

*Gene Editing/methods
Plants, Genetically Modified/genetics
*CRISPR-Cas Systems/genetics
Gene Expression Regulation, Plant
Solanum tuberosum/genetics
Fragaria/genetics/growth & development
*Genome, Plant
Populus/genetics
Plant Roots/genetics
Transcriptional Activation
Plant Shoots/genetics/growth & development
Plant Proteins/genetics/metabolism
Genes, Plant

@article {pmid42786170,
year = {2026},
author = {Liu, YV and Suryatenggara, J and Wong, H and Jayasinghe, MK and Tang, JP and Tan, HK and Kwon, J and Zhou, Q and Zhang, J and Moein, S and Ummarino, S and Le, MTN and Doench, JG and Benoukraf, T and Chai, L and Hiwase, D and Thomas, D and Di Ruscio, A and Tenen, DG and Bassal, MA},
title = {Methylation Mesa define functional regulatory elements for targeted gene activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42786170},
issn = {2041-1723},
support = {P01 HL158688/HL/NHLBI NIH HHS/United States ; 2016-0476//Fondazione Cariplo (Cariplo Foundation)/ ; P01HL158688//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; P01 HL131477/HL/NHLBI NIH HHS/United States ; R00 CA188595/CA/NCI NIH HHS/United States ; R35 CA197697/CA/NCI NIH HHS/United States ; R01 DK136116/DK/NIDDK NIH HHS/United States ; },
mesh = {Humans ; Animals ; *DNA Methylation/genetics ; Mice ; Promoter Regions, Genetic/genetics ; *Transcriptional Activation ; Epigenesis, Genetic ; CpG Islands/genetics ; Chromatin/metabolism ; CRISPR-Cas Systems ; Epigenome Editing ; 5' Untranslated Regions/genetics ; *Regulatory Sequences, Nucleic Acid/genetics ; },
abstract = {DNA methylation regulates transcription, yet the demethylation of canonical elements like promoter CpG islands exhibits inconsistent correlations with gene activation. We hypothesize that causal regulatory elements are defined by biophysical hypersensitivity. Profiling 24 whole-genome bisulfite sequencing samples across diverse human and murine models, we identify Methylation Mesa, narrow (~45-300 bp), structurally conserved epigenetic regulatory elements. Mesa show enrichment in 5' untranslated regions and associate with transcriptional activation significantly better than canonical promoters. To investigate causal regulatory dynamics, we develop CRISPR-DiR, an RNA-based targeted demethylation technology offering greater spatial precision, higher potency, and reduced toxicity compared to CRISPR-TET1. While proximal promoter demethylation initiated limited early transcription, focal demethylation of the Mesa seed acts as the primary driver of exponential CDKN2A (p16) reactivation and robust in vivo tumor suppression. We demonstrate that precise demethylation of a Mesa locus triggers localized demethylation, subsequent activation histone mark deposition, and long-range three-dimensional chromatin rewiring. Thus, Methylation Mesa act as precise, dominant epigenetic regulatory hubs, and CRISPR-DiR as a potent high-resolution tool, establishing a structural framework for biomarker discovery and targeted therapies.},
}

Humans
Animals
*DNA Methylation/genetics
Mice
Promoter Regions, Genetic/genetics
*Transcriptional Activation
Epigenesis, Genetic
CpG Islands/genetics
Chromatin/metabolism
CRISPR-Cas Systems
Epigenome Editing
5' Untranslated Regions/genetics
*Regulatory Sequences, Nucleic Acid/genetics

@article {pmid42786391,
year = {2026},
author = {Ghasemi, R and Heidari, H},
title = {Nucleic Acid-Based Antimicrobial Strategies Against Drug-Resistant Staphylococcus aureus: A Review of Mechanisms of Action and Delivery Approaches.},
journal = {Infectious diseases and therapy},
volume = {},
number = {},
pages = {},
pmid = {42786391},
issn = {2193-8229},
abstract = {Drug-resistant Staphylococcus aureus remains one of the major clinical challenges in both community and hospital settings. Increasing rates of resistance to conventional antibiotics have prompted the development of alternative therapeutic strategies capable of selectively targeting essential bacterial functions. Nucleic acid-based antimicrobial strategies have emerged as promising and precise approaches, enabling the inhibition of gene expression, suppression of virulence, restoration of antibiotic susceptibility, and inhibition of bacterial growth in a sequence-specific manner. This review investigates various studies on antisense oligonucleotides (ASOs), small RNA-based approaches, and CRISPR-Cas systems against S. aureus, with a focus on their antimicrobial efficacy, mechanisms of action, and delivery strategies. These platforms can target genes involved in cell division, transcription, quorum sensing, biofilm formation, and antibiotic resistance. As efficient intracellular delivery remains a major barrier, multiple carriers-including cell-penetrating peptides, nanoparticles, liposomes, DNA nanostructures, and phagemid-based platforms-have been developed to enhance therapeutic efficacy. It has been demonstrated that optimized delivery systems can substantially improve the stability, cellular uptake, and antibacterial activity of nucleic acid therapeutics. Furthermore, the programmability and high target specificity of these agents is a step forward, facilitating the development of precision antimicrobial therapies with the potential for reduced effects on non-target bacterial populations. Overall, the available data support the potential of nucleic acid-based antimicrobials as promising preclinical adjuncts to conventional antibiotics for combating multidrug-resistant S. aureus.},
}

@article {pmid42788230,
year = {2026},
author = {Kumar Mandal, R and Halder, J and Kumar, A and Kurmi, BD and Kaur, K and Sharma, A},
title = {Smart nanocarriers against MDR-tuberculosis: stimuli-responsive strategies for granuloma penetration and next generation therapy.},
journal = {Therapeutic delivery},
volume = {},
number = {},
pages = {1-24},
doi = {10.1080/20415990.2026.2732635},
pmid = {42788230},
issn = {2041-6008},
abstract = {Multidrug-resistant tuberculosis (MDR-TB) remains a global health concern due to long treatment durations, limited drug penetration into granulomatous lesions, systemic toxicity, and the persistence of dormant Mycobacterium tuberculosis. The granuloma microenvironment, characterized by hypoxia, acidic pH, enzymatic activity, dense extracellular matrix, and redox imbalance, acts as a barrier to the effectiveness of conventional antitubercular therapy. Recent advances in nanotechnology have led to the development of stimuli-responsive nanocarriers that enable targeted, controlled, and on-demand drug delivery in response to disease-specific stimuli. This narrative review summarizes peer-reviewed literature published between 2015 and 2025 on stimuli-responsive nanocarrier systems for MDR-TB therapy, retrieved from major scientific databases, including PubMed, Scopus, Google Scholar, ScienceDirect, Web of Science, and ResearchGate, focusing on granuloma-targeted drug delivery and nanomedicine strategies. Stimuli-responsive liposomes, polymeric nanoparticles, dendrimers, and metallic nanocarriers demonstrate enhanced pulmonary accumulation with improved bacterial targeting and reduced systemic toxicity compared to free drugs. In addition, co-delivery strategies incorporating host-directed immunomodulators further improve bactericidal efficacy. Emerging approaches, including CRISPR-Cas‑based nano-therapies, RNA therapeutics, regenerative and stem cell-based strategies, artificial intelligence‑guided nanocarrier design, and biohybrid delivery systems, represent important future directions. Overall, stimuli-responsive nanocarriers, integrated with next-generation technologies, offer a promising pathway toward precise and effective MDR-TB treatment.},
}

@article {pmid42213070,
year = {2026},
author = {Nakanishi, K and Takano, Y and Yamamoto, K and Matsuda, Y and Yano, M and Mito, K and Ichino, T and Tatsumi, K and Li, H and Ohara, K and Munakata, R and Suzuki, H and Sakurai, N and Shibata, D and Osakabe, K and Watanabe, B and Okada, T and Shimomura, K and Takanashi, K and Sugiyama, A and Yazaki, K},
title = {CRISPR/Cas9-mediated genome editing reveals the involvement of a polyphenol oxidase in the shikonin-specific biosynthesis in Lithospermum erythrorhizon.},
journal = {Plant & cell physiology},
volume = {67},
number = {9},
pages = {1557-1572},
doi = {10.1093/pcp/pcag074},
pmid = {42213070},
issn = {1471-9053},
support = {19H05638//Japan Society for the Promotion of Science KAKENHI/ ; 22K05438//Japan Society for the Promotion of Science KAKENHI/ ; 25K08838//Japan Society for the Promotion of Science KAKENHI/ ; JPMJSP2110//Japan Science and Technology Agency (JST) SPRING/ ; NEDO 16100890-0//New Energy and Industrial Technology Development Organization/ ; //RIKEN Cluster for Science, Technology and Innovation Hub/ ; //Institute for the Future of Human Society of Kyoto University for 2023/ ; //Jikei University Research Fund/ ; //Humanosphere Science Research on Sustainable Humanosphere Science/ ; //Kyoto University/ ; },
mesh = {*Lithospermum/genetics/metabolism/enzymology ; *Naphthoquinones/metabolism ; *CRISPR-Cas Systems/genetics ; *Catechol Oxidase/metabolism/genetics ; Plant Roots/metabolism/genetics ; Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; },
abstract = {Shikonin, a 1,4-naphthoquinone derivative produced by some limited Boraginaceae species, exhibits unique pharmacological properties and is also used as a natural dye. The regulatory factors of shikonin production have been demonstrated using a cell culture system of Lithospermum erythrorhizon. Among these factors, copper is known to be the strongest enhancer of shikonin production, and the copper-requiring biosynthetic step is shikonin-specific, unlike the byproduct benzoquinones. Despite the long history of shikonin biosynthesis studies for over 40 years, the copper-involved reaction has been still unknown. Here, we explored candidate genes associated with shikonin production using a PCR-select subtraction experiment. Genes encoding PPO, a dicopper-dependent oxidoreductase, was highlighted by the strong synchronous expression with shikonin production. Transcriptome analysis of hairy roots and cultured cells of this plant revealed that, of the five PPO genes expressed in L. erythrorhizon (LePPOs), only LePPO1 showed a close correlation with shikonin production. Then, we generated genome-edited hairy roots of LePPO1 using CRISPR/Cas9-mediated mutagenesis to analyze its impact on shikonin derivative and other specialized metabolite production. The results showed that shikonin content was markedly reduced in all LePPO1-ge lines, while the content of deoxyshikonofuran, a hydroquinone-type shunt product that branches after 3''-hydroxygeranylhydroquinone in the shikonin biosynthetic pathway, remained unaffected in the LePPO1-ge lines. These findings address the question of why a copper ion is crucial for shikonin biosynthesis and suggest that LePPO1 participates in naphthalene ring formation. Interestingly, LePPO1 is localized in plastids, whereas shikonin accumulates in the apoplast.},
}

*Lithospermum/genetics/metabolism/enzymology
*Naphthoquinones/metabolism
*CRISPR-Cas Systems/genetics
*Catechol Oxidase/metabolism/genetics
Plant Roots/metabolism/genetics
Plant Proteins/metabolism/genetics
Gene Expression Regulation, Plant

@article {pmid42487027,
year = {2026},
author = {Kam, KM and Shiu, TE and Hsieh, CM and Lu, WT and Pan, YY and Lin, CI and Lin, PH and Chuang, YH and Yu, IS and Chen, PL},
title = {Generation and characterization of humanized CD4 knock-in mice expressing chimeric mouse/human CD4 protein.},
journal = {Lab animal},
volume = {55},
number = {10},
pages = {402-412},
pmid = {42487027},
issn = {1548-4475},
support = {112-S0309//National Taiwan University Hospital (NTUH)/ ; },
mesh = {Animals ; Humans ; *Gene Knock-In Techniques ; Mice ; *CD4 Antigens/genetics/metabolism ; Mice, Transgenic/genetics ; CD4-Positive T-Lymphocytes ; CRISPR-Cas Systems ; Female ; Male ; },
abstract = {Humanized mouse models have become indispensable tools for investigating human gene function and disease modeling. However, conventional transgenic approaches carry the risk of unforeseen biological consequences. Here, to address this concern, we developed a novel human CD4 knock-in (hCD4 KI) mouse model using CRISPR-Cas9 gene-editing technology. We replaced the region encoding the first two major extracellular domains of the mouse Cd4 gene, which are critical for interaction with major histocompatibility complex (MHC) class II, with the corresponding human CD4 sequence. Subsequently, we conducted comprehensive physiological and immune system analyses on hCD4 KI mice, including both heterozygous (CD4[m/h]) and homozygous (CD4[h/h]) genotypes. Our investigations revealed a dosage-dependent impact of the hCD4 KI, resulting in a decreased percentage of CD4[+] single-positive cells, accompanied by a corresponding increase in CD8[+] single-positive cells within the thymus. These developmental alterations, evident in the thymus, were also observed in the peripheral lymphatic system such as the spleen and in the peripheral blood, exhibiting an increased population of mature CD8[+] T cells and a decreased proportion of mature CD4[+] T cells. Despite these changes, hCD4 KI mice exhibited normal biological characteristics, including T cell activation and proliferation functions, blood composition, tissue structure and body weight, closely resembling those of wild-type (CD4[m/m]) mice. Our study underscores hCD4 KI mice as a valuable tool for exploring CD4 and MHC class II interactions, with potential for future integration with humanized MHC class II KI mice, offering insights into immune disease mechanisms.},
}

Animals
Humans
*Gene Knock-In Techniques
Mice
*CD4 Antigens/genetics/metabolism
Mice, Transgenic/genetics
CD4-Positive T-Lymphocytes
CRISPR-Cas Systems
Female
Male

@article {pmid42615605,
year = {2026},
author = {Kim, LM and Koo, B-M and Gross, CA and Todor, H},
title = {Elevated temperature undermines the efficacy of Streptococcus pyogenes dCas9-based CRISPRi systems.},
journal = {mSphere},
volume = {11},
number = {9},
pages = {e0037926},
doi = {10.1128/msphere.00379-26},
pmid = {42615605},
issn = {2379-5042},
support = {R35 GM118061/NH/NIH HHS/United States ; },
mesh = {*Streptococcus pyogenes/genetics/enzymology ; Bacillus subtilis/genetics ; *CRISPR-Cas Systems ; *Hot Temperature ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Temperature ; },
abstract = {UNLABELLED: Targeted gene repression using CRISPRi has become a foundational technique for microbiology. The effector protein in most CRISPRi systems is a catalytically inactivated version of Cas9, an RNA-guided DNA endonuclease from Streptococcus pyogenes (SpCas9). The application of SpCas9-based CRISPRi systems in increasingly ambitious experiments in diverse bacteria raises important questions about the effects of growth temperature on CRISPRi efficacy, particularly because SpCas9 loses cutting efficacy at elevated temperatures. Here, we test the effect of temperature on SpCas9-based CRISPRi knockdown efficacy by performing a series of genome-wide screens using a well-characterized CRISPRi system in the model firmicute Bacillus subtilis. We observe very little repression at high (45°C) growth temperatures and confirm this observation using single cell measurements of GFP fluorescence in CRISPRi strains grown at different temperatures. Our data suggest that Streptococcus pyogenes-based CRISPRi systems are ill-suited for thermophiles and should not be used for elevated-temperature or heat shock experiments in mesophilic bacteria.

IMPORTANCE: CRISPRi is a common technique for inducible gene depletion in bacteria and has been used for many different types of experiments in increasingly diverse bacteria. We identify increased temperatures as an important limitation of the most common CRISPRi effector (Streptococcus pyogenes dCas9). This finding both affects experimental design and informs the kinds of bacterial species amenable to manipulation by S. pyogenes-based CRISPRi systems.},
}

*Streptococcus pyogenes/genetics/enzymology
Bacillus subtilis/genetics
*CRISPR-Cas Systems
*Hot Temperature
*CRISPR-Associated Protein 9/genetics/metabolism
*Temperature

@article {pmid42627152,
year = {2026},
author = {Danzeisen, EL and Lihon, MV and Milholland, KL and Bias, TR and Bates, AF and Hall, MC},
title = {Expansion and optimization of the auxin-inducible degron 2 (AID2) system in Candida pathogens.},
journal = {mSphere},
volume = {11},
number = {9},
pages = {e0035926},
doi = {10.1128/msphere.00359-26},
pmid = {42627152},
issn = {2379-5042},
support = {AI168050, AI174123//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Indoleacetic Acids/pharmacology ; Degrons ; CRISPR-Cas Systems ; *Candida albicans/genetics/drug effects ; *Fungal Proteins/genetics ; *Gene Editing/methods ; Genetic Vectors ; },
abstract = {UNLABELLED: The auxin-inducible degron (AID) technology is a convenient and powerful tool for protein functional characterization in a broad array of eukaryotic species. We recently demonstrated that the original AID and improved AID2 systems are very effective at rapid protein depletion in Candida albicans, and described a limited set of reagents for their use in certain auxotrophic lab strains. With an eye toward broader applicability with improved flexibility, we report here a new series of template vectors suitable for employing AID2 technology in prototrophic C. albicans strains, such as clinical isolates and the reference strain SC5314. We adapted a common recyclable antibiotic marker system for the required genome editing steps, and developed a strategy for simultaneous CRISPR/Cas9-mediated tagging of both target alleles. We also developed a composite all-in-one tagging cassette that combines the degron tag and the OsTIR1[F74A] gene for single-step strain engineering. We added a fluorescent protein tag option and designed and validated an approach for N-terminal tagging that retains natural promoter control. We also compared the effectiveness of the two commonly used synthetic auxins, 5-phenyl-indole-3-acetic acid and 5-adamantyl-indole-3-acetic acid, and the two common OsTIR1 variants, F74A and F74G, and provide guidelines for using the new AID2 system. Finally, using the novel all-in-one cassette, we demonstrate that the AID2 system also works in Candida auris, albeit less effectively under some conditions. The new reagents should enhance the convenience and accessibility of the AID2 system for the Candida research community.

IMPORTANCE: Invasive fungal infections, including those caused by Candida species, are a persistent global health problem, and their treatment is hindered by limited antifungal options and the emergence of drug resistance. There is an urgent need for tools and methods to accelerate the discovery of novel therapeutic targets. The expanded and optimized auxin-inducible degron system described herein provides a versatile platform for characterizing protein function and dissecting pathways governing important traits like virulence, stress tolerance, and antifungal resistance. The new reagents make AID technology applicable to any strain. Ultimately, this enhanced toolkit has the potential to help identify and validate new high‑value drug targets and deepen our understanding of molecular mechanisms that drive pathogenicity of Candida and other fungal pathogen species.},
}

*Indoleacetic Acids/pharmacology
Degrons
CRISPR-Cas Systems
*Candida albicans/genetics/drug effects
*Fungal Proteins/genetics
*Gene Editing/methods
Genetic Vectors

@article {pmid42677878,
year = {2026},
author = {Qin, H and Liu, J and Luo, Y and Yu, J and Xiao, W and Tang, Y and Quan, Q},
title = {Universal CRISPR/Cas12a-G4 DNAzyme biosensing platform for rapid detection of nucleic acid and non-nucleic acid targets.},
journal = {The Analyst},
volume = {151},
number = {19},
pages = {5774-5782},
doi = {10.1039/d6an00567e},
pmid = {42677878},
issn = {1364-5528},
mesh = {*DNA, Catalytic/chemistry/metabolism ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Associated Proteins/chemistry/metabolism ; *CRISPR-Cas Systems ; Limit of Detection ; *SARS-CoV-2/genetics/isolation & purification ; Prostate-Specific Antigen/analysis/blood ; *RNA, Viral/analysis ; *Endodeoxyribonucleases/metabolism/chemistry ; Benzidines/chemistry ; COVID-19/diagnosis/virology ; G-Quadruplexes ; Hydrogen Peroxide/chemistry ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) system enables sensitive and specific biomolecular detection due to its programmability, high fidelity, and signal amplification. Herein, a novel universal CRISPR/Cas12a-G4 DNAzyme-TMB (Cas-GT) enzymatic biosensing platform was constructed. This platform regulates the catalytic function of G4 DNAzyme through the trans-cleavage activity of Cas12a, achieving an "off/on" response of the TMB-H2O2 enzymatic signal for nucleic acid, protein, and metal ion targets. In proof-of-concept experiments, the detection limit of Cas-GT for in vitro transcribed SARS-CoV-2 RNA reached as low as 100 aM and it distinguished clinical positive from negative patients with good diagnostic performance (AUC = 0.9420). It is also suitable for protein targets, enabling quantitative analysis of prostate-specific antigen (PSA) within the range of 0-100 ng mL[-1], with results highly consistent with clinical chemiluminescence immunoassay (CLIA). For small-molecule targets, Cas-GT exhibited good universality, achieving quantitative detection of Hg[2+] within the range of 0.06-4 ng mL[-1], with recovery rates of 93%-107.4% in spiked river water samples, showing no significant difference from ICP-MS (P = 0.9968). In summary, Cas-GT is a simple, sensitive, rapid, and label-free enzymatic biosensing platform with significant potential for clinical biomarker detection and environmental pollution monitoring.},
}

*DNA, Catalytic/chemistry/metabolism
*Biosensing Techniques/methods
Humans
*CRISPR-Associated Proteins/chemistry/metabolism
*CRISPR-Cas Systems
Limit of Detection
*SARS-CoV-2/genetics/isolation & purification
Prostate-Specific Antigen/analysis/blood
*RNA, Viral/analysis
*Endodeoxyribonucleases/metabolism/chemistry
Benzidines/chemistry
COVID-19/diagnosis/virology
G-Quadruplexes
Hydrogen Peroxide/chemistry
*Bacterial Proteins/metabolism/chemistry

@article {pmid42716355,
year = {2026},
author = {Wang, X and Huang, Y and Zhang, L and Zheng, L},
title = {Emerging strategies for regulating Cas12a and Cas13a trans-cleavage activity in biosensing.},
journal = {Biotechnology advances},
volume = {93},
number = {},
pages = {109033},
doi = {10.1016/j.biotechadv.2026.109033},
pmid = {42716355},
issn = {1873-1899},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas12a and Cas13a have become widely used platforms for molecular diagnostics and biosensing, because target recognition can trigger collateral nucleic acid cleavage for signal amplification. Leveraging this mechanism, numerous biosensing platforms have been developed with high sensitivity, high specificity, and compatibility with point-of-care testing. However, their analytical performance depends strongly on the regulation of trans-cleavage activity. This review summarizes recent advances in the molecular basis and activity regulation of CRISPR-Cas12a- and Cas13a-based biosensing systems. We discuss the target-recognition and conformational activation mechanisms underlying Cas12a and Cas13a trans-cleavage. We then examine how Cas effector properties, crRNA architecture and composition, activator accessibility and structure, reaction conditions, and reporter design regulate target-induced activation and signal output. We further highlight the use of these strategies for controlled activation, tunable signal generation, and broader biosensing applications. By organizing these strategies across different regulatory layers, this review aims to provide a framework for designing more controllable and adaptable CRISPR trans-cleavage biosensing systems.},
}

*Biosensing Techniques
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism/genetics
*Bacterial Proteins/genetics/metabolism

@article {pmid42734156,
year = {2026},
author = {Lyu, C and Hall, SD and Stamnes, MA and Chen, S},
title = {In vivo CRISPR screening links NFKB1 to endocrine resistance in ER+ breast cancer.},
journal = {Endocrine-related cancer},
volume = {33},
number = {9},
pages = {},
doi = {10.1530/ERC-25-0463},
pmid = {42734156},
issn = {1479-6821},
mesh = {Humans ; Animals ; *Breast Neoplasms/genetics/drug therapy/metabolism/pathology ; Female ; *Drug Resistance, Neoplasm/genetics ; Mice ; *NF-kappa B p50 Subunit/genetics/metabolism ; Tamoxifen/pharmacology/therapeutic use ; CRISPR-Cas Systems ; *Antineoplastic Agents, Hormonal/therapeutic use/pharmacology ; Mice, Nude ; Receptors, Estrogen/metabolism ; MCF-7 Cells ; },
abstract = {Resistance to endocrine therapy (ET) remains a major clinical challenge in the treatment of estrogen receptor-positive (ER+) breast cancer, underscoring the need for novel therapeutic targets. To identify genetic drivers of ET resistance, we conducted an in vivo genome-wide CRISPR-Cas9 screen in MCF7 cells implanted into ovariectomized nude mice under estrogen-deprived conditions. Despite the bottlenecks inherent to in vivo pooled screening, recurrent enrichment analysis identified NFKB1 as a candidate regulator of estrogen-independent tumor progression. Functional studies confirmed that NFKB1 deficiency enhanced tumorigenicity and conferred resistance to tamoxifen and fulvestrant both in vitro and in vivo. Mechanistically, transcriptomic and biochemical analyses revealed that NFKB1 deficiency activated canonical NF-κB signaling, leading to inflammatory gene induction and enhanced ER signaling. Furthermore, pharmacologic inhibition of NF-κB signaling restored ET sensitivity in NFKB1-deficient cells. Analysis of TCGA breast cancer datasets revealed reduced NFKB1 expression in luminal breast tumors, whereas expression of other NF-κB family members was largely unchanged. Further analysis showed that low NFKB1 expression was associated with poorer clinical outcomes in patients with ER+ breast cancer. Collectively, these findings identify NFKB1 as a negative regulator of NF-κB signaling and endocrine resistance in ER+ breast cancer and provide mechanistic evidence linking NF-κB activation to ligand-independent ER signaling. Our results support further investigation of NFKB1 as a candidate biomarker and of NF-κB pathway inhibition as a potential therapeutic strategy in endocrine therapy-resistant breast cancer. These findings also illustrate the utility of in vivo CRISPR screening for identifying candidate regulators of endocrine resistance in breast cancer.},
}

Humans
Animals
*Breast Neoplasms/genetics/drug therapy/metabolism/pathology
Female
*Drug Resistance, Neoplasm/genetics
Mice
*NF-kappa B p50 Subunit/genetics/metabolism
Tamoxifen/pharmacology/therapeutic use
CRISPR-Cas Systems
*Antineoplastic Agents, Hormonal/therapeutic use/pharmacology
Mice, Nude
Receptors, Estrogen/metabolism
MCF-7 Cells

@article {pmid42782117,
year = {2026},
author = {Chhanda, MS and Ladhari, E and Zerillo, L and Paquet, VE and St-Laurent, RE and Marcoux, P-É and Turmel, A and Denicourt, M and Lambert, M-È and Vincent, AT and Charette, SJ},
title = {Complete genome sequences of 22 Staphylococcus spp. bacteriophages isolated from a single swine farm environment in Quebec, Canada.},
journal = {Microbiology resource announcements},
volume = {},
number = {},
pages = {e0088626},
doi = {10.1128/mra.00886-26},
pmid = {42782117},
issn = {2576-098X},
abstract = {Twenty-two bacteriophages infecting Staphylococcus spp. were isolated from water and manure collected on a Quebec swine farm affected by exudative epidermitis. Phylogenetic analysis of their genomes showed that 20 of them clustered into two groups, Twort- and ShyM-like. No CRISPR-Cas systems, antimicrobial resistance determinants, or virulence genes were identified.},
}

@article {pmid42782528,
year = {2026},
author = {Madhankumar, P and Sharma, GK and Balaji, KGS and Singh, K and Dixit, S and Khandelwal, S and Mahesh, PR and Manju, R and Mathesh, K and Gupta, PK and Agrawal, RK and Mahajan, S},
title = {A field deployable duplex RPA-CRISPR/Cas12a assay for rapid and sensitive detection of African swine fever virus.},
journal = {Archives of virology},
volume = {171},
number = {10},
pages = {},
pmid = {42782528},
issn = {1432-8798},
mesh = {*African Swine Fever Virus/genetics/isolation & purification ; Animals ; *African Swine Fever/diagnosis/virology ; Swine ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {African swine fever (ASF) is a notifiable transboundary disease of domestic pigs and wild suids that causes severe haemorrhagic fever, with high mortality, and substantial socio-economic losses worldwide. In the absence of effective vaccines or therapeutics, ASF control relies primarily on early diagnosis, strict biosecurity, and stamping-out strategies. In this study, we developed a rapid, field-deployable recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay for the detection of African swine fever virus using both fluorescence- and lateral flow strip (LFS)-based readouts. The assay operates isothermally at 37 °ºC and enables rapid, equipment-free visual detection suitable for on-site testing. To improve diagnostic sensitivity and robustness, a dual-target approach was employed targeting the highly conserved C-terminal region of the p72 (B646L) gene and the early-expressing p22 (KP177R) gene. In the uniplex format, fluorescence-based assays achieved limits of detection of 10.18 and 13.68 copies/reaction for p72 and KP177R, respectively, whereas the LFS format detected 1,018 and 1,368 copies/reaction. Notably, the duplex RPA-CRISPR/Cas12a assay demonstrated enhanced sensitivity with detection limits of 1.02 copies/reaction in the fluorescence format and 102 copies/reaction in the LFS format. The assay showed 100% analytical specificity against other porcine viral pathogens and strong agreement with real-time PCR. Furthermore, lyophilized reagents formulated in a two-tube format retained their analytical performance after storage at 4 °C for one week, highlighting the platform's potential for decentralized ASF surveillance and outbreak response.},
}

*African Swine Fever Virus/genetics/isolation & purification
Animals
*African Swine Fever/diagnosis/virology
Swine
Sensitivity and Specificity
Rapid Diagnostic Tests
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods

@article {pmid42782748,
year = {2026},
author = {Wieland, J and Jackson, P and Penrod, W and Nadauld, S and Barrott, J},
title = {Beyond Precision: A Multidimensional Framework for Selecting Genetic Medicine Platforms.},
journal = {Cells},
volume = {15},
number = {18},
pages = {},
pmid = {42782748},
issn = {2073-4409},
mesh = {Humans ; *Genetic Therapy/methods ; *Precision Medicine/methods ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Gene therapy is undergoing continued clinical translation and technological development. This progress has been marked by regulatory approvals and broadened therapeutic indications across genetic, metabolic, and oncologic diseases and disorders. The field has evolved over decades from early viral-mediated gene addition to approaches capable of targeted editing, regulation, or replacement of genetic information. These systems include base and prime editors, epigenetic modulators, CRISPR-Cas, RNA therapeutics and programmable integration platforms. When paired with increasingly sophisticated viral and nonviral delivery strategies, these technologies enable greater control over tissue targeting, duration of activity, and therapeutic exposure. Recent clinical successes, including approved ex vivo CRISPR-based therapies for hemoglobinopathies, in vivo CRISPR editing for transthyretin amyloidosis, and emerging clinical applications of base and prime editing, provide growing clinical evidence for the feasibility of genetic medicines. However, technological advancement has also made platform selection increasingly complex. Therapeutic performance is determined not by editing efficiency alone, but by the interaction among genetic precision, temporal control, dosage tunability, delivery efficiency, durability, and disease-specific safety requirements. A molecularly efficient platform may still have limited therapeutic value if it cannot reach the disease-relevant cell population at sufficient and safe exposure. In this review, we examine recent technological and clinical advances in genetic medicine with particular emphasis on developments during the past approximately five years. We propose a multidimensional framework in which gene therapy platforms are evaluated according to three intrinsic properties-genetic precision, temporal control, and dosage tunability-while delivery, clinical maturity, and disease context act as major translational constraints. This framework highlights that no single platform is universally optimal; rather, successful therapeutic design depends on matching the biological characteristics of the intervention to the requirements of the disease and target tissue. Remaining challenges in extrahepatic delivery, genomic safety, immunogenicity, manufacturing, and long-term monitoring remain important determinants of broader clinical implementation.},
}

Humans
*Genetic Therapy/methods
*Precision Medicine/methods
Gene Editing/methods
CRISPR-Cas Systems/genetics
Animals

@article {pmid42783150,
year = {2026},
author = {Ali, NB and Xiao, Y and Jin, K and Wang, Y and Zhao, Y},
title = {Microfluidic-Integrated CRISPR-Cas Biosensor for Marine Pollutant and Pathogen Monitoring: A Review.},
journal = {Biosensors},
volume = {16},
number = {9},
pages = {},
pmid = {42783150},
issn = {2079-6374},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; *Environmental Monitoring ; *Microfluidics ; Seawater ; },
abstract = {Marine ecosystems face escalating threats from heavy metals, harmful algal bloom toxins, pathogens, and antibiotic resistance genes, yet conventional detection methods remain laboratory-dependent and incapable of real-time, multiplexed field monitoring. CRISPR-Cas diagnostics, leveraging programmable Cas12a/Cas13a trans-cleavage for attomolar-level sensitivity, offers a transformative solution when integrated with microfluidic platforms that provide the automation and miniaturisation required for field deployment. This review systematically examines this emerging convergence across four marine target classes: heavy metals, biotoxins, pathogens, and resistance genes alongside integration architectures, signal readout strategies, and comparative performance benchmarking. We identify that only a small fraction of reported platforms have been validated in authentic seawater, with cross-class multiplexing, biofouling resistance during autonomous deployment, and regulatory standardisation remaining largely unaddressed. By synthesising this rapidly developing literature and articulating these unresolved challenges, this review provides a foundational reference and research agenda for translating microfluidic-CRISPR biosensors from laboratory proof-of-concept to operational marine environmental surveillance.},
}

*Biosensing Techniques
*CRISPR-Cas Systems
*Environmental Monitoring
*Microfluidics
Seawater

@article {pmid42783548,
year = {2026},
author = {Tentler, K and Snider, PL and Matias, C and Brault, JJ and Conway, SJ},
title = {Diaphragmatic Mitochondrial Myopathy in a Patient-Derived Mouse Model of Barth Syndrome.},
journal = {Journal of developmental biology},
volume = {14},
number = {3},
pages = {},
pmid = {42783548},
issn = {2221-3759},
support = {R01 HL159436/HL/NHLBI NIH HHS/United States ; 1R01HL159436-05/NH/NIH HHS/United States ; },
abstract = {Barth syndrome (BTHS) is a rare, X-linked genetic disorder caused by mutations in the enzyme TAFAZZIN (TAZ), resulting in insufficient cardiolipin (CL) remodeling and mitochondrial dysfunction. While BTHS respiratory distress and breathing difficulties are commonly reported, the precise role of intrinsic respiratory tissue vulnerabilities has only recently begun to be appreciated. Historically, BTHS respiratory distress is frequently attributed to secondary consequences like cardiomyopathy or generalized skeletal myopathy, leaving the intrinsic vulnerability of vital respiratory muscles poorly understood. Using a patient-tailored point mutant knock-in mouse model (Taz[PM]) harboring a stable but enzymatically deficient Taz[D75H] protein, we investigated the autonomous physiological and metabolic responses in the diaphragm and lungs. Contrary to the paradigm that respiratory muscles are unaffected, Taz[PM] diaphragms exhibit structurally abnormal mitochondria and undergo a survival-critical, bifurcated compensatory remodeling response to prevent fatal respiratory failure under severe bioenergetic stress. The Taz[PM] adaptive mechanism is orchestrated by chronic activation of the mitochondrial Integrated Stress Response (ISR) via the Gcn2/eIF2α signaling pathway. This stress pathway halts global translation to conserve cellular ATP at the expense of reduced NAD+ levels, while selectively upregulating defensive mitokines and metabolic sirtuins and structural muscle remodeling. Furthermore, the Taz[PM] diaphragm transitions into a highly specialized, slow-twitch motor system that is expected to reduce the energy cost per contraction. Concurrently, despite Taz[PM] lungs exhibiting structurally abnormal mitochondria, they resist generalized mitochondrial collapse despite ADP reduction, executing tissue-specific metabolic reprogramming and localized biochemical adaptations to sustain respiratory homeostasis.},
}

@article {pmid42399777,
year = {2026},
author = {Bachler, A and Walsh, TK and Andrews, D and Williams, M and Tay, WT and Gordon, K and James, B and Fang, C and Wang, L and Wu, Y and Stone, EA and Padovan, A},
title = {Transposable element disruption of a second thyroglobulin-like gene confers Vip3Aa resistance in Helicoverpa armigera.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {42399777},
issn = {1471-2164},
mesh = {Animals ; *Helicoverpa armigera/genetics/drug effects ; *DNA Transposable Elements ; *Bacterial Proteins/genetics ; CRISPR-Cas Systems ; *Thyroglobulin/genetics ; *Insect Proteins/genetics ; },
abstract = {BACKGROUND: The cotton bollworm Helicoverpa armigera is a major global pest controlled by genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins, including Vip3Aa. While Vip3Aa is widely deployed, the genetic basis of resistance remains poorly understood. Previous work identified disruption of a thyroglobulin-like gene (HaVipR1) as one mechanism of resistance, suggesting additional loci may be involved.

RESULTS: Using linkage analysis, transcriptomics, long-read sequencing, and CRISPR-Cas9 gene editing, we identify a second thyroglobulin-like gene, HaVipR2, as a novel mediator of Vip3Aa resistance. Resistance in a field-derived H. armigera line was shown to be monogenic, recessive, and autosomal, mapping to chromosome 29. Long-read sequencing revealed a ~ 16 kb transposable element insertion disrupting HaVipR2, which was undetectable using standard short-read approaches. CRISPR-Cas9 knockout of HaVipR2 conferred > 900-fold resistance, confirming its causal role. Comparative analyses show that HaVipR1 and HaVipR2 share conserved domain architecture, indicating that thyroglobulin-domain proteins represent a recurrent target of resistance evolution.

CONCLUSIONS: Our findings establish thyroglobulin-domain proteins as a new class of Bt resistance genes in Lepidoptera and demonstrate that transposable element insertions can drive adaptive resistance while evading detection by conventional methods. These results highlight the importance of long-read sequencing and accurate genome annotation for resistance monitoring and provide new insights into the molecular basis and evolution of Vip3Aa resistance.},
}

Animals
*Helicoverpa armigera/genetics/drug effects
*DNA Transposable Elements
*Bacterial Proteins/genetics
CRISPR-Cas Systems
*Thyroglobulin/genetics
*Insect Proteins/genetics

@article {pmid42634427,
year = {2026},
author = {Safaei, Z and Bellizzi, A and Liu, H and Huang, Y and Sariyer, IK and Khalili, K},
title = {Triple-Target CRISPR Strategy to Block HIV Entry and Replication in Permissive Cells.},
journal = {Human gene therapy},
volume = {37},
number = {21-22},
pages = {1027-1044},
doi = {10.1177/10430342261463565},
pmid = {42634427},
issn = {1557-7422},
support = {P30 MH092177/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; *Virus Replication/genetics ; *HIV-1/genetics/physiology ; *CRISPR-Cas Systems ; Receptors, CCR5/genetics ; *Virus Internalization ; *Gene Editing ; *HIV Infections/virology/genetics/therapy ; HIV Envelope Protein gp120/genetics/metabolism ; Proviruses/genetics ; },
abstract = {Total elimination of replication-competent human immunodeficiency virus type 1 (HIV-1) remains a major clinical challenge, in part due to random integration of the proviral DNA into host cell chromosomes, which enables lifelong persistence and production of progeny. Although antiretroviral therapies (ARTs) suppress viral replication, they cannot eliminate integrated proviral DNA, which remains a fundamental obstacle to achieving a cure. To overcome this problem, we developed a combinatorial clustered regularly interspaced short palindromic repeats-Cas9 gene editing strategy to disrupt viral replication and inactivate host factors essential for HIV-1 entry and spread. This approach targets C-C chemokine receptor type 5 (CCR5), a chemokine receptor central to HIV-1 host cell entry, and mannosyl-oligosaccharide glucosidase (MOGS), a key enzyme in glycoprotein processing that modifies the HIV-1 envelope glycoprotein gp120, facilitating receptor engagement, viral entry, and morphogenesis of infectious virion. We demonstrate that our strategy, which includes editing of the integrated proviral DNA, in concert with two cellular genes whose products facilitate viral entry, results in robust suppression of viral replication in vitro and in ex vivo-infected cells. Using transmission electron microscopy, HIV-1 p24 ELISA, and GFP-based viral infection assays, we show that the combination knockout of CCR5, MOGS, and viral sequences profoundly reduces HIV-1 replication in an ex vivo cellular model, that is, HIV-1-infected peripheral blood mononuclear human cells, thus offering a pathway to launch further preclinical studies.},
}

Humans
*Virus Replication/genetics
*HIV-1/genetics/physiology
*CRISPR-Cas Systems
Receptors, CCR5/genetics
*Virus Internalization
*Gene Editing
*HIV Infections/virology/genetics/therapy
HIV Envelope Protein gp120/genetics/metabolism
Proviruses/genetics

@article {pmid42781469,
year = {2026},
author = {Jiao, X and Chen, W and Lv, Z and Chen, W},
title = {Transposon-derived genome editors in plants: from compact nucleases to large-fragment integration and regeneration strategies.},
journal = {Engineering in life sciences},
volume = {26},
number = {5-6},
pages = {e001},
pmid = {42781469},
issn = {1618-0240},
abstract = {CRISPR/Cas technology has been transformative for genome editing, enabling the precise integration of large DNA fragments-a capability essential for advanced genome (re)writing in plants, such as for trait stacking and pathway engineering. However, several new RNA-guided systems derived from transposable elements (TEs) have shown promise for plant genome editing, thereby enriching the toolkit available for plant engineering. This review comprehensively analyzes these emerging TE-based editors, including OMEGA nucleases (TnpB, IscB, Fanzor), CRISPR-associated transposases (CASTs), and R2 retrotransposon-derived systems. We detail their distinct architectures, evolutionary origins, and unique advantages over traditional Cas9, such as their compact size, simplified guide RNAs, and staggered DNA cleavage. We critically evaluate their nascent applications in plant models, highlighting ongoing challenges in editing efficiency, delivery, and specificity. Notably, CASTs and R2 retrotransposon-derived systems show particular promise for programmable large-fragment DNA integration, offering potential solutions for next-generation plant genome writing applications. We also examine the critical link between genome editing and plant regeneration, discussing phytohormonal and morphogenic regulators (e.g., BBM, WUS), and innovative tissue-culture-free methods, such as Cut-Dip-Budding. By comparing their merits and limitations, we position these systems not as replacements but as complementary tools within an expanding genome-editing toolkit. Integrating optimized TE-based editors with robust regeneration strategies is poised to unlock new frontiers in plant synthetic biology and crop improvement.},
}

@article {pmid42781958,
year = {2026},
author = {Wen, Z and Su, J},
title = {CRISPR-Cas9 and precision editing technologies linking functional genomics to clinical translation in genetic diseases.},
journal = {Clinical and translational medicine},
volume = {16},
number = {9},
pages = {e70764},
pmid = {42781958},
issn = {2001-1326},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods/trends ; *Genomics/methods/trends ; *Genetic Diseases, Inborn/therapy/genetics ; *Translational Research, Biomedical/methods/trends ; Animals ; },
abstract = {BACKGROUND: CRISPR-Cas9 and derivative precision-editing platforms increasingly connect pathogenic variant interpretation with functional genomics and therapeutic development in genetic diseases. This narrative review focuses on a variant-mechanism-driven framework for matching editing strategies to mutation structure, functional consequence, disease-model evidence, delivery feasibility, safety risk, and translational readiness.

MAIN BODY: The review summarizes how monogenic, polygenic, coding, non-coding, mitochondrial, and complex disease contexts influence the choice of canonical Cas9 editing, base editing, prime editing, Cas variants, CRISPR interference/activation, epigenome editing, and disease-model systems. It further compares ex vivo and in vivo delivery routes, safety assessment strategies, immunogenicity and genotoxicity concerns, and clinical implementation barriers, including CMC/manufacturing scalability, long-term follow-up, affordability, and regulatory oversight. Current evidence supports the clinical maturity of ex vivo hematopoietic editing, whereas most in vivo and precision-repair approaches remain constrained by delivery, durability, product heterogeneity, and safety uncertainties.

CONCLUSION: The central conclusion is that future CRISPR-based interventions should be judged not only by editability, but by whether molecular correction can be translated into durable, safe, manufacturable, and clinically meaningful benefit.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods/trends
*Genomics/methods/trends
*Genetic Diseases, Inborn/therapy/genetics
*Translational Research, Biomedical/methods/trends
Animals

@article {pmid42781968,
year = {2026},
author = {Wenjing, Z and Zhen, W and Xinyue, D and Ning, X and Lili, Z and Zhaoming, Q and Xue, H},
title = {Integrated Strategies for Saline-Alkali Soil Remediation and the Development of Salt-Alkali-Tolerant Crops.},
journal = {Plant, cell & environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70913},
pmid = {42781968},
issn = {1365-3040},
support = {LH2023C005//Natural Science Foundation of Heilongjiang/ ; CARS-09-P13//National Potato Industry Technology System/ ; },
abstract = {Soil salinization is a major threat to global food security and sustainable agriculture, soil salinization affects approximately 8.31 × 10[8] hm[2] of soil resources globally. The remediation and productive use of saline-alkali land have therefore become urgent global challenges. This review summarises the classification and global distribution of saline-alkali soils and critically compares physical, chemical, microbial, and phytoremediation strategies. It then examines the physiological, cellular, and molecular mechanisms that support plant salt and alkali tolerance, with emphasis on ion transporters that maintain ion homoeostasis, the interactions among phytohormone pathways, including ABA, JA, and GA, in balancing growth and stress responses, and the contribution of epigenetic regulation to stress adaptation. Although substantial progress has been made in identifying salt-alkali-tolerance genes and developing soil amendments, a considerable gap remains between laboratory-based studies and field-level yield improvement. Future progress will depend on moving from single-factor seedling assays to field-based yield validation, combining CRISPR/Cas-mediated genome editing with microbiome engineering, and addressing the socioeconomic feasibility of large-scale remediation. Integrated strategies that link soil improvement, crop genetics, microbial regulation, and field management can provide both theoretical insights and practical guidance for sustainable agriculture in saline-alkali environments.},
}

@article {pmid42776750,
year = {2026},
author = {Zhou, K and Hepler, NK and Jia, M and Cosgrove, DJ},
title = {Functional divergence of plant expansins as revealed by genetic complementation of root-hair tip growth in Arabidopsis thaliana.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {39},
pages = {e2623483123},
doi = {10.1073/pnas.2623483123},
pmid = {42776750},
issn = {1091-6490},
support = {DE-FG02-84ER13179//U.S. Department of Energy (DOE)/ ; DGE1255832//NSF | National Science Foundation Graduate Research Fellowship Program (GRFP)/ ; },
mesh = {*Arabidopsis/genetics/growth & development/metabolism ; *Plant Roots/growth & development/genetics/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Phylogeny ; Genetic Complementation Test ; Cell Wall/metabolism/genetics ; Gene Expression Regulation, Plant ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Plant cell wall expansion is essential for plant growth, with expansins promoting wall creep during diffuse growth. Expansin's role in tip growth, such as root hair elongation, is less clear. Here, we used CRISPR/Cas9 to knock out root hair-specific α-expansins EXPA7 and EXPA18 in Arabidopsis thaliana, which abolished root hair elongation. Complementation with expansin genes from various clades (driven by the EXPA7 promoter) revealed functional differences: Some fully restored root hair growth, others only partially restored growth, while others failed, notably EXPA13 (clade-VIII) and EXPA20 (clade-IX)-both lacking a conserved Asp considered essential, but not sufficient, for expansin-induced wall enlargement. Phylogenetic analysis suggests loss of this Asp may have predated angiosperms. Mutation of this Asp in EXPA7 confirmed its necessity for wall loosening, but its restoration in EXPA13 did not restore activity. Other expansin families (EXPB, EXLA, and EXLB) failed to restore root hair growth. Chimeric fusions of EXPAs with mCherry revealed differences in trafficking patterns and wall binding among EXPA clades. These results demonstrate functional differences among EXPA clades and among expansin families, establishing a genetic platform to analyze functionalities (trafficking, binding, activity) of EXPA and other wall-modifying proteins during root-hair tip growth.},
}

*Arabidopsis/genetics/growth & development/metabolism
*Plant Roots/growth & development/genetics/metabolism
*Arabidopsis Proteins/genetics/metabolism
*Plant Proteins/genetics/metabolism
Phylogeny
Genetic Complementation Test
Cell Wall/metabolism/genetics
Gene Expression Regulation, Plant
Mutation
CRISPR-Cas Systems

@article {pmid42777884,
year = {2026},
author = {Li, L and Shen, C and Yu, M and Fan, C and Deng, C and Li, W},
title = {Bacteriophage-based antibiotic resistant bacteria control in wastewater treatment: advances and prospects.},
journal = {Bioresource technology},
volume = {},
number = {},
pages = {135939},
doi = {10.1016/j.biortech.2026.135939},
pmid = {42777884},
issn = {1873-2976},
abstract = {Wastewater treatment plants (WWTPs) are critical hotspots for antibiotic resistance dissemination due to residual antibiotics promoting the proliferation of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs). While conventional disinfection methods (e.g., chlorination, UV irradiation) are widely employed, they often fail to simultaneously inactivate ARB and degrade both intracellular antibiotic-resistance genes (iARGs) and extracellular antibiotic-resistance genes (eARGs), while potentially generating toxic by-products. Bacteriophages (phages) offer a promising eco-friendly alternative, applicable during biological treatment or pre-disinfection. They can specifically target pathogenic or ARB strains without disrupting wastewater biological processes, and notably, ARGs released via phage-mediated ARB lysis are more susceptible to subsequent disinfection, lowering dissemination risks. To illustrate the growing research interest in this field, a statistical analysis of the literature was conducted based on 2377 articles retrieved from the Web of Science database (2015-2025), revealing publication trends in research on phage-mediated control of ARB and ARGs in WWTPs. More importantly, this review summarizes the mechanisms and applications of lytic and lysogenic (temperate) phages for ARB control in wastewater, analyzing key challenges hindering phage application including bacterial anti-phage defenses (e.g., CRISPR-Cas systems, biofilm shielding) and adverse wastewater conditions (e.g., pH fluctuations, particulate adsorption), as well as major application risks such as phage-mediated ARGs transduction. Corresponding mitigation strategies are discussed, including phage cocktails (to expand host range), genetic engineering (to boost lytic activity), and targeted delivery systems (e.g., magnetic nanoparticles). Furthermore, extending beyond host-phage interactions, this review evaluates engineering compatibility, offering novel perspectives to bridge the gap between biological potential and practical application.},
}

@article {pmid42778074,
year = {2026},
author = {Raihan, M and Younas, I and Su, S and Xu, L},
title = {Molecular mechanisms of WRKY transcription factors integrating environmental signals to regulate flowering time in brassica crops.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {373},
number = {},
pages = {113465},
doi = {10.1016/j.plantsci.2026.113465},
pmid = {42778074},
issn = {1873-2259},
abstract = {Flowering time is a critical developmental process that directly influences reproductive success, environmental adaptation, and agricultural productivity in Brassica crops. Recent climate instability, including drought, temperature fluctuations, salinity, and irregular photoperiods, has intensified the need to understand the molecular mechanisms regulating flowering adaptation. WRKY transcription factors are key integrators of flowering time. They connect environmental cues, hormone networks, and circadian rhythms to floral regulator genes. This review summarizes the structural, evolutionary, and functional characteristics of WRKY transcription factors involved in flowering regulation in Brassica species. Current evidence indicates that WRKY transcription factors contribute to flowering-time regulation through direct or indirect modulation of floral regulators, including FT, SOC1, and LFY. However, much of the mechanistic evidence originates from Arabidopsis, and the direct regulatory relationships between WRKY proteins and major flowering genes, particularly FLC, remain insufficiently characterized in Brassica species. Transcriptomics, epigenomics, proteomics, single-cell sequencing, and CRISPR/Cas genome editing further reveal that WRKY proteins participate in regulatory networks associated with photoperiodic signalling, vernalization, gibberellin pathways, stress-responsive flowering, and hormonal crosstalk. Comparative genomics demonstrates substantial expansion of WRKY gene families through genome triplication and polyploidization. However, limited functional validation and incomplete regulatory network mapping remain major challenges. Integrating multi-omics, artificial intelligence-assisted systems biology, genomic selection, and precision genome editing will accelerate development of climate-resilient, early-maturing Brassica cultivars.},
}

@article {pmid42298770,
year = {2026},
author = {Li, S and Sun, T and Liu, D and Zhang, T and Chen, L and Zhang, W},
title = {Iterative Genome Engineering Platform Enables Efficient Sucrose Biosynthesis From CO2 in Photosynthetic Synechococcus elongatus UTEX 2973.},
journal = {Plant biotechnology journal},
volume = {24},
number = {10},
pages = {5284-5299},
pmid = {42298770},
issn = {1467-7652},
support = {2024YFA0919700//National Key Research and Development Program of China/ ; },
mesh = {*Synechococcus/genetics/metabolism ; *Sucrose/metabolism ; *Carbon Dioxide/metabolism ; *Genetic Engineering/methods ; Photosynthesis/genetics ; Homologous Recombination ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {The single crossover occurring via homologous recombination is a common phenomenon existing among microbes like Escherichia coli, Bacillus subtilis, Vibrio natriegens, Gluconobacter oxydans and most cyanobacteria species, threatening the stability of engineered strains and challenging iterative genetic engineering. Among them, we take the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) as a representative study due to its promising roles for CO2 fixation and bioconversion. We established three marker-free platforms to achieve stable genome recombination: (i) T4CROSS, which employs two plasmids and four rounds of single crossover; (ii) TRIPLEARM, which uses a single plasmid containing three homologous arms for three rounds of single crossover; and (iii) CRISPRARM, which integrates CRISPR/Cpf1-mediated genome editing with homologous recombination. As proof of concept, we employed the CRISPRARM platform for a three-step sequential engineering of the sucrose biosynthetic pathway. The final engineered strain produced 7.12 g L[-1] of sucrose within 4 days.},
}

*Synechococcus/genetics/metabolism
*Sucrose/metabolism
*Carbon Dioxide/metabolism
*Genetic Engineering/methods
Photosynthesis/genetics
Homologous Recombination
Gene Editing
CRISPR-Cas Systems

@article {pmid42470166,
year = {2026},
author = {Mirzaee, M and Best, C and Wachowski, EV and Maliga, P},
title = {RNA-Guided Engineering of the Chloroplast Genome Enabled by Plastid-Expressed Guide RNAs.},
journal = {Plant biotechnology journal},
volume = {24},
number = {10},
pages = {5498-5509},
pmid = {42470166},
issn = {1467-7652},
support = {IOS 2224861//National Science Foundation/ ; FI-613-2021//United States-Israel Binational Agricultural Research and Development Fund/ ; },
mesh = {*Genome, Chloroplast/genetics ; *Nicotiana/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Chloroplasts/genetics ; *Plastids/genetics ; Plants, Genetically Modified ; Gene Editing/methods ; *Genetic Engineering/methods ; },
abstract = {Our goal is to develop RNA-guided engineering of the chloroplast genome using the CRISPR/Cas9 system. We designed chloroplast minigenes to obtain properly sized single guide RNAs (sgRNAs) in tobacco chloroplasts. The sgRNA 5' end is defined by transcription from an rRNA operon promoter, and its 3' end by processing a downstream tRNA (trnG) or a hepatitis delta virus (HDV) ribozyme. Cas9 is expressed from a nuclear gene and is targeted to chloroplasts by fusion to a transit peptide. Cas9 incorporated the sgRNA and introduced double-strand breaks in the plastid DNA (ptDNA). We report here that the double-strand DNA break in the ndhA and rpoC1 genes was repaired by microhomology-mediated end joining (MMEJ), resulting in deletions in the ptDNA. We further showed that nuclear-expressed sgRNA can be delivered into chloroplasts by fusion with a viroid RNA, as one possible approach for RNA-guided engineering of the ptDNA without direct chloroplast genome transformation. These results are the first step of RNA-guided editing of the chloroplast genome in any crop.},
}

*Genome, Chloroplast/genetics
*Nicotiana/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
CRISPR-Cas Systems/genetics
Chloroplasts/genetics
*Plastids/genetics
Plants, Genetically Modified
Gene Editing/methods
*Genetic Engineering/methods

@article {pmid42660094,
year = {2026},
author = {Huang, Z and Li, W and Zhang, J and Hao, H and Niu, M and Dong, X and Sun, W and Wu, X and Sun, Y and Li, H},
title = {Multiplex crRNAs powered CRISPRCas13a detection for rifampicin-resistant mycobacterium tuberculosis.},
journal = {Journal of infection and public health},
volume = {19},
number = {10},
pages = {103348},
doi = {10.1016/j.jiph.2026.103348},
pmid = {42660094},
issn = {1876-035X},
mesh = {*Mycobacterium tuberculosis/genetics/drug effects/isolation & purification ; *Rifampin/pharmacology ; Humans ; Sensitivity and Specificity ; *Drug Resistance, Bacterial/genetics ; DNA-Directed RNA Polymerases/genetics ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; *Molecular Diagnostic Techniques/methods ; *Tuberculosis, Multidrug-Resistant/diagnosis/microbiology ; Sputum/microbiology ; Rapid Diagnostic Tests ; Mutation ; *CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Rapid and accessible detection of Mycobacterium tuberculosis (MTB) and rifampicin resistance is essential for timely treatment and transmission control. However, widely used drug-resistance testing methods, including qPCR and gene sequencing, are time-consuming and require specialized instrumentation, limiting their utility for rapid testing in primary-care and resource-limited settings. Therefore, rapid, sensitive and readily deployable assays are urgently needed for the simultaneous diagnosis of tuberculosis (TB) and screening for drug resistance.

METHODS: A multiplex crRNA system was integrated with isothermal amplification, lyophilized reagents and a lateral flow readout to enable detection of 4 common rpoB mutation sites in a single reaction. Analytical sensitivity and specificity were evaluated using mutant plasmids, rifampicin-resistant clinical isolates, clinically relevant bacterial pathogens and non-tuberculous mycobacteria (NTM). Clinical performance was assessed using sputum-derived nucleic acids, with qPCR and Sanger sequencing as reference methods.

RESULTS: This detection assay can accurately identify nucleic acids from 10 clinical rifampicin-resistant isolates carrying four types of target mutations. For nucleic acids extracted from 21 clinical sputum samples, the assay achieved a sensitivity and specificity of 100%, with detection results fully consistent with those of qPCR and gene sequencing. The limit of detection (LOD) of the assay for all mutant plasmids was 10 copies/μL. In addition, this study realized highly sensitive and specific detection of MTB, with a LOD as low as 1 copy/μL, and successfully identified nucleic acids from 42 clinical TB samples. No cross-reactivity was observed between this assay and 5 pathogenic bacteria with clinical symptoms overlapping with MTB, as well as 8 NTM strains. Amplification and visual lateral flow readout were completed within 1 h.

CONCLUSIONS: This multiplex CRISPR-Cas13a assay enables rapid, sensitive and visually interpretable detection of MTB and common rifampicin resistance-associated mutations with limited instrumentation, offers a novel detection method for rifampicin-resistant tuberculosis (RR-TB) and exhibits potential in improving TB diagnosis.},
}

*Mycobacterium tuberculosis/genetics/drug effects/isolation & purification
*Rifampin/pharmacology
Humans
Sensitivity and Specificity
*Drug Resistance, Bacterial/genetics
DNA-Directed RNA Polymerases/genetics
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins/genetics
*Molecular Diagnostic Techniques/methods
*Tuberculosis, Multidrug-Resistant/diagnosis/microbiology
Sputum/microbiology
Rapid Diagnostic Tests
Mutation
*CRISPR-Cas Systems

@article {pmid42771321,
year = {2027},
author = {Papaioannou, NY and Papasavva, PL and Patsali, P and Lederer, CW},
title = {Electroporation for High-Efficiency Delivery of CRISPR to Hematopoietic Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {341-366},
pmid = {42771321},
issn = {1940-6029},
mesh = {*Electroporation/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Ribonucleoproteins/genetics ; RNA, Messenger/genetics ; Transfection/methods ; Electroporation Therapies ; },
abstract = {Recent advances in genome editing technologies have enabled transformative therapeutic strategies for hematological disorders. Efficient implementation of these approaches requires reliable delivery of genome editing components into primary hematopoietic stem and progenitor cells (HSPCs), which are particularly sensitive and resistant to conventional transfection methods. Electroporation has emerged as the most widely used strategy for ex vivo delivery of genome editing reagents into CD34[+] hematopoietic cells. Genome editing tools can be delivered in multiple formats, including plasmid DNA, messenger RNA (mRNA), and ribonucleoprotein (RNP) complexes. In HSPCs, mRNA- and RNP-based approaches are generally better tolerated than plasmid DNA and allow transient expression with reduced cytotoxicity. In this chapter, we describe optimized protocols for electroporation-based delivery of CRISPR/Cas9 RNP complexes and mRNA-encoded base editors into HSPCs using the Amaxa™ 4D-Nucleofector™ system. Detailed procedures are provided for cell preparation, guide RNA design, assembly of Cas9 RNPs, in vitro transcription and purification of base editor mRNA, electroporation parameters, and assessment of editing efficiency. Emphasis is placed on maximizing editing performance while preserving stem cell viability and functionality.},
}

*Electroporation/methods
*Hematopoietic Stem Cells/metabolism/cytology
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Ribonucleoproteins/genetics
RNA, Messenger/genetics
Transfection/methods
Electroporation Therapies

@article {pmid42771322,
year = {2027},
author = {Peña-Gutierrez, I and Mazzeo, D and Bassons-Bascuñana, A and Haider, S and Garcia-Garcia, L and Olalla-Sastre, B and Río, P and López-Manzaneda, S and Mataix, M},
title = {An Electroporation-Based Protocol for Ex Vivo Base Editing: From Design to Quantitative Assessment.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {367-388},
pmid = {42771322},
issn = {1940-6029},
mesh = {*Electroporation/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Base editing is a CRISPR variant approach that enables single-nucleotide conversions without generating double-strand breaks. Cytosine and adenine base editors mediate C•G to T•A and A•T to G•C transitions, respectively, by coupling a deaminase to a catalytically impaired Cas9, a modified nuclease that lacks DNA cleavage activity but retains DNA binding capability. By avoiding double-strand breaks, base editing limits reliance on unpredictable end-joining pathways and facilitates more precise outcomes. This chapter provides a practical protocol for base editing by electroporation in adherent and suspension stem cells. The workflow spans guide design aligned with the editor activity window, in vitro synthesis of mRNA-based editor components, and electroporation for transient delivery. We detail essential steps for cell handling and recovery, followed by standard readouts. Analytical endpoints focus on targeted sequencing to quantify base conversion and assess editing specificity, with guidance for basic interpretation of results. Troubleshooting notes address frequent pitfalls and practical remedies to optimize performance across cell types.},
}

*Electroporation/methods
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
Animals
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42771323,
year = {2027},
author = {Ortiz-Bueno, M and Millán-López, A and Labun, K and Benabdellah, K},
title = {Nucleofection-Based CRISPR/Cas Delivery in Human T Cells for Immunotherapy Applications.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {389-409},
pmid = {42771323},
issn = {1940-6029},
mesh = {Humans ; *T-Lymphocytes/metabolism/immunology ; *Electroporation/methods ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Receptors, Chimeric Antigen/genetics ; *Immunotherapy/methods ; Gene Transfer Techniques ; Immunotherapy, Adoptive/methods ; Electroporation Therapies ; },
abstract = {Electroporation-based delivery of CRISPR/Cas systems has emerged as a powerful and versatile approach for gene editing in primary human T cells, enabling efficient, transient, and nonviral modification while minimizing genomic integration risks. This chapter focuses on the principles and practical implementation of electroporation (nucleofection) for the delivery of Cas9 ribonucleoprotein (RNP) complexes into human T cells, highlighting critical parameters that influence editing efficiency, cell viability, and scalability for research and clinical applications. We provide a comprehensive protocol for multiplex gene editing in primary human T cells using Cas9 RNP electroporation, including optimization of cell activation status, buffer composition, electroporation settings, and post-electroporation recovery. Particular emphasis is placed on strategies to achieve high-efficiency disruption of target loci such as TRAC and B2M, enabling the generation of edited T cell products with defined functional properties. As a representative application, we describe how this delivery platform can be integrated with chimeric antigen receptor (CAR) engineering to produce edited CAR-T cells, including universal "off-the-shelf" designs with reduced risks of graft-versus-host disease and immune rejection. Downstream evaluation methods, including multiparameter flow cytometry for assessing editing efficiency and immunophenotype, are also outlined.},
}

Humans
*T-Lymphocytes/metabolism/immunology
*Electroporation/methods
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Receptors, Chimeric Antigen/genetics
*Immunotherapy/methods
Gene Transfer Techniques
Immunotherapy, Adoptive/methods
Electroporation Therapies

@article {pmid42771324,
year = {2027},
author = {Vaskovicova, M and Dolejs, V and Dostalova, P and Michalikova, CS and Prochazka, J and Sedlacek, R},
title = {Delivery of Targeting Constructs into Zygotes Using Electroporation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {411-421},
pmid = {42771324},
issn = {1940-6029},
mesh = {*Zygote/metabolism ; *Electroporation/methods ; Animals ; CRISPR-Cas Systems ; Mice ; Gene Editing/methods ; *Gene Transfer Techniques ; Animals, Genetically Modified ; *Gene Targeting/methods ; Female ; Blastocyst/metabolism ; },
abstract = {Zygote electroporation (EP) is a widely used technique for the delivery of targeting constructs into the embryos and has become very popular in the last few years in transgenic facilities all over the world. Zygote electroporation is technically simple, as it does not require equipment for microinjection. Moreover, zygote electroporation is less invasive, resulting in higher survival rates in comparison to pronuclear microinjection, leading to a reduction in the number of animals needed. Another advantage of zygote electroporation is a higher number of zygotes that can be targeted simultaneously, which minimizes the time required for zygote manipulation. Zygote electroporation is easily combined with the use of the CRISPR-Cas9 system.},
}

*Zygote/metabolism
*Electroporation/methods
Animals
CRISPR-Cas Systems
Mice
Gene Editing/methods
*Gene Transfer Techniques
Animals, Genetically Modified
*Gene Targeting/methods
Female
Blastocyst/metabolism

@article {pmid42772585,
year = {2026},
author = {Javadi, K and Zebardast, A},
title = {Targeting host factors in antiviral therapy: new frontiers in combating viral infections.},
journal = {Gene},
volume = {},
number = {},
pages = {150416},
doi = {10.1016/j.gene.2026.150416},
pmid = {42772585},
issn = {1879-0038},
abstract = {The rapid emergence of antiviral resistance and recurrent viral outbreaks have exposed limitations of conventional direct-acting antivirals (DAAs), prompting increasing interest in host-directed antiviral therapies (HDAs). Unlike virus-targeted agents, HDAs target host cellular factors and pathways required for viral replication, potentially providing broader activity and a higher genetic barrier to resistance. This review summarizes advances in host-targeted antiviral strategies and their therapeutic potential against established and emerging viral pathogens. We examine host dependency and restriction factors, together with virus-host interactions, as key therapeutic targets. Functional genomic approaches, including CRISPR-based screening and RNA interference (RNAi), have enabled systematic identification and modulation of host factors. We discuss CRISPR-mediated genome editing, RNAi therapeutics, metabolic reprogramming, and small-molecule host-targeting antivirals, highlighting their mechanisms, therapeutic development, and advantages over conventional approaches. Major challenges include host toxicity, pathway redundancy, delivery limitations, off-target effects, and viral adaptation. Emerging directions, including artificial intelligence-assisted target discovery, multi-omics integration, programmable nucleic acid therapeutics, and precision medicine, may accelerate the development of broad-spectrum and pandemic-ready antivirals. Overall, host-directed strategies represent a paradigm shift in antiviral drug development, offering opportunities for durable, potentially less susceptible to classical resistance mechanisms, and potentially broad-spectrum host-targeting approacheswhile complementing existing virus-directed therapies. Their successful translation will depend on identifying safe non-essential host targets, improving tissue-specific delivery, integrating combination therapies, and establishing robust safety and efficacy profiles for clinical use across diverse populations.},
}

@article {pmid42773127,
year = {2026},
author = {Walter, M and Finocchio, G and Oberli, S and Hammerschmid, IC and Lampe, GD and Karan, J and Swartjes, T and Sternberg, SH and Jinek, M and Querques, I},
title = {Transposon end recognition and excision mechanisms of type I-F CRISPR-associated transposases.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42773127},
issn = {2041-1723},
support = {820152 CRISPR2.0//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; DP2 HG011650/HG/NHGRI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; Vallee Scholar Award//Vallee Foundation (Bert L. & N. Kuggie Vallee Foundation)/ ; 101115765 BROADCAST//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; R01 EB027793/EB/NIBIB NIH HHS/United States ; R01 EB031935/EB/NIBIB NIH HHS/United States ; R01EB031935//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; RM1HG009490//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01EB027793//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DOC fellowship//Österreichischen Akademie der Wissenschaften (Austrian Academy of Sciences)/ ; 320030-228089//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; DP2HG011650//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Transposases/metabolism/genetics/chemistry ; *DNA Transposable Elements/genetics ; Cryoelectron Microscopy ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics/chemistry ; Humans ; Models, Molecular ; },
abstract = {CRISPR-associated transposons (CASTs) are Tn7-like elements that have co-opted RNA-guided CRISPR effectors for targeted DNA insertion. CASTs have been adapted as genome editing tools for programmable, site-specific integration. Among them, the type I-F system from Pseudoalteromonas (PseCAST) shows exceptionally robust activity in human cells, yet its mechanistic basis remains poorly understood. Here, we present structural and biochemical analysis of the PseCAST transposase TnsAB. Biochemical reconstitution of transposon DNA excision defines key characteristics of the transposition mechanism. Cryogenic electron microscopy (cryo-EM) structures of PseTnsAB paired-end complexes reveal molecular determinants of transpososome assembly, transposon end recognition and cleavage. We validate these findings using biochemical and in vivo assays of structure-based transposase mutants, and provide mechanistic insights into the enhanced activity of a laboratory-evolved TnsAB variant. Together, our studies highlight molecular features underlying the efficiency of natural and engineered type I-F transposases and establish a mechanistic framework for their continued rational optimization.},
}

*Transposases/metabolism/genetics/chemistry
*DNA Transposable Elements/genetics
Cryoelectron Microscopy
*CRISPR-Cas Systems
*Bacterial Proteins/metabolism/genetics/chemistry
Humans
Models, Molecular

@article {pmid42773193,
year = {2026},
author = {Xu, R and Cong, T and Yuan, J and Chen, X and Li, Y and Lan, X and Zhu, M},
title = {Author Correction: Tracking-seq: a universal off-target detection approach for CRISPR-Cas genome editing.},
journal = {Nature protocols},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41596-026-01462-7},
pmid = {42773193},
issn = {1750-2799},
}

@article {pmid42773653,
year = {2026},
author = {Han, Z and Wang, L and Fang, L and Cao, Y},
title = {[Advances and applications of microbial genome evolution engineering].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {9},
pages = {3905-3923},
doi = {10.13345/j.cjb.260147},
pmid = {42773653},
issn = {1872-2075},
support = {22478294 and 22308256//the National Natural Science Foundation of China/ ; QN20230234//the Tianjin Municipal Young Science and Technology Talents Training Program/ ; 25JCQNJC01890//the Tianjin Natural Science Foundation/ ; },
mesh = {*Directed Molecular Evolution/methods ; *Metabolic Engineering/methods ; *Genome, Microbial/genetics ; CRISPR-Cas Systems ; DNA-Directed RNA Polymerases/genetics ; *Genetic Engineering/methods ; },
abstract = {The construction of microbial cell factories frequently encounters bottlenecks due to the complexity of metabolic pathways, insufficient enzyme functionality, and poor environmental tolerance. Directed evolution, as a pivotal technology for overcoming these engineering constraints, can significantly enhance the titer, productivity, and overall robustness of target-producing strains. Its development originated from traditional random mutagenesis, a method that generates genetic diversity through the imposition of artificial growth pressures and is currently in widespread use. However, owing to the uncontrollable direction of mutagenesis and the low frequency of beneficial mutations, this approach suffers from low screening efficiency and is both time-consuming and labor-intensive. To accelerate the evolutionary rate, global accelerated evolution strategies based on tools such as DNA repair deficiencies and cytidine deaminases have emerged, achieving orders-of-magnitude increases in genome-wide mutation rates. Targeted continuous evolution strategies centered on CRISPR/Cas and T7 RNA polymerase systems enable the precise introduction of mutations within predefined genomic regions, thereby greatly improving the enrichment efficiency of beneficial mutations while minimizing non-productive mutations. This review will focus on three core strategies-traditional random evolution, global accelerated evolution, and targeted continuous evolution-systematically elucidating their developmental trajectories, applications in the construction of microbial cell factories, and future directions. Collectively, this review aims to provide methodological guidance for overcoming the engineering bottlenecks in the construction of microbial cell factories.},
}

*Directed Molecular Evolution/methods
*Metabolic Engineering/methods
*Genome, Microbial/genetics
CRISPR-Cas Systems
DNA-Directed RNA Polymerases/genetics
*Genetic Engineering/methods

@article {pmid42773905,
year = {2026},
author = {Meulenberg, A and Pavez, M and Gowing, EK and Mayo-Muñoz, D and Birkholz, N and Suhono, G and Fineran, PC and Fagerlund, RD and Gumy, LF},
title = {Comparison of CRISPR-Cas-Based Knockdown of Endogenous mRNA in Sensory Neurons.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {25731599261489437},
doi = {10.1177/25731599261489437},
pmid = {42773905},
issn = {2573-1602},
abstract = {Ribonucleic acid (RNA)-targeting clustered regularly interspaced short palindromic repeats-CRISPR-associated (CRISPR-Cas) systems enable modulation of gene expression without permanent genome modification, making them useful for sensitive cell types such as neurons. While CRISPR-Cas technologies have been most extensively applied and validated in primary hippocampal and cortical neurons, their use in sensory neurons remains largely unexplored. Sensory neurons are an established cellular model for studying axon growth and regeneration, pain mechanisms, sensory transduction, and neuron-environment interactions. Here, we evaluated the performance of compact RNA-targeting CRISPR-Cas effectors Cas7-11S, hfCas13X, and hfCas13d in primary rat sensory neurons in culture. Using an endogenous mRNA as the target, we compared knockdown efficiency and assessed the effects of CRISPR-Cas expression on neuronal health. The systems showed distinct differences in performance, with Cas7-11S inducing toxicity, hfCas13X showing minimal knockdown, and hfCas13d providing robust gene silencing with minimal adverse effects on neuronal health. These findings identify hfCas13d as the most effective and well-tolerated RNA-targeting CRISPR-Cas tool for sensory neurons and provide important insight into its suitability for neuroscience research and potential therapeutic applications.},
}

@article {pmid42774032,
year = {2026},
author = {Beaver, D and Cioanca, AC and Barnett, NL},
title = {Reporter-guided photoreceptor differentiation in 2D culture using PGP1-hIPSCs: A comparable alternative to 3D organoid systems?.},
journal = {Molecular vision},
volume = {32},
number = {},
pages = {232-253},
pmid = {42774032},
issn = {1090-0535},
mesh = {Humans ; *Cell Differentiation/genetics ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Organoids/cytology/metabolism ; *Photoreceptor Cells, Vertebrate/cytology/metabolism ; *Cell Culture Techniques/methods ; *Genes, Reporter ; Gene Expression Profiling ; Cell Culture Techniques, Three Dimensional ; CRISPR-Cas Systems ; Cell Line ; Retina/cytology/metabolism ; Biomarkers/metabolism ; Flow Cytometry ; Transcriptome ; },
abstract = {PURPOSE: To develop and characterize a xeno-free, two-dimensional (2D) differentiation protocol for directing human induced pluripotent stem cells (hIPSCs) toward photoreceptor (PhR)-like cells, using a live-reporter system and transcriptomic analysis to evaluate lineage fidelity and maturation compared to the three-dimensional (3D) culture paradigm.

METHODS: A CRISPR/Cas9-engineered PGP1 hIPSC line expressing fluorescent reporters for retinal markers (VSX2, BRN3B, and RCVRN) was differentiated in adherent culture using chemically defined media supplemented with small molecules (T3, DAPT, taurine, and retinoic acid). Differentiation was assessed over time by immunocytochemistry, flow cytometry, reverse transcription quantitative polymerase chain reaction, ultrastructural imaging (transmission electron microscopy and scanning electron microscopy), and bulk RNA sequencing. Comparative transcriptomic analysis with 3D retinal organoid data was conducted to evaluate developmental kinetics and pathway enrichment.

RESULTS: The 2D protocol reproducibly generated PhR-like cells expressing PhR-associated protein markers, including CRX, NR2F2, RCVRN, THRβ OPSIN-S, OPSN-M/L, and ARR3. Flow cytometric analysis demonstrated 93.6%, 96.2%, and 70.3% of RCVRN, OPSN-M/L, and OPSN-S positive populations at day (D) 42, while up to 97.4% of cells stained positive for RCVRN by D52. Early commitment to a PhR lineage was evident by D30, supported by transcriptional profiles consistent with PhR-like ontogeny. Ultrastructural analyses revealed features of putative inner and outer segments, including developing cilia and disc-like "whorls" supported by expression of gap junction protein markers and cilium markers (TMEM138 and CX36). Bulk RNA sequencing demonstrated faithful temporal regulation of PhR gene networks and highlighted accelerated differentiation compared to 3D cultures.

CONCLUSIONS: This pilot xeno-free 2D differentiation protocol offers a timely and scalable method for generating PhR-like cells from hIPSCs comparable to standard 3D culture systems. The results validate its downstream utility for retinal cell therapy development, high-throughput screening, and transplantation outputs pending further investigation, while supporting the transcriptome dominance model as a framework for evaluating photoreceptor fate acquisition in this culture paradigm.},
}

Humans
*Cell Differentiation/genetics
*Induced Pluripotent Stem Cells/cytology/metabolism
*Organoids/cytology/metabolism
*Photoreceptor Cells, Vertebrate/cytology/metabolism
*Cell Culture Techniques/methods
*Genes, Reporter
Gene Expression Profiling
Cell Culture Techniques, Three Dimensional
CRISPR-Cas Systems
Cell Line
Retina/cytology/metabolism
Biomarkers/metabolism
Flow Cytometry
Transcriptome

@article {pmid42774937,
year = {2026},
author = {Mollaev, TD and Bruskin, SA and Pushkova, EN and Dmitriev, AA and Melnikova, NV},
title = {Genetic transformation and genome editing of flax (Linum usitatissimum L.): current status and future perspectives.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1923497},
pmid = {42774937},
issn = {1664-462X},
abstract = {Flax (Linum usitatissimum L.) is an important multipurpose crop cultivated for fiber, oil (edible and industrial), and bioactive compounds used in medicine and cosmetics, making reliable transformation methods essential for targeted product quality improvement. This review compares three classic delivery platforms (Agrobacterium-mediated transformation, protoplast transformation, and particle bombardment) regarding efficiency, chimerism frequency, reproducibility, and suitability for genome editing, while also discussing virus-mediated delivery as a developing alternative. Currently, Agrobacterium-mediated transformation of hypocotyls followed by callus induction is widely used, but untransformed escapes and chimerism complicate the production of fully transgenic plants. This issue is mitigated when anther-derived calli are used as explants instead of hypocotyls and is absent in floral dip and protoplast transformation methods. Promising genome editing approaches now target the generation of non-transgenic flax plants. Although transgene-free lines were reportedly obtained through Cas-mediated oligonucleotide-directed mutagenesis (a single-stranded oligonucleotide template combined with transient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and transcription activator-like effector nucleases (TALEN) expression) in protoplasts, the lack of data confirming the absence of transgenes undermines these findings, therefore, verification of the obtained plants is essential. In theory, ribonucleoprotein (RNP) complexes could achieve a sufficient editing outcome via particle bombardment or delivery to protoplasts. Similarly, virus-induced genome editing (VIGE) utilizing viral vectors to deliver CRISPR/Cas components is also suggested as a viable approach to generate non-transgenic genome-edited plants, which is particularly advantageous as it bypasses the highly challenging plant regeneration stage.},
}

@article {pmid42775062,
year = {2026},
author = {Yamashita, T and Naito, Y and Yamamoto, T and Yoshida, T and Uchida, Y and Uchida, E and Inoue, T},
title = {Digenome-Detect: Accurate, statistics-based analysis software for identifying off-target cleavage sites in genome editing.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {4},
pages = {201837},
pmid = {42775062},
issn = {3117-387X},
abstract = {Off-target mutations pose major safety concerns in genome-editing therapy owing to their potential for leading to serious adverse events such as carcinogenesis. Thus, accurate prediction and evaluation of off-target mutations are critical in ensuring the safety of genome-editing therapeutics. Among the methods available for predicting off-target mutations, cell-free assays, which detect cleavage sites in extracted genomic DNA treated with genome-editing tools in vitro, are valuable because of their genome-wide, unbiased, and sensitive detection capabilities. However, cell-free assays are prone to identifying false-positive off-target cleavage sites, limiting their practical utility. To address this issue, we developed "Digenome-Detect," a highly accurate and sensitive data analysis software tool for Digenome-seq, which is the simplest cell-free assay. Digenome-Detect calculates a statistically derived score for genomic cleavage sites, followed by additional filters to reduce false positives. Compared with the current standard software, Digenome-toolkit, Digenome-Detect identified more off-target cleavage sites with fewer obvious false positives. Digenome-Detect enables accurate and sensitive prediction of off-target cleavage sites and can thereby contribute to ensuring the safety of genome-editing therapeutics.},
}

@article {pmid42775150,
year = {2026},
author = {Liyanage, R and Jin, L and Chen, SJ},
title = {Machine-learning in optimization of CRISPR technology.},
journal = {Machine learning. Health},
volume = {2},
number = {2},
pages = {021001},
pmid = {42775150},
issn = {3049-477X},
support = {R35 GM134919/GM/NIGMS NIH HHS/United States ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has become an indispensable tool in modern gene-engineering applications over recent years. Despite its rapid adoption, further development and broader applicability are hindered by several inherent limitations. This review surveys a range of machine-learning based approaches that aim to address these challenges. In particular, we focus on the optimization of key components of the CRISPR system, including protospacer adjacent motif recognition, Cas-protein-engineering, guide RNA sequence design and extension of the approaches to alternative editing modalities. Applied machine-learning methodologies, their underlying rationale, and comparisons with experimental observations are critically discussed. Special emphasis is placed on the role of machine-learning frameworks in advancing biophysical research, where complex, high-dimensional data increasingly demand integrative computational approaches. Finally, we outline current limitations, draw overarching conclusions, and propose perspectives for future developments and applications in CRISPR-based technologies.},
}

@article {pmid42776479,
year = {2026},
author = {Granata, G and Petrosillo, N and Taglietti, F},
title = {Novel options for the management of C. difficile: a look into the future.},
journal = {Expert review of anti-infective therapy},
volume = {},
number = {},
pages = {1-12},
doi = {10.1080/14787210.2026.2739273},
pmid = {42776479},
issn = {1744-8336},
abstract = {INTRODUCTION: Clostridioides difficile infection (CDI) remains one of the leading causes of healthcare-associated diarrhea; additionally, evidence suggests a growing incidence of community-acquired CDI worldwide. CDI is characterized by substantial morbidity, mortality, and risk of recurrence. CDI underdiagnosis and recurrent CDI represent a major unmet clinical need, highlighting the need for innovative diagnostic, preventive and therapeutic strategies.

AREAS COVERED: This perspective article summarizes emerging approaches that may shape the future management of CDI, including novel microbiome-sparing antimicrobials, fecal microbiota transplantation (FMT), live biotherapeutic products, C. difficile vaccines, bacteriophage-derived therapies, CRISPR-Cas technology and artificial intelligence (AI) applications.

EXPERT OPINION: Future CDI management is expected to evolve toward precision medicine focused on microbiome preservation, prevention of recurrence, and individualized patient care. Novel antimicrobials such as ibezapolstat and CRS3123, phage-derived approaches, and CRISPR-guided antimicrobials may provide highly targeted alternatives to conventional treatments. Microbiota-based therapies will evolve to assume an increasingly central role in reducing microbiota disruption. Simultaneously, advances in diagnostics, vaccine development, and AI-driven predictive tools may improve risk stratification, therapeutic selection, and infection prevention and control. All these innovative strategies have the potential to redefine CDI prevention and treatment, although robust clinical validation and long-term safety data remain essential.},
}

@article {pmid42468709,
year = {2026},
author = {Righetto, F and Bosaro, M and Sartori, G and Granuzzo, S and Antoniali, P and Lopreiato, R},
title = {CRISPR/Cas9-assisted heterothallic conversion enables rapid hybridization of industrial Saccharomyces strains.},
journal = {Bioresource technology},
volume = {461},
number = {},
pages = {135432},
doi = {10.1016/j.biortech.2026.135432},
pmid = {42468709},
issn = {1873-2976},
mesh = {*CRISPR-Cas Systems/genetics ; *Saccharomyces/genetics/metabolism ; *Hybridization, Genetic ; Fermentation ; *Industrial Microbiology/methods ; Phenotype ; *Saccharomyces cerevisiae/genetics ; },
abstract = {Efficient yeast breeding remains a major bottleneck in industrial biotechnology, particularly in food and beverage applications, where strain improvement must balance phenotypic innovation with regulatory constraints. Traditional hybridization procedures for natural Saccharomyces strains are experimentally limited by their diploid and homothallic nature, often resulting in labor-intensive processes of unpredictable duration. Here, we developed and validated a CRISPR/Cas9-assisted breeding framework that enables rapid, systematic, and informative hybridization by transiently converting homothallic strains into stable heterothallic mating partners through targeted HO gene inactivation. Using this strategy, we generated and characterized multiple intraspecific and interspecific hybrids challenging some industrially relevant traits, as sulfur dioxide release, acetic acid production, and the ability to restart stuck fermentations. In all cases, we generated the hybrids and assessed their performance within three months. Hybrid phenotypes followed inheritance patterns including parental-like, intermediate, and combinatorial traits, enabling rapid evaluation of cross utility. Moreover, cisgenic hybrids were generated by the restoration of the native HO locus, maintaining identical fermentative performance. Application to Saccharomyces cerevisiae × Saccharomyces uvarum interspecific crosses further demonstrated the versatility of the approach while revealing intrinsic biological constraints on trait combination. Overall, CRISPR/Cas9-assisted heterothallic conversion emerges as a versatile platform for accelerated yeast breeding, providing decision-enabling insights that support informed strain development across fermentation-based industries.},
}

*CRISPR-Cas Systems/genetics
*Saccharomyces/genetics/metabolism
*Hybridization, Genetic
Fermentation
*Industrial Microbiology/methods
Phenotype
*Saccharomyces cerevisiae/genetics

@article {pmid42610192,
year = {2026},
author = {Zhao, Y and Ma, P and Huang, J and Zhou, L and Chen, W and Li, B and Liu, S and Zhao, Z and Liu, S},
title = {Ultrasensitive detection of CYN based on the cascade amplification strategy of RCA-DNAzyme and Cas12a.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {36},
pages = {7992-7999},
doi = {10.1039/d6ay01017b},
pmid = {42610192},
issn = {1759-9679},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *DNA, Catalytic/chemistry/metabolism ; Cyanobacteria Toxins ; Alkaloids/analysis ; *CRISPR-Associated Proteins/chemistry ; Limit of Detection ; *Bacterial Proteins/chemistry ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/chemistry/metabolism ; *Uracil/analogs & derivatives/analysis ; Biosensing Techniques/methods ; },
abstract = {Cylindrospermopsin (CYN) is a class of cyanotoxins found widely across the globe, primarily in environments such as lake water, river water and drinking water. Ingestion can severely impair the function of organs such as the liver, kidneys and lungs. Consequently, the detection of CYN in the environment is crucial for safeguarding human health and public health safety. However, traditional detection methods are time-consuming and cumbersome to operate, and are prone to producing false-positive results. Consequently, there is an urgent need to establish a highly specific and accurate method for the ultra-sensitive detection of CYN. This study has developed a highly specific and ultra-sensitive detection method for CYN toxins based on a strategy combining rolling circle amplification DNAzyme with CRISPR/Cas12a cascade amplification. This technique utilises isothermal nucleic acid amplification technology and the trans-cleavage activity of CRISPR/Cas12a to amplify the signal. In the presence of the target algal toxin, the toxin binds to the arch-shaped probe, releasing an activator that triggers the amplified DNAzyme, thereby generating a large number of CRISPR/Cas12a activation sequences. This activates the trans-cleavage activity of CRISPR/Cas12a, which cleaves the reporter probe to produce a significantly enhanced fluorescent signal. The detection limit for CYN using this strategy is 2.4 nM. This method enables ultra-sensitive and highly specific detection of CYN and is suitable for the identification of CYN in real-world samples. The fluorescence detection of CYN achieved through the coupling of DNAzymes with CRISPR/Cas12a via rolling circle amplification provides an innovative solution for the ultra-sensitive detection of CYN.},
}

*Nucleic Acid Amplification Techniques/methods
*DNA, Catalytic/chemistry/metabolism
Cyanobacteria Toxins
Alkaloids/analysis
*CRISPR-Associated Proteins/chemistry
Limit of Detection
*Bacterial Proteins/chemistry
*CRISPR-Cas Systems
*Endodeoxyribonucleases/chemistry/metabolism
*Uracil/analogs & derivatives/analysis
Biosensing Techniques/methods

@article {pmid42771312,
year = {2027},
author = {Llanos-Ardaiz, A and Zabaleta, N and Torella, L and Gonzalez-Aseguinolaza, G},
title = {Adeno-Associated Viral Vector (AAV)-Mediated In Vivo CRISPR-Cas9 Delivery.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {175-197},
pmid = {42771312},
issn = {1940-6029},
mesh = {*Dependovirus/genetics ; Animals ; *Genetic Vectors/genetics ; *CRISPR-Cas Systems ; Mice ; *Gene Editing/methods ; Liver/metabolism ; *Gene Transfer Techniques ; Humans ; Plasmids/genetics ; Staphylococcus aureus/genetics/enzymology ; },
abstract = {CRISPR-based gene editing is a growing therapeutic strategy for modifying or silencing disease-causing genes. This chapter focuses on the in vivo delivery of CRISPR systems using recombinant adeno-associated viral (rAAV) vectors. Specifically, we detail a methodology using a rAAV vector carrying Staphylococcus aureus Cas9 (SaCas9) to target the murine Hao1 gene in liver parenchymal cells as a curative treatment for primary hyperoxaluria type 1 (PH1). We provide comprehensive protocols for plasmid design and cloning, rAAV production, animal administration, and evaluation of editing efficacy.},
}

*Dependovirus/genetics
Animals
*Genetic Vectors/genetics
*CRISPR-Cas Systems
Mice
*Gene Editing/methods
Liver/metabolism
*Gene Transfer Techniques
Humans
Plasmids/genetics
Staphylococcus aureus/genetics/enzymology

@article {pmid42771313,
year = {2027},
author = {Olsen, AL and Brandt, CB and Larsen, RK and Luo, Y},
title = {Lipid Nanoparticle-Mediated Delivery of CRISPR-Cas9 Components for Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {201-218},
pmid = {42771313},
issn = {1940-6029},
mesh = {*Nanoparticles/chemistry ; *Gene Editing/methods ; *Lipids/chemistry ; *CRISPR-Cas Systems/genetics ; Humans ; *Gene Transfer Techniques ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are a clinically validated nonviral platform for the delivery of CRISPR-associated components. Composed of ionizable lipids, phospholipids, cholesterol, and PEG-lipids, LNPs enable the efficient encapsulation, protection, and cytosolic delivery of therapeutic cargo such as DNA, RNA, or proteins. The clinical relevance of LNPs has already been shown by multiple FDA-approved therapies, including siRNA-based treatments and mRNA vaccines. Compared with viral vectors, LNPs offer several advantages, including reduced immunogenicity, absence of genomic integration, scalable manufacturing, and flexibility in cargo size, while supporting transient expression, which is desirable for genome editing applications. However, challenges remain, including limited tissue specificity and inefficient endosomal escape. Recent advances in lipid chemistry optimization and surface modification have improved delivery performance. Among available formulation techniques, microfluidic mixing has emerged as a preferred method due to its reproducibility, scalability, and precise control over particle properties. This protocol describes a standardized microfluidic workflow for reproducible LNP formulation, providing practical guidance on lipid preparation, nanoparticle assembly, and quality control.},
}

*Nanoparticles/chemistry
*Gene Editing/methods
*Lipids/chemistry
*CRISPR-Cas Systems/genetics
Humans
*Gene Transfer Techniques
Liposomes

@article {pmid42771314,
year = {2027},
author = {Pečan, P and Manček-Keber, M},
title = {CRISPR/Cas9 Delivery Using Extracellular Vesicles.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {219-235},
pmid = {42771314},
issn = {1940-6029},
mesh = {*Extracellular Vesicles/metabolism/genetics ; *CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Ribonucleoproteins/genetics/metabolism ; *Gene Transfer Techniques ; Plasmids/genetics ; },
abstract = {Extracellular vesicles (EVs), particles released from cells, have the potential to become an important in vivo delivery vehicle for CRISPR machinery. Unlike viral vectors such as Adeno-associated virus (AAV), EVs reduce risks associated with immunogenicity, long-term expression, and potential off-target effects. EVs can efficiently deliver CRISPR/Cas9 ribonucleoprotein (RNP) complexes, which provide transient, ready-to-function editing machinery with reduced off-target risk compared with plasmid DNA or mRNA delivery. RNP loading into EVs can occur without specific targeting signals, although strategies such as membrane anchoring, inducible dimerization systems, or fusion with EV-associated proteins can enhance cargo enrichment. EVs are typically produced by transfecting producer cells with plasmids encoding Cas9 and sgRNA, followed by vesicle release into culture media. Purification requires removal of cellular debris and enrichment of vesicles using methods such as ultracentrifugation, ultrafiltration, chromatography, or precipitation. As no single gold-standard purification approach exists, method selection should balance yield and purity, and characterization using vesicle markers and size distribution profiling is recommended.},
}

*Extracellular Vesicles/metabolism/genetics
*CRISPR-Cas Systems
Humans
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Animals
Ribonucleoproteins/genetics/metabolism
*Gene Transfer Techniques
Plasmids/genetics

@article {pmid42771316,
year = {2027},
author = {Vasti, C and Valenti, LE and Giacomelli, CE},
title = {Quantitative Analysis of CRISPR Encoding Plasmid DNA-Nanocarrier Interactions by Gel Electrophoresis and Densitometry.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {257-273},
pmid = {42771316},
issn = {1940-6029},
mesh = {*Plasmids/genetics/chemistry ; *Densitometry/methods ; Electrophoresis, Agar Gel/methods ; *Nanoparticles/chemistry ; *DNA/genetics/chemistry ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; },
abstract = {Nanoparticle-based carriers are key systems for the transport and delivery of nucleic acids in gene therapy, RNA therapeutics, and genome editing applications. The development of efficient delivery systems for CRISPR-Cas technologies requires reliable methods to evaluate nanocarrier-nucleic acid interactions, binding efficiency, and loading capacity. Agarose gel electrophoresis is commonly used to study these interactions through gel retardation assays, but results are often interpreted qualitatively based on visual inspection of DNA band migration. In this chapter, we present a quantitative approach that combines agarose gel electrophoresis with densitometric analysis of plasmid DNA (pDNA) bands to evaluate the interaction between pDNA and inorganic nanocarriers. The method relies on quantifying the fraction of free pDNA that migrates through the gel, while nanocarrier-pDNA systems remain near the loading wells. Band intensities are analyzed using ImageJ software to determine the amount of free pDNA and to estimate binding efficiency through depletion analysis. This protocol provides a simple and accessible strategy to quantify the loading of pDNA encoding CRISPR components, guide RNA constructs, or other nucleic acid cargos, facilitating the development and optimization of nanoparticle-based delivery systems for genome editing applications.},
}

*Plasmids/genetics/chemistry
*Densitometry/methods
Electrophoresis, Agar Gel/methods
*Nanoparticles/chemistry
*DNA/genetics/chemistry
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing/methods

@article {pmid42771317,
year = {2027},
author = {Paul, A and Lee, HY and Tuma, J},
title = {Cell-Penetrating Peptide Delivery for CRISPR Applications.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {275-297},
pmid = {42771317},
issn = {1940-6029},
mesh = {*Cell-Penetrating Peptides/chemistry/metabolism/genetics ; *CRISPR-Cas Systems ; Humans ; Animals ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; Ribonucleoproteins/genetics/metabolism ; *Gene Transfer Techniques ; },
abstract = {Cell-penetrating peptides (CPPs) provide a modular nonviral strategy for intracellular delivery of CRISPR/Cas9 components, particularly preassembled Cas9 ribonucleoprotein (RNP) complexes. This chapter summarizes key principles of CPP-mediated Cas9 RNP delivery, including noncovalent complexation and covalent CPP-Cas9 conjugation strategies. A representative protocol is provided for the preparation and evaluation of Cas9-K5 RNP, including in vitro uptake and reporter-editing assays, nucleofection-based functional controls, local intracranial delivery, immunofluorescence staining, and quantitative analysis of reporter activation in brain tissue.},
}

*Cell-Penetrating Peptides/chemistry/metabolism/genetics
*CRISPR-Cas Systems
Humans
Animals
*Gene Editing/methods
*CRISPR-Associated Protein 9/genetics/metabolism
Ribonucleoproteins/genetics/metabolism
*Gene Transfer Techniques

@article {pmid42771319,
year = {2027},
author = {Carusillo, A and Naseem, A and Cavazza, A},
title = {Virus-Like Particles as a Nonviral CRISPR/Cas Editing Delivery Tool.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {311-324},
pmid = {42771319},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Virion/genetics ; Genetic Vectors/genetics ; *Gene Transfer Techniques ; Viral Envelope Proteins/genetics ; HEK293 Cells ; Membrane Glycoproteins ; },
abstract = {Virus-like particles (VLPs) are self-assembling viral protein complexes that mimic native virions while lacking replicative and infectious capacity. By retaining efficient cellular entry and cargo protection, they overcome key limitations of viral vectors, including insertional mutagenesis and strong immunogenicity. Their ease of production, flexible cargo capacity, and tunable tropism make them highly versatile tools. Here, we describe a protocol for generating VSV-G-pseudotyped VLPs that deliver CRISPR/Cas9 RNPs and their functional validation in surrogate cell lines.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Humans
*Virion/genetics
Genetic Vectors/genetics
*Gene Transfer Techniques
Viral Envelope Proteins/genetics
HEK293 Cells
Membrane Glycoproteins

@article {pmid42771320,
year = {2027},
author = {Skrbinek, M and Bohinc, J},
title = {Production and Purification of Cas9 Protein for Ribonucleoprotein Complex Formation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {327-339},
pmid = {42771320},
issn = {1940-6029},
mesh = {*Ribonucleoproteins/genetics/metabolism/isolation & purification/chemistry ; Escherichia coli/genetics/metabolism ; Chromatography, Affinity/methods ; *CRISPR-Associated Protein 9/isolation & purification/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Cas Systems ; Chromatography, Gel ; Gene Editing/methods ; },
abstract = {Cas9-guide RNA ribonucleoprotein (RNP) complexes are widely used for genome editing because they provide rapid, transient nuclease activity without introducing exogenous DNA into target cells. Efficient RNP delivery requires a highly purified, correctly folded Cas9 protein that is free of contaminating nucleases and small-molecule impurities. This chapter describes the expression of N-terminally His-tagged Cas9 from a pET28b vector in Escherichia coli Rosetta strains, followed by purification using Ni-NTA immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) on a Superdex 200 Increase column. The protocol covers bacterial fermentation, cell lysis, IMAC with high-salt and graded-imidazole washes, SEC polishing, dialysis into a storage buffer, and concentration to RNP-ready stocks, with quality control by SDS-PAGE and optional western blotting and BCA assay. Finally, a short procedure for assembling Cas9 RNPs with in vitro-transcribed guide RNAs is provided, making the protocol directly applicable to CRISPR RNP delivery in mammalian cells.},
}

*Ribonucleoproteins/genetics/metabolism/isolation & purification/chemistry
Escherichia coli/genetics/metabolism
Chromatography, Affinity/methods
*CRISPR-Associated Protein 9/isolation & purification/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
CRISPR-Cas Systems
Chromatography, Gel
Gene Editing/methods

@article {pmid41601258,
year = {2026},
author = {Zhang, J and Zhang, L and Hou, L and Li, W and Geng, S and Wang, X and Wang, T},
title = {OAZ1/ CASP8AP2 double knockout enhances recombinant protein production in HEK293 cells through metabolic reprogramming and antiapoptotic effects.},
journal = {Acta biochimica et biophysica Sinica},
volume = {58},
number = {9},
pages = {2033-2043},
doi = {10.3724/abbs.2025196},
pmid = {41601258},
issn = {1745-7270},
mesh = {Humans ; HEK293 Cells ; *Apoptosis/genetics ; *Recombinant Proteins/biosynthesis/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Metabolic Reprogramming ; *Apoptosis Regulatory Proteins/genetics/metabolism ; },
abstract = {Human embryonic kidney (HEK) 293 cells are widely used for recombinant protein production because of their efficient posttranslational modification capabilities. However, their large-scale culture is often limited by metabolic stress and early apoptosis, leading to insufficient protein yields. In this study, we aim to increase protein expression through the coordinated modulation of metabolic and apoptotic pathways. Using CRISPR/Cas9 technology, we target and knockout the genes of ornithine decarboxylase antizyme 1 (OAZ1), which regulates polyamine metabolism, and caspase 8-associated protein 2 (CASP8AP2), an apoptosis-related protein. We successfully construct an OAZ1/ CASP8AP2 double-knockout HEK293 cell line. Following transfection with the knockout vector and screening of single-cell clones, multiple levels of validation confirm the successful gene knockout. The results show that the double-knockout cells exhibit significantly reduced apoptosis rates. Furthermore, the production of recombinant secreted alkaline phosphatase (SEAP) and vitronectin (VN) increases by 2.1 folds and 2.9 folds, respectively, compared with those in wild-type cells. Metabolic profiling reveals that the cell cycle is arrested in the G1/G0 phase, accompanied by increased specific consumption and production rates of key metabolites. This study demonstrates that concurrent inhibition of apoptosis and optimization of metabolism effectively enhances recombinant protein production in HEK293 cells, suggesting a novel strategy for improving HEK293 cell-based expression.},
}

Humans
HEK293 Cells
*Apoptosis/genetics
*Recombinant Proteins/biosynthesis/genetics
Gene Knockout Techniques
CRISPR-Cas Systems
Metabolic Reprogramming
*Apoptosis Regulatory Proteins/genetics/metabolism

@article {pmid42696756,
year = {2026},
author = {Cadeau, M and de Lima, JR and Sabou, AM and Robert, E and Le Pape, P and Ourliac-Garnier, I and Morio, F},
title = {A new MRR1 gain-of-function mutation involved in cross-resistance to antifungal agents in the fungal priority pathogen Candida parapsilosis.},
journal = {Medical mycology},
volume = {64},
number = {9},
pages = {},
doi = {10.1093/mmy/myag094},
pmid = {42696756},
issn = {1460-2709},
support = {//Mundipharma to investigate antifungal resistance mechanisms in yeasts/ ; },
mesh = {*Candida parapsilosis/drug effects/genetics/pathogenicity ; *Antifungal Agents/pharmacology ; Microbial Sensitivity Tests ; Animals ; *Gain of Function Mutation ; Humans ; Fluconazole/pharmacology ; *Drug Resistance, Fungal/genetics ; Virulence ; *Fungal Proteins/genetics ; Candidiasis/microbiology/drug therapy ; Disease Models, Animal ; Moths/microbiology ; *Transcription Factors/genetics ; Amino Acid Substitution ; CRISPR-Cas Systems ; },
abstract = {OBJECTIVES: Candida parapsilosis is a leading cause of invasive candidiasis globally, with rising reports of fluconazole resistance threatening its clinical management. Among the mechanisms involved, gain-of-function mutations in the MRR1 gene have emerged as key drivers of antifungal resistance. We aimed to investigate a novel amino acid substitution (G982E) in the Mrr1 zinc cluster transcription factor, identified in a fluconazole-resistant C. parapsilosis isolate from a patient exposed to fluconazole.

METHODS: Using CRISPR-Cas9 genome editing, we introduced the G982E variant into two fluconazole-susceptible C. parapsilosis genetic backgrounds. The antifungal susceptibility of the engineered mutants was assessed in vitro against a broad panel of systemic antifungal agents. A Galleria mellonella infection model was also used to evaluate the impact of the G982E variant on antifungal treatment efficacy and virulence in vivo.

RESULTS: Acquisition of the G982E substitution dramatically altered the antifungal susceptibility profile, particularly for fluconazole for which the minimum inhibitory concentration (MIC) increased to >256 µg/mL. However, the magnitude of the MIC increase varied by azole, with the greatest increase seen for fluconazole (>9-10 log-fold), followed by voriconazole (5 log-fold), isavuconazole (3 log-fold), but also flucytosine (1.5 log-fold). In contrast, susceptibility to posaconazole remained largely unchanged. In vivo, this new variant conferred fluconazole treatment failure but was associated with a significant reduction in virulence.

CONCLUSIONS: The G982E is a novel Mrr1 gain-of-function mutation driving high-level fluconazole resistance in C. parapsilosis. These findings reinforce the central role of Mrr1 in antifungal resistance, underscore the functional diversity of its mutational landscape, with potential implications for fungal fitness and transcriptional regulation.},
}

*Candida parapsilosis/drug effects/genetics/pathogenicity
*Antifungal Agents/pharmacology
Microbial Sensitivity Tests
Animals
*Gain of Function Mutation
Humans
Fluconazole/pharmacology
*Drug Resistance, Fungal/genetics
Virulence
*Fungal Proteins/genetics
Candidiasis/microbiology/drug therapy
Disease Models, Animal
Moths/microbiology
*Transcription Factors/genetics
Amino Acid Substitution
CRISPR-Cas Systems

@article {pmid42726641,
year = {2026},
author = {Desjardins, J and Bowlan, J and Bernier, C and Jain, S and Goullet de Rugy, T and Gallo, D and Orcholski, ME and Laterreur, N and Rajah, A and Miller, J and Lafontaine, J and Bhaskaran, V and Li, L and Ling, A and Leblanc, JH and Mathieu, MC and Zinda, M and Morris, SJ and Durocher, D and Zimmermann, M and Roulston, A and Veloso, A and Fiore, C and Álvarez-Quilón, A and Young, JTF},
title = {Genome-wide CRISPR screens map synthetic lethal interactions across recurrent cancer driver alterations.},
journal = {Cell reports},
volume = {45},
number = {9},
pages = {117961},
doi = {10.1016/j.celrep.2026.117961},
pmid = {42726641},
issn = {2211-1247},
mesh = {Humans ; *Neoplasms/genetics ; *Synthetic Lethal Mutations/genetics ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {Synthetic lethality (SL) provides a treatment paradigm for targeting cancer with alterations in driver genes that are not conventionally druggable, including tumor suppressor genes. We execute a series of genome-wide CRISPR screens using functionally validated isogenic cell lines and conduct a large-scale SL analysis using data from the cancer dependency map (DepMap). We chart SL interactions across 15 driver alterations: FBXW7, CCNE1, CDK12, ARID1A, KMT2D, DNMT3A, TET2, KEAP1, STK11, IDH1, SF3B1, SRSF2, U2AF1, chromosome 18q loss, and chromosome 13q loss. We show validation of several SL interactions, including ARID1A and the hexosamine biosynthetic pathway aminotransferase GFPT1, STK11 with CAMK protein kinase MARK2, FBXW7 and the CDK1 regulatory kinase PKMYT1, and CCNE1 amplification and the anaphase-promoting complex or cyclosome (APC/C). In summary, this study offers a rich resource of genetic interactions across cancer drivers enabling the discovery of biological insights and drug targets for future therapeutic development.},
}

Humans
*Neoplasms/genetics
*Synthetic Lethal Mutations/genetics
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics

@article {pmid42771155,
year = {2027},
author = {Wang, Y and Fan, H},
title = {Conditional dCas12-Mediated Gene Knockdown and Complementation in Chlamydia.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3073},
number = {},
pages = {243-258},
pmid = {42771155},
issn = {1940-6029},
mesh = {*Gene Knockdown Techniques/methods ; *CRISPR-Cas Systems ; *Chlamydia trachomatis/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Complementation Test/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; },
abstract = {We describe an oligonucleotide RNA-guided, DNase-dead CRISPR-associated protein 12 (dCas12)-mediated transcriptional interference (CRISPRi) system optimized for gene knockdown in Chlamydia trachomatis. In this system, while guide RNAs are expressed constitutively, dCas12 expression is conditional and depends on the presence of the small molecule anhydrotetracycline (ATC). This design enables temporal control of gene repression, allowing the functional analysis of a wide range of genes, including essential genes, without permanent genetic disruption. Due to its T-rich protospacer adjacent motif (PAM) requirement, dCas12 is particularly well-suited for targeting genes in the highly A/T-rich genomes of pathogenic chlamydiae. We further detail strategies for genetic complementation through co-expression of dCas12-resistant alleles, thereby permitting validation of knockdown phenotypes.},
}

*Gene Knockdown Techniques/methods
*CRISPR-Cas Systems
*Chlamydia trachomatis/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Genetic Complementation Test/methods
*CRISPR-Associated Proteins/genetics/metabolism
Humans

@article {pmid42771304,
year = {2027},
author = {Cakiroglu, E and Senturk, S},
title = {CRISPR/Cas Systems: Biological Basis and Genome Editing Applications.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {3-18},
pmid = {42771304},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; Epigenome Editing ; Genomics/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and associated (Cas) systems have revolutionized the field of genome engineering by providing versatile, efficient, and programmable tools for precise genetic manipulation. Originally identified as an adaptive immune mechanism in prokaryotes, CRISPR/Cas systems have been extensively repurposed for a wide range of applications across molecular biology, biotechnology, and medicine. This chapter provides a comprehensive overview of the molecular mechanisms underlying CRISPR/Cas immunity. Furthermore, the classification of CRISPR/Cas systems into distinct types and subtypes is discussed, highlighting their structural and functional diversity. Advances in genome editing technologies, including CRISPR-mediated knockout, base editing, and prime editing, are explored with an emphasis on their mechanisms and applications. The chapter also examines emerging CRISPR-based platforms for transcriptional regulation, epigenome editing, and RNA targeting, which enable precise and reversible modulation of gene expression without altering genomic DNA. In addition, the transformative impact of CRISPR technologies on functional genomics is addressed, particularly through high-throughput screening approaches that facilitate the identification of gene function and genetic vulnerabilities. CRISPR-based diagnostic tools and therapeutic strategies are also reviewed, underscoring their potential in disease detection and treatment. Despite significant progress, challenges such as off-target effects, delivery limitations, and safety concerns remain critical considerations. Overall, this chapter highlights the expanding capabilities of CRISPR/Cas systems and their growing importance in both fundamental research and clinical applications.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Humans
Animals
Epigenome Editing
Genomics/methods
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42771306,
year = {2027},
author = {García-Tenorio, EM and Alvarez, M and Richard, E and Desviat, LR},
title = {Prime Editing: An Overview.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {41-57},
pmid = {42771306},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR tools are revolutionizing the landscape of genetic therapies, with the potential to cure a range of previously untreatable diseases. Among all the available genome editing technologies, prime editing is an especially versatile tool that enables precise genetic modifications, including point mutations, insertions, and deletions, without inducing double-strand breaks or requiring a donor DNA template. Through structural modifications and the development of novel systems with additional functionalities, prime editing has expanded its applicability with improved precision, efficacy, and safety. It is already being tested in clinical trials for chronic granulomatous disease, and many preclinical studies are underway. However, significant challenges remain for its broad applicability as a potential curative therapy for human genetic diseases, mainly related to ensuring efficient and safe delivery to target tissues.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
Animals
*Genetic Therapy/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42771307,
year = {2027},
author = {Catalano, F},
title = {Base Editing: Mechanisms and Therapeutic Applications.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {59-74},
pmid = {42771307},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *Genetic Therapy/methods ; Nanoparticles/chemistry ; CRISPR-Cas Systems ; Cytosine/metabolism ; },
abstract = {Base editing enables the direct, programmable conversion of one nucleotide into another at a defined genomic site without introducing a double-strand break. First reported in 2016, a decade later, it has expanded into a broad family of molecular tools that has now entered clinical trials. This chapter reviews the development of base editing from its origins, including the early transition of cytosine and adenine base editors, to the more recent emergence of transversion editors. For each class, this section describes the mechanism, the optimization of on-target efficiency, product purity, and specificity, as well as the key strengths and limitations. The following discussion focuses on delivery, which remains one of the central bottlenecks for clinical translation, with particular attention to lipid nanoparticles, engineered virus-like particles, and other emerging strategies. Finally, a review of the current clinical landscape is presented. This already includes the first ex vivo multiplex base-edited cell therapy in T-cell leukemia, ex vivo hematopoietic stem cell transplantation targeting hemoglobin disorders, the first systemic in vivo base editing in humans, and, importantly, the first personalized N-of-1 in vivo base-editing therapy, which was developed within a remarkably short time. These developments show that base editing has moved from a proof-of-concept to a clinically-ready platform with incredible speed, and that the central questions for the field, perhaps, concern the pace at which the surrounding technology, for example delivery, can keep up with base editors themselves.},
}

Humans
*Gene Editing/methods
Animals
*Genetic Therapy/methods
Nanoparticles/chemistry
CRISPR-Cas Systems
Cytosine/metabolism

@article {pmid42771308,
year = {2027},
author = {Djordjevič, M and Dinič, S and Sarič, A and Uskokovič, A and Vidakovič, M},
title = {An Overview of Programmable Epigenetic Editing Based on CRISPR Tools for Gene Expression Regulation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {75-92},
pmid = {42771308},
issn = {1940-6029},
mesh = {Humans ; *Epigenome Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Epigenesis, Genetic ; DNA Methylation ; *Gene Expression Regulation ; *Gene Editing/methods ; },
abstract = {Epigenetic regulation provides a dynamic and reversible layer of gene control that functions independently of changes in DNA sequence, primarily mediated by DNA methylation, histone modifications, and higher-order chromatin organization. Aberrant epigenetic states contribute to a wide range of human diseases. However, conventional epigenetic therapies based on small-molecule inhibitors lack locus specificity and often cause global chromatin disturbances. The emergence of programmable epigenetic editing technologies has transformed the field by enabling targeted rewriting of chromatin states at defined genomic loci. Catalytically inactive CRISPR/Cas9 platforms fused to transcriptional activators, repressors, or chromatin-modifying enzymes now allow precise addition or removal of epigenetic marks without altering the underlying DNA sequence. This chapter provides an overview of the conceptual and technical foundations of CRISPR-based epigenetic editing, including tools for gene activation and repression, DNA methylation, histone modifications, and multiplexed systems that permit coordinated regulation of multiple genomic loci or epigenetic marks. Delivery methods for in vitro and in vivo applications are discussed, with an emphasis on viral and nonviral platforms that enable tissue-specific, durable gene regulation. Finally, recent preclinical and clinical studies highlight the potential of programmable epigenetic editing as a next-generation therapy for precise and reversible gene control.},
}

Humans
*Epigenome Editing/methods
*CRISPR-Cas Systems
Animals
*Epigenesis, Genetic
DNA Methylation
*Gene Expression Regulation
*Gene Editing/methods

@article {pmid42771309,
year = {2027},
author = {Debelec Butuner, B},
title = {Overview of Delivery Methods for Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {93-123},
pmid = {42771309},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *Gene Transfer Techniques ; Animals ; CRISPR-Cas Systems ; Nanoparticles/chemistry ; Genetic Therapy/methods ; },
abstract = {The clinical success of CRISPR-based interventions depends primarily on the efficient delivery of editing components into target cells. While base and prime editing have refined genomic precision, achieving therapeutic efficacy requires specialized vehicles that can navigate systemic circulation, escape endosomes, and ensure cell-specific entry. Delivery platforms are traditionally categorized into viral and nonviral systems. Viral vectors, namely adeno-associated vectors (AAV), lentiviral vectors, and adenoviral vectors, employ evolved mechanisms to achieve high transduction efficiency and predictable biodistribution, yet remain constrained by immunogenicity and the risk of insertional mutagenesis. In contrast, nonviral approaches, including synthetic nanoparticles and physical methods, offer superior scalability and transient expression profiles, reducing long-term genomic risks. Next-generation platforms such as virus-like particles (VLPs), engineered extracellular vesicles (EVs), and functionalized nanoparticles have emerged to bridge this gap. These hybrid systems synergize the entry efficiency of viral proteins with the low-immunogenicity profiles of synthetic carriers. Ultimately, the optimal delivery method is determined by the CRISPR cargo format and target cell characteristics, balancing safety, immunogenicity, and scalability. This chapter emphasizes that the strategic selection of a delivery vehicle must be harmonized with the specific cargo and the unique biological requirements of the target tissue to ensure therapeutic success.},
}

Humans
*Gene Editing/methods
*Genetic Vectors/genetics
*Gene Transfer Techniques
Animals
CRISPR-Cas Systems
Nanoparticles/chemistry
Genetic Therapy/methods

@article {pmid42771310,
year = {2027},
author = {Stilhano, R and Martin, L},
title = {Production of Lentiviral Vectors Encoding the CRISPR-Cas13d System for RNA Targeting.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {127-141},
pmid = {42771310},
issn = {1940-6029},
mesh = {*Lentivirus/genetics ; Humans ; *Genetic Vectors/genetics ; *CRISPR-Cas Systems ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transfection/methods ; Gene Knockdown Techniques ; *Gene Editing/methods ; Transduction, Genetic ; Plasmids/genetics ; },
abstract = {The CRISPR-Cas13d system has been shown to be a potent tool for target RNA knockdown, offering advantages over DNA-editing systems by providing a non-genotoxic method to modulate gene expression. Efficient delivery of Cas13d and its guide RNAs into target cells is essential to achieve a strong knockdown effect, particularly in cell lines that are difficult to transfect and primary cells. Lentiviral vectors (LVs) are ideal for delivering large gene-editing tools due to their large packaging capacity and ability to transduce both dividing and nondividing cells. Here, we describe a detailed protocol for the production of high-titer, third-generation LVs with a dual-expression cassette for Cas13d and a customizable guide RNA. The method describes the cloning of target-specific guide RNAs, the transfection of HEK293T cells with packaging and transfer plasmids, the harvest and concentration of viral supernatants, and the determination of viral titer. The generated lentiviral particles can be used for stable transduction and efficient RNA knockdown in a broad range of mammalian cell types.},
}

*Lentivirus/genetics
Humans
*Genetic Vectors/genetics
*CRISPR-Cas Systems
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics
Transfection/methods
Gene Knockdown Techniques
*Gene Editing/methods
Transduction, Genetic
Plasmids/genetics

@article {pmid42771311,
year = {2027},
author = {Wang, X and Liu, J and Janssen, JM and Gonçalves, MAFV},
title = {Production and Storage of High-Capacity Adenoviral Vectors for the Delivery of Advanced CRISPR Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3075},
number = {},
pages = {143-174},
pmid = {42771311},
issn = {1940-6029},
mesh = {*Genetic Vectors/genetics ; *Adenoviridae/genetics ; Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Gene Transfer Techniques ; },
abstract = {Genome editing based on engineered CRISPR systems is advancing rapidly, with the field increasingly moving toward approaches that avoid the induction of mutagenic double-stranded DNA breaks (e.g., RNA-programmable base editing, prime editing, and donor DNA transposition). These nuclease-free strategies often rely on large or multi-component molecular assemblies that can include gene-sized donor DNA substrates. There is, nonetheless, a paucity of vehicles capable of delivering such large and complex genome-editing components effectively and, ideally, in defined stoichiometric ratios. High-capacity adenoviral vector particles (AdVPs) offer an attractive set of features to address these challenges, including robust cell transduction levels regardless of mitotic status, exceptional payload capacity (up to ~36 kb), strict chromosomal nonintegrating character, and the complete absence of viral coding sequences. Hence, AdVPs can serve as biological nanoparticles suitable for the evaluation and application of next-generation CRISPR technologies in physiologically relevant cellular contexts, regardless of the size and number of the attendant tools. Here, after summarizing the key characteristics of earlier- and latest-generation adenoviral vector platforms, we describe protocols for producing AdVPs, including vectors that deliver multiplexing, prime-editing, and orthogonal nuclease constructs. Finally, we highlight important considerations for designing AdVP production reagents and validate a storage buffer that preserves AdVP functionality after repeated freeze-thaw cycles.},
}

*Genetic Vectors/genetics
*Adenoviridae/genetics
Humans
*CRISPR-Cas Systems
*Gene Editing/methods
Gene Transfer Techniques

@article {pmid25977940,
year = {2015},
author = {Vyas, VK and Barrasa, MI and Fink, GR},
title = {A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families.},
journal = {Science advances},
volume = {1},
number = {3},
pages = {e1500248},
pmid = {25977940},
issn = {2375-2548},
support = {F32 AI072935/AI/NIAID NIH HHS/United States ; R01 GM035010/GM/NIGMS NIH HHS/United States ; },
mesh = {*Candida albicans/genetics ; *Genetic Engineering/methods ; *Genes, Essential ; *CRISPR-Cas Systems ; *Multigene Family ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genes, Fungal ; Mutation ; Gene Knockout Techniques ; },
abstract = {Candida albicans is a pathogenic yeast that causes mucosal and systematic infections with high mortality. The absence of facile molecular genetics has been a major impediment to analysis of pathogenesis. The lack of meiosis coupled with the absence of plasmids makes genetic engineering cumbersome, especially for essential functions and gene families. We describe a C. albicans CRISPR system that overcomes many of the obstacles to genetic engineering in this organism. The high frequency with which CRISPR-induced mutations can be directed to target genes enables easy isolation of homozygous gene knockouts, even without selection. Moreover, the system permits the creation of strains with mutations in multiple genes, gene families, and genes that encode essential functions. This CRISPR system is also effective in a fresh clinical isolate of undetermined ploidy. Our method transforms the ability to manipulate the genome of Candida and provides a new window into the biology of this pathogen.},
}

*Candida albicans/genetics
*Genetic Engineering/methods
*Genes, Essential
*CRISPR-Cas Systems
*Multigene Family
*Clustered Regularly Interspaced Short Palindromic Repeats
*Genes, Fungal
Mutation
Gene Knockout Techniques

@article {pmid42508764,
year = {2026},
author = {Ghosh, U and Tay, A},
title = {Biophysical considerations for designing viruses, lipid nanoparticles and virus-like particles for CRISPR-based genome editing.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {398},
number = {},
pages = {115204},
doi = {10.1016/j.jconrel.2026.115204},
pmid = {42508764},
issn = {1873-4995},
mesh = {Humans ; *Gene Editing/methods ; *Nanoparticles/chemistry/administration & dosage ; Animals ; *Lipids/chemistry ; *CRISPR-Cas Systems ; *Viruses/genetics ; Genetic Vectors ; Liposomes ; },
abstract = {CRISPR-based genome editing has opened new pathways towards precision medicine, but its success depends on more than just molecular engineering. Cargo and carrier dynamics are profoundly influenced by the underlying biophysical properties of cells and vectors. Consequently, this domain is moving beyond simple "lock and key" approaches, and towards disease-customised fits. In this review, we examine how biophysical properties of viral vectors, lipid nanoparticles, and hybrid virus-like particles influence editor delivery performance. We examine parameters such as size, cargo capacity, charge, shape, stiffness, membrane composition, internalisation strategies, tropism, endosomal escape, protein corona formation, and immune recognition as key drivers of intelligent, modular engineering. Finally, we explore how the notion of carrier systems shifts in diseased states like solid tumour cancers, autoimmune psoriasis of the skin, and the autosomal monogenic cystic fibrosis, where altered biophysical landscapes demand adaptable, context-informed genome editing solutions.},
}

Humans
*Gene Editing/methods
*Nanoparticles/chemistry/administration & dosage
Animals
*Lipids/chemistry
*CRISPR-Cas Systems
*Viruses/genetics
Genetic Vectors
Liposomes

@article {pmid42763006,
year = {2026},
author = {Patel, V and Singh, P and Dutta, SP and Pande, S and Langstang, R and Kushwaha, A},
title = {CRISPR and Gene Editing Approaches in Prostate Cancer: Clinical Applications and Therapeutic Potential.},
journal = {Critical reviews in oncology/hematology},
volume = {},
number = {},
pages = {105610},
doi = {10.1016/j.critrevonc.2026.105610},
pmid = {42763006},
issn = {1879-0461},
abstract = {Prostate cancer is one of the leading causes of cancer-related morbidity and mortality among men worldwide and is characterized by substantial molecular heterogeneity and the development of therapeutic resistance. Recent advances in genome-editing technologies, particularly CRISPR-Cas systems, have expanded opportunities for precise investigation and modification of genetic and epigenetic determinants involved in prostate cancer progression. This review comprehensively describes the evolution of CRISPR-based genome-editing tools, including Cas9 nucleases, base editing, prime editing, and CRISPR interference/activation systems, and their applications in prostate cancer models. Particular emphasis is placed on androgen receptor (AR) signaling and DNA damage repair (DDR) pathways, as well as genomic alterations such as PTEN loss and TMPRSS2-ERG fusion, which represent important molecular determinants and therapeutic targets in prostate cancer. The review further examines the application of CRISPR in functional genomic screening, disease modeling, and the identification of synthetic lethal interactions that may reveal novel therapeutic vulnerabilities. Emerging therapeutic strategies, including gene correction, targeting mechanisms underlying resistance to androgen deprivation and AR-directed therapies, sensitization to chemotherapy and radiotherapy, epigenome editing, and immunotherapy engineering, are critically discussed. Advances in CRISPR delivery modalities, including viral vectors, lipid-based nanoparticles, polymeric systems, and extracellular vesicles, are also evaluated with emphasis on tumor targeting, delivery efficiency, safety, and translational challenges. Overall, CRISPR-based technologies show considerable potential to support precision oncology in prostate cancer by enabling molecularly informed therapeutic strategies and addressing treatment resistance, although challenges related to delivery, off-target effects, tumor heterogeneity, immunogenicity, and clinical translation remain to be resolved.},
}

@article {pmid42763149,
year = {2026},
author = {Cheng, L and Yang, Q and Yang, Y and Wang, Y and He, Q and Zou, Z and Su, C and Chen, J and Liu, Y and Hong, G},
title = {Amplification-free electrochemiluminescence biosensors based on peptide-templated gold nanocluster and CRISPR-Cas12a for Mycobacterium tuberculosis IS6110 detection.},
journal = {Analytica chimica acta},
volume = {1422},
number = {},
pages = {346090},
doi = {10.1016/j.aca.2026.346090},
pmid = {42763149},
issn = {1873-4324},
mesh = {*Gold/chemistry ; *Mycobacterium tuberculosis/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Metal Nanoparticles/chemistry ; *Electrochemical Techniques/methods ; Luminescent Measurements/methods ; *CRISPR-Cas Systems/genetics ; *Peptides/chemistry ; *DNA, Bacterial/analysis/genetics ; Limit of Detection ; Humans ; Ferrous Compounds/chemistry ; },
abstract = {BACKGROUND: Tuberculosis (TB) nucleic acid diagnosis urgently requires rapid, amplification-free methods to overcome limitations of quantitative real-time polymerase chain reaction (qPCR), including instrument dependency, prolonged time, and contamination risks from nucleic acid amplification.

RESULTS: Here, an amplification-free electrochemiluminescence (ECL) biosensor based on peptide-templated gold nanoclusters and the clustered regularly interspaced short palindromic repeats-Cas12a system (CRISPR-Cas12a) has been constructed for the detection of Mycobacterium tuberculosis (MTB)-specific IS6110 sequences. Peptide-templated gold nanoclusters with low background and high ECL response can be used as sensitive signal probes. When CRISPR-Cas12a recognizes the target IS6110 DNA, it exhibits non-specific cleavage activity, repeatedly cleaving ferrocene-labelled DNA. This results in the restoration of ECL signals quenched by ferrocene, thereby achieving signal amplification. There is a linear relationship between the signal and the target concentration range from 10 CFU/mL to 10[4] CFU/mL, with a detection limit of 7 CFU/mL (S/N = 3). Clinical validation (n = 40) showed strong agreement with qPCR (κ = 0.90).

SIGNIFICANCE: Critically, this amplification-free strategy eliminates the need for temperature cycling equipment, reduces detection time to 1 h, and completely avoids the risk of amplicon contamination, providing an ideal solution for rapid screening of highly infectious diseases such as TB.},
}

*Gold/chemistry
*Mycobacterium tuberculosis/isolation & purification/genetics
*Biosensing Techniques/methods
*Metal Nanoparticles/chemistry
*Electrochemical Techniques/methods
Luminescent Measurements/methods
*CRISPR-Cas Systems/genetics
*Peptides/chemistry
*DNA, Bacterial/analysis/genetics
Limit of Detection
Humans
Ferrous Compounds/chemistry

@article {pmid42763165,
year = {2026},
author = {Xiao, S and Liang, Y and Zhang, Y and Liang, L and Xu, B and Mu, X and Zhao, S and Tian, J},
title = {A double-key responsive TDNs-HC/Cas13a DNA circuit for free-amplified detection and precise imaging of miRNAs in living cell.},
journal = {Analytica chimica acta},
volume = {1422},
number = {},
pages = {346106},
doi = {10.1016/j.aca.2026.346106},
pmid = {42763165},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; DNA Nanostructures ; *DNA/chemistry/genetics ; Nucleic Acid Hybridization ; Biosensing Techniques/methods ; },
abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated (Cas) proteins is an RNA-guided gene editing system with high targeting specificity. Its exceptional recognition capability for target genes has demonstrated immense potential in the field of biosensing. However, the effective integration and delivery of CRISPR/Cas systems and nucleic acid hybridization for precise imaging and detection of lowly expressed analyte in cellulo remains a critical challenge. Here, an integrated TDNs-HC/Cas13a DNA circuit was constructed for free-amplified detection and precise imaging of miRNAs in living cell. The novel design of this method utilizes DNA Tetrahedrons (TDNs) as nanoscaffolds, with three vertices assembled to incorporate miRNA-155-responsive CRISPR/Cas13a and the lock that recognizes miRNA-21, enabling precise molecular recognition through an AND logic gate mechanism.

RESULTS: This TDNs-HC/Cas13a strategy integrated target recognition module, logical operations module, and signal output module, enabling intracellular co-delivery of elements of module without external vectors. The dual target recognition and synergistically signal-amplification of CRISPR/Cas13a enabled the sensitive and free-amplified detection of miRNA-155 and miRNA-21, and the limit of detection is 32 pM and 5 pM, respectively. At the same time it can be applied for the expression level analysis and single-cell imaging of miRNA-155 and miRNA-21 in cells. Experimental results show that the TDNs-HC/Cas13a system effectively discriminates between normal cells and cancer cells based on fluorescence intensity, confirming its capability for specific imaging of cancer cells.

SIGNIFICANCE AND NOVELTY: This design likes a dual-password safe lock, precisely excluding other cells that express only a single marker or ingest a small number of probe molecules, significantly improving the signal-to-noise ratio and accuracy of detection and imaging.},
}

*MicroRNAs/analysis/genetics
Humans
*CRISPR-Cas Systems/genetics
DNA Nanostructures
*DNA/chemistry/genetics
Nucleic Acid Hybridization
Biosensing Techniques/methods

@article {pmid42763168,
year = {2026},
author = {Zhang, X and Pan, Z and Wen, J and Wang, C and Tang, C and Tang, Z},
title = {A purification-free one-pot CRISPR/Cas13a assay for detection of porcine epidemic diarrhea virus.},
journal = {Analytica chimica acta},
volume = {1422},
number = {},
pages = {346109},
doi = {10.1016/j.aca.2026.346109},
pmid = {42763168},
issn = {1873-4324},
mesh = {*Porcine epidemic diarrhea virus/isolation & purification/genetics ; Animals ; Swine ; *CRISPR-Cas Systems/genetics ; *RNA, Viral/genetics/analysis ; *Coronavirus Infections/veterinary/diagnosis/virology ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) causes up to 80-100% mortality in neonatal piglets, yet field surveillance remains constrained by RT-qPCR's dependence on column-based nucleic acid purification and centralized laboratory infrastructure-a bottleneck especially severe in the inhibitor-rich matrices typical of swine clinical samples. Here we report an integrated sample-to-answer methodology coupling a purification-free thermal lysis step with one-pot RT-RPA-CRISPR/Cas13a chemistry, delivering PEDV detection within 35 min. Reverse transcription, recombinase polymerase amplification, T7 in vitro transcription, and Cas13a collateral cleavage are confined to a single sealed tube, eliminating open-tube transfer and aerosol contamination. Optimized thermal lysis (80°C, 7 min) liberates amplifiable viral RNA directly from crude anal swabs and feces, obviating column purification. The assay attained 10[1] copies/μL analytical sensitivity with high analytical specificity against six non-target porcine pathogens under the tested conditions. Critically, matrix-tolerance profiling showed the CRISPR-based workflow suppressed inhibition to 7.16% (anal swabs) and 11.89% (feces), versus 63.65% and 69.39% for RT-qPCR (P < 0.01), demonstrating markedly superior robustness under authentic matrices. In a double-blind evaluation of 297 clinical specimens, the platform reached 98.65% concordance with national reference standards (Cohen's κ = 0.941), correctly identifying all 37 RT-qPCR-confirmed positives and additionally resolving four low-titer infections missed by RT-qPCR. By converting an inhibitor-sensitive, infrastructure-bound assay into a purification-free, contamination-resistant workflow with validated clinical reliability, this work provides a robust sample-to-answer analytical strategy for PEDV detection in complex clinical matrices.},
}

*Porcine epidemic diarrhea virus/isolation & purification/genetics
Animals
Swine
*CRISPR-Cas Systems/genetics
*RNA, Viral/genetics/analysis
*Coronavirus Infections/veterinary/diagnosis/virology

@article {pmid42763182,
year = {2026},
author = {Zhou, L and Wei, K and Liu, Q and Wu, G and Wang, K},
title = {Signal transduction and engineering strategies of CRISPR-Cas biosensors: A review.},
journal = {Analytica chimica acta},
volume = {1422},
number = {},
pages = {346016},
doi = {10.1016/j.aca.2026.346016},
pmid = {42763182},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Signal Transduction ; Humans ; Protein Engineering ; },
abstract = {BACKGROUND: Class 2 CRISPR-Cas systems are characterized by their single-component architecture and RNA-guided effector proteins such as Cas9, Cas12, and Cas13. They have established a versatile molecular framework for developing a new generation of biosensing platforms. The programmability of these systems, combined with their unique enzymatic properties, particularly the target-activated trans-cleavage of reporters, allows molecular detection with exceptional specificity and sensitivity. However, translation of these advantages into practical applications and resource-limited settings remains challenging due to complex signal readout, engineering constraints, and integration hurdles.

RESULTS: This review systematically examines the current CRISPR-Cas biosensing landscape, focusing on the operational mechanisms of major effector proteins and the broad spectrum of signal transduction methodologies that convert molecular recognition into measurable signals. These approaches encompass optical techniques (fluorescence, colorimetry, surface-enhanced Raman scattering, chemiluminescence) and electrochemical-based methods (conventional electrochemistry, photoelectrochemistry, electrochemiluminescence). We further highlight engineering advances that enhance performance through protein engineering, amplification-free strategies, and expansion to non-nucleic acid targets and multiplexed assays. Integration with miniaturized platforms, digital readouts, and artificial intelligence is accelerating the transition toward practical use. We conclude that CRISPR-Cas biosensors hold considerable potential to advance decentralized diagnostics, biomedical research, and global health surveillance.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Signal Transduction
Humans
Protein Engineering

@article {pmid42764010,
year = {2026},
author = {Shao, F and Hu, J and Traylor, A and Lei, H and Zhang, P and Akarapipad, P and Park, JS and Hsieh, K and Wang, TH},
title = {CRISPR-AMPED: A CRISPR/Cas-based immunoassay with attomolar sensitivity enabled by magnetic proximity extension and detection.},
journal = {Biosensors & bioelectronics},
volume = {315},
number = {},
pages = {119227},
doi = {10.1016/j.bios.2026.119227},
pmid = {42764010},
issn = {1873-4235},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-associated systems have emerged as powerful tools for next-generation molecular diagnostics, particularly for nucleic acid detection. However, ultrasensitive protein detection is equally critical across diverse applications in biology and medicine, especially for diagnosing and prognosing diseases such as cancer, traumatic brain injury (TBI), Alzheimer's disease, and cardiovascular diseases. Despite recent efforts to adapt CRISPR/Cas systems for protein detection, these methods have typically achieved sensitivity in the femtomolar to picomolar range, underscoring the need for enhanced detection capabilities. Here, we developed CRISPR-AMPED, a CRISPR/Cas-based immunoassay enhanced by magnetic proximity extension and detection. This approach combines proximity extension assay (PEA) with magnetic beads to convert protein targets into DNA barcodes while enabling effective washing to reduce background noise. The resulting DNA barcodes are detected through recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12a, eliminating thermocycling and providing simultaneous target and signal amplification. CRISPR-AMPED achieves attomolar-level sensitivity, surpassing ELISA by over three orders of magnitude and outperforming existing immunoassays and CRISPR/Cas-based protein detection systems. As an initial demonstration of clinical utility, we applied CRISPR-AMPED to detect the inflammatory biomarker interleukin-8 (IL-8) in serum samples from patients with TBI and healthy controls. Further integration with a smartphone-based detection device demonstrates its potential for portable testing, while the digital format extends the dynamic range and enhances quantitation precision. Together, these results establish CRISPR-AMPED as a sensitive protein detection approach using IL-8 as an initial model target and provide a framework for future adaptation to additional protein biomarkers.},
}

@article {pmid42766049,
year = {2026},
author = {Janthabut, T and Pongjaroenkit, S and Khemkladngoen, N and Lithanatudom, SK and Chanchay, P and Vuttipongchaikij, S and Sakulsingharoj, C},
title = {Multiplex CRISPR/Cas12a editing of ROC5, GS3, GW2, and LARGE2 reveals effects on yield-related traits in rice cultivar Kasalath.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {42766049},
issn = {1573-9368},
support = {FF(KU-SRIU)22.67//Kasetsart University Research and Development Institute/ ; Maejo University Fundamental Fund 2025 (MJ.1-68-01-002)//Thailand Science Research and Innovation/ ; },
mesh = {*Oryza/genetics/growth & development ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plants, Genetically Modified/genetics/growth & development ; *Plant Proteins/genetics ; Phenotype ; Alleles ; },
abstract = {Sustainable improvement of rice yield requires coordinated modification of multiple agronomic traits, yet the agronomic outcomes of multiplex genome editing remain difficult to predict. Here, we applied CRISPR/Cas12a-mediated multiplex editing in the indica rice cultivar Kasalath to target six yield-related genes, Gn1a, TAD1, ROC5, GS3, GW2, and LARGE2, using a single Agrobacterium-delivered construct. Sanger sequencing of 28 T0 plants detected edits in Gn1a (3.57%), ROC5 (7.14%), GS3 (35.71%), and GW2 (10.71%), whereas TAD1 and LARGE2 showed no detectable T0 edits. Single, double, and triple edits were recovered, and homozygous lines were established for four single mutants, two double mutants, and two triple mutants. Molecular analysis showed that the recovered GW2 alleles were intronic and did not alter the GW2 coding sequence. Phenotypic evaluation across 13 agronomic traits showed that allele type and locus combination, rather than simple additive effects, determined agronomic outcome. The gs3-13d frameshift allele caused strong pleiotropic effects, including increased tillering and panicle number but reduced grain length and yield. The roc5-7d gw2-8d gs3-3i triple mutant maintained wild-type-level yield performance with altered leaf architecture, whereas gw2-8d gs3-3i large2-10d showed increased grain width, grain thickness, and 1000-grain weight but severe reductions in plant height, tillering, seed setting, yield per panicle, and yield per plant. These results demonstrate the feasibility of CRISPR/Cas12a multiplex editing in Kasalath and highlight the importance of allele design, locus combination, and source-sink balance in multiplex genome editing targeting yield-related traits.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Plants, Genetically Modified/genetics/growth & development
*Plant Proteins/genetics
Phenotype
Alleles

@article {pmid42766112,
year = {2026},
author = {Shang, J and Li, L and Dong, C and Wei, Y},
title = {Bacteriophage-bacteria coevolution: from molecular arms races to ecological and applied perspectives.},
journal = {Archives of microbiology},
volume = {208},
number = {12},
pages = {},
pmid = {42766112},
issn = {1432-072X},
mesh = {*Bacteriophages/genetics/physiology ; *Bacteria/virology/genetics ; *Biological Coevolution ; Gene Transfer, Horizontal ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; *Biological Evolution ; Evolution, Molecular ; },
abstract = {Bacteriophages are the most abundant biological entities, driving bacterial evolution through long-term coevolution. Bacteria have evolved diverse defense strategies against phage, including receptor modification, restriction-modification systems, CRISPR-Cas, abortive infection systems, and newly discovered systems such as BREX, DISARM, CBASS, Thoeris, and Zorya. In response, phages deploy countermeasures such as receptor-binding diversification, anti-CRISPR proteins, DNA modification, and inhibitors targeting host immunity. These interactions generate distinct evolutionary dynamics-arms race and fluctuating selection-shaping microbial population structure and ecological stability. Phage-host coevolution promotes microbial diversity, horizontal gene transfer, and regulates community composition across ecosystems. Understanding these processes is critical for applications like phage therapy, microbiome engineering, and biotechnology. This review summarizes molecular mechanisms of bacterial defense and phage counter-defense, discusses coevolutionary models, highlights ecological and applied implications, and outlines future research directions.},
}

*Bacteriophages/genetics/physiology
*Bacteria/virology/genetics
*Biological Coevolution
Gene Transfer, Horizontal
Host-Pathogen Interactions
CRISPR-Cas Systems
*Biological Evolution
Evolution, Molecular

@article {pmid42766154,
year = {2026},
author = {Shabbir, AQ and Idrees, J and Khan, AA and Alvi, IA and Sana, S and Rehman, SU and Asif, M},
title = {Technical limitations of CRISPR-Cas9 genome editing in bacteria: challenges and future directions.},
journal = {Archives of microbiology},
volume = {208},
number = {12},
pages = {},
pmid = {42766154},
issn = {1432-072X},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Bacteria/genetics ; Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR-Cas system, which originated as an adaptive immune system in bacteria and archaea, has been repurposed as a precise and programmable tool for genetic manipulation in both prokaryotes and eukaryotes. Its applications in bacteria include targeted genome modifications, antimicrobial resistance studies, functional genomics studies, and the development of engineered strains for industrial and synthetic biology applications. Therefore, the understanding of technical aspects of CRISPR systems and their underlying molecular mechanisms is essential for experimental accuracy, reproducibility, and biosafety. CRISPR editing introduces several challenges in bacteria, including off-target effects, DNA repair limitations, cytotoxicity of Cas nucleases, and host-specific restriction-modification barriers. In addition to these specific challenges, metabolic burden, sgRNA design, and delivery challenges further introduce limitations. Such issues compromise editing efficiency, genomic stability, and cell viability. Recent studies have focused on improved guide RNA design, alternative Cas variants, refined delivery strategies, and host-adapted engineering as promising directions to enhance editing. This review discusses the principal barriers to CRISPR-Cas9 genome editing of bacteria, evaluates the current strategies for addressing these barriers, and highlights emerging approaches aimed at improving the efficiency, reliability and precision in bacterial genome engineering.},
}

*CRISPR-Cas Systems
*Gene Editing/methods/trends
*Bacteria/genetics
Genome, Bacterial
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42768305,
year = {2026},
author = {Avershina, E and Birkeland, EE and Bucher-Johannessen, C and Rounge, TB},
title = {CRISPR-Cas immune repertoires as an ecological record of bacterial interactions with mobile genetic elements in the human gut.},
journal = {Gut microbes},
volume = {18},
number = {1},
pages = {2734649},
pmid = {42768305},
issn = {1949-0984},
mesh = {Humans ; *Interspersed Repetitive Sequences ; *Bacteria/genetics/classification/immunology/isolation & purification/virology ; *Gastrointestinal Microbiome/genetics ; *CRISPR-Cas Systems ; Plasmids/genetics ; Feces/microbiology ; Middle Aged ; Metagenome ; Norway ; Female ; Diet ; Bacteriophages/genetics ; Gastrointestinal Tract/microbiology ; },
abstract = {Bacteria in the human gut influence host physiology and disease risk, but their ecology is strongly shaped by mobile genetic elements (MGEs) such as phages and plasmids. Past interactions between bacteria and MGEs can be inferred from CRISPR-Cas cassettes, which contain short DNA fragments derived from invading elements. To lay the groundwork for research on the impact of such interactions on the human host, we constructed an extended microbiome resource comprising 1.7 K prokaryotic mOTUs, 19.5 K viral vOTUs, and 24.2 K plasmid PTUs, using fecal shotgun metagenomes from 1034 adults over 55 y of age residing in South-East Norway. We also recovered 74.2 K unique CRISPR-Cas cassettes to map past bacteria-MGE interactions and assessed their associations with the human diet and lifestyle factors. CRISPR-Cas spacers, and which viruses and plasmids they targeted, varied substantially within bacterial species, but were predominantly directed towards cohort-specific MGEs. Moreover, bacteria were more likely to target MGEs present in the same sample, consistent with local exposure. Plasmid MGEs were more often targeted by Type II CRISPR-Cas cassettes, whereas viruses were more likely to be targeted by Type I CRISPR-Cas cassettes. Bacteria also shared more targets within taxonomic families than across families, where mobilizable plasmids were more frequent among the targets. CRISPR-Cas cassettes mirrored microbiome associations to human demographic and lifestyle factors and enabled the recovery of dairy-associated B. animalis. Together, this research provides a large-scale resource and a structured analysis of bacteria-MGE interactions in the gut microbiome and their contribution to microbial ecosystem dynamics.},
}

Humans
*Interspersed Repetitive Sequences
*Bacteria/genetics/classification/immunology/isolation & purification/virology
*Gastrointestinal Microbiome/genetics
*CRISPR-Cas Systems
Plasmids/genetics
Feces/microbiology
Middle Aged
Metagenome
Norway
Female
Diet
Bacteriophages/genetics
Gastrointestinal Tract/microbiology

@article {pmid42769418,
year = {2026},
author = {Natarajan, PM and Ebenezer, V and Varma, SR and Ganesh, P},
title = {Novel biofilm-targeted therapeutics for oral infections: enzymes, EPS disruptors, phage/CRISPR, photodynamic and cold-plasma approaches - a systematic review.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1915920},
pmid = {42769418},
issn = {2235-2988},
mesh = {*Biofilms/drug effects ; Humans ; *Photochemotherapy/methods ; *Plasma Gases/therapeutic use/pharmacology ; Animals ; Bacteriophages ; *Extracellular Polymeric Substance Matrix/drug effects ; *Mouth Diseases/therapy/microbiology ; Phage Therapy ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Microbial biofilms underpin the chronicity, recurrence and antimicrobial tolerance of most oral infections. As mechanical and antibiotic strategies are constrained by antimicrobial resistance and by the protective biofilm matrix, non-antibiotic, biofilm-targeted therapeutics have attracted intense interest. We systematically mapped and appraised five mechanistically distinct modalities - matrix-degrading (anti-biofilm) enzymes, extracellular polymeric substance (EPS) disruptors, bacteriophage and CRISPR-based therapy, antimicrobial photodynamic therapy (aPDT) and cold atmospheric plasma (CAP) - selected because each targets a different, non-antibiotic vulnerability of the biofilm.

METHODS: Following a PRISMA 2020 protocol (PROSPERO), PubMed, Embase, Web of Science and Scopus were searched from inception to January 2026. In vitro, animal and clinical studies reporting a quantitative anti-biofilm outcome for any modality against oral or oral-relevant pathogens were included, appraised with RoB 2, SYRCLE and a modified in vitro checklist, and the certainty of evidence rated with GRADE. Prespecified subgroup (biofilm maturity, species complexity) and quality-based sensitivity analyses were performed.

RESULTS: Seventy-eight studies met the criteria; 58% (45/78) were in vitro/ex vivo, 16 animal and only 17 (22%) clinical, so clinical evidence was limited and concentrated in aPDT. aPDT provided small but consistent adjunctive gains over scaling and root planing (SRP): pooled additional probing-pocket-depth reduction ≈0.35-0.45 mm and clinical-attachment gain ≈0.25-0.34 mm at 3-6 months (low-moderate certainty). EPS disruptors reduced biofilm biomass by 58-94% and CAP rendered ≈90% of treated samples culture-negative in vitro, but both rested on preclinical data (low-very-low certainty). Enzymes and phage/CRISPR acted mainly by dispersal or targeted killing (representative reductions ≈1.5-4.5 log10 CFU). Efficacy fell consistently against mature, multispecies biofilms; sensitivity analysis excluding high-risk studies changed estimates minimally.

CONCLUSION: On current evidence these modalities are best positioned as adjuncts that enhance, rather than replace, mechanical and antimicrobial therapy. Only aPDT currently has sufficient clinical evidence for consideration as an adjunct to conventional therapy; the remaining modalities remain investigational and require further translational and clinical development. Combination (matrix-first) strategies, targeted delivery, and standardised oral-biofilm models and clinical trials are priorities.

https://www.crd.york.ac.uk/PROSPERO/, identifier CRD420261428848.},
}

*Biofilms/drug effects
Humans
*Photochemotherapy/methods
*Plasma Gases/therapeutic use/pharmacology
Animals
Bacteriophages
*Extracellular Polymeric Substance Matrix/drug effects
*Mouth Diseases/therapy/microbiology
Phage Therapy
CRISPR-Cas Systems

@article {pmid42770331,
year = {2026},
author = {Li, X and Wu, C and Guo, J and Zheng, R and Kong, J and Du, L and She, Q and Ji, S},
title = {BaCas12a3 represents a new subtype of type V CRISPR effector with collateral activity toward tRNA.},
journal = {Nucleic acids research},
volume = {54},
number = {18},
pages = {},
pmid = {42770331},
issn = {1362-4962},
support = {2021YFA0717000//National Key Research and Development Program of China/ ; SKLMTFCP-2023-01//Frontiers and Challenges Projects/ ; SKLMTIJP-2024-05//Intramural Joint Program Fund/ ; //State Key Laboratory of Microbial Technology/ ; //Shandong University/ ; },
mesh = {*RNA, Transfer/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; Models, Molecular ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; Catalytic Domain ; },
abstract = {The CRISPR-Cas12 family encompasses diverse RNA-guided nucleases with both DNA- and RNA-targeting subtypes. They can trigger antiviral activities through either direct elimination of invading nucleic acids or activating broad collateral cleavage to induce abortive infection. Here, we report a novel type V CRISPR effector BaCas12a3 that causes growth inhibition through a unique tRNA-cleavage mechanism. Plasmid interference and western blot assays showed that BaCas12a3 induces host growth arrest without DNA damage response, suggestive of the absence of double-strand DNA breaks. Indeed, biochemical characterization of the BaCas12a3-crRNA ribonucleoprotein unraveled that the effector is an RNA-activating nuclease that cleaves the 3' terminal CCA of tRNAs. Cryo-EM structures of BaCas12a3 reveal a conserved bilobed architecture featuring a unique tRNA-loading domain (tRLD) adjacent to the RuvC catalytic center. Structural and mutagenesis analyses show that the tRLD domain, together with a zinc ribbon domain, form a gated substrate groove. Target RNA binding induces conformational changes that open the groove and expose the RuvC active site, enabling specific tRNA 3' end cleavage while preventing other non-specific degradation. Our findings identify the tRLD domain aside the RuvC active site responsible for the tRNA recognition in BaCas12a3, expanding the functional diversity of CRISPR immunity.},
}

*RNA, Transfer/metabolism/genetics/chemistry
*CRISPR-Cas Systems
Models, Molecular
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
*Bacterial Proteins/metabolism/genetics/chemistry
Cryoelectron Microscopy
Catalytic Domain

@article {pmid42449099,
year = {2026},
author = {Huang, T and He, Y and Wang, X and Liu, Z and Qiao, E and Sun, T and Mei, J and Dai, S and Wang, Z and Feng, C and Cao, H and Wang, Y and Xia, Y and Liu, E and Guo, J and Lu, J},
title = {CRISPR/Cas9 screen identifies DCAF4 as a novel protector of hepatocellular carcinoma against brachytherapy via stress granule-dependent NRF2 activation.},
journal = {Cell death & disease},
volume = {17},
number = {1},
pages = {},
pmid = {42449099},
issn = {2041-4889},
support = {82572339//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82372066//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics/radiotherapy/metabolism/pathology ; *Liver Neoplasms/radiotherapy/genetics/metabolism/pathology ; Animals ; *NF-E2-Related Factor 2/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Kelch-Like ECH-Associated Protein 1/metabolism ; *Stress Granules/metabolism ; Oxidative Stress ; Cell Line, Tumor ; Mice ; Ubiquitin-Protein Ligases/metabolism ; Ubiquitination ; Mice, Nude ; },
abstract = {Hepatocellular carcinoma (HCC) cells sustain viability and radioresistance by actively countering oxidative stress. Understanding the mechanisms regulating reactive oxygen species (ROS) homeostasis is therefore crucial for developing novel therapies. Using integrated genome-wide CRISPR-Cas9 screening coupled with transcriptomic and metabolomic profiling, we identified DDB1 and CUL4-associated factor 4 (DCAF4) as an essential regulator of oxidative stress resistance in HCC. Mechanistically, DCAF4 functions as a CRL4 E3 ligase adapter that promotes KEAP1 ubiquitination and degradation. Notably, under oxidative stress, cytoplasmic stress granules (SGs) form a localized platform that facilitates the DCAF4-KEAP1 interaction, accelerating KEAP1 degradation and leading to NRF2 activation and upregulation of antioxidant genes. We further identified that the transcription factor XBP1 enhances DCAF4 expression. Targeting this axis, we performed computational screening to identify a small-molecule inhibitor that disrupts the DCAF4-KEAP1 interaction. This compound effectively enhanced brachytherapy (BT) sensitivity and inhibited tumor growth in preclinical HCC models.},
}

Humans
*Carcinoma, Hepatocellular/genetics/radiotherapy/metabolism/pathology
*Liver Neoplasms/radiotherapy/genetics/metabolism/pathology
Animals
*NF-E2-Related Factor 2/metabolism/genetics
*CRISPR-Cas Systems/genetics
Kelch-Like ECH-Associated Protein 1/metabolism
*Stress Granules/metabolism
Oxidative Stress
Cell Line, Tumor
Mice
Ubiquitin-Protein Ligases/metabolism
Ubiquitination
Mice, Nude

@article {pmid42745954,
year = {2026},
author = {Nguyen, ANT and Zhang, J and Zhang, S and Pitt, ME and Ganesamoorthy, D and Fritzlar, S and Chang, JJ and Londrigan, SL and Coin, LJM},
title = {Customizable host and viral transcript enrichment using CRISPR-Cas9 long-read sequencing for characterization of low-to-moderate abundance isoforms.},
journal = {NAR genomics and bioinformatics},
volume = {8},
number = {3},
pages = {lqag111},
pmid = {42745954},
issn = {2631-9268},
mesh = {*CRISPR-Cas Systems ; Humans ; Protein Isoforms/genetics ; High-Throughput Nucleotide Sequencing/methods ; *Sequence Analysis, RNA/methods ; },
abstract = {One of the main challenges of whole transcriptome sequencing is the difficulty in detecting and quantifying low-to-moderate abundance transcripts. Methods that address this are either complicated to scale or customize; long-range PCR is problematic to scale, and probe hybridization panels are expensive to customize. In this study, we developed an RNA-guided CRISPR-Cas9 nuclease-based enrichment strategy combined with long-read sequencing, which achieved up to 60-fold enrichment of the target. Our findings demonstrate that the CRISPR-Cas system is a highly effective method for customizable long-read sequencing of target transcripts, which preserves estimation of relative abundance.},
}

*CRISPR-Cas Systems
Humans
Protein Isoforms/genetics
High-Throughput Nucleotide Sequencing/methods
*Sequence Analysis, RNA/methods

@article {pmid42746213,
year = {2026},
author = {Xedzro, C and Shimamoto, T and Ahmed, AM and Yu, L and Sugawara, Y and Sugai, M and Shimamoto, T},
title = {Genomic Insights Into Multidrug-Resistant Foodborne Serratia liquefaciens Strains Carrying mcr-9 and Comparative Genomic Analysis of Novel Biosynthetic Gene Clusters.},
journal = {International journal of food science},
volume = {2026},
number = {},
pages = {5035164},
pmid = {42746213},
issn = {2314-5765},
abstract = {Serratia liquefaciens is an opportunistic nosocomial pathogen with a wide range of antibiotic resistance patterns. This study reports the characterization of the first mcr-9-positive S. liquefaciens strains, 35E-19E1 and CST-066, isolated from meat products in Japan. The strains were screened for the presence of β-lactamases, plasmid-mediated mobile colistin resistance (mcr) genes, and carbapenemase-encoding genes using PCR. Antimicrobial susceptibility was tested using the broth microdilution method. The strains exhibited multidrug resistance (MDR) phenotypes to third-generation cephalosporins, cephamycin, fosfomycin, and other clinically important antimicrobials. Genomic DNA sequencing showed that the genome sizes of CST-066 and 35E-19E1 are 5,529,704 and 5,261,506 bps, respectively. mcr-9 was identified on a chromosome within a genetic environment that included the two-component system qseBC, which plays a key role in the signaling network that triggers colistin resistance in Enterobacterales. Downstream genome analysis revealed a 1695-bp eptB-like kdo2-lipid phosphoethanolamine transferase, which is involved in intrinsic polymyxin resistance mechanisms in Serratia spp. The strain 35E-19E1 carries five CRISPR-Cas enzymes that are essential for adaptive immunity in bacteria, allowing defense against invading elements. Functional analysis using subsystem technology revealed that both strains possess subsystem features responsible for invasion and adhesion within the host biomes. Genome mining using antiSMASH and BAGL4 revealed various biosynthetic gene clusters, responsible for secondary metabolite synthesis. Notably, we identified novel gene clusters, mainly nonribosomal peptide synthetases, in both the strains, indicating their potential to produce bioactive compounds. Although the presence of mcr-9 in Serratia may not be of clinical significance because of natural resistance of the strain to polymyxins, we shed light on the genomic characteristics of this MDR pathogen and the potential spread of mcr-9 among other bacterial species. The emergence of mcr-9 in drug-resistant S. liquefaciens provides significant insights, underscoring the need for increased surveillance of this pathogen.},
}

@article {pmid42747042,
year = {2026},
author = {Stoker, C and Mustafa, Y and Liu, Y and Patrick, T and Perisse, IV and Polejaeva, IA},
title = {Generation of Genetically Engineered Embryos Using Gene Editing and Somatic Cell Nuclear Transfer for Production of Sheep Models of Human Disease.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {235},
pages = {},
doi = {10.3791/72709},
pmid = {42747042},
issn = {1940-087X},
mesh = {Animals ; *Nuclear Transfer Techniques ; Sheep/genetics/embryology ; Humans ; *Disease Models, Animal ; Female ; *Animals, Genetically Modified/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Engineering/methods ; },
abstract = {Large animal models are valuable tools for investigating human disease. Sheep, pigs, and goats often better recapitulate the anatomy and physiology of human organs and the complexity of human disease, thereby enhancing their clinical relevance compared to rodents. CRISPR-Cas9 and somatic cell nuclear transfer (SCNT) enable the generation of large animal models with greater precision, versatility, and genetic uniformity. The primary benefit of this approach, compared with zygote microinjection, is the ability to confirm in vitro whether the desired genetic modification and potential off-target mutations are present in gene-edited cells prior to animal production. Moreover, SCNT eliminates the chance of genetic mosaicism, which frequently results from zygote microinjection. Here, we describe the generation of gene-edited ovine cells through non-homologous end-joining (NHEJ) and homology-directed repair (HDR), followed by the production of cloned embryos carrying the mutations of interest. Genetic modifications are introduced by transfecting cultured somatic cells, typically fetal fibroblasts, with the CRISPR-Cas9 system. Mutation efficiency in pooled cells is assessed by polymerase chain reaction (PCR) and Sanger sequencing of edited genes and analyzed using Tracking of Indels by DEcomposition (TIDE)/Tracking of Insertions, Deletions, and Recombination events (TIDER) software. Limiting dilution of the pooled cells is performed to obtain single-cell-derived colonies, which are screened by PCR and DNA sequencing of edited genes. Donor cells with the edit(s) of interest are subsequently expanded and used for the generation of embryos by SCNT. After limiting dilution and cell screening, 22/114 (19.3%) of colonies modified through NHEJ contained knockout (KO) mutations and 4/56 (7.1%) of colonies modified with HDR contained the F508del mutation. A total of 370 genetically modified embryos were created from four colonies. These methods are successfully used for precise gene editing in fetal fibroblasts and generation of genetically engineered embryos to produce ovine models of human disease.},
}

Animals
*Nuclear Transfer Techniques
Sheep/genetics/embryology
Humans
*Disease Models, Animal
Female
*Animals, Genetically Modified/genetics
CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Engineering/methods

@article {pmid42747551,
year = {2026},
author = {Gopukumar, ST and Saha, M and Soni, TK and Alatwi, ES and Khan, GA and Asad, MR and Shamshad, S and Tadakod, S and Sharma, A and Das, U},
title = {CRISPR-Enabled functional genomics in hPSCs-derived neural models for autism spectrum disorder.},
journal = {Metabolic brain disease},
volume = {41},
number = {1},
pages = {},
pmid = {42747551},
issn = {1573-7365},
support = {DBTHRDPMU/JRF/BET-24/I/2024-25/376//Department of Biotechnology, Ministry of Science and Technology, India/ ; 24J/01/00130//Council of Scientific and Industrial Research, India/ ; 3/1/3/BRET-2024/HRD (L1)//Indian Council of Medical Research/ ; },
mesh = {Humans ; *Genomics/methods ; *Autism Spectrum Disorder/genetics/metabolism ; *Pluripotent Stem Cells/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Neurons/metabolism ; *Neural Stem Cells/metabolism ; *CRISPR-Cas Systems ; },
abstract = {Autism Spectrum Disorder (ASD) is a genetically heterogeneous neurodevelopmental condition in which hundreds of individually rare risk variants converge on a small number of shared biological pathways, including synaptic scaffolding, chromatin remodeling, excitation-inhibition balance, and cellular energy metabolism. Translating this genetic heterogeneity into mechanistic insight requires experimental systems capable of interrogating individual gene functions in human-relevant neural contexts at scale. CRISPR-enabled functional genomics in human pluripotent stem cell (hPSC)-derived neural models, spanning neural progenitors, cortical and inhibitory neurons, astrocytes, microglia, and brain organoids, provides precisely this capability. By integrating pooled perturbation screens with multimodal readouts including single-cell and spatial transcriptomics, chromatin accessibility profiling, proximity labeling proteomics, multi-electrode array electrophysiology, and metabolic flux analysis, these platforms enable systematic, causal mapping of ASD gene function at system resolution. Early applications have already revealed convergent mechanisms: BAF complex disruption expands the ventral progenitor pool and biases its fate toward oligodendrocyte and interneuron lineages; ADNP loss impairs microglial synaptic pruning through altered endocytic trafficking; and mTOR pathway dysregulation in PTEN- and TSC2-perturbed models links genetic risk directly to metabolic and mitochondrial dysfunction. Computational frameworks including MIMOSCA and SCEPTRE enable causal network reconstruction and pseudotime inference from these datasets, moving the field from gene lists toward pathway-level models of ASD pathobiology. Translational applications leverage isogenic iPSC panels and variant-level base and prime editing to stratify ASD variants by functional impact, informing gene therapy design for haploinsufficient targets such as CHD8 and SCN2A via AAV or antisense oligonucleotide delivery. Remaining challenges, including model developmental immaturity, batch variability, and the difficulty of modeling polygenic risk, are addressed by a roadmap integrating spatial perturbomics, AI-driven causal inference, and population-scale standardized biobanks. This review synthesizes the current state of CRISPR-based functional genomics in human stem cell neural models as a coherent experimental framework for converting ASD genetic associations into mechanistic understanding and therapeutic opportunity.},
}

Humans
*Genomics/methods
*Autism Spectrum Disorder/genetics/metabolism
*Pluripotent Stem Cells/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Neurons/metabolism
*Neural Stem Cells/metabolism
*CRISPR-Cas Systems

@article {pmid42747567,
year = {2026},
author = {Hilkmann, M and Welsch, N and Felle, MF and Schweder, T and Appelbaum, M},
title = {An inducer-independent, single-plasmid CRISPR-Cas9 system for genome editing in Bacillus species.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {42747567},
issn = {1432-0614},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Bacillus/genetics ; *Plasmids/genetics ; Promoter Regions, Genetic ; Genome, Bacterial ; Gene Deletion ; },
abstract = {Advances in molecular biology tools are essential for streamlining and accelerating genetic engineering of cells across industrial and academic applications. While CRISPR-Cas improves genome editing efficiency, current systems have limitations and are often host specific, which restricts their versatility. This study describes a versatile CRISPR-Cas9 system for genome editing in industrially relevant Bacillus species. By adapting the well-established pJOE8999 vector-based CRISPR-Cas9 genome editing system, we constructed an inducer-independent, broad-host-range genome editing system. It maintains the benefits of low toxicity to the target cell and the cloning host as well as the ease to use of a single-plasmid CRISPR-Cas9 system. We utilized the constitutive Sigma70-type promoter from the conserved veg gene of Bacillus, to develop and test the suitability of promoter variants of different strengths for Cas9 expression. Successful gene deletions in three different Bacillus species demonstrated the versatility of the modified system for this industrially important genus. This was further confirmed by the integration of a reporter gene fusion and the introduction of a single point mutation in the genome of Bacillus licheniformis. This one-step CRISPR-based transformation protocol developed in this study enables fast genome editing workflows with minimal hands-on time. KEY POINTS: • Editing and screening of promoter variants for balanced Cas9 expression in Bacillus. • Development of a versatile inducer-independent, single-plasmid CRISPR-Cas-based system. • Verification of the modified CRISPR-based system for genome editing in different Bacilli.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Bacillus/genetics
*Plasmids/genetics
Promoter Regions, Genetic
Genome, Bacterial
Gene Deletion

@article {pmid42748215,
year = {2026},
author = {Gou, H and Chen, L and Eick, KL and Bao, D and Miner, TA and Tran, D and Gan, M and Zheng, W and Wang, S and Wu, Z and Huang, Z and Dial, CN and Seo, S and Mollin, S and Miller, MB and Zelazny, AM and Lyon, CJ and Fan, J and Wu, X and Yu, F and Zhu, X and Liu, Q and Ning, B and Hu, T},
title = {A rapid CRISPR-based nanodroplet assay enables direct clinical identification of mycobacteria species.},
journal = {Science translational medicine},
volume = {18},
number = {867},
pages = {eaef2648},
doi = {10.1126/scitranslmed.aef2648},
pmid = {42748215},
issn = {1946-6242},
mesh = {Humans ; *Mycobacterium/genetics/classification/isolation & purification ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Species Specificity ; Mycobacterium tuberculosis/genetics ; CRISPR-Cas Systems/genetics ; *Nanoparticles/chemistry ; },
abstract = {The global incidence and mortality of nontuberculous mycobacterial infections have risen sharply with population aging. In some regions, they are now surpassing Mycobacterium tuberculosis complex infections, imposing a substantial clinical and economic burden. Because nontuberous mycobacteria exhibit species-level heterogeneity and require prolonged culture for identification, their diagnosis remains slow and is frequently inaccurate. Here, we describe a multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-assisted nanodroplet differential identification (CANDI) diagnostic platform that integrates species-agnostic target amplification with species-specific CRISPR-associated protein 12a (Cas12a) detection in fluorescence-barcoded nanodroplets. By spatially compartmentalizing CRISPR reactions into color-encoded nanodroplets, CANDI overcomes the multiplexing limitations of conventional CRISPR diagnostics and enables simultaneous interrogation of multiple mycobacterial targets in a single assay. We designed a 16-plex panel that distinguishes 15 clinically relevant Mycobacterium species and subspecies. CANDI achieved high analytical sensitivity and accurate discrimination in samples containing coinfections with multiple species or subspecies. When applied to 230 clinical specimens, including sputum, tracheal aspirates, and other respiratory fluids, CANDI delivered subspecies-level results within 3.5 hours, achieving 97.08% sensitivity and 99.7% specificity relative to culture-based identification. By combining multiplexed, high-specificity CRISPR detection with scalable droplet-based engineering, CANDI has the potential to overcome the culture dependency of current diagnostics and enable species- and subspecies-level identification across the genetically complex Mycobacterium genus, offering a clinically adaptable framework for rapid, precision diagnosis of mycobacterial infections.},
}

Humans
*Mycobacterium/genetics/classification/isolation & purification
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Species Specificity
Mycobacterium tuberculosis/genetics
CRISPR-Cas Systems/genetics
*Nanoparticles/chemistry

@article {pmid42749264,
year = {2026},
author = {Rostami, S and Dos Santos, AM and Van, RS and Long, K and Shao, Y and Rajan, R},
title = {Active-site arginines differentially control Cas12a DNA cleavage and specificity.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {113560},
doi = {10.1016/j.jbc.2026.113560},
pmid = {42749264},
issn = {1083-351X},
abstract = {Cas12a is a CRISPR-Cas nuclease with biochemical features that make it useful for genome editing and nucleic acid diagnostics. However, its off-target and non-specific trans and CRISPR RNA-independent DNA cleavages can reduce the accuracy and limit applications requiring high fidelity. Here, we analyzed the role of two conserved arginine residues, R918 and R921, found in the RuvC active site pocket of Francisella novicida Cas12a. Through amino acid substitutions, biochemical assays, kinetic analysis, and computational study, we establish that a positive charge at 921 is required for CRISPR RNA-dependent DNA cleavage (cis cleavage), whereas R918 primarily enhances cleavage efficiency. Replacing R918 with lysine or alanine eliminates trans activity while retaining cis cleavage, whereas replacing R921 with lysine eliminates trans activity and replacing with alanine abolishes cis and trans cleavages. Furthermore, these changes significantly decrease RNA-independent cleavage and improve mismatch discrimination during cis cleavage, especially at PAM-distal sites. Structural analysis shows that R918 assists in the conversion of the lid covering the RuvC active site to an alpha helical form, while R921 stabilizes the DNA in the active site. Molecular dynamics simulations reveal that while R921 is critical in supporting the positioning of scissile phosphate, R918 is essential in maintaining catalytic-site organization through lid's conformational change as well as in positioning DNA through its role in stabilizing the active site framework. Together, our results highlight the importance of R918 and R921 in Cas12a's activity and the potential of modifying active pocket residues to reduce unwanted DNA cleavage while increasing on-target specificity.},
}

@article {pmid42749951,
year = {2026},
author = {Rahbari, M and Lohrasbi, R and Amiri-Yekta, A},
title = {From scissors to editors: how the evolution of precision is redefining therapeutic genome editing.},
journal = {Molecular genetics and genomics : MGG},
volume = {301},
number = {1},
pages = {},
pmid = {42749951},
issn = {1617-4623},
mesh = {*Gene Editing/methods/trends ; Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; *Precision Medicine/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; },
abstract = {Since its introduction as a genome-editing tool, CRISPR-based technology has undergone rapid refinement, with precision emerging as a central focus of development. Early CRISPR-Cas9 systems demonstrated unprecedented ease and efficiency in targeting specific DNA sequences, but concerns over off-target effects and variable editing outcomes limited their broader application. This review outlines the progression of CRISPR from its discovery in prokaryotes to its application as a versatile tool in precision medicine, where it supports targeted therapies for genetic disorders in various ways. Although technical challenges, including off-target editing and delivery inefficiencies, persist alongside ethical considerations of accessibility and long-term consequences, CRISPR's ongoing refinements and innovations reflect a clear trajectory toward greater specificity, safety, and predictability, positioning CRISPR as an increasingly precise platform for both fundamental research and therapeutic use.},
}

*Gene Editing/methods/trends
Humans
*CRISPR-Cas Systems/genetics
*Genetic Therapy/methods
*Precision Medicine/methods
Clustered Regularly Interspaced Short Palindromic Repeats
Animals

@article {pmid42752133,
year = {2026},
author = {Yoon, PH and Loi, KJ and Zhang, ZT and Docter, TA and Lopez, SC and Langeberg, CJ and Ur-Rehman, MM and Vohra, K and Zhou, Z and Esain-Garcia, I and Trinidad, MI and Shi, H and Boger, R and Wang, PY and Adler, BA and Brohawn, SG and Doudna, JA},
title = {A noncontiguous code for RNA-guided DNA recognition at the origin of CRISPR-Cas.},
journal = {Science (New York, N.Y.)},
volume = {393},
number = {6817},
pages = {1230-1235},
doi = {10.1126/science.aei0498},
pmid = {42752133},
issn = {1095-9203},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry/metabolism/genetics ; Evolution, Molecular ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; *RNA, Viral/genetics/chemistry ; *Viral Proteins/genetics/chemistry ; *Viral Interference ; Bacteria/virology ; },
abstract = {CRISPR-Cas provides RNA-mediated adaptive immunity, but how its first RNA-guided effector arose is unclear. In this study, we report the discovery of Viral Interference Programmable Repeat (VIPR) systems consisting of a Vipr protein ancestral to the earliest CRISPR-Cas effectors and VIPR RNAs (vrRNAs) comprising alternating GGY/NN motifs. Unlike canonical guide RNAs that pair with target nucleic acids through contiguous complementarity, vrRNAs recognize double-stranded DNA through a noncontiguous code in which the variable NN dinucleotides collectively specify a gapped target sequence. Natural vrRNA targets suggest that VIPR systems act against competing phages, and we demonstrate programmable phage defense by redirecting the complex for transcriptional repression. These results suggest that adaptive immunity originated from ancient warfare between viruses, revealing a previously unidentified logic for encoding information in sequence.},
}

*Bacteriophages/genetics
*CRISPR-Cas Systems/genetics
*DNA/chemistry/metabolism/genetics
Evolution, Molecular
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism
*RNA, Viral/genetics/chemistry
*Viral Proteins/genetics/chemistry
*Viral Interference
Bacteria/virology

@article {pmid42752153,
year = {2026},
author = {Bravo, JPK},
title = {A viral origin for RNA-guided immunity.},
journal = {Science (New York, N.Y.)},
volume = {393},
number = {6817},
pages = {1187-1188},
doi = {10.1126/science.ael0758},
pmid = {42752153},
issn = {1095-9203},
mesh = {*Bacteria/genetics/virology ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/virology ; Viruses/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Ancient viral warfare could be at the root of modern class 1 CRISPR bacterial defense systems.},
}

*Bacteria/genetics/virology
*CRISPR-Cas Systems/genetics
Escherichia coli/genetics/virology
Viruses/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42753192,
year = {2026},
author = {Jackson, KM and Morales, MM and Szewczyk, E and Homer, CM and Itogawa, AN and Brem, RB and Sil, A and Barker, BM},
title = {Protoplasting and Transformation Using CRISPR/Cas9 in Coccidioides.},
journal = {Current protocols},
volume = {6},
number = {9},
pages = {e70444},
pmid = {42753192},
issn = {2691-1299},
support = {1U19AI166798/AI/NIAID NIH HHS/United States ; U10AI166058/AI/NIAID NIH HHS/United States ; R21AI193607/AI/NIAID NIH HHS/United States ; },
mesh = {*Coccidioides/genetics ; *Protoplasts/metabolism ; *Transformation, Genetic ; *CRISPR-Cas Systems/genetics ; Gene Deletion ; Coccidioidomycosis/microbiology ; },
abstract = {Coccidioides posadasii and C. immitis are human fungal pathogens endemic to the American Southwest. C. posadasii and C. immitis are the causative agents of coccidioidomycosis, or Valley fever. They are pathogens of growing concern, as reported cases of coccidioidomycosis have increased 20-fold in the last two decades. Despite their importance as pathogens, Coccidioides spp. are understudied, especially compared to other human fungal pathogens with similar infectious burdens. The reasons Coccidioides spp. are understudied are multifactorial, including requirement of high biocontainment, technical difficulties in lab-based culture growth, and a historic lack of genetic tools. Previous methods of genetic manipulation have been technically challenging, time consuming, and inefficient. Here, we present protocols for designing gene deletion constructs, generating protoplasts from both genetically engineered biosafety level 2 (BSL2) and wildtype biosafety level 3 (BSL3) strains, and performing transformations with CRISPR/Cas9. The protoplasting protocol described here uses a cell wall digestion enzyme employed in the wine-making industry and results in high-quality protoplasts that have the potential to be used for applications beyond transformations. We also present a high-efficiency transformation method using CRISPR/Cas9. The protocols described here will allow for genetic manipulation of Coccidioides, using both BSL2 and BSL3 strains. This resource can be applied to expand research done in Coccidioides spp., build molecular tools, and expand overall knowledge of these important pathogens. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Construction of gene deletion construct Alternate Protocol 1: Construction of gene deletion construct Basic Protocol 2: Creation of protoplasts in BSL3 Alternate Protocol 2: Creation of protoplasts in BSL2 Basic Protocol 3: CRISPR/Cas9 transformation in BSL3 Alternate Protocol 3: CRISPR/Cas9 transformation in BSL2 Support Protocol: Passaging of mutants and PCR confirmation.},
}

*Coccidioides/genetics
*Protoplasts/metabolism
*Transformation, Genetic
*CRISPR-Cas Systems/genetics
Gene Deletion
Coccidioidomycosis/microbiology

@article {pmid42753721,
year = {2026},
author = {Schargel, RD and Chacon Machado, L and Kumaran, S and Thesier, JE and Krishnan, SS and Guarné, A and Peters, JE},
title = {De novo-engineered guide RNA-directed transposition with TnpB-family proteins.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
pmid = {42753721},
issn = {1097-4164},
support = {R35 GM152260/GM/NIGMS NIH HHS/United States ; },
abstract = {Programmable DNA integration using CRISPR-associated transposase elements (CASTs) offers powerful capabilities for genome engineering. The large single effector Cas12k CAST examples evolved from a minimal TnpB nuclease protein. Here, we engineer a de novo RNA-guided transposition systems in bacteria, where the single guide RNA effector components are repurposed nuclease-dead TnpB-family proteins. These compact systems mediate high-efficiency guide-RNA-directed DNA insertion with preserved orientation control, target immunity, and release of a host factor requirement and can be paired with an exonuclease domain to mediate cut-and-paste transposition. In this engineered context, the TnpB derivatives show features not predicted from the original enzymes, suggesting untapped avenues for improvement. In parallel, we show that mutations at the TniQ-TnsC interface in the Cas12k CAST system selectively attenuate off-site insertions while enhancing on-site activity. These results establish how Cas12 proteins and antecedent TnpB proteins can be engineered for high performance and specificity with guide-RNA-directed systems.},
}

@article {pmid42754810,
year = {2026},
author = {Toma, L and Barbălată, T and Hărătău, JIC and Sanda, GM and Fuior, EV and Fenyo, IM and Niculescu, LŞ and Dăian, LM and Sasson, S and Sima, AV and Stancu, CS},
title = {CRISPR/dCas9-induced upregulation of endogenous apolipoprotein A1 and paraoxonase 1 genes reduces the aortic lipid deposits in apoE[-/-] mice.},
journal = {Molecular biomedicine},
volume = {7},
number = {1},
pages = {},
pmid = {42754810},
issn = {2662-8651},
support = {PN-III-P2-2.1-PED-2021-1929//UEFISCDI/ ; 10-CoEx/2026 PN-IV-P6-6.1-CoEx-2024-0029//UEFISCDI/ ; 760059/23.05.2023//PNRR Program/ ; GAR2023 Project No. 83//Academia Româna/ ; },
mesh = {Animals ; *Aryldialkylphosphatase/genetics/metabolism ; *Apolipoprotein A-I/genetics/metabolism ; Humans ; *Up-Regulation/genetics ; *Apolipoproteins E/deficiency/genetics ; *Aorta/metabolism/pathology ; Mice ; Atherosclerosis/genetics/metabolism/pathology ; Tumor Necrosis Factor-alpha ; Hepatocytes/metabolism ; *CRISPR-Cas Systems/genetics ; Mice, Knockout ; Male ; },
abstract = {High-density lipoproteins (HDL) are essential to alleviate the progression of atherosclerosis by mediating reverse-cholesterol transport, antioxidant and anti-inflammatory effects. We aimed to enhance the expression of endogenous HDL components, apolipoprotein A1 (APOA1) and antioxidant enzyme paraoxonase 1 (PON1), and to investigate their athero-protective effects. The CRISPR/dCas9 technology was used to activate the transcription of endogenous APOA1/PON1 in human hepatocytes (Huh7 line) and Apoa1/Pon1 in apoE[-/-] mice. The expression of APOA1/PON1 genes was successfully upregulated in hepatocytes, and their proteins were secreted in the culture medium in the presence/absence of tumor necrosis factor-α (TNFα). APOA1-rich Huh7-derived conditioned medium exerted antioxidant and anti-inflammatory effects in TNFα-activated EA.hy926 endothelial cells. A single dose of the CRISPR/dCas9 plasmids i.v. injected in apoE[-/-] mice increased the expression of hepatic Apoa1/Pon1 and their serum levels up to four weeks. FPLC analysis showed that increased serum APOA1 was distributed between HDL, LDL, and in lipid-free form. These mice also exhibited high levels of hepatic, gallbladder and feces cholesterol, in part due to the upregulation of hepatic scavenger receptor class-B1, cholesterol 7-alpha-hydroxylase, and ATP-binding cassette sub-family-G-member-8 transporter. In apoE[-/-] mice with upregulated Apoa1/Pon1, no increased inflammatory stress or innate immune activation were detected, while lipid peroxides were decreased in PON1 mice. Of major interest, the area of aortic lipid deposits was halved in the treated mice. Our findings demonstrate the successful upregulation of endogenous Apoa1/Pon1 in apoE[-/-] mice by using the CRISPR/dCas9 system, and highlight new mechanisms for APO1/PON1 anti-atherosclerotic action, explaining the reduction of aortic lipid deposits.},
}

Animals
*Aryldialkylphosphatase/genetics/metabolism
*Apolipoprotein A-I/genetics/metabolism
Humans
*Up-Regulation/genetics
*Apolipoproteins E/deficiency/genetics
*Aorta/metabolism/pathology
Mice
Atherosclerosis/genetics/metabolism/pathology
Tumor Necrosis Factor-alpha
Hepatocytes/metabolism
*CRISPR-Cas Systems/genetics
Mice, Knockout
Male

@article {pmid42754893,
year = {2026},
author = {Höijer, I and van Schendel, R and Emmanouilidou, A and Östlund, R and Bunikis, I and Tijsterman, M and den Hoed, M and Ameur, A},
title = {Accurate characterization of CRISPR-Cas9 genome editing outcomes and mosaicism with near-perfect long reads.},
journal = {Genome medicine},
volume = {18},
number = {1},
pages = {},
pmid = {42754893},
issn = {1756-994X},
mesh = {*Mosaicism ; Animals ; Zebrafish/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; High-Throughput Nucleotide Sequencing ; Genome ; },
abstract = {BACKGROUND: Genetic mosaicism is a well-recognized consequence of CRISPR-Cas9 genome editing, yet its characterization remains challenging, especially when it involves low-frequency structural variants. A comprehensive analysis of mosaicism requires deep and unbiased sequencing of the target loci, with accurate single-molecule reads.

METHODS: We performed amplification-free PureTarget PacBio sequencing to investigate CRISPR-Cas9 outcomes at on-target and off-target sites in genome edited zebrafish and their offspring. CRISPR-Cas9 genome editing was performed by micro-injection in fertilized eggs at the single-cell stage.

RESULTS: Thirty samples from pooled larvae and individual zebrafish were successfully sequenced, resulting in > 1100x average target coverage. The PacBio reads reached an exceptional accuracy (QV39) over the target regions, with every read originating from a unique DNA molecule. The two haplotypes of the target loci displayed a balanced depth of coverage, while long-range PCR of the same samples resulted in skewed data. Further analysis of the PureTarget data revealed widespread genetic mosaicism in individual founder (F0) fish, with up to 18 distinct on-target events and 11 off-target events present in a single adult founder. Several CRISPR-Cas9 editing outcomes, including large structural variants and off-target mutations, were inherited to the F1 generation. Notably, as many as seven unique editing events were found among sibling F1 juvenile offspring derived from a single founder pair, thereby confirming the presence of genetic mosaicism in germ cells of founder zebrafish. This implies that some consequences of CRISPR-Cas9 editing may emerge only in the second generation. We also analyzed DNA methylation signals in the PureTarget data but did not observe altered 5mC CpG levels in genome edited samples.

CONCLUSIONS: PureTarget enables efficient, accurate, and unbiased profiling of genetic mosaicism and DNA methylation at pre-defined genomic regions. Our results show that CRISPR-Cas9-induced mosaicism is widespread and represents an important factor to consider in genome editing experiments.},
}

*Mosaicism
Animals
Zebrafish/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
High-Throughput Nucleotide Sequencing
Genome

@article {pmid42755327,
year = {2026},
author = {Wong Castro, DA and Morocho Perugachi, AC and Fuel Herrera, MO},
title = {CRISPR-Based Mediated Reactivation of Fetal Hemoglobin as a Therapeutic Strategy for Hemoglobinopathies: Evidence from Preclinical to Clinical Trials in Sickle Cell Disease and β-Thalassemia.},
journal = {Hemoglobin},
volume = {},
number = {},
pages = {1-10},
doi = {10.1080/03630269.2026.2729189},
pmid = {42755327},
issn = {1532-432X},
abstract = {β-Hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia, are inherited disorders caused by mutations in the β-globin gene (HBB), leading to defective production of adult hemoglobin (HbA), vaso-occlusive crises, and rapid destruction of erythrocytes as they leave the bone marrow, resulting in hemolytic anemia. In recent years, CRISPR-based genome-editing technologies have emerged as promising therapeutic strategies to reactivate fetal hemoglobin (HbF) expression by targeting key regulatory elements, including the BCL11A enhancer and the HBG1/HBG2 promoters. This review aimed to synthesize available scientific evidence from PubMed, Scopus, Web of Science, and ClinicalTrials.gov on preclinical and clinical studies evaluating CRISPR-based genome-editing approaches for the treatment of SCD and β-thalassemia. This review was conducted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) Statement. A total of 247 records were identified; 20 studies met the inclusion criteria and were included in the qualitative synthesis. The included studies investigated various CRISPR-based platforms, including nuclease-mediated editing and base-editing strategies, to reactivate HbF expression. The studies reported durable increases in HbF levels, with therapeutic effects persisting for up to 22 months after treatment. Clinically, these outcomes were associated with the elimination or marked reduction of vaso-occlusive crises in patients with SCD and with transfusion independence in patients with β-thalassemia. Overall, the available evidence suggests that CRISPR-based HbF reactivation is a promising therapeutic approach for β-hemoglobinopathies, although long-term efficacy and safety data remain necessary.},
}

@article {pmid42755359,
year = {2026},
author = {Martella, A and Matreyek, KA and Fisher, DI},
title = {Large serine recombinase-mediated gene insertion for high-throughput screens: advantages, design principles, and applications.},
journal = {Nucleic acids research},
volume = {54},
number = {17},
pages = {},
pmid = {42755359},
issn = {1362-4962},
mesh = {Humans ; Animals ; *DNA Nucleotidyltransferases/metabolism/genetics ; *Mutagenesis, Insertional/methods ; *High-Throughput Screening Assays/methods ; *Recombinases/metabolism/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods ; },
abstract = {High-throughput functional assays, such as multiplexed assays of variant effect (MAVE), increasingly demand stable, precise integration of large DNA libraries into mammalian genomes. While CRISPR-based technologies excel at localized, small-scale edits, they are constrained by payload size limits, heterogeneous editing outcomes, and, depending on the specific modality and repair pathway utilized, potential variability in junction fidelity during multikilobase insertions. In this review, we highlight large serine recombinases (LSRs) as highly efficient, single-enzyme alternatives for unidirectional, site-specific integration of large payloads with deterministic junctions. We survey targeted genomic integration strategies and detail best practices for implementing recombinase-based landing pad architectures. By enforcing single-copy, orientation-fixed integration at defined loci, landing pads decouple variant delivery from local chromatin effects to ensure the uniform, isogenic expression required for quantitative genotype-phenotype mapping. We further outline scalable applications of LSR-mediated integration across pooled and arrayed MAVE, CRISPR screens, and precise gene expression tuning. Finally, we assess current technological bottlenecks, particularly large donor delivery and the requisite pre-installation of canonical att recognition sites, while exploring emerging innovations in virus-like particle delivery, one-step CRISPR-recombinase systems, and computationally engineered programmable recombinases that promise to bypass these limitations and broaden mammalian genome engineering.},
}

Humans
Animals
*DNA Nucleotidyltransferases/metabolism/genetics
*Mutagenesis, Insertional/methods
*High-Throughput Screening Assays/methods
*Recombinases/metabolism/genetics
CRISPR-Cas Systems
*Gene Editing/methods

@article {pmid42758019,
year = {2026},
author = {Boneza, MM and Keller, T and Mostek, J and Smanski, MJ},
title = {Strain-specific outcomes of cytosine-base editing in Streptomyces.},
journal = {Journal of bacteriology},
volume = {},
number = {},
pages = {e0024126},
doi = {10.1128/jb.00241-26},
pmid = {42758019},
issn = {1098-5530},
abstract = {UNLABELLED: The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has facilitated gene editing of different organisms. Specifically, programmable base editing enables stable conversion of a single nucleotide to another nucleotide without causing DNA double-strand breaks. Cas9-derived base editors, including adenine and cytidine base editors, catalyze the formation of transition mutations with high efficiency. The third-generation cytidine base editors comprise a nickase-Cas9 fused to a cytidine deaminase and uracil DNA glycosylase inhibitor to enable the transition from C:G to T:A. We observed that in certain Streptomyces spp., this cytidine base editor produced C:G to G:C transversions at a surprisingly high rate of 44, while in other strains, it yielded the expected C:G to T:A transition mutations. There was also a notable timing difference for base editing between distinct strains. Bioinformatics analysis revealed differences in DNA mismatch repair and nucleotide excision repair pathways, including the presence of uvrD helicase gene only in the C:G to G:C transversion strain. Expression of uvrD in the C:G to T:A transition strain led to higher rates of C:G to G:C transversions. These discoveries will aid in the development of efficient C:G to G:C base editors.

IMPORTANCE: Base editors are useful tools that allow genetic engineers to precisely change single bases in an organism's genome. Most base editors catalyze transition mutations (e.g., A-to-G or C to-T). Transversion mutations, which convert a purine base to a pyrimidine base, have been described but operate at much lower efficiency. Here, we describe a surprising discovery that a transition base editor produced high-efficiency transversion mutations in a species of Streptomyces. This information was used to engineer a high-efficiency transversion base editor.},
}

@article {pmid42758354,
year = {2026},
author = {Islam, MN and Islam, MM and Feng, H and Bellah, SF and Zaman Miah, MM and Hossain, MS},
title = {CRISPR-Cas9 genome editing: technological advances, delivery strategies and precision engineering of primary cells for therapeutic application in genetic disorders and diabetes mellitus.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42758354},
issn = {1573-4978},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Diabetes Mellitus/genetics/therapy ; Animals ; *Genetic Diseases, Inborn/therapy/genetics ; *Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR-Cas9 system has revolutionized modern life sciences, driving a paradigm shift in biomedical research and becoming an indispensable tool in molecular biology due to its remarkable precision, efficiency, and simplicity. Originating from a bacterial adaptive immune mechanism, CRISPR-Cas9 has evolved rapidly, providing a versatile framework for manipulating genetic material and addressing a wide spectrum of human diseases. Recent progress includes the discovery of novel Cas orthologs and the rational engineering of Cas9 variants to enhance editing fidelity, broaden target range, and minimize off-target effects. Structural and functional optimization of single-guide RNAs (sgRNAs) has further improved target binding affinity, stability, and Cas9-sgRNA complex formation, thereby increasing overall editing performance. The development of high-fidelity Cas9 derivatives and next-generation platforms such as base editors and prime editors has enabled precise single-nucleotide substitutions and small insertions or deletions without generating double-stranded DNA breaks. Advanced delivery systems-including viral vectors, lipid nanoparticles, and ribonucleoprotein electroporation-have facilitated efficient CRISPR-mediated editing across in vitro, ex vivo, and in vivo models. Remarkable therapeutic milestones have been achieved in treating monogenic disorders such as sickle cell disease, β-thalassemia, and cystic fibrosis, where long-term clinical benefits have been documented. Furthermore, CRISPR-Cas9 technology is redefining diabetes research by enabling precise modeling of disease mechanisms, uncovering molecular pathways involved in glucose homeostasis, and opening new avenues for cellular and gene-based therapies. This review highlights recent advances in CRISPR-Cas9-mediated genome editing, emphasizing breakthroughs in primary cell editing and the development of translational models for genetic diseases and diabetes mellitus.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Diabetes Mellitus/genetics/therapy
Animals
*Genetic Diseases, Inborn/therapy/genetics
*Genetic Therapy/methods
RNA, Guide, CRISPR-Cas Systems/genetics