@article {pmid42364585,
year = {2027},
author = {Wang, T and Wen, Y and Ma, H and Wang, Y and Liao, K and Xue, F},
title = {RAA-CRISPR/Cas12a-mediated SERS magnetic biosensor combined with a portable Raman spectrometer for rapid and ultrasensitive detection of Lumpy skin disease virus.},
journal = {Talanta},
volume = {311},
number = {},
pages = {130222},
doi = {10.1016/j.talanta.2026.130222},
pmid = {42364585},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *Spectrum Analysis, Raman/methods ; Animals ; *CRISPR-Cas Systems ; *Lumpy skin disease virus/isolation & purification/genetics ; Cattle ; Nucleic Acid Amplification Techniques/methods ; DNA, Viral/genetics ; Rapid Diagnostic Tests ; Recombinases/metabolism ; Gold/chemistry ; },
abstract = {Lumpy skin disease virus (LSDV) poses a severe threat to global cattle farming, causing significant economic losses. Current laboratory-based methods such as qPCR are accurate procedures; however, they are time-consuming and require sophisticated instrumentation, thus hindering onsite outbreak control. We report a novel magnetic biosensor integrating recombinase-aided amplification (RAA), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a, and surface-enhanced Raman scattering (SERS) for ultrasensitive and portable detection of LSDV. The biosensor operates through a synergistic cascade: (i) RAA isothermally amplifies the viral DNA target; (ii) the amplified product activates the trans-cleavage activity of CRISPR/Cas12a, thus generating numerous short ssDNA fragments; and (iii) these fragments cleave linker DNA, thereby releasing AuNS@DTNB SERS nanoprobes from the magnetic bead surface and enabling signal readout via a handheld Raman spectrometer. This triple-amplification strategy yielded an ultrahigh sensitivity at 1.2 copies/μL, with a total assay time of only 90 min. The platform exhibited exceptional specificity, discriminating LSDV from genetically homologous Capripoxviruses (up to 97% similarity). In blind tests on 30 beef samples spiked with LSDV nucleic acid, it achieved 100% concordance with qPCR results, thus demonstrating excellent robustness. By integrating a handheld Raman spectrometer and a portable thermostat, this platform delivers laboratory-grade performance in a field-deployable format. This biosensor represents a powerful new tool for onsite LSDV surveillance; given its modular design, it can be readily adapted for detecting other emerging nucleic acid targets.},
}

*Biosensing Techniques/methods
*Spectrum Analysis, Raman/methods
Animals
*CRISPR-Cas Systems
*Lumpy skin disease virus/isolation & purification/genetics
Cattle
Nucleic Acid Amplification Techniques/methods
DNA, Viral/genetics
Rapid Diagnostic Tests
Recombinases/metabolism
Gold/chemistry

@article {pmid42379036,
year = {2027},
author = {Chen, Y and Liu, S and Yuan, Z and He, X and Zhu, X and Wang, G and Wang, X},
title = {Dual-engine amplification: Integrating catalytic hairpin assembly with CRISPR-Cas12a for ultrasensitive point-of-care testing of Chikungunya virus RNA.},
journal = {Talanta},
volume = {311},
number = {},
pages = {130191},
doi = {10.1016/j.talanta.2026.130191},
pmid = {42379036},
issn = {1873-3573},
mesh = {*RNA, Viral/genetics/analysis ; *Chikungunya virus/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Testing ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; *Chikungunya Fever/diagnosis/virology ; Rapid Diagnostic Tests ; Limit of Detection ; Sensitivity and Specificity ; },
abstract = {Chikungunya virus (CHIKV) has emerged as a globally important arthropod-borne pathogen with rapid geographical expansion, urging high-performance yet simple diagnostic tools. Herein, we develop an isothermal dual-engine amplification biosensor by integrating catalytic hairpin assembly (CHA) with CRISPR-Cas12a for ultrasensitive and rapid detection of CHIKV RNA. This strategy combines the enzyme-free isothermal amplification of CHA and the high-specificity trans-cleavage activity of CRISPR-Cas12a to realize cascade signal enhancement. By systematically optimizing reaction parameters including probe ratio, temperature, buffer, and incubation time, we achieve superior analytical performance. The biosensor exhibits a low limit of detection of 4 fM, a wide linear range from 10 fM to 1 μM (R[2] > 0.98), and excellent specificity against dengue virus, Zika virus, and other common blood-borne viruses. It also shows strong resistance to interference from hemolysis, icterus, and lipemia. The whole assay can be finished within 60 min under isothermal conditions without sophisticated thermal cyclers. In clinical serum samples, this method achieves 97.5% concordance with RT-qPCR, with a sensitivity of 96.8% and a specificity of 100%. This CHA-CRISPR-Cas12a dual-amplification platform provides a sensitive, specific, rapid, and cost-effective approach for CHIKV detection and holds great promise for point-of-care testing in resource-limited settings.},
}

*RNA, Viral/genetics/analysis
*Chikungunya virus/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*Point-of-Care Testing
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Humans
*Chikungunya Fever/diagnosis/virology
Rapid Diagnostic Tests
Limit of Detection
Sensitivity and Specificity

@article {pmid42402225,
year = {2027},
author = {Jia, X and Wang, J},
title = {Aptamer-CRISPR Glucose Transducer for point-of-care IgE detection.},
journal = {Talanta},
volume = {311},
number = {},
pages = {130221},
doi = {10.1016/j.talanta.2026.130221},
pmid = {42402225},
issn = {1873-3573},
mesh = {*Immunoglobulin E/analysis/blood ; Humans ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *Point-of-Care Systems ; *Glucose/analysis ; *CRISPR-Cas Systems ; *Point-of-Care Testing ; Limit of Detection ; Rapid Diagnostic Tests ; },
abstract = {Immunoglobulin E (IgE) is a critical biomarker for the diagnosis and therapeutic monitoring of allergic diseases. Conventional IgE detection methods (e.g., ELISA, ImmunoCAP) generally offer satisfactory sensitivity but require hours to days, rely on specialized equipment and trained personnel, and are unsuitable for point-of-care or home-based testing. Here, we developed a portable point-of-care testing platform termed Aptamer-CRISPR Glucose Transducer (ACGT), based on aptamer competition and CRISPR-Cas12a trans-cleavage. The platform employs the D17.4 aptamer as the molecular recognition element. In the presence of target IgE, the aptamer specifically binds IgE, triggering a strand displacement reaction that releases the blocker as ssDNA. This released blocker (now serving as the trigger) activates the trans-cleavage of Cas12a, which cleaves ssDNA linkers on magnetic beads, thereby releasing invertase into the supernatant. The released invertase hydrolyzes sucrose into glucose, which is quantitatively measured using a personal glucose meter. The method achieves a detection limit as low as 76.5 kU/L, with a total assay time of approximately 80 min. By integrating the signal amplification capability of CRISPR with the accessibility of glucose-based transduction, the ACGT platform provides a sensitive, cost-effective, and user-friendly solution for allergen diagnosis and biomarker monitoring in decentralized settings, offering great potential for primary care and home-based testing.},
}

*Immunoglobulin E/analysis/blood
Humans
*Aptamers, Nucleotide/chemistry
*Biosensing Techniques/methods
*Point-of-Care Systems
*Glucose/analysis
*CRISPR-Cas Systems
*Point-of-Care Testing
Limit of Detection
Rapid Diagnostic Tests

@article {pmid42420522,
year = {2026},
author = {Price, D and Zemlyanskiy, G and Trakarnphornsombat, W and Forne, I and Volkova, N and Dubusse, L and Cooper, AJ and Imhof, A and Radzisheuskaya, A},
title = {Identifying critical lysines in mammalian histone H3 with high-throughput CRISPR prime editing.},
journal = {Nature genetics},
volume = {58},
number = {9},
pages = {2303-2319},
pmid = {42420522},
issn = {1546-1718},
support = {UKRI698//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; EP/Y000331/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; RG\R1\241175//Royal Society/ ; CRC1309/325871075 and SPP2191/419067076//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Histones/genetics/metabolism/chemistry ; Animals ; *Lysine/genetics/metabolism ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Protein Processing, Post-Translational/genetics ; Mouse Embryonic Stem Cells/metabolism ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mutation ; Genomic Instability ; Saccharomyces cerevisiae/genetics ; },
abstract = {Histone post-translational modifications are fundamental to genome regulation, yet dissecting the functions of individual histone marks in mammals remains challenging due to the presence of multiple histone gene copies. Here we develop a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) prime editing platform enabling precise, reversible and combinatorial mutagenesis of canonical and noncanonical histone H3 genes within their native genomic context. Using systematic lysine-to-arginine substitutions benchmarked against synonymous controls, we identify key residues, including H3K4, H3K9, H3K14, H3K18 and H3K79, whose mutation compromises fitness in mouse embryonic stem cells. We further show that H3K56, linked to genome stability in yeast and Drosophila, has a conserved role in mammalian cells. Through analysis of selected double mutants, we uncover functional crosstalk across residues, with combinations such as H3K27R + H3K36R impairing stem cell self-renewal and altering transcription. Altogether, this study establishes a functional map of histone H3 lysines in mammals and provides a broadly applicable platform for systematic dissection of chromatin regulation.},
}

*Histones/genetics/metabolism/chemistry
Animals
*Lysine/genetics/metabolism
Mice
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Protein Processing, Post-Translational/genetics
Mouse Embryonic Stem Cells/metabolism
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mutation
Genomic Instability
Saccharomyces cerevisiae/genetics

@article {pmid42664856,
year = {2026},
author = {Huang, J and Zhang, Q and Yu, FW and Liu, MQ and Jing, HK and Wang, SY and Wang, HY and Li, JQ and Zheng, L and Shao, H and He, XL and Shen, RF and Zhu, XF},
title = {Disruption of OsGONST3 reduces grain cadmium accumulation without yield penalty in field-grown rice.},
journal = {Journal of hazardous materials},
volume = {516},
number = {},
pages = {143397},
doi = {10.1016/j.jhazmat.2026.143397},
pmid = {42664856},
issn = {1873-3336},
mesh = {*Oryza/metabolism/genetics/growth & development ; *Cadmium/metabolism ; *Plant Proteins/genetics/metabolism ; *Soil Pollutants/metabolism ; Plants, Genetically Modified ; Plant Roots/metabolism ; CRISPR-Cas Systems ; },
abstract = {Cadmium (Cd) pollution in paddy soils threatens global food safety. Identifying molecular gateways controlling grain Cd accumulation is essential for breeding low-Cd rice. A field-based screen of a large-scale CRISPR/Cas9 mutant library identified OsGONST3, a Golgi nucleotide sugar transporter family member localized to the plasma membrane. After 1 μM Cd treatment for 7 d, OsGONST3 transcript abundance increased approximately 3.4-fold in root and 5.7-fold in shoot, accompanied by increased OsGONST3-GFP protein accumulation. Heterologous OsGONST3 expression increased yeast Cd content by approximately 23% relative to the empty-vector control. Under hydroponic exposure to 1 μM Cd for 7 d, two independent osgonst3 mutants contained 22-25% less Cd in root, approximately 13% less in shoot, and 16-21% less in xylem sap than Nipponbare. Conversely, OsGONST3-overexpressing lines contained 40-42%, 38-40%, and 28-37% more Cd in root, shoot, and xylem sap, respectively. Under tested field condition, OsGONST3 disruption reduced grain Cd by approximately 25-26% without a significant yield penalty and was associated with secondary transcriptional adjustments in Cd-homeostasis genes. These findings identify OsGONST3 as a plasma membrane-localized Cd influx-associated protein and a promising target for breeding low-Cd rice.},
}

*Oryza/metabolism/genetics/growth & development
*Cadmium/metabolism
*Plant Proteins/genetics/metabolism
*Soil Pollutants/metabolism
Plants, Genetically Modified
Plant Roots/metabolism
CRISPR-Cas Systems

@article {pmid42690973,
year = {2026},
author = {Qin, Y and Zhang, G and Hu, Y},
title = {CRISPR/Cas- and Argonaute-Based In Vivo Nucleic-Acid Imaging Technologies: Strategies, Challenges, and Perspectives.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01856},
pmid = {42690973},
issn = {2379-3694},
support = {32401261//National Natural Science Foundation of China/ ; },
abstract = {Live-cell monitoring of sequence-specific nucleic acids is essential to understanding genome organization, RNA regulation, and disease progression. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) systems provide programmable, guide-directed recognition of DNA or RNA and are increasingly used as platforms for in vivo bioimaging. This review summarizes the structural and mechanistic features of representative CRISPR and Ago effectors and discusses design strategies for sensitive, specific, and multiplexed imaging of genomic loci, extrachromosomal DNA, and endogenous RNA in living cells. We compare the analytical performance and limitations of CRISPR- and Ago-based imaging, with particular emphasis on the major technical and biological challenges affecting their accuracy, applicability, and reliability. Finally, this review offers insights into developing high-resolution and user-friendly bioimaging platforms for fundamental biology and future translational applications.},
}

@article {pmid42692561,
year = {2026},
author = {Zheng, Z and Xu, D},
title = {CRISPR-Cas9 screen to identify genes regulating cell death.},
journal = {Methods in cell biology},
volume = {210},
number = {},
pages = {83-96},
doi = {10.1016/bs.mcb.2026.05.010},
pmid = {42692561},
issn = {0091-679X},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Cell Death/genetics ; Animals ; *Apoptosis/genetics ; },
abstract = {Regulated cell death mediated by dedicated molecular machines, known as programmed cell death, plays important roles in health and disease. Understanding the mechanisms of cell death is crucial for elucidating the control of cellular homeostasis and developing therapies for related diseases. Despite extensive research efforts spanning decades, many aspects of cell death mechanisms remain elusive, highlighting the need for continued exploration. Here, we describe how to identify novel regulators involved in cell death pathways using a genome-wide screening approach.},
}

*CRISPR-Cas Systems/genetics
Humans
*Cell Death/genetics
Animals
*Apoptosis/genetics

@article {pmid42692894,
year = {2026},
author = {Gaizauskaite, U and Songailiene, I and Sasnauskas, G and Siksnys, V},
title = {Building CRISPR immunity: evolution and mechanisms of spacer acquisition.},
journal = {Trends in biochemical sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibs.2026.08.004},
pmid = {42692894},
issn = {0968-0004},
abstract = {CRISPR-Cas systems in prokaryotes serve as adaptive immune systems that neutralize phage infections through RNA-guided nucleases. Immunization is achieved during the adaptation stage through Cas1-Cas2 integrase-mediated insertion of short foreign DNA snippets, termed spacers, into a CRISPR array in the host genome. This review examines the evolutionary origins of Cas1-Cas2 and the mechanisms of spacer acquisition in DNA-targeting CRISPR-Cas systems. Particular emphasis is placed on the recently characterized effector-assisted adaptation pathways, in which CRISPR effector proteins, such as Cascade and Cas9, typically involved in target interference, are repurposed for prespacer capture and integration into a CRISPR array.},
}

@article {pmid42694431,
year = {2026},
author = {Putignani, L and Marsiglia, R and Turco, L and Russo, A and Pane, S and Fusco, A and Lopetuso, L and Trecarichi, EM},
title = {The gut reservoir of carbapenem-resistant Enterobacterales: from dysbiosis and colonization to infection and decolonization, with a focus on patients with hematologic malignancies - a narrative review.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1939690},
pmid = {42694431},
issn = {2235-2988},
mesh = {Humans ; *Hematologic Neoplasms/complications/microbiology ; *Dysbiosis/microbiology ; *Enterobacteriaceae Infections/microbiology/therapy ; *Carbapenem-Resistant Enterobacteriaceae/drug effects ; *Gastrointestinal Microbiome ; Fecal Microbiota Transplantation ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Probiotics ; },
abstract = {Carbapenem-resistant Enterobacterales (CRE) remain among the highest-priority antimicrobial-resistant pathogens worldwide, and intestinal colonization is increasingly recognized as the key precursor of invasive infections, particularly in patients with hematological malignancies. Increasing evidence indicates that disruption of the gut microbial ecosystem, reflected in reduced diversity, depletion of beneficial anaerobic taxa, intestinal barrier dysfunction, immune dysregulation, and expansion of Enterobacterales, plays a central role in the transition from colonization to infection. Consequently, restoring colonization resistance through microbiome-targeted interventions has emerged as a promising preventive strategy. This narrative review summarizes the current evidence on the epidemiology and clinical impact of CRE colonization and infection, with particular emphasis on the ecological alterations of the gut microbiome linking gut dysbiosis to epithelial barrier dysfunction, immune dysregulation, and loss of colonization resistance to CRE persistence and invasive infection. We critically discuss both conventional and emerging decolonization approaches, including selective digestive decontamination, probiotics, prebiotics and synbiotics, fecal microbiota transplantation (FMT), bacteriophage therapy, and CRISPR-Cas-based technologies, highlighting their mechanisms of action, available clinical evidence, and current limitations. Particular attention is given to patients with hematological malignancies, in whom the clinical need for effective decolonization strategies is greatest. Although FMT currently represents the most promising microbiome-based intervention, the available evidence remains heterogeneous and largely derived from small studies. Overall, durable and standardized decolonization strategies have yet to be established, underscoring the need for well-designed multicenter randomized clinical trials to define effective microbiome-directed approaches for preventing CRE-related infections in high-risk populations.},
}

Humans
*Hematologic Neoplasms/complications/microbiology
*Dysbiosis/microbiology
*Enterobacteriaceae Infections/microbiology/therapy
*Carbapenem-Resistant Enterobacteriaceae/drug effects
*Gastrointestinal Microbiome
Fecal Microbiota Transplantation
Anti-Bacterial Agents/pharmacology/therapeutic use
Probiotics

@article {pmid42694879,
year = {2026},
author = {Huang, CW and Chen, HH and Jao, TM and Li, CJ and Sung, JM and Tsai, YS and Chen, JS},
title = {CRISPR/Cas9-Driven Fndc5 Knockout Reveals Augmented Mitochondrial Structural and Dynamic Alterations in Diabetic Nephropathy.},
journal = {International journal of medical sciences},
volume = {23},
number = {9},
pages = {2884-2898},
pmid = {42694879},
issn = {1449-1907},
mesh = {Animals ; Mice ; *Diabetic Nephropathies/pathology/genetics ; *Mitochondria/pathology/metabolism/genetics/ultrastructure ; Mice, Knockout ; *Diabetes Mellitus, Experimental/pathology/genetics/chemically induced/complications ; *Fibronectins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Male ; Diet, High-Fat/adverse effects ; Mitochondrial Dynamics/genetics ; Kidney/pathology ; Mice, Inbred C57BL ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism ; Oxidative Stress/genetics ; Humans ; Streptozocin/toxicity ; Lipid Peroxidation/genetics ; },
abstract = {FNDC5 has been implicated in glucose homeostasis and is associated with mitochondrial function. Its role in diabetes and diabetic nephropathy (DN) remains unclear. This study hypothesizes that FNDC5 deficiency predisposes the kidney to accelerated mitochondrial dysfunction in diabetes and DN. Systemic Fndc5 knockout (KO) C57BL/6 mice were generated using CRISPR/Cas9. DN were induced in six-week-old Fndc5 wild-type (WT) and KO mice using high-fat diet combined with streptozotocin injection. Weekly blood and urine analyses assessed glucose, cholesterol, triglycerides, blood urea nitrogen, creatinine, and proteinuria. At 15 weeks, kidneys and metabolic tissues including pancreas, muscle and adipose were collected for histological and molecular analyses. Results showed that while both Fndc5 WT and KO mice were successfully induced with hyperglycemia, the Fndc5 KO DN group exhibited a slightly lower cumulative glycemic burden compared with the WT DN group. Despite this milder metabolic stress, proteinuria remained comparable between the two groups. Furthermore, histological analysis revealed that Fndc5 KO DN mice displayed more severe mesangial expansion, glomerular basement membrane thickening, and podocyte effacement compared with WT DN mice. The elevated lipid peroxidation, reduced PGC-1α expression, and increased DNA fragmentation were also evident in Fndc5 KO DN. More swollen mitochondria with a significantly higher percentage of disrupted cristae were observed in Fndc5 KO DN mice compared with WT DN mice. This was accompanied by the upregulation of mitochondrial fission-related genes (Dnm1l and Fis1), downregulation of the fusion-related gene (Mfn1), and reduced expression of ATP synthase subunits (ATP5A1 and ATP5B). Systemic analysis of other metabolic tissues, including the pancreas and muscle and adipose tissues, revealed increased lipid peroxidation and decreased PGC-1α expression. These findings underscore FNDC5's role in maintaining mitochondrial integrity and cellular health under diabetic conditions, positioning FNDC5 as a potential therapeutic target.},
}

Animals
Mice
*Diabetic Nephropathies/pathology/genetics
*Mitochondria/pathology/metabolism/genetics/ultrastructure
Mice, Knockout
*Diabetes Mellitus, Experimental/pathology/genetics/chemically induced/complications
*Fibronectins/genetics/metabolism
CRISPR-Cas Systems/genetics
Male
Diet, High-Fat/adverse effects
Mitochondrial Dynamics/genetics
Kidney/pathology
Mice, Inbred C57BL
Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism
Oxidative Stress/genetics
Humans
Streptozocin/toxicity
Lipid Peroxidation/genetics

@article {pmid42695329,
year = {2026},
author = {Chowdhury, S and Nayak, SP and Pattanayak, R and Sardar, S and Majhi, A and Yadav, B and Dasari, A and Mandlik, R and Sonah, H and Deshmukh, R and Gupta, I and Bauer, P and Ram, H},
title = {Establishment of a CRISPR-Cas9 Library for Indica Rice and Identification of OsOPR5 (LOC_Os06g11210) as a Regulator of Root Architecture.},
journal = {Physiologia plantarum},
volume = {178},
number = {5},
pages = {e71097},
pmid = {42695329},
issn = {1399-3054},
support = {SRGJ2021/001495//Department of Biotechnology, Ministry of Science and Technology, Govt of India/ ; BT/PR53626/BSA/33/96/2024//Department of Biotechnology, Ministry of Science and Technology, Govt of India/ ; BT/PR56697/AMRIT/165/21/2025//Department of Biotechnology, Ministry of Science and Technology, Govt of India/ ; //BRIC-National Institute of Plant Genome Research/ ; //Alexander von Humboldt Foundation/ ; },
mesh = {*Oryza/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Plant Roots/genetics/anatomy & histology/growth & development/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Gene Library ; Gene Expression Regulation, Plant ; Mutation ; Cyclopentanes/metabolism ; Oxylipins/metabolism ; Gene Editing ; },
abstract = {Functional characterization of a large number of rice genes remains a major challenge despite the availability of genome sequences and large-scale transcriptomic datasets. CRISPR-Cas9 library is a powerful approach for high-throughput targeted mutagenesis; however, its application in indica rice cultivars remains limited due to low transformation and regeneration efficiencies. In this study, we developed a CRISPR-Cas9 library targeting 12,000 rice genes and evaluated its utility for functional genomics in the indica cultivar MTU-1010. Sanger sequencing and NGS analysis of the plasmid library revealed high sgRNA coverage and more than 80% accuracy. Transformation of the developed library into the indica cultivar MTU-1010 resulted in a high target editing efficiency, with 90% of analyzed transgenic plants carrying mutations at the intended target site. Functional analysis of one homozygous mutant identified a previously uncharacterized role for OsOPR5 (LOC_Os06g11210), a member of the 12-oxophytodienoate reductase family in root architecture. The opr5 mutants exhibited significant reductions in lateral root number, seminal and crown root number, and root length, demonstrating that OsOPR5 positively regulates root system architecture in rice. Notably, endogenous jasmonic acid (JA) and JA-isoleucine levels were not significantly altered in the mutant, suggesting potential functional specialization or redundancy among rice OPR family members for JA accumulation. The root system architecture is a key determinant of water and nutrient acquisition; our results suggest that OsOPR5 may play an important role in adaptation under adverse environmental conditions. Collectively, this study establishes an efficient genome-editing platform for indica rice and identifies OsOPR5 as a novel regulator of root development.},
}

*Oryza/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Plant Roots/genetics/anatomy & histology/growth & development/metabolism
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Gene Library
Gene Expression Regulation, Plant
Mutation
Cyclopentanes/metabolism
Oxylipins/metabolism
Gene Editing

@article {pmid42695976,
year = {2026},
author = {de Kreek, F and Hertzberger, R and van Eeden, F and Illidge, S and Teunis, EJ and Hanemaaijer, M and Lievens, E and Rienstra, F and Wiedhaup, DE and Lisotto, P and Butler, D and Molenaar, D and Kort, R},
title = {Intra-individual genetic diversity of vaginal Lactobacillus crispatus revealed through citizen science-driven isolation and pangenome analysis.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxag218},
pmid = {42695976},
issn = {1365-2672},
abstract = {AIMS: A vaginal microbiome dominated by Lactobacillus crispatus is associated with positive reproductive and sexual health outcomes, yet intra-individual genetic diversity within this species remains largely unexplored. This study characterised inter- and intra-individual genomic variation in L. crispatus strains isolated through a citizen science initiative and assessed implications for multi-strain probiotic development.

METHODS AND RESULTS: Fifty-three women participated in this citizen science project. Self-sampling resulted in 48 shotgun metagenomes. Twenty-two participants isolated their own L. crispatus strains using selective enrichment and LAMP-based species confirmation, resulting in 53 whole-genome-sequenced isolates. L. crispatus dominated 20 of 48 metagenomes (50.1-99.6% relative abundance). Pangenome analysis revealed 3 456 gene families, of which 43.7% were core and 56.3% accessory. A 14-kb plasmid harbouring a Fic-domain toxin-antitoxin protein, but devoid of antimicrobial resistance genes, was present in 44 of 53 strains. Strains from the same individual clustered closely together yet harboured 1-123 gene differences. Intra-individual variation was observed in the pullulanase type I gene required for glycogen degradation: 40 strains were predicted to grow on glycogen, six showed genetic disruptions with unknown consequences, and seven were predicted to lack this ability entirely. Variation within individuals was also found for bacteriocin classes and CRISPR-Cas genes.

CONCLUSIONS: Substantial functional diversity exists within L. crispatus, even among strains from the same individual, supporting the rationale for multi-strain vaginal probiotics. This citizen science approach enabled discovery of host-specific adaptations while ensuring participant ownership of their strains.},
}

@article {pmid42696079,
year = {2026},
author = {Mitra, S and Damini, and Devi, M and Niyogi, SG and Singh, UP and Lo, D and Bishtania, HC and Thakur, S},
title = {Unlocking the potential of bacteriophage-based therapeutic gene delivery in hepatocellular carcinoma.},
journal = {Journal of the Egyptian National Cancer Institute},
volume = {38},
number = {1},
pages = {},
pmid = {42696079},
issn = {2589-0409},
mesh = {Humans ; *Liver Neoplasms/therapy/genetics ; *Carcinoma, Hepatocellular/therapy/genetics ; *Bacteriophages/genetics ; *Genetic Therapy/methods ; *Gene Transfer Techniques ; Animals ; CRISPR-Cas Systems ; Genetic Vectors/genetics/administration & dosage ; Gene Therapy Agents ; },
abstract = {Liver cancer, mainly hepatocellular carcinoma (HCC), remains a global health burden marked by poor prognosis with limited therapeutic efficacy, and high recurrence rates. HCC remains one of the most lethal malignancies worldwide, with limited therapeutic options and high resistance to conventional treatments. Despite low therapeutic efficacy, molecular heterogeneity, treatment resistance and high recurrence rate, hepatocellular carcinoma (HCC) is still a significant health problem worldwide. These restrictions have stimulated the research of focused methods for delivering therapeutic genetic payload into cancer cells. Bacteriophages have been gaining growing attention as an emerging delivery platform due to their genetic versatility, ease of engineering, ability to be surface modified and payload targeted. In this narrative review, the therapeutic potential of engineered bacteriophages in the context of HCC therapy is critically analyzed focusing on phage display-mediated tumor targeting, phage-mediated intracellular gene delivery, TRAIL gene delivery, and CRISPR/Cas-based therapeutic strategies. It has been previously noted in the literature that phage display can be used to attach tumor-targeting ligands to the surface of a phage, which may aid in the recognition of receptors at the tumor site and promote targeted delivery to the receptor. Therapeutic application is stunted by inefficient trafficking to the cytosol, endosomal degradation, immune recognition and clearance, vector stability, manufacturing scalability and regulatory issues. In conclusion, engineered bacteriophages are a promising and versatile tool for targeted gene delivery in HCC but more mechanistic, preclinical and translational research is needed to prove their therapeutic effectiveness and clinical usefulness for this purpose.},
}

Humans
*Liver Neoplasms/therapy/genetics
*Carcinoma, Hepatocellular/therapy/genetics
*Bacteriophages/genetics
*Genetic Therapy/methods
*Gene Transfer Techniques
Animals
CRISPR-Cas Systems
Genetic Vectors/genetics/administration & dosage
Gene Therapy Agents

@article {pmid40923633,
year = {2026},
author = {Ramakrishnan, M and Kaul, R and Sharma, A and Ahmad, Z and Vijayakanth, V and Keerthana, K and Gao, Z and Zhou, M and Wei, Q},
title = {CRISPR RNP-Mediated Transgene-Free Genome Editing in Plants: Advances, Challenges and Future Directions for Tree Species.},
journal = {Plant, cell & environment},
volume = {49},
number = {10},
pages = {7636-7656},
doi = {10.1111/pce.70176},
pmid = {40923633},
issn = {1365-3040},
support = {//The preparation of this review was supported by grants from the National Natural Science Foundation of China (32471977 and 32071848); a grant from the Natural Science Foundation of Jiangsu Province (BK20231289); the Natural Science Foundation for Distinguished Young Scholars of Nanjing Forestry University (JC2019004); the Project for Groundbreaking Achievements of Nanjing Forestry University (202211); and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors are also grateful for the Young Foreign Talent Program (Y20240114) and the support of Metasequoia Faculty Research Start-up Funding (163100028 and 163100036) at the Bamboo Research Institute, Nanjing Forestry University./ ; },
mesh = {*Gene Editing/methods ; *Ribonucleoproteins/genetics ; *Trees/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Transgenes ; Plants, Genetically Modified/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR ribonucleoprotein (RNP)-mediated genome editing offers a transgene-free platform for precise genetic modification in diverse herbaceous and tree species, including rice, wheat, apple, poplar, oil palm, rubber tree and grapevine. However, its application in woody plants faces distinct challenges, notably inefficient delivery and regeneration difficulties, particularly in species such as bamboo. While some of these issues also occur in herbaceous plants, they are often significantly more complex in woody species due to factors such as intricate cell wall architecture, widespread recalcitrant genotypes and inherent limitations of current delivery platforms. This review presents the first in-depth, critical re-evaluation of recent advancements in RNP-mediated editing in woody plants, highlighting these obstacles that warrant focused attention. Unlike plasmid-based CRISPR systems, RNP editing utilises Cas9/Cas12a protein-guide RNA complexes without integrating foreign DNA. This enables a DNA-free editing strategy that simplifies regulatory approval and minimises off-target effects due to the transient presence and rapid degradation of RNPs within plant cells. While PEG-mediated protoplast transfection and particle bombardment remain the primary reported methods for RNP delivery in trees, we evaluate promising alternative strategies such as lipofection, electroporation, cell-penetrating peptides and nanoparticle-based systems for targeted RNP delivery. Despite their promise, these advanced methods remain largely untested in woody species. Finally, we outline future research directions, including the development of tree-specific RNP delivery systems and regeneration protocols to enhance efficiency and minimise cytotoxicity. These innovations are essential for unlocking the full potential of RNP-mediated genome editing in long-lived tree species. This review provides a focused and timely roadmap for expanding the application of RNP technology across diverse woody plants.},
}

*Gene Editing/methods
*Ribonucleoproteins/genetics
*Trees/genetics
*CRISPR-Cas Systems/genetics
*Genome, Plant/genetics
Transgenes
Plants, Genetically Modified/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42285341,
year = {2027},
author = {Nejatinejad, M and Maleki-Aram, A and Namavar Abibiglou, A and Vahedi, C and Panahian, A and Ebrahimi, A and Yaghoubi, R and Parsaei, H and Vafaei, S and Jafari, D},
title = {Molecular Landscape and Advanced Diagnostic Technologies for BRAF Mutations in Cancer: From Quantitative PCR and ddPCR to CRISPR-Based Platforms.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {592},
number = {},
pages = {121178},
doi = {10.1016/j.cca.2026.121178},
pmid = {42285341},
issn = {1873-3492},
mesh = {Humans ; *Proto-Oncogene Proteins B-raf/genetics ; *Mutation ; *Neoplasms/genetics/diagnosis ; *Polymerase Chain Reaction/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {BRAF mutations are key oncogenic alterations across multiple malignancies, including melanoma, thyroid carcinoma, colorectal cancer, non-small cell lung cancer, glioma, and hairy cell leukemia. The most prevalent variant, BRAF-V600E, induces constitutive activation of the MAPK signaling pathway, promoting tumor progression and influencing therapeutic responsiveness. Accurate detection of BRAF alterations is therefore essential for molecular classification, prognostic assessment, treatment selection, and resistance surveillance. This review summarizes the molecular heterogeneity of BRAF mutations and critically evaluates current diagnostic methodologies. Conventional approaches such as allele-specific PCR and Sanger sequencing are compared with advanced quantitative platforms, including high-resolution melting analysis, droplet digital PCR, and next-generation sequencing, with emphasis on analytical sensitivity, mutation coverage, and clinical applicability. Emerging technologies such as CRISPR-based assays, rolling circle amplification systems, and nanoparticle-based biosensors and point-of-care diagnostic platforms are also discussed for their potential to enhance ultra-sensitive detection, particularly in liquid biopsy settings. These emerging tools are highlighted for their potential to enable ultra-sensitive, rapid, and decentralized mutation detection, particularly in liquid biopsy settings. Key challenges, including intratumoral heterogeneity, low allele-frequency variants, FFPE-associated artifacts, and clonal evolution under therapeutic pressure, are examined within a translational framework. In addition, we examine critical barriers to clinical implementation, including standardization, cost, and global accessibility of molecular diagnostics, and outline potential solutions through scalable technologies and decentralized testing strategies. We propose that optimal BRAF testing requires a mutation subclass-informed and clinically integrated strategy combining comprehensive baseline profiling with longitudinal molecular monitoring. Future diagnostic paradigms will likely integrate multi-omics data and artificial intelligence (AI)-assisted interpretation to refine precision oncology implementation. Looking forward, we propose that optimal BRAF testing will require integration of multi-omics profiling with AI-assisted interpretation, enabling automated variant classification, real-time clinical decision support, and improved prediction of therapeutic response and resistance.},
}

Humans
*Proto-Oncogene Proteins B-raf/genetics
*Mutation
*Neoplasms/genetics/diagnosis
*Polymerase Chain Reaction/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics

@article {pmid42379464,
year = {2027},
author = {Zhang, Y and Lin, X and Shi, H and Chen, Z and Luo, Q and Wu, H and Zeng, T},
title = {CRISPR-Cas12a-based liquid biopsy technology: challenges in large-scale clinical application.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {592},
number = {},
pages = {121210},
doi = {10.1016/j.cca.2026.121210},
pmid = {42379464},
issn = {1873-3492},
mesh = {Humans ; Liquid Biopsy/methods ; *CRISPR-Cas Systems/genetics ; Biomarkers, Tumor/analysis/blood/genetics ; },
abstract = {Liquid biopsy technology, which enables the acquisition of tumor-related biomarkers in a minimally invasive and reproducible manner, has been widely applied in clinical scenarios such as early tumor detection, treatment response assessment, minimal residual disease (MRD) monitoring, and recurrence surveillance. However, its clinical utility is still limited by the extremely low abundance of clinically relevant targets, the inhibitory effects of complex matrices, and the high variability in pre-analytical stages. As a next-generation programmable nucleic acid diagnostic platform, CRISPR-Cas12a combines the unique mechanisms of sequence-specific recognition and trans-cleavage signal amplification, demonstrating significant technical advantages and translational potential in the detection of low-abundance targets in liquid biopsies. Nevertheless, a mere improvement in analytical sensitivity is insufficient to support its large-scale clinical application; the robustness of detection methods, the reproducibility of results, and clinical interpretability remain the key factors currently restricting its clinical translation. This review summarizes the mechanism of CRISPR-Cas12a. Focusing on seven representative clinical biofluids, including blood, urine, and cerebrospinal fluid, it analyzes the technical difficulties and adaptation strategies associated with different matrices, and summarizes the performance differences of detection methods alongside the most clinically rational application scenarios across various biofluids. Furthermore, it explores the primary bottlenecks this technology faces when transitioning from laboratory proof-of-concept to routine clinical application, and provides a preliminary discussion on its future development directions based on existing research. Ultimately, this review aims to provide a reference for promoting the clinical translation of CRISPR-Cas12a liquid biopsy technologies and the broader implementation of precision medicine.},
}

Humans
Liquid Biopsy/methods
*CRISPR-Cas Systems/genetics
Biomarkers, Tumor/analysis/blood/genetics

@article {pmid42538409,
year = {2026},
author = {Song, L and Song, Y and Nguyen, V and Xu, S and Ho, K and Mohammed, A and Shoemaker, RH and Hoang, BH and Yu, J and Uchio, E and Zi, X},
title = {The establishment of prostate-specific, SKP2 humanized mice by CRISPR knock-in method reveals neoplastic initiation and microenvironmental reprogramming.},
journal = {Oncogene},
volume = {45},
number = {37},
pages = {3984-3996},
pmid = {42538409},
issn = {1476-5594},
support = {R01CA255643//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; I01 BX005105/BX/BLRD VA/United States ; UG3 CA290368/CA/NCI NIH HHS/United States ; R01 CA260351/CA/NCI NIH HHS/United States ; I01BX005105//U.S. Department of Veterans Affairs (Department of Veterans Affairs)/ ; P30 CA062203/CA/NCI NIH HHS/United States ; UG3 CA290368//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA260351//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Animals ; Male ; *S-Phase Kinase-Associated Proteins/genetics/metabolism ; Humans ; *Prostatic Neoplasms/pathology/genetics/metabolism ; Mice ; *Tumor Microenvironment/genetics ; Gene Knock-In Techniques ; Disease Models, Animal ; Prostate/pathology/metabolism ; Mice, Transgenic ; Gene Expression Regulation, Neoplastic ; Cell Transformation, Neoplastic/genetics ; Epithelial-Mesenchymal Transition/genetics ; CRISPR-Cas Systems ; *Carcinogenesis/genetics ; Cell Movement/genetics ; },
abstract = {Genetic inactivation of SKP2 has been shown to effectively prevent cancer initiation and block tumorigenesis. However, direct in vivo evidence for SKP2 on cancer initiation and prostatic microenvironment is still lacking and a SKP2 humanized mouse model is critical for developing prostate cancer immunoprevention approaches through targeting SKP2. We therefore have established a prostate-specific human SKP2 knock-in mouse model driven by an endogenous mouse probasin promoter. Overexpression of hSKP2 induces PIN and low-grade carcinoma. RNA-sequencing analysis revealed significant gene expression alterations in EMT, extracellular matrix, and interferon signaling. Single-cell deconvolution showed an increase of fibroblast population and a decrease of CD8[+] T cell and B cell populations. Consistent with these results from the SKP2 humanized mouse, SKP2 protein is overexpressed in human prostatic hyperplasia, PIN and prostate adenocarcinoma compared to normal prostate tissues. Overexpression of SKP2 markedly increased cell migration and invasion and induced the gene expression of EMT and interferon pathways. Inhibition of SKP2 signaling by Flavokawain A and C1 reverses EMT and affects EMT and interferon-related gene expression. In addition, paired prostate organoids were derived from SKP2 humanized and wild-type mice for drug screening and validated by known SKP2 inhibitors, Flavokawain A and C1. Both of which selectively decreased viability and altered the morphologies of organoids of hSKP2 knock-in rather than wild-type mice. Our studies provide a well-characterized prostate-specific hSKP2 knock-in mouse model and offer new mechanistic insights for understanding the oncogenic role of SKP2 in shaping the prostatic microenvironment during early carcinogenesis.},
}

Animals
Male
*S-Phase Kinase-Associated Proteins/genetics/metabolism
Humans
*Prostatic Neoplasms/pathology/genetics/metabolism
Mice
*Tumor Microenvironment/genetics
Gene Knock-In Techniques
Disease Models, Animal
Prostate/pathology/metabolism
Mice, Transgenic
Gene Expression Regulation, Neoplastic
Cell Transformation, Neoplastic/genetics
Epithelial-Mesenchymal Transition/genetics
CRISPR-Cas Systems
*Carcinogenesis/genetics
Cell Movement/genetics

@article {pmid42571853,
year = {2026},
author = {Nasir, U and Yasmeen, A and Awais, M and Latif, A and Bakhsh, A and Ahmad, N and Shahid, N and Azam, S and Rao, AQ and Shahid, AA},
title = {Multiplexed CRISPR/Cas9 mediated knockdown of BCH gene in potato enhances beta-carotene to combat vitamin A deficiency.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {372},
number = {},
pages = {113370},
doi = {10.1016/j.plantsci.2026.113370},
pmid = {42571853},
issn = {1873-2259},
mesh = {*Solanum tuberosum/genetics/metabolism ; *beta Carotene/metabolism ; *CRISPR-Cas Systems ; *Mixed Function Oxygenases/genetics/metabolism ; Plants, Genetically Modified/metabolism ; *Vitamin A Deficiency/prevention & control/genetics ; Gene Knockdown Techniques ; *Plant Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {The inadequate amounts of provitamin A carotenoids in crops contribute to the widespread vitamin A deficiency, leading to malnutrition and blindness in humans. Suppression of the β-carotene hydroxylase (BCH) increases β-carotene levels. In the current study, we utilized the multiplexed CRISPR/Cas9 approach by designing three targets against the BCH gene in a local potato cultivar. Transformation efficiency was recorded as 15%, the successful integration of the CRISPR/Cas9-BCH multiplex construct in potatoes was confirmed through PCR. When analysed using TIDE software, Sanger sequencing revealed the highest indel efficacy of 92.1% in plant 7 and 26.6% in plant 1. qRT-PCR (quantitative real-time PCR) analysis indicated a significant 89-fold reduction in BCH transcript levels in genome-edited potato lines compared to control plants. Spectrophotometry demonstrated a notable increase in beta-carotene levels in genome-edited potato plants, ranging from 0.831 µg/mL FW to 4.236 µg/mL FW, compared to the control plant with the lowest beta-carotene concentration (0.344 µg/mL FW). HPLC analysis further confirmed increased beta-carotene levels in genome-edited potato plants, ranging from 0.11 mg/mL FW to 0.36 mg/mL FW, compared to the unmodified control plant with a minimum beta-carotene value of 0.09 mg/mL. Our results revealed that the multiplexed CRISPR-Cas9 approach targeting the BCH gene results in enhanced beta-carotene contents in potato tubers.},
}

*Solanum tuberosum/genetics/metabolism
*beta Carotene/metabolism
*CRISPR-Cas Systems
*Mixed Function Oxygenases/genetics/metabolism
Plants, Genetically Modified/metabolism
*Vitamin A Deficiency/prevention & control/genetics
Gene Knockdown Techniques
*Plant Proteins/genetics/metabolism
Gene Editing

@article {pmid42660118,
year = {2026},
author = {Tong, X and Visscher, M and Riemers, FM and Versluis, D and Geijsen, N and Shang, P and Tryfonidou, MA and Poramba-Liyanage, DW},
title = {CRISPR activation reveals SOX5/6/9 as key transcriptional regulators directing iPSC-derived cells toward a notochordal lineage.},
journal = {Stem cell reports},
volume = {21},
number = {9},
pages = {103060},
doi = {10.1016/j.stemcr.2026.103060},
pmid = {42660118},
issn = {2213-6711},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Notochord/cytology/metabolism ; Cell Differentiation/genetics ; *Cell Lineage/genetics ; *SOXD Transcription Factors/genetics/metabolism ; *SOX9 Transcription Factor/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; Gene Expression Regulation ; },
abstract = {Intervertebral disc (IVD) degeneration, a leading cause of chronic lower back pain, is associated with loss of vacuolated notochordal cells (NCs) and fibrotic remodeling of the nucleus pulposus. Emerging therapies increasingly focus on NCs, which are rare but therapeutically relevant cells for regenerating degenerated IVDs. In this study, we used CRISPR-based transactivation (CRISPRa) to direct the differentiation of human induced pluripotent stem cells (iPSCs) into the NC lineage. We tested CRISPRa-mediated activation of NOTO, TBXT, FOXA2, SOX5, SOX6, and SOX9, coupled with single-cell sequencing of Aggrecan-2A-mScarlet reporter iPSCs. This approach identified the SOX5/6/9 combination (SOX-trio) as critical for promoting NC lineage commitment. The SOX-trio yielded the largest cell population expressing a range of genes previously associated with NC identity, including SHH, FOXA1, FOXA2, FOXJ1, FN1, ALCAM, KRT8, and KRT18. Our study demonstrates the integration of CRISPRa with single-cell technologies as a powerful platform for investigating and enriching iPSC-derived NCs, supporting future regenerative strategies across various fields.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Notochord/cytology/metabolism
Cell Differentiation/genetics
*Cell Lineage/genetics
*SOXD Transcription Factors/genetics/metabolism
*SOX9 Transcription Factor/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems
Gene Expression Regulation

@article {pmid42687278,
year = {2026},
author = {Han, W and Wei, P and Wang, L and Zhu, G and Xie, L and Zhu, L and He, B and Ji, X and Cao, X},
title = {Tube-Anchored Dual-Atom Nanozyme Hydrogel Microreactors with Dual-Interface Engineering for Portable Biosensing.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {7426-7434},
doi = {10.1021/acssensors.6c01841},
pmid = {42687278},
issn = {2379-3694},
support = {2024TXTD11//Henan University of Technology/ ; 232300421080//Natural Science Foundation of Henan Province/ ; 242300421035//Natural Science Foundation of Henan Province/ ; 23ZX008//Key Scientific Research Project of Colleges and Universities in Henan Province/ ; },
mesh = {*Biosensing Techniques/methods/instrumentation ; *Hydrogels/chemistry ; DNA/chemistry ; *Nanostructures/chemistry ; Cobalt/chemistry ; Nickel/chemistry ; Colorimetry/methods ; Electrochemical Techniques/methods/instrumentation ; CRISPR-Cas Systems ; Nucleic Acid Hybridization ; Limit of Detection ; Polymers/chemistry ; Indoles ; },
abstract = {DNA hydrogels are promising platforms for portable biosensing, but their combination with high-activity catalytic nanomaterials remains hindered by interfacial incompatibility and inconsistent device-level retention. Here, a tube-anchored hydrogel microreactor is developed, coupling Co-Ni dual-atom nanozymes (CoNi DANs), interfacial stabilization, and CRISPR-triggered release within a standard centrifuge tube. CoNi DANs are synthesized on a defect-rich nitrogen-doped carbon scaffold, achieving high metal loading and improved peroxidase-like activity relative to those of their single-atom counterparts. Carboxymethyl cellulose-mediated interfacial stabilization then suppresses CoNi DAN aggregation through electrostatic complementarity and polymeric steric shielding, enabling uniform dispersion of CoNi DANs within the DNA hydrogel. A polydopamine-based dual-anchoring strategy further combines covalent grafting with sequence-specific DNA hybridization, providing a chemically reinforced hydrogel-device interface. Upon target recognition, CRISPR/Cas12a trans-cleavage activity induces controlled hydrogel degradation and on-demand release of CoNi DANs for colorimetric and electrochemical dual-mode readout. Using atrazine as a proof-of-concept analyte, the platform achieves limits of detection of 6.1 and 2.1 pg mL-1 for colorimetric and electrochemical modes, respectively, with satisfactory recoveries in real samples. A smartphone-assisted digital readout module further improves the portability of the colorimetric mode. The dual-interface design, uniting material-level compatibilization with device-level dual-anchoring, provides a generalizable framework for embedding high-activity nanozymes into responsive DNA hydrogel microreactors for portable on-site biosensing.},
}

*Biosensing Techniques/methods/instrumentation
*Hydrogels/chemistry
DNA/chemistry
*Nanostructures/chemistry
Cobalt/chemistry
Nickel/chemistry
Colorimetry/methods
Electrochemical Techniques/methods/instrumentation
CRISPR-Cas Systems
Nucleic Acid Hybridization
Limit of Detection
Polymers/chemistry
Indoles

@article {pmid42687281,
year = {2026},
author = {Lu, M and Cheng, J and Guo, J},
title = {Advances in Single-Molecule Immunoassay: From Counting Strategies to CRISPR-Enhanced Biosensing.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {6600-6616},
doi = {10.1021/acssensors.6c01523},
pmid = {42687281},
issn = {2379-3694},
support = {CSTB2024NSCQ-JQX0012//Natural Science Foundation of Chongqing Municipality/ ; 2023YFF0724300//National Key Research and Development Program of China/ ; 2025ZD01902700//National Science and Technology Major Project/ ; },
mesh = {Immunoassay/methods ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Single-molecule immunoassays (SMIs) overcome the sensitivity limitations of conventional bulk measurements by enabling a paradigm shift from analog to digital signal readouts, thereby facilitating highly sensitive quantification of ultra-low-abundance biomarkers for precision diagnostics. This review provides a systematic overview of recent advances in SMI technologies and the conceptual framework underlying their evolution. First, discretization strategies for single-molecule counting are classified into hard discretization, based on physical confinement, and soft discretization, based on spatiotemporal isolation, within heterogeneous and homogeneous assay systems, respectively. The fundamental mechanisms by which these strategies mitigate diffusion limitations and enhance signal-to-noise ratios are discussed. Second, the integration of SMIs with CRISPR-based diagnostic systems (CRISPR-dx) is examined, with particular emphasis on their complementary roles in target recognition and signal amplification. Finally, recent applications of SMIs in the diagnosis of oncological, neurological, infectious, and cardiovascular diseases are summarized, along with a critical discussion of current engineering challenges and future directions toward clinical translation.},
}

Immunoassay/methods
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42688545,
year = {2026},
author = {Song, Z and Li, H and Zhao, Y and Li, M},
title = {[Research Progress on the Application of CRISPR/Cas System in Rapid Testing of Drug-Resistant Bacteria in Clinical Practice].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {57},
number = {4},
pages = {1212-1220},
pmid = {42688545},
issn = {1672-173X},
mesh = {*CRISPR-Cas Systems ; Humans ; *Drug Resistance, Bacterial/genetics ; *Bacteria/genetics/drug effects/isolation & purification ; *Bacterial Infections/diagnosis/microbiology ; Rapid Diagnostic Tests ; },
abstract = {The misuse of antimicrobial agents has made antimicrobial resistance one of the major threats to global public health. Efficient and rapid detection of drug-resistant bacteria and resistance genes is crucial for controlling infection spread and safeguarding human health. However, traditional detection methods (such as culture isolation, polymerase chain reaction, etc.) exhibit significant limitations in detection speed, cost-effectiveness, and convenience, increasingly failing to meet current clinical testing demands. In this context, the CRISPR/Cas system, as an emerging molecular diagnostic technology, offers innovative solutions for this field. This review details the developmental status of CRISPR/Cas systems and their research progress in rapid clinical detection of drug-resistant bacteria. It systematically explains the working principles and efficacy of various CRISPR/Cas-based detection platforms (e.g., SHERLOCK, DETECTR, HOLMES), highlighting the system's advantages in sensitivity, specificity, efficiency, real-time capability, portability, and low cost. Additionally, the article discusses challenges faced by CRISPR/Cas systems, including direct clinical sample detection, multiplex testing, cost reduction, clinical validation, and standardization. Finally, it outlines future development directions, emphasizing technological innovation and clinical translation to establish CRISPR/Cas systems as efficient tools for drug-resistant bacteria prevention and control.},
}

*CRISPR-Cas Systems
Humans
*Drug Resistance, Bacterial/genetics
*Bacteria/genetics/drug effects/isolation & purification
*Bacterial Infections/diagnosis/microbiology
Rapid Diagnostic Tests

@article {pmid42689566,
year = {2026},
author = {Zhang, R and Wen, Z and Zhao, Y and Cheng, L and Liu, C and Bian, S and Xu, L and Pei, R},
title = {Phosphorothioate-Enabled Cas12a Autocatalytic Network for Amplification-Free Ultrasensitive Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {25454-25462},
doi = {10.1021/acs.analchem.6c05179},
pmid = {42689566},
issn = {1520-6882},
support = {NA//Yinuosai Biotech Co., Ltd./ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry ; *DNA, Single-Stranded/chemistry/analysis ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; *DNA/analysis ; Limit of Detection ; *Phosphorothioate Oligonucleotides/chemistry ; *Phosphates/chemistry ; },
abstract = {Autocatalytic CRISPR/Cas systems offer a promising route toward amplification-free nucleic acid detection, yet their performance is limited by indiscriminate transcleavage of ssDNA activators and the reliance on structurally complex, thermodynamically unstable DNA architectures. A phosphorothioate (PS)-enabled Cas12a autocatalytic network (SCAN) is presented to address these challenges by leveraging the unique compatibility of F. novicida Cas12a (FnCas12a) with fully PS-modified ssDNA activators. An ssDNA mediator is engineered with fully PS-modified activator domains flanking a cleavable, unmodified spacer. The intact mediator remains sterically inactive toward Cas12a ribonucleoproteins, whereas trace-target-induced cleavage liberates split activators that trigger a self-propagating amplification network. The SCAN platform achieves ultrasensitive detection with limits of detection of 889 aM and 341 aM for ssDNA and dsDNA targets, respectively. The platform further enables amplification-free detection of Salmonella enterica and Escherichia coli in real samples, with detection limits of 30 and 20 CFU/mL, respectively. This work establishes fully PS-modified ssDNA as a robust and generalizable design element for CRISPR-based autocatalytic systems, providing a simple, flexible, and efficient strategy to enhance stability, suppress background, and improve analytical performance.},
}

*CRISPR-Associated Proteins/metabolism/chemistry
*DNA, Single-Stranded/chemistry/analysis
CRISPR-Cas Systems
*Endodeoxyribonucleases/metabolism/chemistry
*Bacterial Proteins/metabolism/chemistry
*DNA/analysis
Limit of Detection
*Phosphorothioate Oligonucleotides/chemistry
*Phosphates/chemistry

@article {pmid42689567,
year = {2026},
author = {Yan, Y and Chen, M and Li, H and Zhou, Y and Chen, X and Dong, X and Liu, F and Xiao, M and Zhu, L},
title = {AND-Logic Gated Electrochemical Sensor for Separate Detection of Aflatoxin B1 and Ochratoxin A via Split-Activator CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {25286-25298},
doi = {10.1021/acs.analchem.6c03714},
pmid = {42689567},
issn = {1520-6882},
support = {22404119//National Natural Science Foundation of China/ ; JWC20250202//Teaching Reform Project of Sichuan Normal University/ ; },
mesh = {*Ochratoxins/analysis ; *Aflatoxin B1/analysis ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; DNA, Catalytic/metabolism/chemistry ; Limit of Detection ; Food Contamination/analysis ; G-Quadruplexes ; Electrodes ; },
abstract = {To address the frequent co-occurrence of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in real food matrices, as well as the difficulty of achieving both high-specificity recognition and rapid quantification with existing detection methods, this study developed an AND logic-gated electrochemical sensor based on split-activator CRISPR/Cas12a. The sensor constructed an ordered signaling layer on the electrode interface using a DNA tetrahedron. Through DNAzyme-catalyzed reactions triggered separately by the two targets, two split DNA fragments were generated. Only when both fragments coexisted could they jointly activate the trans-cleavage activity of CRISPR/Cas12a, leading to extensive cleavage of the G-quadruplex sequence supported by the DNA tetrahedron. This cleavage caused the loss of the G-quadruplex/hemin signal-reporting group, resulting in a sharp attenuation of the electrochemical signal. By optimizing the heparin sodium concentration, a one-pot integration of the DNAzyme catalytic cycle and the CRISPR cleavage reaction was successfully achieved. The sensor achieved detection limits of 1.12 pg/mL for AFB1 and 0.26 pg/mL for OTA, which are significantly lower than international regulatory limits. For real corn flour, wheat flour, rice flour, and soybean flour samples, spike recoveries ranged from 90.99% to 107.29%, and the results were highly correlated with those obtained using a commercial enzyme-linked immunosorbent assay method (R2 > 0.996) and high-performance liquid chromatography-tandem mass spectrometry (R2 > 0.998). This work not only provides a new, ultrasensitive, and highly specific tool for copresence reporting of dual mycotoxins, but also offers an innovative paradigm for constructing logic-based biosensors designed for intelligent analysis of complex matrices.},
}

*Ochratoxins/analysis
*Aflatoxin B1/analysis
*Electrochemical Techniques/methods
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
DNA, Catalytic/metabolism/chemistry
Limit of Detection
Food Contamination/analysis
G-Quadruplexes
Electrodes

@article {pmid42689595,
year = {2026},
author = {Liu, Y and Yu, L and Shen, Y and Gan, Z and Tong, J and Zhao, F and Xiao, Y},
title = {Lanthanide Metal-Organic Frameworks Enable Target-Triggered CRISPR-Cas12a Activator Release for Ultrasensitive Non-nucleic Acid Biomarker Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {24865-24875},
doi = {10.1021/acs.analchem.6c01959},
pmid = {42689595},
issn = {1520-6882},
support = {82273895//National Natural Science Foundation of China/ ; 82572684//National Natural Science Foundation of China/ ; 2025HBBSHXF006//Hubei Provincial Postdoctoral Pioneer Talent Tracking Program/ ; WHYC202503//Wuhan Top-Notch Talent Program/ ; ZNJY202607//Hubei Provincial Clinical Research Center for Molecular Diagnostics/ ; },
mesh = {*CRISPR-Cas Systems ; *Lanthanoid Series Elements/chemistry ; Humans ; Biomarkers/blood/analysis ; Aptamers, Nucleotide/chemistry ; *Metal-Organic Frameworks/chemistry ; Limit of Detection ; *Biosensing Techniques/methods ; *Troponin I/blood/analysis ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism ; },
abstract = {Rapid, ultrasensitive detection of cardiac troponin I (cTnI) is critical for the early diagnosis of acute myocardial infarction (AMI). However, CRISPR-Cas systems, despite their unparalleled nucleic acid detection performance, face inherent bottlenecks in protein sensing, including inefficient signal transduction, high background noise, and insufficient anti-interference capability in complex biological matrices. Herein, we report a modular fluorescence signal transduction platform integrating aptamer-functionalized magnetic beads, lanthanide metal-organic frameworks (Ln-MOFs) with high nucleic acid affinity, and CRISPR-Cas12a-mediated collateral cleavage amplification for sensitive cTnI detection. Target cTnI binding to magnetic bead-immobilized aptamers drives the formation of sandwich complexes with CRISPR activator DNA (act)-functionalized Ln-MOFs, triggering phosphate-mediated release of act. The liberated act initiates robust Cas12a trans-cleavage activity, generating an amplified fluorescence readout. This platform achieves a limit of detection (LOD) of 0.1 pg mL-1 in serum samples, with excellent specificity against off-target interfering proteins. Validation in 28 clinical serum samples demonstrates near-perfect agreement with standard clinical ELISA measurements and yields an area under the curve (AUC) of 0.995, confirming its clinical diagnostic accuracy. Furthermore, the modular design enables facile reconfiguration for diverse protein biomarkers via aptamer substitution, providing a universal strategy to expand CRISPR-Cas systems toward non-nucleic acid target detection.},
}

*CRISPR-Cas Systems
*Lanthanoid Series Elements/chemistry
Humans
Biomarkers/blood/analysis
Aptamers, Nucleotide/chemistry
*Metal-Organic Frameworks/chemistry
Limit of Detection
*Biosensing Techniques/methods
*Troponin I/blood/analysis
*CRISPR-Associated Proteins/metabolism/chemistry
*Bacterial Proteins/metabolism/chemistry
*Endodeoxyribonucleases/metabolism

@article {pmid42689621,
year = {2026},
author = {Fu, H and Wang, Z and Guo, J and Wang, T and Li, X and Su, Y and Chen, Z and Yang, H and Xie, Z and Li, D},
title = {High-Sensitivity Molecular Detection of Viral Bioaerosols Using a Composite Collection System and Optimized RPA-CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {98},
number = {34},
pages = {24876-24887},
doi = {10.1021/acs.analchem.6c01983},
pmid = {42689621},
issn = {1520-6882},
support = {12274197//National Natural Science Foundation of China/ ; 12304487//National Natural Science Foundation of China/ ; 2023ZDZX2071//Department of Education of Guangdong Province/ ; GJHZ20220913143207014//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20220818102618040//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20230807093808017//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20241202130558075//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; 2022A0505030024//Science and Technology Planning Project of Guangdong Province/ ; 2022B1515020093//Science and Technology Planning Project of Guangdong Province/ ; 2022B1515120012//Science and Technology Planning Project of Guangdong Province/ ; 2026A1515010034//Science and Technology Planning Project of Guangdong Province/ ; NA//Guangdong Provincial Intelligent Diagnosis Engineering Research Center for Molecular Instant Detection of Children?s Infectious Diseases/ ; },
mesh = {Aerosols/analysis ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {Highly pathogenic airborne microorganisms pose a significant public health threat, particularly because their small particle size can support prolonged suspension and long-range transport. Here, we present an integrated platform that combines a composite bioaerosol collection system with a three-stage enrichment mechanism and recombinase polymerase amplification-CRISPR/Cas12a (RPA-CRISPR/Cas12a) detection. The platform incorporates model-based constant-temperature control, stacked optical filters, and multichannel fluorescence imaging. The temperature-control model accurately described the thermal response of the reaction module, while the optical and imaging subsystems supported stable fluorescence readout. Using aerosolized monkeypox virus (MPXV) pseudovirus as a controlled test target, the platform detected target nucleic acid in collection liquid down to 10 copies/μL under the tested conditions. These results support the feasibility of integrating aerosol enrichment with isothermal molecular detection in a compact prototype. The main contributions lie in innovations and the engineering integration of bioaerosol collection and enrichment, thermal control of biochemical reactions in molecular detection, and precision fluorescence detection techniques for molecular detection. Regarding molecular detection methods, the biochemical reaction parameters were experimentally optimized rather than introducing new biochemical reaction mechanisms. Validation with authentic clinical or environmental viral aerosols remains necessary before diagnostic or field-use claims can be made.},
}

Aerosols/analysis
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods

@article {pmid42690503,
year = {2026},
author = {Indurthi, S and Kaur, G and Dutta, R and Madhu, S and Chodasani, B and Yadav, A and Singh, M and Meena, TK and Singh, G},
title = {Integrating genomics, multi-omics, CRISPR and speed breeding for stress-resilient vegetable legume improvement.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42690503},
issn = {1438-7948},
mesh = {*Fabaceae/genetics/metabolism ; *Plant Breeding ; Multiomics ; *Stress, Physiological/genetics ; Genomics ; Quantitative Trait Loci ; Genome, Plant ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Vegetable legumes are nutritionally and ecologically important crops. However, their genetic improvement has not kept pace with the increasing challenges posed by climate change due to the polygenic nature of stress tolerance, narrow genetic diversity, and the persistent gap between molecular discoveries and field-level cultivar development. Although recent reviews have examined individual genomic tools or specific stress responses, a comprehensive synthesis integrating genomics-assisted breeding, multi-omics technologies, genome editing, and speed breeding within a unified crop improvement framework has been lacking. This review addresses that gap by critically evaluating how these complementary approaches can accelerate the development of stress-resilient vegetable legumes, including pea, common bean, cowpea, faba bean, cluster bean, yard-long bean, and hyacinth bean. This review synthesizes advances in QTL mapping, genome-wide association studies, transcriptomics, metabolomics, and CRISPR-based functional genomics that have identified key regulators and pathways underlying resistance to major biotic and abiotic stresses. Rather than considering these technologies independently, the review emphasizes their convergence into a systems-level breeding framework integrating genomic discovery, functional validation, predictive breeding, and accelerated generation advancement to improve breeding efficiency. Speed breeding, enabling up to seven to eight generations annually under optimized controlled-environment experimental conditions in cowpea, is discussed as a complementary strategy with genomic selection and genome editing. The review further identifies major translational bottlenecks, including transformation recalcitrance, limited genomic resources for underutilized vegetable legumes, inadequate multi-environment validation, and fragmented omics integration, and presents an integrated systems-breeding framework to bridge the gap between gene discovery and cultivar development.},
}

*Fabaceae/genetics/metabolism
*Plant Breeding
Multiomics
*Stress, Physiological/genetics
Genomics
Quantitative Trait Loci
Genome, Plant
CRISPR-Cas Systems
Gene Editing

@article {pmid42272442,
year = {2026},
author = {Luo, X and Qu, Y and Ye, Z and Li, Z and Zhang, Y and Luo, L and Li, S and Zhao, W and Wang, M and Bock, R and Wan, J and Tan, J},
title = {Synergistic HMGN1 and VP64 Fusions Potentiate High-Precision and PAM-Flexible Base Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {50},
pages = {e76047},
pmid = {42272442},
issn = {2198-3844},
support = {BF2025302//Frontier Technologies R&D Program of Jiangsu/ ; ZSBBL-KY2023-04//Zhongshan Biological Breeding Laboratory/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Oryza/genetics ; Humans ; *HMGN Proteins/genetics ; },
abstract = {RNA-guided CRISPR-derived base editors (BEs) have revolutionized genome editing by enabling targeted base substitutions. However, their application is frequently constrained by the stringent requirement for PAM sequences and low editing precision (bystander editing). Here, we present a robust strategy to overcome these limitations by coupling SpRY, a near-PAM-less Cas9 variant, with truncated CDA1 cytidine deaminases. While this combination enables precise editing of virtually any cytosine in the genome, it initially exhibited suboptimal efficiency. To address this, we systematically screened a diverse panel of candidate DNA-binding proteins and identified that the synergistic fusion of HMGN1 and VP64 substantially enhances editing activity without compromising precision. Importantly, this enhanced editing efficiency was achieved without markedly increasing off-target effects. Our new BEs demonstrated robust performance not only in yeast but also in rice, suggesting broad applicability in gene therapy, precision breeding, and fundamental research.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Oryza/genetics
Humans
*HMGN Proteins/genetics

@article {pmid42315549,
year = {2026},
author = {Sekine, T and Shinzawa, N and Kubota, R and Kobayashi, D and Okubo, Y and Itokawa, K and Isawa, H and Amino, H and Ishino, T},
title = {Versatile, marker-free platform for life cycle-wide imaging of Plasmodium falciparum by integrating an exogenous gene cassette into a conserved intergenic locus.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42315549},
issn = {2045-2322},
support = {JPMJSP2120//Japan Science and Technology Corporation/ ; JP20wm0325018//Japan Agency for Medical Research and Development/ ; JP24wm0325074//Japan Agency for Medical Research and Development/ ; 24K02272//Japan Society for the Promotion of Science/ ; 24KK0149//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Plasmodium falciparum/genetics/growth & development ; *Life Cycle Stages/genetics ; Mice ; Humans ; Genetic Loci ; Gene Editing ; Sporozoites ; Luminescent Proteins/genetics ; CRISPR-Cas Systems ; *DNA, Intergenic/genetics ; Liver/parasitology ; Malaria, Falciparum/parasitology ; },
abstract = {The creation of transgenic Plasmodium falciparum lines with robust fluorescence across the entire life cycle is essential for advancing our understanding of parasite biology, which in turn informs the development of new drugs and vaccines. In this study, we utilized Plasmodium-optimized genome editing to integrate an mCherry expression cassette into a selected intergenic locus without gene disruption. The resulting marker-free line, NF54-mCh, exhibited intense fluorescence throughout all developmental stages, including asexual and sexual blood stages, as well as mosquito (ookinete, oocyst, and sporozoite) and liver stages. NF54-mCh showed normal proliferation, gametocytogenesis, and efficient transmission to mosquitoes. The ultra-high brightness in salivary gland sporozoites allowed for the non-invasive identification of infected mosquitoes. Sporozoites remained highly infectious to humanized mouse livers, thus enabling the completion of the full life cycle. NF54-mCh serves as a parental line for performing additional genetic modifications, because the CRISPR/Cas9-based genome editing method is free of introduced drug resistance markers. The broader applicability of this strategy was validated by generating similar reporter lines in Plasmodium species utilized in rodent malaria models. In summary, NF54-mCh represents a unique, versatile platform that will accelerate fundamental research and support the future development of malaria control strategies, including new vaccines and drugs.},
}

Animals
*Plasmodium falciparum/genetics/growth & development
*Life Cycle Stages/genetics
Mice
Humans
Genetic Loci
Gene Editing
Sporozoites
Luminescent Proteins/genetics
CRISPR-Cas Systems
*DNA, Intergenic/genetics
Liver/parasitology
Malaria, Falciparum/parasitology

@article {pmid42332291,
year = {2026},
author = {Al Madhoun, A and Malik, MZ and Al-Beloushi, S and Abukhalaf, N and Miranda, L and Jacob, S and Nizam, R and John, S and George, P and Channanath, A and Bahman, F and Liao, M and Ahmad, R and Chen, W and Bekri, A and Drapeau, P and Thanaraj, TA and Al-Mulla, F},
title = {Targeting glucagon signaling in metabolic disorders, functional insights from zebrafish receptor knockouts.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {83},
number = {1},
pages = {},
pmid = {42332291},
issn = {1420-9071},
support = {RA-CB-2021-007//Kuwait Foundation for the Advancement of Sciences/ ; },
mesh = {Animals ; *Zebrafish/metabolism/genetics ; *Receptors, Glucagon/genetics/metabolism ; *Signal Transduction ; *Glucagon/metabolism ; *Metabolic Diseases/metabolism/genetics ; Protein Isoforms/genetics/metabolism ; Lipid Metabolism ; Gene Knockout Techniques ; *Zebrafish Proteins/genetics/metabolism ; Energy Metabolism ; Glucose/metabolism ; Transcriptome ; CRISPR-Cas Systems ; },
abstract = {Glucagon receptor (GCGR) signaling is essential for glucose and lipid homeostasis, making it a potential therapeutic target for metabolic disorders. Zebrafish possess two GCGR co-orthologs, GCGRa and GCGRb, however, their distinct function remain unclear. In this study we employed CRISPR/Cas9 gene editing to generate GCGRa[-]/[-], GCGRb[-]/[-], and double-knockout (GCGR[-]/[-]) zebrafish to dissect isoform-specific functions. RNA-Seq analysis was performed to characterize transcriptomic alterations, while an overfeeding protocol was used to assess metabolic tolerance, and ligand-response assays in cell lines evaluated isoform activation dynamics. Transcriptomic analysis revealed that both isoforms regulate overlapping but distinct metabolic pathways. Functional enrichment analysis linked GCGRa to lipid and energy metabolism, cholesterol biosynthesis and glucose homeostasis, through key signaling cascades such as glucagon, PPARγ and PI3K-AKT. In contrast, GCGRb loss altered fatty acid β-oxidation, GPCR signaling, and oxidative phosphorylation networks, implicating roles in metabolism and cellular stress. The GCGR[-]/[-] primarily impacted core metabolic networks including lipid, gluconeogenesis and energy metabolism, indicating complementary and overlapping functions of both receptors in maintaining hepatic metabolic homeostasis. Ligand-response assays revealed that GCGRb, but not GCGRa, is activated by both glucagon (GCGa) and glucagon like-peptide-1 (GLP1a), supporting the post-duplication receptor diversification theory. Notably, all knockouts exhibited impaired growth under high-nutrient conditions, confirming GCGR's role in diet-responsive development. This study provides the first systematic functional comparison of zebrafish GCGR isoforms, establishing zebrafish as a valuable model for investigating glucagon-based metabolic regulation and therapeutic interventions.},
}

Animals
*Zebrafish/metabolism/genetics
*Receptors, Glucagon/genetics/metabolism
*Signal Transduction
*Glucagon/metabolism
*Metabolic Diseases/metabolism/genetics
Protein Isoforms/genetics/metabolism
Lipid Metabolism
Gene Knockout Techniques
*Zebrafish Proteins/genetics/metabolism
Energy Metabolism
Glucose/metabolism
Transcriptome
CRISPR-Cas Systems

@article {pmid42412303,
year = {2026},
author = {Khatun, M and Lundin, K and Tuuri, T and Piltonen, T and Tapanainen, JS and Salumets, A},
title = {From Gene Function to Precision Intervention: CRISPR/Cas9 and Stem Cell-Based Strategies as Emerging Disease-Modifying Approaches in PMOS.},
journal = {Stem cell reviews and reports},
volume = {22},
number = {7},
pages = {3056-3080},
pmid = {42412303},
issn = {2629-3277},
mesh = {Humans ; Female ; *CRISPR-Cas Systems/genetics ; Animals ; Induced Pluripotent Stem Cells/metabolism ; Gene Editing/methods ; Mesenchymal Stem Cells/metabolism ; *Metabolic Syndrome/therapy/genetics ; *Precision Medicine/methods ; },
abstract = {Polyendocrine metabolic ovarian syndrome (PMOS) is a complex endocrine-metabolic disorder affecting up to 18% of women worldwide and remains the leading cause of anovulatory infertility. Despite extensive research, current treatments primarily target symptoms, including menstrual irregularities, hyperandrogenism, and metabolic dysfunction, without addressing the underlying molecular and tissue-level disturbances. Advances in multi‑omic profiling have identified disruptions across neuroendocrine, metabolic, inflammatory, and extracellular matrix pathways, alongside genetic susceptibility at loci such as DENND1A, CYP17A1, LHCGR, FSHR, IRS1, and PPARG. However, the functional roles of many variants remain unresolved. CRISPR/Cas9 gene editing enables precise interrogation of these pathways, while stem cell-based platforms, including mesenchymal stem cells (MSCs), exosomes, and gene-edited induced pluripotent stem cells (iPSCs), may serve as complementary platforms for regeneration and disease modeling. Preclinical studies demonstrate that MSCs and their derivatives modulate inflammation, restore ovarian structure, and improve metabolic parameters, while iPSC-based models enable patient-specific investigation of steroidogenic and metabolic abnormalities. Translational challenges remain, including targeted delivery, off-target effects, phenotypic heterogeneity, and regulatory considerations. Integrating CRISPR‑based functional genomics with stem cell research may shift PMOS management from symptom‑focused care to targeted, mechanism‑driven interventions that could modify the course of PMOS (Graphical Abstract).},
}

Humans
Female
*CRISPR-Cas Systems/genetics
Animals
Induced Pluripotent Stem Cells/metabolism
Gene Editing/methods
Mesenchymal Stem Cells/metabolism
*Metabolic Syndrome/therapy/genetics
*Precision Medicine/methods

@article {pmid42681397,
year = {2026},
author = {Savash Ishanzadeh, MC and Wells, D},
title = {Clustered Regularly Interspaced Palindromic Repeats (CRISPR) Applied to Gametes and Embryos.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3038},
number = {},
pages = {447-475},
doi = {10.1007/978-1-0716-5292-3_24},
pmid = {42681397},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; Female ; *Preimplantation Diagnosis/methods ; *CRISPR-Cas Systems ; *Blastocyst/metabolism ; Fertilization in Vitro/methods ; *Germ Cells/metabolism ; Pregnancy ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Preimplantation genetic testing (PGT) facilitates the identification of embryos affected by specific types of genetic abnormalities. However, PGT does not seek to treat the genetic abnormality, rather it is an embryo selection tool, employing a strategy of detection and exclusion. Until recently, the notion that mutations and aneuploidies could be corrected in gametes, or in embryos produced using in vitro fertilization (IVF), seemed improbable. However, rapid progress in the evolution of gene editing technologies may make this a realistic possibility in the near future. Not only would such an approach help to avoid the discard of human embryos, which some find challenging from ethical or religious perspectives, but it would also increase the number of embryos considered suitable for transfer, potentially leading to higher pregnancy rates than achieved in PGT cycles. This chapter considers the use of genome editing applied to human preimplantation embryos, describing a protocol that can be used to inactivate genes for research purposes, and which might, in the future, allow for the correction of pathogenic mutations.},
}

Humans
*Gene Editing/methods
Female
*Preimplantation Diagnosis/methods
*CRISPR-Cas Systems
*Blastocyst/metabolism
Fertilization in Vitro/methods
*Germ Cells/metabolism
Pregnancy
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42681488,
year = {2026},
author = {Fernandes, LGV and Nally, JE},
title = {Gene Knockout in Leptospira spp. by CRISPR/Cas9-NHEJ (Non-Homologous End-Joining) and CRISPR-Prime Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3068},
number = {},
pages = {139-156},
pmid = {42681488},
issn = {1940-6029},
mesh = {*Leptospira/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *DNA End-Joining Repair/genetics ; *Gene Editing/methods ; DNA Breaks, Double-Stranded ; },
abstract = {Genetic manipulation of Leptospira spp. has progressed significantly in recent years. Like most prokaryotes, leptospires are unable to survive double-strand breaks (DSBs) induced by the Cas9 endonuclease, prompting the development of alternative strategies for gene knockout. We have established two systems for targeted mutagenesis of Leptospira spp.: CRISPR/Cas9-NHEJ and CRISPR-Prime Editing (PE). The CRISPR/Cas9-NHEJ approach involves coexpression of the CRISPR/Cas9 machinery alongside the DNA repair proteins LigD and Ku from Mycobacterium smegmatis, facilitating error-prone repair of DSBs that results in indel mutations. In contrast, CRISPR-PE is a DSB-free strategy that utilizes a Cas9-nickase fused to a reverse transcriptase that facilitates precise single-nucleotide edits in the genome. This expanded toolbox has placed Leptospira spp. at the forefront of bacterial genetic manipulation.},
}

*Leptospira/genetics
*CRISPR-Cas Systems
*Gene Knockout Techniques/methods
*DNA End-Joining Repair/genetics
*Gene Editing/methods
DNA Breaks, Double-Stranded

@article {pmid42683944,
year = {2026},
author = {Schulman, J and Egan, R and Kandari, L and Madayiputhiya, N and Mahon, D and Tao, L and Luo, H and Condon, K and Feder, JN and Huang, D and Khetan, A},
title = {TPST Gene Knock-Out Eliminates Tyrosine Sulfation on a Recombinant Antibody Produced in CHO Cells.},
journal = {Biotechnology journal},
volume = {21},
number = {9},
pages = {e70302},
pmid = {42683944},
issn = {1860-7314},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Tyrosine/metabolism ; *Sulfotransferases/genetics/metabolism ; *Recombinant Proteins/metabolism/genetics ; *Antibodies, Monoclonal/metabolism/genetics ; Gene Knockout Techniques ; Protein Processing, Post-Translational ; CRISPR-Cas Systems ; Cricetinae ; Antibodies, Bispecific/metabolism/genetics ; },
abstract = {Tyrosine sulfation is a post-translational modification that has been reported to occur infrequently on recombinant monoclonal antibodies (mAbs). We recently demonstrated that tyrosine sulfation occurred on a bispecific antibody (bsAb) produced in Chinese hamster ovary (CHO) cells, using a multi-enzymatic approach in combination with intact mass and peptide-based mass spectrometry analysis supplemented with the use of synthetic peptides. Tyrosine sulfation needs to be controlled during the manufacturing process due to potential undesired effects, such as impact on potency and immunogenicity. Here, we report that tyrosine sulfation was not significantly inhibited by the addition of chemical inhibitors, such as sodium chlorate. Individual knockout and double knockout (DKO) of two key genes in the tyrosine sulfation pathway were carried out sequentially. Tyrosyl protein sulfotransferase 1/2 (TPST1/2) DKO by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing eliminated tyrosine sulfation while maintaining cell growth, antibody production, and overall product quality.},
}

Animals
CHO Cells
Cricetulus
*Tyrosine/metabolism
*Sulfotransferases/genetics/metabolism
*Recombinant Proteins/metabolism/genetics
*Antibodies, Monoclonal/metabolism/genetics
Gene Knockout Techniques
Protein Processing, Post-Translational
CRISPR-Cas Systems
Cricetinae
Antibodies, Bispecific/metabolism/genetics

@article {pmid42684483,
year = {2026},
author = {Wang, K and Chen, J and Bai, S and Wang, Q and Zhang, W and Qin, Z and Zhang, Z and Zhang, Z and Ma, Y},
title = {Efficient Microinjection and CRISPR/Cas9 Mediated Genome Editing in Urechis unicinctus.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {28},
number = {5},
pages = {},
pmid = {42684483},
issn = {1436-2236},
support = {42176122//National Natural Science Foundation of China/ ; 32170373//National Natural Science Foundation of China/ ; ZR2025MS368//Natural Science Foundation of Shandong Province/ ; 2022120090//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Microinjections/methods ; Caveolin 1/genetics/metabolism ; Cilia/genetics/metabolism ; *Gene Editing/methods ; Tubulin/genetics ; Base Sequence ; },
abstract = {Urechis unicinctus is an economically important aquaculture species and an emerging model for developmental and evolutionary research; however, progress in functional genetic studies in this species has remained limited by the absence of effective genome editing approaches. Here, we establish a CRISPR/Cas9-mediated genome editing platform in U. unicinctus based on an optimized microinjection procedure. Targeting the cilia-associated gene Caveolin-1, Cas9/sgRNA ribonucleoprotein complexes were delivered into oocytes, generating mosaic G0 larvae harboring insertion-deletion mutations, as confirmed by ICE analysis and cloning-based Sanger sequencing with mutation detection rates of 68.51% and 64.86%, respectively. Ciliary defects were frequently observed in edited larvae, including shortening of the circumoral ciliary ring and reduction of apical ciliary tufts, accompanied by impaired swimming performance. Whole-mount in situ hybridization further revealed altered spatial expression patterns of Caveolin-1 in edited embryos, with 78% of individuals showing detectable changes in expression patterns. To independently assess the robustness of the platform, the conserved cytoskeletal gene α-tubulin was additionally targeted, resulting in successful mutagenesis and associated ciliary abnormalities. Together, these findings establish the first CRISPR/Cas9 genome editing workflow for U. unicinctus, providing a technical framework for functional genomics in echiurans and facilitating future studies of developmental mechanisms and molecular breeding in this species.},
}

Animals
*CRISPR-Cas Systems
Microinjections/methods
Caveolin 1/genetics/metabolism
Cilia/genetics/metabolism
*Gene Editing/methods
Tubulin/genetics
Base Sequence

@article {pmid42686754,
year = {2026},
author = {Dinesh, RK and Wang, X and Mohammad, IA and Gunasekaran, P and Stiklioraitis, K and Villafuerte, JR and Rao, A and Hernandez-Lopez, RA and Sunwoo, JB and Cong, L},
title = {Surfaceome CRISPR activation screening uncovers ligands regulating tumor sensitivity to NK cell killing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42686754},
issn = {2041-1723},
support = {R35GM155437//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35HG011316//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35DE030054//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35 GM155437/GM/NIGMS NIH HHS/United States ; R35 HG011316/HG/NHGRI NIH HHS/United States ; R01 GM141627/GM/NIGMS NIH HHS/United States ; 1R01GM141627//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35 DE030054/DE/NIDCR NIH HHS/United States ; },
mesh = {*Killer Cells, Natural/immunology/metabolism ; Humans ; Animals ; Mice ; Ligands ; Cell Line, Tumor ; *Cytotoxicity, Immunologic ; CRISPR-Cas Systems ; Leukosialin/metabolism/genetics/immunology ; Hyaluronan Receptors/genetics/metabolism/immunology ; Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics/immunology ; Female ; Immunotherapy/methods ; Coculture Techniques ; },
abstract = {Natural killer (NK) cell-based immunotherapies are promising for cancer treatment due to their ability to eliminate cancer cells independently of antigen presentation and "off-the-shelf" utility. However, molecular determinants governing tumor susceptibility to NK cytotoxicity remain incompletely understood. Here we employ CRISPR activation (CRISPRa) screening to identify cancer cell surface regulators of NK killing. Using a surfaceome-focused library, we screen human and murine cancer cell lines co-cultured with NK cells, identifying known and novel ligands modulating NK cytotoxicity. Screens reveal established factors including CD43 and previously uncharacterized regulators CD44, PDPN, and Siglec-1/CD169. Validation with orthogonal approaches confirm that disruption of these factors alters NK killing susceptibility in vitro and in humanized mouse models. Mechanistically, we find that CD43-mediated NK resistance operates independently of its proposed interaction with Siglec-7, and that targeting CD43 on NK cells or CAR T cells substantially enhances cytotoxic activity against leukemia. These results establish gain-of-function surfaceome screening as a powerful tool for identifying therapeutic targets for NK cell-based immunotherapy.},
}

*Killer Cells, Natural/immunology/metabolism
Humans
Animals
Mice
Ligands
Cell Line, Tumor
*Cytotoxicity, Immunologic
CRISPR-Cas Systems
Leukosialin/metabolism/genetics/immunology
Hyaluronan Receptors/genetics/metabolism/immunology
Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics/immunology
Female
Immunotherapy/methods
Coculture Techniques

@article {pmid41945283,
year = {2026},
author = {Chu, NH and Le, HD and Quach, NT and Pham, TKL and Ho, NA and Nguyen, THH and Tran, XK and Pham, BN and Chu, HH},
title = {CRISPR/Cas9-Mediated Knockout of the NAD-Dependent Lactate Dehydrogenases for Altered Stereospecific Lactic Acid Production in Lacticaseibacillus paracasei NC4.},
journal = {Biochemical genetics},
volume = {64},
number = {5},
pages = {7016-7031},
pmid = {41945283},
issn = {1573-4927},
support = {TĐNSH0.06/22-24//Vietnam Academy of Science and Technology/ ; },
mesh = {*Lactic Acid/biosynthesis ; *CRISPR-Cas Systems ; *Lacticaseibacillus paracasei/genetics/enzymology/metabolism ; Gene Knockout Techniques ; *L-Lactate Dehydrogenase/genetics/metabolism ; *NAD/metabolism ; *Lactate Dehydrogenases/genetics/metabolism ; Stereoisomerism ; },
abstract = {The optical purity of lactic acid is a critical parameter for producing high-performance polylactic acid (PLA). To investigate the genetic basis of stereospecific lactic acid biosynthesis, the present study aimed to functionally characterize the roles of ldh1 and ldh2 in Lacticaseibacillus paracasei NC4 through targeted gene disruption. A CRISPR/Cas9 nickase-based system was employed to construct three mutant strains (Δldh1, Δldh2, and Δldh1Δldh2). Fermentation experiments were conducted under identical conditions, and the concentrations of D- and L-lactic acid were quantified using HPLC. Three mutant strains, Δldh1, Δldh2, and Δldh1Δldh2, were successfully constructed from the wild-type NC4 using the CRISPR-Cas9 system. Compared with the wild-type NC4, which produced 89.31 ± 0.21 g/L L-lactic acid and 10.74 ± 0.19 g/L D-lactic acid, the Δldh1 mutant produced 76.31 ± 2.22 g/L L-lactic acid and 7.72 ± 0.36 g/L D-lactic acid, while the Δldh2 mutant yielded 81.73 ± 0.46 g/L L-lactic acid and ND (not detected) D-lactic acid. The Δldh1Δldh2 double mutant generated 75.57 ± 2.96 g/L L-lactic acid and ND (not detected) D-lactic acid. The Δldh1Δldh2 double mutant similarly exhibited no detectable D-lactic acid formation, supporting the role of ldh2 in D-lactate biosynthesis. These results indicate that targeted deletion of ldh genes significantly alters the stereospecificity of lactic acid biosynthesis. In particular, deletion of ldh2 was sufficient to eliminate detectable D-lactic acid formation, whereas deletion of ldh1 alone did not completely abolish D-lactate production. Overall, this study provides functional genetic insight into the roles of ldh1 and ldh2 in controlling lactic acid stereospecificity in L. paracasei NC4 and establishes a genetic basis for future metabolic and process-oriented optimization of optically pure lactic acid production.},
}

*Lactic Acid/biosynthesis
*CRISPR-Cas Systems
*Lacticaseibacillus paracasei/genetics/enzymology/metabolism
Gene Knockout Techniques
*L-Lactate Dehydrogenase/genetics/metabolism
*NAD/metabolism
*Lactate Dehydrogenases/genetics/metabolism
Stereoisomerism

@article {pmid42189436,
year = {2026},
author = {Mendoza, A and Simon, I and Hasan, S},
title = {A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function.},
journal = {Biochemical genetics},
volume = {64},
number = {5},
pages = {7759-7786},
pmid = {42189436},
issn = {1573-4927},
mesh = {Animals ; *Microinjections/methods ; *Oryzias/genetics/embryology ; *Zebrafish/genetics/embryology ; CRISPR-Cas Systems ; *Embryo, Nonmammalian/metabolism ; Gene Knockdown Techniques/methods ; Female ; Morpholinos/genetics/administration & dosage ; },
abstract = {Zebrafish (Danio rerio) and medaka (Oryzias latipes) are popular teleost models used in developmental biology and functional genomics. To achieve high-quality and reproducible microinjections, it is essential to have robust protocols for breeding, egg collection, and the precise delivery of genetic material. In this protocol, we present a comprehensive and optimized methodology for setting up breeding tanks under controlled photoperiod conditions to maximize egg yield while minimizing contamination. We provide detailed procedures for sex identification, pair selection, the use of grated breeding inserts, and methods to increase egg collection efficiency. We outline procedures for making injection gel beds, pulling needles, and calibration using one-microliter microcapillaries to achieve consistent nanoliter-scale injections. Our protocol outlines settings for the pico-liter injector that are optimized to deliver a precise amount per pulse with minimal variability. Finally, we demonstrate the application of these methods for gene knockdown using morpholino antisense oligonucleotides, gene knockout using CRISPR-Cas9, and gain-of-function mRNA overexpression experiments. Phenotypic assessments conducted at various developmental stages to evaluate gene-specific effects reveal consistent phenotypic outcomes between the morpholino and CRISPR-Cas9 approaches. This easy and comprehensive protocol enables efficient, precise, and scalable genetic manipulation of zebrafish and medaka embryos, thereby supporting advanced functional studies in developmental biology and disease modeling. To our knowledge, this is the first unified protocol for both zebrafish and medaka microinjection systems achieving 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with precision together with a triple validation approach that confirms gene function across multiple techniques.},
}

Animals
*Microinjections/methods
*Oryzias/genetics/embryology
*Zebrafish/genetics/embryology
CRISPR-Cas Systems
*Embryo, Nonmammalian/metabolism
Gene Knockdown Techniques/methods
Female
Morpholinos/genetics/administration & dosage

@article {pmid42537911,
year = {2026},
author = {Liu, K and Fan, X and Tong, Z and Zhang, J and Zhao, M and Chen, Y and Wu, C and Wang, T and Wei, S and Liu, Y and Xue, Z and Zheng, Y},
title = {Reprogrammed Komagataella phaffii for enhanced secretory expression of human lactoferrin.},
journal = {Journal of biotechnology},
volume = {419},
number = {},
pages = {1-10},
doi = {10.1016/j.jbiotec.2026.07.011},
pmid = {42537911},
issn = {1873-4863},
mesh = {*Lactoferrin/genetics/metabolism ; Humans ; *Saccharomycetales/genetics/metabolism ; CRISPR-Cas Systems ; Recombinant Proteins/genetics/metabolism ; Endoplasmic Reticulum/metabolism/genetics ; Bioreactors ; Vacuoles/metabolism/genetics ; },
abstract = {Human lactoferrin (hLF) is a multifunctional glycoprotein of the transferrin family derived from milk and mucosal secretions, which exhibits antibacterial, anti-tumor, and immunomodulatory functions, and is an important component of infant formula. Conventional methods for lactoferrin expression are often inefficient, primarily due to inadequate protein synthesis capabilities and poor stability within microbial hosts. Herein, a Komagataella phaffii yeast strain capable of high-level secretory expression of hLF was constructed by reprogramming the endoplasmic reticulum (ER) and vacuole using CRISPR/Cas9 technology. A dual-expression cassette containing the AOX1 promoter, an α-secretion signal peptide, the hLF gene, and a terminator was integrated into three different sites of the K. phaffii genome. The stepwise strategy combining expansion of the ER membrane involved in protein synthesis with knockout of vacuolar proteases further enhanced hLF production. Subsequently, 0.1 g/L FeCl3 was added to the medium to reduce the toxicity of hLF and improve its stability. After high-density cultivation of K. phaffii through optimization of cultivation conditions in shake flasks and a 5 L bioreactor, the secretory intact hLF titer reached 2214 mg/L, representing a 76.3-fold increase achieved through these engineering strategies. In addition, antibacterial experiments demonstrated that this secretory hLF had a significant inhibitory effect on Escherichia coli, Staphylococcus aureus, and yeast. Overall, the developed K. phaffii protein expression platform enabled efficient production of lactoferrin, demonstrating its potential for expressing other lactoproteins.},
}

*Lactoferrin/genetics/metabolism
Humans
*Saccharomycetales/genetics/metabolism
CRISPR-Cas Systems
Recombinant Proteins/genetics/metabolism
Endoplasmic Reticulum/metabolism/genetics
Bioreactors
Vacuoles/metabolism/genetics

@article {pmid42574952,
year = {2026},
author = {Zhu, X and Zhang, W and Sun, T and Chen, L},
title = {Advances in combinatorial CRISPRi screening and applications: Decoding higher-order interactions for next-generation microbial cell factories in synthetic biology.},
journal = {Microbiological research},
volume = {313},
number = {},
pages = {128663},
doi = {10.1016/j.micres.2026.128663},
pmid = {42574952},
issn = {1618-0623},
mesh = {*Synthetic Biology/methods ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; Gene Regulatory Networks ; Bacteria/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Biological systems possess high robustness and intricate genetic redundancy, rendering traditional single-gene perturbations largely inadequate for comprehensively elucidating the true regulatory mechanisms underlying complex phenotypes. To address this, combinatorial CRISPR interference (CRISPRi) has emerged as an essential tool in systems and synthetic biology, offering reversible epigenetic control, multi-target regulation, and an absence of DNA toxicity. Unlike pooled single-gene screens, combinatorial CRISPRi facilitates the systematic dissection of buffering, synergy, and metabolic trade-offs by targeting multiple loci simultaneously. This review explores the latest advancements in pairwise and higher-order combinatorial CRISPRi screening, beginning with design strategies for multiplex guide RNA (gRNA) arrays and orthogonal systems. Computational techniques utilized for analyzing high-dimensional screening data and visualizing complex genetic networks are subsequently examined. Building upon these methodological foundations, crucial applications within microbial engineering are highlighted. Specifically, the review details the optimization of carbon flux in microbial cell factories to circumvent production bottlenecks, alongside the elucidation of protective multigenic networks against severe environmental stress. Furthermore, it addresses current obstacles, such as system noise and library construction challenges, and outlines future research directions. Ultimately, this review provides a comprehensive guide for decoding complex traits and driving rational designs of next-generation cell factories.},
}

*Synthetic Biology/methods
*CRISPR-Cas Systems
*Metabolic Engineering/methods
Gene Regulatory Networks
Bacteria/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41047874,
year = {2026},
author = {Ji, Y and Sun, Y and Zhou, H and Liu, Z and Sun, Z and Xu, G and Wen, H and Zheng, Z and Tu, L and Yang, Z and Zhang, Y and Liu, X and Zhou, S and Dong, X and Wang, Y and Li, C and Wan, J},
title = {Engineer the eukaryotic OMEGA-Fanzor systems for genome editing in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3160-3162},
doi = {10.1111/jipb.70049},
pmid = {41047874},
issn = {1744-7909},
support = {2023ZD04074//the Biological Breeding-Major Projects/ ; 2023YFD1202900//the National Key Research and Development Program/ ; ZSBBL-KY2023-04//the Zhongshan Biological Breeding Laboratory/ ; NAUSY-ZZ03//the Guidance Foundation of the Sanya Institute of Nanjing Agricultural University/ ; BK20230038//the Jiangsu Province Natural Science Foundation/ ; //the Nanjing U35 program/ ; 2023AB006-02//the Bingtuan Key Science and Technology Program of Xinjiang Province/ ; KYT2024005//the Fundamental Research Funds for the Central Universities/ ; 31872806//the National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics ; *Genome, Plant/genetics ; *Genetic Engineering/methods ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; },
abstract = {The activity of the eukaryotic OMEGA-Fanzor genome editing system remains limited in plants. We engineered the Fanzor nucleases SpuFz1, GtFz1, NlovFz2, and MmeFz2 in plants, with NlovFz2 being the most efficient, achieving up to 50.0% editing in regenerated rice plants, making it a promising tool for plant genome editing.},
}

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

@article {pmid41566884,
year = {2026},
author = {Zhang, R and Tang, X and He, Y and Wang, W and Ren, Q and Qi, Y and Zhang, Y},
title = {Enhanced exonuclease-Cas9 systems promote multiple nucleotide deletions with higher efficiency and broader targeting scope in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3316-3328},
doi = {10.1111/jipb.70155},
pmid = {41566884},
issn = {1744-7909},
mesh = {*CRISPR-Cas Systems/genetics ; *Oryza/genetics ; *Sequence Deletion/genetics ; Gene Editing/methods ; *Exonucleases/metabolism/genetics ; Plants, Genetically Modified ; },
abstract = {CRISPR-Cas9 is a widely used platform for plant genome editing, but its outcomes are typically dominated by small insertions and deletions (indels). Such limited mutation profiles restrict its utility in functional studies of non-coding RNAs and regulatory elements, such as microRNAs (miRNAs), untranslated regions (UTRs), and promoter sequences, where larger sequence disruptions are often required. Here, we developed enhanced exonuclease-Cas9 platforms, termed multiple nucleotide deletion Cas9 (MND-Cas9) systems, for efficient generation of large deletions in rice. By screening four exonucleases (RecJ, T5, TREX2, and SbcB), we established MND-Cas9v1 systems based on TREX2 or SbcB that produced substantially larger deletions without reducing editing efficiency. Further optimization with an inserted DNA-binding domain (DBD) between Cas9 and exonuclease yielded MND-Cas9v2, which simultaneously enhanced efficiency and deletion size. To expand PAM compatibility, we introduced PAM-relaxed Cas9-NG and SpG variants, generating MND-Cas9-NG/SpGv2 systems with broader targeting scope and superior performance compared to their parental nucleases. Finally, we demonstrated the utility of these systems in two applications: MND-Cas9v2 efficiently knocked out the miRNA gene OsMIR530, producing larger seeds, and generated extended deletions in the 3'UTR of OsGhd2, which upregulated its expression and increased grain size. These results demonstrate that MND-Cas9 systems enable high-efficiency generation of extended deletions and facilitate functional analyses of non-coding RNAs and regulatory sequences. Overall, this work establishes a versatile and expandable exonuclease-Cas9 platform that substantially broadens the mutational spectrum and application potential of CRISPR-Cas9 for plant genome engineering.},
}

*CRISPR-Cas Systems/genetics
*Oryza/genetics
*Sequence Deletion/genetics
Gene Editing/methods
*Exonucleases/metabolism/genetics
Plants, Genetically Modified

@article {pmid41588854,
year = {2026},
author = {Zhang, C and Li, J and Li, Y and Yan, L and Yong, CSY and Li, S and He, Y and Xia, L},
title = {Coupling of both a transactivation module and a double-stranded DNA-binding domain boosts Cas12i3 variant-based cytosine and adenine editing in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3196-3207},
doi = {10.1111/jipb.70154},
pmid = {41588854},
issn = {1744-7909},
mesh = {*Gene Editing/methods ; *Oryza/genetics ; *Cytosine/metabolism ; *Adenine/metabolism ; *Transcriptional Activation/genetics ; CRISPR-Cas Systems/genetics ; Protein Domains ; *DNA/metabolism ; },
abstract = {CRISPR/Cas12i3 belongs to the type V-I Cas system, characterized by its smaller protein size and less restricted canonical "TTN" protospacer adjacent motif. Developments of Cas12i3-mediated base editing systems for either C-to-T or A-to-G transitions will expand the editing scope and enrich the plant base editing toolkits for crop improvement. However, while the Cas12i3-based cytosine base editor (CBE) only shows very low editing efficiency in plants, its adenine base editor (ABE) has not been documented as yet. Here, we engineered a series of Cas12i3 (5M)-based CBEs (V0-V5) and ABEs (V0-V5) by fusing a deactivated dCas12i3 (5M) with a transactivation module VP64, a single-stranded DNA-binding domain Rad51, or a double-stranded DNA-binding domain HMG-D, or in combinations, and systemically evaluated their performance in rice protoplasts. Our results demonstrated that synergistic combinations of both VP64 and HMG-D outperformed other architectures and significantly boosted the efficiencies of Cas12i3 (5M)-based CBE and ABE for C-to-T and A-to-G base editing and expanded the editing window. In stable lines, in comparison to the non-fusion control, the optimized Cas12i3 (5M)-based CBE-V5 and ABE-V5 enabled up to 4.78- and 3.35-fold higher editing efficiencies, with the maximum C-to-T and A-to-G efficiencies reaching 32.35% and 38.24%, respectively, and a higher proportion of homozygous mutants in the T0 generation. Furthermore, we generated herbicide-resistant rice germplasm by using CBE-V5 and ABE-V5, demonstrating their potential for precision breeding in crops. Together, here, we report novel Cas12i3 (5M)-based CBE and ABE that substantially enrich base editing toolkits for improvement of rice and potentially other crops.},
}

*Gene Editing/methods
*Oryza/genetics
*Cytosine/metabolism
*Adenine/metabolism
*Transcriptional Activation/genetics
CRISPR-Cas Systems/genetics
Protein Domains
*DNA/metabolism

@article {pmid41664360,
year = {2026},
author = {Liu, X and Jin, S and Xiao, Z and Ma, C and Wang, Q and Wang, H and Zhou, R and Gu, D and Xu, R and Qin, R and Li, J and Wei, P},
title = {Optimizations of Cas12a- and Cas12i-based adenine base editors for efficient precision editing in the plant genome.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3169-3171},
doi = {10.1111/jipb.70177},
pmid = {41664360},
issn = {1744-7909},
support = {//This work was supported by the Agriculture Science and Technology Major Project, the Science and Technology Major Project of Anhui Province (No. 202423110050063, No. 202423m10050002, and No. 2023n06020020), the Natural Science Foundation of China (No. 32300343, No.32572441, and No. 32570484), and Yangtze River Delta Science and Technology Innovation Community Joint Research (Basic Research) Project (No. 2024CSJZN01100)./ ; },
mesh = {*Adenine/metabolism ; *Genome, Plant/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; },
abstract = {A strategy coupling high-activity nucleases with dimeric TadA-8e optimizes plant Cas12-based adenine base editors, boosts editing efficiency, and provides precise editors for crop breeding and genomics research.},
}

*Adenine/metabolism
*Genome, Plant/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods

@article {pmid41814562,
year = {2026},
author = {Zhong, D and Dong, Y and Pan, H and Fu, Y and Zhao, Y and Ruan, S and Yu, W and Wang, Y and Yin, Q and Zhang, Y and Huang, Y and Shen, J and Zhang, H and Wu, Y and Xu, J and Lu, Y},
title = {CasY7: An optimized Cas12i system for enhanced genome editing in monocot crops.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3208-3219},
doi = {10.1111/jipb.70181},
pmid = {41814562},
issn = {1744-7909},
support = {CAIC (2024) 003//Construction project of Changzhou Modern Agricultural Science and Technology Innovation Center/ ; K2023001//Shanghai Agricultural Science and Technology Innovation Program/ ; 2021YFD1201300//National Key R&D Program of China/ ; },
mesh = {*Zea mays/genetics ; Plants, Genetically Modified ; Oryza/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Crops, Agricultural/genetics ; *Gene Editing/methods ; Triticum/genetics ; Mutation/genetics ; *CRISPR-Associated Proteins/metabolism ; Base Sequence ; },
abstract = {The CRISPR-Cas12 family nucleases, particularly the Cas12i subtypes, are considered promising alternatives to Cas9 for genome editing in plants. We previously developed a new Cas12i variant, CasY7, which has been successfully applied in clinical trials; its performance in plants remains to be investigated. Initial testing in stable transgenic maize and rice showed that the codon-optimized CasY7 (pCasY7e1) achieved average editing efficiencies of 58.7% and 62.3% across five target sites, respectively, outperforming the typical Cpf1 (pCpf1) control that targets the same sites. To further enhance activity, we fused T5 exonuclease to CasY7 (pCasY7e2), which shifted mutation profiles toward larger deletions, and subsequently integrated an MS2 aptamer into the crRNA scaffold (pCasY7e3). The optimized pCasY7e3 system increased editing efficiencies to 87.7% in maize and 82.9% in rice-approximately 2.7-fold higher than pCpf1. We further demonstrated multiplexed editing in maize, generating biallelic dwarf mutants, and validated functionality in hexaploid wheat with editing efficiencies up to 58.8%. Overall, our comprehensive validation across 942 transgenic plants confirmed robust editing in maize, rice, and wheat, establishing CasY7 as a high-efficiency addition to the CRISPR toolkit.},
}

*Zea mays/genetics
Plants, Genetically Modified
Oryza/genetics
*CRISPR-Cas Systems/genetics
*Genome, Plant/genetics
*Crops, Agricultural/genetics
*Gene Editing/methods
Triticum/genetics
Mutation/genetics
*CRISPR-Associated Proteins/metabolism
Base Sequence

@article {pmid41834254,
year = {2026},
author = {Ge, F and Peng, C and Du, Y and Chen, Y and Zhao, Z and Yu, M and Feng, H and Xie, Y and Sun, S and Liu, S and Xin, B and Zhao, H and Wu, S and Bian, C and Yang, Z and Lai, J and Chen, J},
title = {Synergistic engineering of Casδ nuclease for robust genome editing.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3231-3242},
doi = {10.1111/jipb.70222},
pmid = {41834254},
issn = {1744-7909},
support = {2024M753551//Project funded by China Postdoctoral Science Foundation/ ; 2022YFF1002800//National Key Research and Development Program of China/ ; 32572347//National Natural Science Foundation of China/ ; Z231100003723004//Agriculture Science and Technology Major Project, the Beijing Rural Revitalization Agricultural Science and Technology Project/ ; },
mesh = {*Gene Editing/methods ; Humans ; Zea mays/genetics ; CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; *Endonucleases/metabolism/genetics ; },
abstract = {Casδ is a recently identified evolutionary transitional CRISPR system characterized by its compact size (~900 amino acids), broad temperature tolerance, and guidance by a short crRNA without the requirement of a tracrRNA. However, the low editing efficiency of Casδ in eukaryotic cells limits its application. Here, we have developed a hierarchical engineering strategy to improve the genome editing activity of Casδ-1, with optimization focused on enhancing its interactions with the crRNA, the protospacer adjacent motif (PAM) duplex, the single-stranded DNA substrate, and the RNA-DNA heteroduplex. Through this strategy, we successfully generated an activity-enhanced Casδ-1 variant, designated enCasδ, which harbors 9 amino acid substitutions that synergistically augment its editing efficiency. In human cell lines, enCasδ showed 1.3- to 29.3-fold higher editing activity than the wild-type Casδ-1 across ten tested genomic loci, with an average editing efficiency of 54.6%. In addition, enCasδ also mediated robust genome editing in maize; its editing efficiency increased by an average of 5.3-fold relative to Casδ-1, and reached up to an average of 80% at the TS4 and PSY1 loci in stable transgenic lines. The overall editing performance of enCasδ was comparable to that of Streptococcus pyogenes Cas9 (SpCas9) and other Cas12 nucleases. Collectively, enCasδ represents a highly optimized Casδ-1 variant that broadens the applicability of the Casδ CRISPR system and facilitates robust genome editing in both animal cells and plants.},
}

*Gene Editing/methods
Humans
Zea mays/genetics
CRISPR-Cas Systems/genetics
*Genetic Engineering/methods
*Endonucleases/metabolism/genetics

@article {pmid41840837,
year = {2026},
author = {Lin, W and Wu, H and Kuang, H and Feng, X and Bai, M and He, F and Liang, R and Zeng, Y and Li, M and Kong, F and Liu, B and Guan, Y},
title = {Developing a robust multiplex CRISPR/Cas12i3-5M system for trait stacking in soybean.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3172-3174},
doi = {10.1111/jipb.70233},
pmid = {41840837},
issn = {1744-7909},
support = {2023YFF1000203//National Key Research and Development Program of China/ ; },
mesh = {*Glycine max/genetics ; *CRISPR-Cas Systems/genetics ; Base Sequence ; Plants, Genetically Modified ; *Quantitative Trait, Heritable ; },
abstract = {The high-efficiency multiplex gene editing technology based CRISPR-Cas12i3-5M is capable of simultaneously editing 13 target sites in soybean, and was used to generate germplasm with high oleic acid content and no beany flavor.},
}

*Glycine max/genetics
*CRISPR-Cas Systems/genetics
Base Sequence
Plants, Genetically Modified
*Quantitative Trait, Heritable

@article {pmid41928060,
year = {2026},
author = {Cheng, Y and Li, G and Zhou, M and Mandlik, R and Wang, D and Qi, Y},
title = {Enhancing CRISPR-Cas12a base editing in plants with LbCas12a variants and introns.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3243-3259},
doi = {10.1111/jipb.70249},
pmid = {41928060},
issn = {1744-7909},
support = {MD-PSLA-24014//McIntire Stennis Forest Research/ ; },
mesh = {*Oryza/genetics ; *Introns/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mutation/genetics ; Base Sequence ; Plants, Genetically Modified ; },
abstract = {Cytosine base editors (CBEs) and adenine base editors (ABEs) are powerful tools for precise genome editing in plants. Conventionally, such base editors are built upon the CRISPR-Cas9 systems where Cas9 nickases are used. To expand the base editing scope and minimize off-target effects, base editors derived from the CRISPR-Cas12a systems are desired. However, the use of deactivated Cas12a (dCas12a) in such base editors constrains the editing activity, preventing the wide use of Cas12a base editors for plant research and trait development. In this study, we demonstrate the use of an ABE based on the efficient LbCas12a-RRV variant to introduce herbicide-resistant mutations in OsACCase in rice. To improve Cas12a CBEs and ABEs, we inserted introns into the coding sequence of dLbCas12a-RRV. This intron-containing Cas12a-CBE shows substantial improvement in editing efficiency in rice, compared to the intron-less counterparts. By contrast, the improvement of ABE with the intron-containing dLbCas12a-RRV is very limited, partly due to the already high baseline editing efficiency of the intron-less dLbCas12a-RRV ABE. Testing of these base editors in poplar shows elevated C-to-T base editing by dLbCas12a-RRV-intron-CBE. For A-to-G editing, ABEs built upon dLbCas12a-RV and dLbCas12a-RRV variants showed significant improvement over ABEs derived from wild-type LbCas12a and the ttLbCas12a variant. The addition of introns to dLbCas12a-RRV does not further improve the base editing efficiency. With whole genome sequencing in rice, we evaluated genome editing specificities with these improved Cas12a base editors. Our analyses show that both intron-containing Cas12a CBE and ABE barely introduce guide RNA-dependent off-target mutations. However, they can generate guide RNA-independent off-target mutations, which are likely attributed to the high enzymatic activities of the deaminases. Collectively, our study demonstrates the successful use of a Cas12a base editor for trait development and reports improved Cas12a CBEs and ABEs for precise base editing in plants.},
}

*Oryza/genetics
*Introns/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Mutation/genetics
Base Sequence
Plants, Genetically Modified

@article {pmid42021470,
year = {2026},
author = {Liu, M and Wang, Y and Zhang, L and Zhang, X and Wang, X and Liu, X and Fu, Y and Li, X and Song, Z and Liu, Y and Wang, R and Zhao, J},
title = {Optimized Cas-SF01 gene-editing toolbox shortens flowering timing in commercial maize inbred JING724.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3178-3180},
doi = {10.1111/jipb.70264},
pmid = {42021470},
issn = {1744-7909},
support = {2024-zz-087//Beijing Postdoctoral Science Foundation/ ; },
mesh = {*Zea mays/genetics/physiology/growth & development ; *Flowers/physiology/genetics ; *Gene Editing/methods ; Time Factors ; Mutation/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; },
abstract = {The gene-editing tool Cas-SF01 was optimized to maximize its efficiency in maize. The Cas-SF01-TREX2 configuration was superior in enabling high-purity gene mutations. This toolkit enabled commercial maize to flower seven days earlier without yield loss, thereby securing harvests and accelerating crop breeding.},
}

*Zea mays/genetics/physiology/growth & development
*Flowers/physiology/genetics
*Gene Editing/methods
Time Factors
Mutation/genetics
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified

@article {pmid42152501,
year = {2026},
author = {Zhou, J and Li, R and Wang, Z and Liu, S and Shi, L and Fu, X and Li, F and Zhang, J and Li, G and Zhu, J and Qian, Q and Dun, B},
title = {Engineering herbicide-resistant sorghum with CRISPR/Cas9-mediated adenine base editing.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3292-3294},
doi = {10.1111/jipb.70298},
pmid = {42152501},
issn = {1744-7909},
mesh = {*Sorghum/genetics/drug effects ; *CRISPR-Cas Systems/genetics ; *Herbicide Resistance/genetics ; *Adenine/metabolism ; Plants, Genetically Modified ; *Gene Editing/methods ; *Herbicides/pharmacology ; },
abstract = {An adenine base-editing system was established to precisely modify the sorghum SbALS gene, generating transgene-free mutant plants. These plants exhibit strong herbicide resistance, with no significant differences in agronomic traits, providing valuable germplasm for herbicide-resistance breeding in sorghum.},
}

*Sorghum/genetics/drug effects
*CRISPR-Cas Systems/genetics
*Herbicide Resistance/genetics
*Adenine/metabolism
Plants, Genetically Modified
*Gene Editing/methods
*Herbicides/pharmacology

@article {pmid42206623,
year = {2026},
author = {Bai, Y and Li, B and Peng, J and Bai, Y and Liu, S and Lai, J and Song, W},
title = {Development of fragrant broomcorn millet (Panicum miliaceum L.) via CRISPR/Cas12i.3-mediated genome editing.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3184-3186},
doi = {10.1111/jipb.70295},
pmid = {42206623},
issn = {1744-7909},
mesh = {*Panicum/genetics ; *CRISPR-Cas Systems/genetics ; Mutation/genetics ; *Genome, Plant/genetics ; *Gene Editing/methods ; },
abstract = {Co-editing the broomcorn millet PmBADH2a and PmBADH2b genes using CRISPR/Cas12i.3 generated double mutants with significantly increased 2-acetyl-1-pyrroline content, producing fragrant broomcorn millet without compromising major agronomic traits.},
}

*Panicum/genetics
*CRISPR-Cas Systems/genetics
Mutation/genetics
*Genome, Plant/genetics
*Gene Editing/methods

@article {pmid42367085,
year = {2026},
author = {Zhang, R and Tang, X and Yang, X and Zhang, Y},
title = {Enhanced Cas12i3 system enables precise OsAUX3 editing for rice grain improvement.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3187-3189},
doi = {10.1111/jipb.70337},
pmid = {42367085},
issn = {1744-7909},
support = {//Agriculture Science and Technology Major Project/ ; },
mesh = {*Oryza/genetics ; Plants, Genetically Modified ; *CRISPR-Cas Systems/genetics ; *Edible Grain/genetics ; Promoter Regions, Genetic/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; },
abstract = {An optimized Cas12i3 genome-editing system enables highly efficient and predictable editing of regulatory sequences in rice. Precise promoter engineering fine-tunes gene expression, improves grain size, and enhances production potential, demonstrating a powerful new approach for crop improvement through targeted regulation rather than gene disruption.},
}

*Oryza/genetics
Plants, Genetically Modified
*CRISPR-Cas Systems/genetics
*Edible Grain/genetics
Promoter Regions, Genetic/genetics
Gene Expression Regulation, Plant
Plant Proteins/genetics/metabolism

@article {pmid42446212,
year = {2026},
author = {Cao, X and Liu, H and Bai, S and Wang, R and Xia, L and Sun, Y},
title = {Optimization of a hypercompact Fanzor2 system for improved genome editing performance in plants.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3190-3192},
doi = {10.1111/jipb.70346},
pmid = {42446212},
issn = {1744-7909},
mesh = {*Genome, Plant/genetics ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; },
abstract = {Native Fanzor2 nucleases exhibit weak editing activity in plants. An optimized Fanzor2 system engineered via ωRNA optimization and structure-based protein mutagenesis achieves robust editing at recalcitrant genomic sites and enables cytosine base editing, supporting versatile crop genome modification via transformation or viral delivery.},
}

*Genome, Plant/genetics
Plants, Genetically Modified
CRISPR-Cas Systems/genetics

@article {pmid42619441,
year = {2026},
author = {Chen, Z and Chen, Y and Wan, G and Dong, H and Pan, T and Yang, W and Zhou, Y and Luo, B and Zhu, Y and Han, X and Lu, L and Wang, X and Lei, C and Zhao, Z and Wang, J and Ren, Y and Wan, J},
title = {Development of low-prolamin rice germplasm via CRISPR/Cas9 editing to improve eating and cooking quality.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {9},
pages = {3313-3315},
doi = {10.1111/jipb.70377},
pmid = {42619441},
issn = {1744-7909},
support = {2021YFF1000200//National Key R&D Program of China/ ; 2026//Agriculture Science and Technology Major Project/ ; Y2021YJ18//Central Public-interest Scientific Institution Basal Research Fund/ ; Y2025YC02//Central Public-interest Scientific Institution Basal Research Fund/ ; CAAS-CSNCB-202302//Basic Research Center, Innovation Program of Chinese Academy of Agricultural Sciences/ ; 2025-2027//Inner Mongolia Innovation Center of Biological Breeding Technology/ ; CARS-01-05//Earmarked Fund for China Agriculture Research System/ ; },
mesh = {*Oryza/genetics/metabolism ; *Cooking ; *CRISPR-Cas Systems/genetics ; *Prolamins/metabolism/genetics ; Plants, Genetically Modified ; *Seeds/genetics/metabolism ; Starch/metabolism ; },
abstract = {Simultaneous editing of two major prolamin-encoding genes in rice using a single-guide RNA suppressed prolamin accumulation and reconfigured seed storage protein composition. This targeted modification markedly improved rice eating and cooking quality while maintaining the levels of total starch, total protein, and key agronomic traits.},
}

*Oryza/genetics/metabolism
*Cooking
*CRISPR-Cas Systems/genetics
*Prolamins/metabolism/genetics
Plants, Genetically Modified
*Seeds/genetics/metabolism
Starch/metabolism

@article {pmid42641742,
year = {2026},
author = {Chandrasekaran, S and Duraisamy, N and Jagadeesan, M and Palukuri, YK and Tamilselvan, S},
title = {Engineering CRISPR nanoplatforms to deplete cancer stem cells: Delivery checkpoints, target plasticity, and clinical viability.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {76},
number = {},
pages = {103007},
doi = {10.1016/j.nano.2026.103007},
pmid = {42641742},
issn = {1549-9642},
mesh = {*Neoplastic Stem Cells/pathology/metabolism ; Humans ; Animals ; *Nanomedicine/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy/pathology ; *Nanoparticles/chemistry ; },
abstract = {Cancer stem cells (CSCs) sustain tumor initiation, therapy resistance, and relapse, yet evade durable control because they switch phenotype, enter quiescence, shelter within protective niches, resist drug efflux, and share markers with normal stem cells. Programmable CRISPR editing can disable intracellular self-renewal dependencies that antibodies and small molecules cannot reach, whereas only nanoscale carriers can confine such editing to intended cells; neither component alone solves the CSC problem. This review reframes CSC-directed CRISPR nanomedicine as an integrated design problem. We examine why target plasticity defeats static single-marker targeting; the sequential delivery checkpoints spanning blood stability, organ selection, tumor penetration, CSC recognition, endosomal escape, and productive editing; and advanced architectures including organ-selective lipid nanoparticles, biomimetic and vesicle carriers, metal-organic frameworks, and logic-gated systems. Genotoxicity, immunogenicity, incomplete depletion, manufacturing reproducibility, and absent CSC-specific clinical evidence remain limiting. Clinical viability, not imminent cure, is the realistic near-term objective.},
}

*Neoplastic Stem Cells/pathology/metabolism
Humans
Animals
*Nanomedicine/methods
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Neoplasms/genetics/therapy/pathology
*Nanoparticles/chemistry

@article {pmid42681183,
year = {2026},
author = {Zhang, S and Hubbard, BP},
title = {Production of Cell Models with Differential Zygosity Using CRISPR Base Editors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {181-196},
pmid = {42681183},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; Humans ; Adenine ; },
abstract = {CRISPR base editors have revolutionized the ease with which single-nucleotide gene editing can be performed in cell and organismal models. Unlike conventional CRISPR/Cas9 gene editing, which induces a double-strand DNA break and relies on endogenous homology-directed repair (HDR) or non-homologous end joining (NHEJ) repair, base editors only induce a single-strand nick and exploit the mismatch repair (MMR) system for repair. In many instances, this results in fewer off-target effects and less cytotoxicity. Moreover, because base editors use an alternative repair mechanism, the zygosity of repair is often distinct from that of traditional HDR-based editing. Here, we provide a detailed protocol for performing gene editing using an adenine base editor (ABE) to induce a single A-G transition mutation in a gene of therapeutic relevance. We also illustrate how to sort and isolate cells with differential zygosity state, enabling the production of cell models suited to the study of both dominant and recessive mutations. This workflow can easily be adapted to the use of other variants of base editors (e.g., cytosine base editors) and can be employed in diverse cell lines.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Animals
Humans
Adenine

@article {pmid42681222,
year = {2026},
author = {Powell, S and Martin, SA},
title = {CRISPR-Mediated Gene Editing: Generating Gene Knockouts or Precision Base Changes in F0 Xenopus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3049},
number = {},
pages = {141-170},
pmid = {42681222},
issn = {1940-6029},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *Xenopus/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Microinjections ; Xenopus laevis/genetics ; Female ; },
abstract = {CRISPR-mediated gene editing has transformed Xenopus research by enabling targeted, heritable gene disruption in both Xenopus laevis and Xenopus tropicalis. The CRISPR/Cas9 system has allowed efficient loss-of-function analysis within days of injection, overcoming the limitations of transient morpholino knockdowns. Recent advances in CRISPR-mediated base editing technology further expands this toolkit, permitting the precision generation of single-base changes in Xenopus. High editing efficiency, external development, and large clutch size make Xenopus embryos exceptionally suited for genome manipulation and phenotype screening. CRISPR-mediated gene editing in Xenopus has reproduced classic developmental phenotypes and generated robust models of human disease, including ciliopathies, congenital heart defects, skeletal dysplasias, and neurodevelopmental disorders. Disease modeling using Xenopus CRISPR mutants has provided critical insights into conserved vertebrate pathways and the pathogenic mechanisms of human gene variants. Here, we describe the complete process of producing a gene knockout or precision base changes in F0 Xenopus: target selection, design and synthesis of sgRNA, base editor selection and synthesis of base editor mRNA, microinjection into fertilized Xenopus eggs, and genotyping to assess whether gene editing has successfully occurred.},
}

Animals
*Gene Editing/methods
*CRISPR-Cas Systems
*Gene Knockout Techniques/methods
*Xenopus/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Microinjections
Xenopus laevis/genetics
Female

@article {pmid42681238,
year = {2026},
author = {Lun, J and Chesneau, A and Borday, C and Perron, M},
title = {Retinal Degeneration and Regeneration in Xenopus laevis Tadpoles.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3049},
number = {},
pages = {415-431},
pmid = {42681238},
issn = {1940-6029},
mesh = {Animals ; *Retinal Degeneration/genetics/physiopathology/pathology/metabolism ; *Xenopus laevis/genetics/physiology ; Larva/physiology ; *Regeneration ; Nitroreductases/genetics/metabolism ; Metronidazole/pharmacology ; *Retina/physiology ; Rhodopsin/genetics ; Animals, Genetically Modified ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; CRISPR-Cas Systems ; Ependymoglial Cells/metabolism ; Monophenol Monooxygenase/genetics ; Cell Proliferation ; },
abstract = {Xenopus laevis offers unique advantages for studying retinal regeneration due to its strong regenerative capacity and amenability to transgenesis and genome editing. We present a detailed protocol for inducing and monitoring rod photoreceptor degeneration and regeneration in Tg(rho:GFP-NTR) tadpoles, where nitroreductase expression under the rhodopsin promoter enables conditional and specific ablation of rods upon metronidazole exposure. In addition, we describe the generation of albino Tg(rho:GFP-NTR); tyr [ -/-] tadpoles using CRISPR/Cas9-mediated knockout of the tyrosinase gene, providing an alternative to existing albino Tg(rho:GFP-NTR) lines and facilitating real-time fluorescence imaging by reducing pigmentation interference. Finally, we detail methods to assess Müller glial proliferation (BrdU/Sox9 colabeling) and rod neurogenesis (BrdU pulse-chase). This manuscript provides a robust experimental model that enables the investigation of the cellular and molecular mechanisms underlying retinal regeneration in X. laevis.},
}

Animals
*Retinal Degeneration/genetics/physiopathology/pathology/metabolism
*Xenopus laevis/genetics/physiology
Larva/physiology
*Regeneration
Nitroreductases/genetics/metabolism
Metronidazole/pharmacology
*Retina/physiology
Rhodopsin/genetics
Animals, Genetically Modified
Retinal Rod Photoreceptor Cells/metabolism/pathology
CRISPR-Cas Systems
Ependymoglial Cells/metabolism
Monophenol Monooxygenase/genetics
Cell Proliferation

@article {pmid42681252,
year = {2026},
author = {Wan, W and Ning, J and Yan, A},
title = {Alanine Scanning Analysis of MexB in Multidrug-Resistant Pseudomonas aeruginosa Using the Native Type I-F CRISPR-Cas-Mediated Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3044},
number = {},
pages = {185-201},
pmid = {42681252},
issn = {1940-6029},
mesh = {*Pseudomonas aeruginosa/genetics/drug effects ; *Drug Resistance, Multiple, Bacterial/genetics ; *Bacterial Outer Membrane Proteins/genetics/metabolism ; *Gene Editing/methods ; *Membrane Transport Proteins/genetics/metabolism/chemistry ; *CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; },
abstract = {The rising multidrug resistance (MDR) of the Gram-negative opportunistic pathogen Pseudomonas aeruginosa poses a global public health threat. One key resistance mechanism employed by this pathogen is the overexpression of the resistance-nodulation-cell division (RND) multidrug efflux transporters. MexAB-OprM, a prototype RND efflux pump, confers resistance to most conventional antibiotics except aminoglycosides. Molecules capable of inhibiting efflux pumps, i.e., efflux pump inhibitors (EPIs), have shown promise as therapeutic agents capable of restoring antibiotic efficacy against these superbugs by targeting MexB. Nevertheless, the structure-activity relationship governing substrate transport of MexB in the native genetic background of clinical MDR P. aeruginosa isolates is poorly characterized due to the lack of efficient genetic tools, hindering targeted EPIs development.In this chapter, we introduce a native Type I-F CRISPR-mediated precise genome editing technique that enables in situ alanine substitutions in MexB in clinical MDR P. aeruginosa genotypes. We outline detailed procedures to construct MexB alanine-substitution mutations and methods to examine the expression levels and efflux activities of the constructed MexB mutants. Methods to evaluate the effect of constructed alanine substitutions on antibiotic susceptibilities are also described. These methodologies are expected to provide advanced insights into the substrate recognition and selection mechanism of MexB in the native genetic background of clinical MDR P. aeruginosa isolates to facilitate the development of effective EPIs.},
}

*Pseudomonas aeruginosa/genetics/drug effects
*Drug Resistance, Multiple, Bacterial/genetics
*Bacterial Outer Membrane Proteins/genetics/metabolism
*Gene Editing/methods
*Membrane Transport Proteins/genetics/metabolism/chemistry
*CRISPR-Cas Systems
Anti-Bacterial Agents/pharmacology

@article {pmid42681339,
year = {2026},
author = {Dong, X and Weiss, RJ},
title = {Detection and Localization of Type II Membrane Proteins by Endogenous Gene Tagging and Confocal Microscopy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3020},
number = {},
pages = {49-64},
pmid = {42681339},
issn = {1940-6029},
mesh = {Microscopy, Confocal/methods ; Humans ; CRISPR-Cas Systems ; *Membrane Proteins/metabolism/genetics ; Animals ; Protein Transport ; HEK293 Cells ; },
abstract = {Type II membrane proteins are single-pass transmembrane proteins distinguished by their N-terminus facing the cytoplasmic side and C-terminus oriented toward the extracellular or luminal side. The localization of type II membrane proteins is crucial for their functional roles, molecular interactions, and biological activity. Studying their dynamics remains challenging due to limited availability of highly specific antibodies and potential artifacts introduced by overexpression systems. In this context, we outline a detailed protocol for monitoring the endogenous localization of type II membrane proteins in mammalian cells. Here, we combine CRISPR/Cas9-mediated endogenous protein tagging with confocal microscopy. This method provides a powerful tool for precisely labeling endogenous proteins and investigating the localization and dynamics of membrane proteins under physiological conditions.},
}

Microscopy, Confocal/methods
Humans
CRISPR-Cas Systems
*Membrane Proteins/metabolism/genetics
Animals
Protein Transport
HEK293 Cells

@article {pmid42288725,
year = {2026},
author = {Coleman, S and Tye, J and Furniss, D and Sainsbury, R and Rastogi, A and Xi, X and Murnyak, B and Bell, J and Skeate, J and Wang, M and Webber, B and Moriarity, B and Clement, K},
title = {CRISPRessoSea: streamlined analysis and comparison of pooled amplicon CRISPR screens.},
journal = {BMC bioinformatics},
volume = {27},
number = {1},
pages = {},
pmid = {42288725},
issn = {1471-2105},
support = {T15LM007124/NH/NIH HHS/United States ; T32HL007062/NH/NIH HHS/United States ; R21CA237789, R21AI163731, P01CA254849, P50CA136393, U54CA268069, R01AI146009/NH/NIH HHS/United States ; R01AI146009, R01AI161017, P01CA254849, P50CA136393, U24OD026641, U54CA232561, P30CA077598, U54CA268069/NH/NIH HHS/United States ; R00HG011658/NH/NIH HHS/United States ; T15LM007124/NH/NIH HHS/United States ; T15LM007124/NH/NIH HHS/United States ; T32HL007062/NH/NIH HHS/United States ; R21CA237789, R21AI163731, P01CA254849, P50CA136393, U54CA268069, R01AI146009/NH/NIH HHS/United States ; R01AI146009, R01AI161017, P01CA254849, P50CA136393, U24OD026641, U54CA232561, P30CA077598, U54CA268069/NH/NIH HHS/United States ; R00HG011658/NH/NIH HHS/United States ; },
mesh = {*Software ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {BACKGROUND: CRISPR genome editing enables precise modification of genomic targets but may also induce unintended edits at off-target sites with similar sequences. Pooled amplicon sequencing can assess on- and off-target editing across many samples, yet analyzing, aggregating, and visualizing results from multiple pooled experiments remains challenging. Tools to simplify and standardize these analyses are needed to provide reproducible and comparable interpretation of editing data.

RESULTS: We developed CRISPRessoSea, a software package that processes, compares, and visualizes genome editing rates from pooled amplicon sequencing experiments. The tool provides standardized workflows for analyzing editing across multiple targets and samples, supports both nuclease- and base-editing modalities, and generates clear, data-rich summaries suitable for downstream interpretation.

CONCLUSIONS: CRISPRessoSea facilitates reproducible, scalable analysis of CRISPR editing outcomes across diverse experimental designs, enabling more efficient and transparent assessment of genome editing specificity. The software is freely available at https://github.com/clementlab/CRISPRessoSea .},
}

*Software
*CRISPR-Cas Systems
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
High-Throughput Nucleotide Sequencing/methods

@article {pmid42310064,
year = {2026},
author = {Davoodi, P and Atapour, M and Shahsavari, A and Kiani, R},
title = {Simulation of CRISPR/Cas9-mediated gene editing for the Vitellogenin gene in Apis mellifera.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42310064},
issn = {2045-2322},
support = {ص/3/9/22600, (~1000$ for one year).//University of Kurdistan/ ; },
mesh = {Animals ; *Vitellogenins/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Bees/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Computer Simulation ; Exons ; },
abstract = {CRISPR/Cas9 genome editing provides a powerful framework for interrogating gene function in Apis mellifera. Yet, empirical application remains challenging due to biological constraints, including haplodiploid genetics, narrow embryonic injection window, and the social rearing requirements that complicate functional validation. These constraints necessitate in silico pre-screening to maximize editing success before resource-intensive wet-lab implementation. Within the omnigenic framework, which distinguishes core regulatory genes from peripheral loci buffered by network effects, vitellogenin (Vg) represents an optimal target which is ancestrally dedicated to yolk provisioning; it has been co-opted to orchestrate diverse non-reproductive functions including longevity, stress resistance, immunity, and social behavior. We developed a computational pipeline to design a list of 57 and 56 candidate guide RNAs (gRNA) for targeted Vg knockout, evaluating candidate sites in both functional exons 2 and 3 based on structural accessibility and frameshift efficiency. Comparative analysis revealed complementary strengths in two top-best candidates from initial target pool of predicted gRNAs. The gRNA targeting exon 2 exhibits weaker secondary structure (ΔG = -0.25 kcal/mol versus -2.10 kcal/mol for exon 3), aligning with empirical evidence that sites with ΔG > -1.0 kcal/mol achieve 2-5 × higher Cas9 binding efficiency. This site yielded moderate frameshift frequency (77.8%; 61.9 percentile). Conversely, the predicted editing outcome for the gRNA targeting exon 3, despite stronger structural constraints, demonstrated superior functional disruption metrics demonstrating very high frameshift frequency (88.3%; 95.2 percentile), high in silico editing precision, minimal microhomology-mediated repair bias, and reproducible outcomes wherein nearly all predicted indels disrupt the coding sequence. Protein structure and domain analyses further predict that frameshift edits will generate a truncated protein missing all downstream functional domains. We recommend parallel empirical validation of both exon 2 and exon 3 targets to resolve the trade-off between structural accessibility (favoring higher editing rates) and frameshift efficacy (favoring complete loss-of-function). This dual-target strategy accommodates uncertainty in in vivo performance while maximizing the probability of generating informative phenotypes. Our in silico framework enables rational CRISPR design in non-model organisms by computationally balancing biophysical accessibility with functional impact, accelerating functional genomics in species where empirical optimization faces substantial biological constraints.},
}

Animals
*Vitellogenins/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Bees/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Computer Simulation
Exons

@article {pmid42485833,
year = {2026},
author = {J, MA and K, AP and A, AE},
title = {Cold-chain-free CRISPR diagnostics enabled by magnesium-tannic acid nanoencapsulation of the complete detection system.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {268},
number = {Pt 1},
pages = {115989},
doi = {10.1016/j.colsurfb.2026.115989},
pmid = {42485833},
issn = {1873-4367},
mesh = {*Magnesium/chemistry ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; DNA, Single-Stranded/chemistry/genetics ; *Metal Nanoparticles/chemistry ; Polyphenols ; },
abstract = {Cold-chain dependency remains the major barrier to the widespread deployment of CRISPR-based diagnostics, particularly in resource-limited settings, where the thermal instability of protein and nucleic acid reagents necessitates refrigerated storage and transport from manufacture to point of use. Here we report a one-step aqueous nanoencapsulation strategy that overcomes this limitation using magnesium-tannic acid metal-phenolic nanoparticles (Mg[2][+]-TA MPNs). The platform simultaneously co-encapsulates all functional components of a CRISPR-LbCas12a detection system, including the nuclease, guide RNA, and ssDNA reporter, within a single protective matrix. Magnesium ions serve as a structural coordinator of the metal-phenolic nanoparticles while maintaining biochemical compatibility with the released CRISPR system. Physicochemical characterization confirmed successful particle assembly and efficient cargo incorporation. Functional studies demonstrated that encapsulated CRISPR reagents retained greater than 70% of diagnostic activity after 8 days at 50 °C, whereas non-encapsulated controls were completely inactivated within 24 h under identical conditions. Released formulations also remained fully compatible with both fluorescence- and lateral flow-based detection formats. This approach requires no lyophilization, no specialized equipment, and no cold chain at any stage of production or storage, thereby offering a scalable and readily deployable route to thermally stable molecular diagnostics.},
}

*Magnesium/chemistry
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
DNA, Single-Stranded/chemistry/genetics
*Metal Nanoparticles/chemistry
Polyphenols

@article {pmid42607405,
year = {2026},
author = {Liu, F and Yang, L and Zhang, J and Wang, Y and Xu, F and Ai, Y and Jiang, X},
title = {High-entropy nanozymes integrated with CRISPR/Cas12a cascade amplification for highly sensitive point-of-care detection of cardiac troponin I.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119129},
doi = {10.1016/j.bios.2026.119129},
pmid = {42607405},
issn = {1873-4235},
mesh = {*Troponin I/blood/isolation & purification ; *Biosensing Techniques/methods ; Electrochemical Techniques/methods ; *Myocardial Infarction/diagnosis/blood ; Point-of-Care Systems ; CRISPR-Cas Systems ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Reproducibility of Results ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Myocardial infarction necessitates rapid and ultrasensitive point-of-care detection of cardiac troponin I (cTnI). In this work, we develop a HEAzyme-enabled interfacial electrocatalytic transduction strategy integrated with CHA-CRISPR/Cas12a amplification framework for ultrasensitive electrochemical detection of cTnI. Capitalizing on the cocktail effect and carbon-shell confinement, HEAzyme amplifies the electrochemical response by catalyzing the redox reaction of surface-confined methylene blue. Target recognition initiates CHA, generating abundant DNA activators for Cas12a trans-cleavage. The synergistic integration of molecular cascade amplification and interfacial electrocatalysis enables a limit of detection of 0.21 fg/mL across a broad linear range from 1 fg/mL to 100 pg/mL. It exhibits good selectivity, maintains good stability within 7 days (RSD = 3.9%), and shows good batch-to-batch reproducibility in different batches (RSD = 3.44 and 5.8% for intra and inter-batch, respectively). This work establishes an effective strategy for integrating CRISPR-based molecular amplification with HEAzyme-enabled interfacial electrocatalytic transduction, providing a promising electrochemical platform for point-of-care diagnosis of myocardial infarction.},
}

*Troponin I/blood/isolation & purification
*Biosensing Techniques/methods
Electrochemical Techniques/methods
*Myocardial Infarction/diagnosis/blood
Point-of-Care Systems
CRISPR-Cas Systems
Humans
Limit of Detection
Nucleic Acid Amplification Techniques/methods
Reproducibility of Results
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42612455,
year = {2026},
author = {Ke, J and Zhang, H and Chen, S and Ma, M and Tang, X and Wei, J and Deng, J and Zhai, J and Luan, T},
title = {Engineering bubble structures as Cas12a activators for highly sensitive monitoring of WRN helicase function.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119123},
doi = {10.1016/j.bios.2026.119123},
pmid = {42612455},
issn = {1873-4235},
mesh = {*Werner Syndrome Helicase/metabolism/genetics/chemistry ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; DNA/chemistry/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; *Endodeoxyribonucleases/chemistry/metabolism/genetics ; R-Loop Structures ; RNA/chemistry/genetics ; DNA Replication ; },
abstract = {The Werner syndrome helicase (WRN) is a critical synthetic lethal target in microsatellite instability cancers, essential for resolving complex genomic structures like replication bubbles and R-loops. However, strategies to simultaneously discriminate WRN activity on DNA versus DNA-RNA substrates in living cells are lacking. Here, we developed a structure-specific CRISPR/Cas12a biosensing strategy to visualize WRN functional activity by engineering bubble-structure probes. These probes were rationally designed to structurally mimic DNA replication bubbles and R-loop associated DNA-RNA hybrids. Upon specific unwinding by WRN, the probes release a sequestered activator strand that triggers Cas12a trans-cleavage, effectively converting the unwinding event into an amplified fluorescent signal. This assay achieves low picomolar sensitivity (LODs: 5.6-6.0 pM) and exceptional selectivity against homologous RecQ helicases. Uniquely, this strategy enables the parallel quantification of WRN activity on both substrate types, providing insights into distinct WRN-mediated pathways for resolving genomic stress. We further demonstrated the strategy's utility by visualizing endogenous WRN dynamics in living cells and profiling the efficacy of small-molecule inhibitors. This work offers a powerful molecular toolkit for dissecting WRN biology and facilitating high-throughput drug screening in targeted cancer therapy.},
}

*Werner Syndrome Helicase/metabolism/genetics/chemistry
Humans
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
CRISPR-Cas Systems
DNA/chemistry/genetics
*Bacterial Proteins/chemistry/metabolism/genetics
*Endodeoxyribonucleases/chemistry/metabolism/genetics
R-Loop Structures
RNA/chemistry/genetics
DNA Replication

@article {pmid42641278,
year = {2026},
author = {Fu, R and Zhu, C and Hou, J and Wang, Z and Xianyu, Y},
title = {Click-chemistry-mediated modulation of CRISPR-Cas12a activity through activator modification.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119144},
doi = {10.1016/j.bios.2026.119144},
pmid = {42641278},
issn = {1873-4235},
mesh = {*Click Chemistry/methods ; *CRISPR-Cas Systems/genetics ; Azides/chemistry ; DNA/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Biosensing Techniques ; Cycloaddition Reaction ; Alkynes/chemistry ; Molecular Docking Simulation ; Cyclooctanes/chemistry ; *Bacterial Proteins/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; },
abstract = {Chemical modification strategies offer a promising route for spatiotemporal regulation of CRISPR-Cas12a activity in molecular diagnostics. However, existing methods involve CRISPR RNA with photolabile groups that suffer from complexity and RNA instability. To address these limitations, we report a simple and robust strategy using dibenzocyclooctyne (DBCO)-mediated click chemistry to modulate CRISPR-Cas12a activity. The copper-free strain-promoted azide-alkyne cycloaddition reaction enables CRISPR-Cas12a modulation with low toxicity, biocompatibility, and high selectivity. Utilizing azide-modified non-target DNA strand sequences at different locations to react with DBCO, we show that DBCO-modified activators can regulate CRISPR-Cas12a cleavage in three distinct states: maintain, enhance, and suppress. Mechanistic studies through cleavage kinetics and molecular docking reveal that the regulatory outcome depends on the modification position, protospacer adjacent motif composition, and DBCO concentration. We further employ asymmetric polymerase chain reaction to generate azide-modified DNA for click-chemistry-mediated modulation of CRISPR-Cas12a. This strategy could be a promising tool for regulating CRISPR-Cas12a activity in molecular diagnostics.},
}

*Click Chemistry/methods
*CRISPR-Cas Systems/genetics
Azides/chemistry
DNA/chemistry/genetics
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
*Biosensing Techniques
Cycloaddition Reaction
Alkynes/chemistry
Molecular Docking Simulation
Cyclooctanes/chemistry
*Bacterial Proteins/chemistry/genetics
*Endodeoxyribonucleases/chemistry/genetics

@article {pmid42641282,
year = {2026},
author = {Wang, Y and Xu, B and Zeng, Z and Wang, H and Shi, L and Liu, W and Chen, Y and Deng, X and Chen, J and Chen, JX},
title = {Cascade-coupled colorimetric-fluorescent dual-signal detection of EGFR-positive extracellular vesicles via bifunctional MOF@Pt nanozyme integrated with a CHA-CRISPR system.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119149},
doi = {10.1016/j.bios.2026.119149},
pmid = {42641282},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/methods ; ErbB Receptors/genetics ; Colorimetry/methods ; *Extracellular Vesicles/chemistry ; Platinum/chemistry ; *Glioma/diagnosis/genetics/blood ; CRISPR-Cas Systems/genetics ; MicroRNAs/genetics ; Metal-Organic Frameworks/chemistry ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Metal Nanoparticles/chemistry ; Phthalic Acids ; },
abstract = {Extracellular vesicle (EV)-based liquid biopsy holds great promise for glioma diagnosis, but its clinical translation remains hindered by inefficient isolation of disease-relevant EV subpopulations and insufficiently integrated signal validation. Herein, we develop a platform that integrates a bifunctional nanozyme system (phosphatase-like MOF and peroxidase-like Pt) with a CHA-CRISPR/Cas12a cascade, achieving selective isolation and ultrasensitive detection of EGFR-positive glioma-derived EVs. Defective UiO-66-NH2 loaded with Pt nanoparticles and functionalized with EGFR aptamers (UiO@Pt@Apt) is immobilized on Hook strand-modified glass 96-well plates via Apt-Hook hybridization. Upon introduction of EGFR-positive EVs, they specifically bind to aptamers on UiO@Pt@Apt and induce the release of UiO@Pt@Apt-EV complexes through perturbation and destabilization of the Apt-Hook interface. After EV lysis, EV-derived miRNA-21 activates the CHA-CRISPR/Cas12a cascade to generate a fluorescent signal, while the nucleotide fragments produced by Cas12a trans-cleavage are proposed to be hydrolyzed by phosphatase-like defective UiO-66-NH2 to generate PO4[3-]. The PO4[3-] may contribute to Pt-mediated TMB oxidation, thereby supporting colorimetric signal amplification. The platform achieved a colorimetric EV detection limit of 427 particles/μL after coupling with the CHA-CRISPR system, representing a 6.9-fold improvement over the UiO@Pt@Apt system alone, and enabled a miRNA-21 detection limit of 87.0 fM. In plasma samples from 25 glioma patients and 20 healthy donors, the combined readout achieved an AUC of 0.968, showing numerically better discriminatory performance than either single readout, although the improvement was not statistically significant by DeLong analysis. This work provides a promising strategy for EV-based glioma liquid biopsy and offers a potentially adaptable framework for cascade-coupled dual-signal biosensing.},
}

Humans
*Biosensing Techniques/methods
ErbB Receptors/genetics
Colorimetry/methods
*Extracellular Vesicles/chemistry
Platinum/chemistry
*Glioma/diagnosis/genetics/blood
CRISPR-Cas Systems/genetics
MicroRNAs/genetics
Metal-Organic Frameworks/chemistry
Aptamers, Nucleotide/chemistry
Limit of Detection
Metal Nanoparticles/chemistry
Phthalic Acids

@article {pmid42648078,
year = {2026},
author = {Ye, T and Xue, M and Zhou, B and Kang, S and Chen, Z and Tang, Z and Yang, S and Zhao, Q and Yuan, M and Yu, J and Cao, H and Hao, L and Wu, X and Yin, F and Xu, F},
title = {Biphasic spatiotemporal regulation of Cas12a substrate cleavage enables one-pot autocatalytic CRISPR biosensing of non-nucleic-acid targets.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119152},
doi = {10.1016/j.bios.2026.119152},
pmid = {42648078},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Associated Proteins/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; DNA, Single-Stranded/chemistry/genetics ; *Bacterial Proteins/chemistry/genetics ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; Catalysis ; },
abstract = {One-pot autocatalytic CRISPR biosensing offers a promising route for signal amplification without nucleic acid pre-amplification, but its efficiency is limited by an intrinsic readout-amplification conflict: the ssDNA reporter required for signal output competes with the autocatalytic mediator required for feedback amplification. Here, we report a biphasic spatiotemporal regulation strategy (BS-Cas12a system) to resolve this substrate competition for one-pot autocatalytic CRISPR biosensing of non-nucleic-acid targets. Mechanistic studies revealed that Cas12a preferentially cleaved the ssDNA reporter over the autocatalytic circular mediator, leading to insufficient mediator linearization and impaired autocatalytic amplification. A glycerol/water biphasic system was therefore constructed to spatially delay reporter access to activated Cas12a, allowing preferential cleavage of the autocatalytic mediator and subsequent generation of additional Cas12a activators. This biphasic system achieved a 3-fold increase in the autocatalytic amplification efficiency. By integrating an aptamer-mediated target-to-activator conversion module, non-nucleic-acid recognition was programmably converted into Cas12a activation. Using sulfadimethoxine as a model target, the platform achieved a detection limit of 65 pM and showed good recoveries in fish samples. The system was further extended to Cd[2+] and thrombin detection with limits of detection of 0.65 nM and 11.63 pM, respectively. Moreover, by replacing the fluorescent reporter with a FAM/biotin-labeled reporter, a lateral-flow readout was achieved. This work provides a kinetic-regulation strategy for modular one-pot Cir DNA autocatalytic biosensing with an independent and exchangeable reporter and expands CRISPR-based detection toward diverse non-nucleic-acid targets.},
}

*Biosensing Techniques/methods
*CRISPR-Associated Proteins/chemistry/genetics
*CRISPR-Cas Systems/genetics
*Endodeoxyribonucleases/chemistry/genetics
DNA, Single-Stranded/chemistry/genetics
*Bacterial Proteins/chemistry/genetics
Limit of Detection
Aptamers, Nucleotide/chemistry
Catalysis

@article {pmid42669262,
year = {2026},
author = {Han, J and Song, Y and Ko, U and Son, SU and Lim, EK and Kim, E},
title = {One-pot dual-toehold RCA-Cas12a biosensor driven by a preassembled three-arm toehold-gated DNA template for sequence-selective miRNA liquid biopsy.},
journal = {Biosensors & bioelectronics},
volume = {313},
number = {},
pages = {119170},
doi = {10.1016/j.bios.2026.119170},
pmid = {42669262},
issn = {1873-4235},
mesh = {*MicroRNAs/blood/isolation & purification/genetics ; *Biosensing Techniques/methods ; Humans ; Nucleic Acid Amplification Techniques/methods ; Liquid Biopsy/methods ; CRISPR-Cas Systems/genetics ; DNA/chemistry/genetics ; Breast Neoplasms/blood/genetics/diagnosis ; Limit of Detection ; Female ; *Endodeoxyribonucleases/chemistry/genetics ; *Bacterial Proteins/chemistry/genetics ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) in blood are promising liquid biopsy biomarkers, yet their short length, low abundance, and high intra-family homology hinder sensitive and specific detection. Combining rolling circle amplification (RCA) with CRISPR-Cas12a enables isothermal detection, but existing methods typically depend on auxiliary enzymes or in-assay ligation and rarely encode sequence discrimination within the template itself. Here, we report a one-pot dual-toehold RCA (dtRCA)-Cas12a biosensor driven by a preassembled three-arm toehold-gated (3TG) DNA template for ultrasensitive and selective miRNA detection. The 3TG template adopts a three-arm dumbbell conformation, eliminating the need for a ligase during the assay, and presents two target-complementary toehold domains with a Cas12a-recognition sequence. Target binding triggers strand displacement, initiating dtRCA via a single polymerase. The resulting amplicons activate Cas12a trans-cleavage for fluorescence or lateral flow assay (LFA) readouts. Crucially, a single-base mismatch within the toehold suppressed amplification, whereas a topology-matched circular template lacking the toehold gate failed to distinguish the target, demonstrating that selectivity arises from the template structure. The one-pot dtRCA-Cas12a system achieved attomolar sensitivity, detecting miR-21, miR-375, and let-7a at 2.5, 114.9, and 8.0 aM, respectively. The paper-based LFA maintained femtomolar sensitivity and enabled an instrument-light readout. In plasma, this platform discriminated breast cancer patients (n = 17) from healthy donors (n = 10) with AUC values of 0.97-0.98. Three-marker classification demonstrated robust performance in leave-one-out cross-validation and correctly classified 30 samples in an independent validation cohort, showing performance comparable to RT-qPCR. By embedding selectivity into a preassembled template, this 3TG-driven dtRCA-Cas12a platform provides a highly sensitive and specific strategy for multi-marker miRNA analysis with simplified readout.},
}

*MicroRNAs/blood/isolation & purification/genetics
*Biosensing Techniques/methods
Humans
Nucleic Acid Amplification Techniques/methods
Liquid Biopsy/methods
CRISPR-Cas Systems/genetics
DNA/chemistry/genetics
Breast Neoplasms/blood/genetics/diagnosis
Limit of Detection
Female
*Endodeoxyribonucleases/chemistry/genetics
*Bacterial Proteins/chemistry/genetics
CRISPR-Associated Proteins

@article {pmid42680688,
year = {2026},
author = {Xhaferri, N and Biswas, S and Davies, B and Lindner, M},
title = {Kcnv2 E151X Mouse Captures Hallmarks of KCNV2-Associated Retinal Dystrophy.},
journal = {Clinical & experimental ophthalmology},
volume = {},
number = {},
pages = {},
doi = {10.1111/ceo.70163},
pmid = {42680688},
issn = {1442-9071},
support = {LI 2846/5-1//Deutsche Forschungsgemeinschaft/ ; LI 2846/6-1//Deutsche Forschungsgemeinschaft/ ; },
abstract = {BACKGROUND: KCNV2-associated retinopathy is a rare inherited retinal dystrophy caused by variants in the KCNV2 gene, leading to disrupted photoreceptor behaviour and progressive deterioration of vision. Patients have characteristic electroretinography abnormalities, including reduced cone response, delayed and reduced rod response to low light flashes and paradoxically large rod-driven response to bright flashes of light. To model this condition, we have generated a Kcnv2 E151X mouse line and assessed its structural and functional retinal features.

METHODS: We have employed CRISPR/Cas 9 gene editing technology to generate a mouse line with an early stop mutation in position E151-orthologous to the commonly encountered E143X mutation in humans-and performed a combination of immunohistochemistry and Western blot to confirm the absence of the full-length KCNV2-encoded protein, Kv8.2. Next, to assess how closely it models the human disease, we have characterised the KCNV2 mutant mouse line at histological and functional levels, via immunohistochemistry and electroretinography experiments, respectively.

RESULTS: Kcnv2 mutant mice showed markedly reduced photopic responses and reproduced the supernormal rod phenotype described in affected individuals. In the morphological context, mutant retinas demonstrated strong glial fibrillary acidic protein upregulation together with reduced cone arrestin positive cell counts and photoreceptor layers, indicating photoreceptor loss.

CONCLUSIONS: The Kcnv2 mutant mouse line replicates key functional and structural hallmarks of KCNV2-associated retinopathy. This model provides a relevant platform for mechanistic studies and preclinical evaluation of gene-based or pharmacological therapies targeting cone and rod photoreceptor dysfunction.},
}

@article {pmid42680732,
year = {2026},
author = {Galal, S and Chaltel Lima, L and Wang, N and Moury, C and Yan, G and Dai, M and Ali, S and Lebrun, JJ},
title = {In vivo CRISPR screening identifies metastasis suppressors in triple-negative breast cancer.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42680732},
issn = {2041-1723},
mesh = {Humans ; Animals ; Female ; *Triple Negative Breast Neoplasms/genetics/pathology ; Cell Line, Tumor ; Mice ; Neoplasm Metastasis/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition/genetics ; Cell Movement/genetics ; Neovascularization, Pathologic/genetics ; *Genes, Tumor Suppressor ; Neoplasm Invasiveness/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Metastatic cancer remains the leading cause of cancer-related mortality, yet tumor cell-intrinsic mechanisms restraining metastatic dissemination remain incompletely defined. Here, we perform an unbiased in vivo genome-wide CRISPR/Cas9 loss-of-function screen in a breast cancer xenograft model to identify regulators of metastatic progression. This approach uncovers clinically relevant metastasis suppressor genes (MSGs), including VPS45, CMTR2, RBSN, and NF2, whose loss enhances lung colonization. Functional validation demonstrates that depletion of these genes promotes epithelial-to-mesenchymal transition, migration, invasion, intravasation, and angiogenesis, whereas CRISPR-mediated activation suppresses metastatic spread. Integration with patient datasets reveals reduced expression in tumors and associations with advanced disease, with higher expression trending toward improved outcomes. Notably, CMTR2 loss induces vascular remodeling and intratumoral heterogeneity, supporting a role in tumor-vascular interactions. Collectively, this study identifies a network of MSGs that constrain tumor dissemination and highlights the power of in vivo CRISPR functional genomics to uncover regulators of metastatic disease.},
}

Humans
Animals
Female
*Triple Negative Breast Neoplasms/genetics/pathology
Cell Line, Tumor
Mice
Neoplasm Metastasis/genetics
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic
Epithelial-Mesenchymal Transition/genetics
Cell Movement/genetics
Neovascularization, Pathologic/genetics
*Genes, Tumor Suppressor
Neoplasm Invasiveness/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42681040,
year = {2026},
author = {Tiefenbacher, J and Sandrock, B},
title = {Generation of Gene Knock-Out Mutants in Ustilago maydis Using Cas9hf Nuclease.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3050},
number = {},
pages = {147-161},
pmid = {42681040},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Ustilago/genetics ; *Gene Knockout Techniques/methods ; Mutation ; Basidiomycota ; },
abstract = {Gene disruption of nonessential genes became more convenient with the adaptation of the CRISPR-Cas9 system for Ustilago maydis by the group of Regine Kahmann (MPI Marburg, Germany) in 2016. In our group, we have developed the system further to create defined marker-free gene deletions. Therefore, we used the CRISPR-Cas9 system together with oligonucleotides composed of 40 nucleotides of upstream and downstream flanking regions. Here, we describe the entire way from the decision, which gene of interest should be deleted, to a marker-free U. maydis mutant strain.},
}

*CRISPR-Cas Systems
*Ustilago/genetics
*Gene Knockout Techniques/methods
Mutation
Basidiomycota

@article {pmid42681148,
year = {2026},
author = {Bao, X and Bornert, O and Nyström, A},
title = {Targeting, Mutagenesis, and Functional Testing Strategies for Large Collagens.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3022},
number = {},
pages = {3-20},
pmid = {42681148},
issn = {1940-6029},
mesh = {Humans ; *Mutagenesis ; *Collagen Type VII/genetics/metabolism/chemistry ; CRISPR-Cas Systems ; Animals ; *Protein Engineering/methods ; *Collagen/genetics/metabolism ; *Gene Targeting/methods ; },
abstract = {Collagens comprise a homogeneous family of cell-surface or extracellular proteins. Many of them are fundamental for health, and consequently, their deficiency or dysregulation occurs in a wide range of diseases, from tissue fragility to neoplastic and fibrosing diseases, to name a few. With the advent of new omics approaches, an increasing number of variants of specific collagens have been discovered in various conditions. Some of these variants lead to a loss of expression, highlighting the need to assess the functions and loss of specific collagens in such conditions, while other variants may involve changes in amino acids of unknown consequences. Thus, with the advent of newer omics technologies, there is an increasing need to engineer specific collagens and their encoding genes. However, for larger collagens, this can be challenging because of their size and repetitive structure. Using collagen VII as an example of a large collagen, we will describe strategies for protein engineering, functional analysis, and collagen gene targeting in cells that naturally express collagens.},
}

Humans
*Mutagenesis
*Collagen Type VII/genetics/metabolism/chemistry
CRISPR-Cas Systems
Animals
*Protein Engineering/methods
*Collagen/genetics/metabolism
*Gene Targeting/methods

@article {pmid42681174,
year = {2026},
author = {Su-Tobon, Q and Niu, J},
title = {CRISPR-Hybrid: Intracellular Selection of CRISPR-Associated Aptamers in Bacteria.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {25-45},
pmid = {42681174},
issn = {1940-6029},
mesh = {*Aptamers, Nucleotide/genetics ; *CRISPR-Cas Systems ; Flow Cytometry ; *SELEX Aptamer Technique/methods ; Gene Library ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics ; },
abstract = {CRISPR technologies have evolved from nuclease-based genome editing to programmable systems for transcriptional and epigenetic regulations. Emerging CRISPR systems expand editing versatility by incorporating CRISPR-associated aptamers (CAPs) into single-guide RNAs (sgRNAs), enabling recruitment of RNA-binding proteins (RBPs) fused to diverse effectors. However, the limited availability of orthogonal aptamer-RBP pairs has hindered broad application, as conventional SELEX-based aptamer discovery is time-intensive and often fails to yield aptamers functional in cells. We developed the CRISPR-Hybrid platform, an intracellular selection method that directly evolves CAPs within bacterial cells. This system links aptamer-RBP interactions to a fluorescent reporter readout, allowing fluorescence-activated cell sorting (FACS) to enrich functional variants from libraries exceeding 10[[7]] sequences. Each selection round can be completed in 2 days, enabling rapid enrichment of aptamers that retain activity in both bacterial and mammalian contexts. This protocol details construction of randomized DNA libraries, preparation of host cells, execution of intracellular selection and FACS enrichment, and recovery of aptamer sequences for downstream analysis. By providing a fast, in-cell, and physiologically relevant approach to aptamer discovery, CRISPR-Hybrid expands the repertoire of CAPs available for modular and multiplexed CRISPR editing.},
}

*Aptamers, Nucleotide/genetics
*CRISPR-Cas Systems
Flow Cytometry
*SELEX Aptamer Technique/methods
Gene Library
RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*Bacteria/genetics

@article {pmid42681177,
year = {2026},
author = {Li, M and Chen, C and Tao, P},
title = {CRISPR-Engineered Bacteriophage T4 for Foot-and-Mouth Disease Nanoparticle Vaccine Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {87-100},
pmid = {42681177},
issn = {1940-6029},
mesh = {*Bacteriophage T4/genetics/immunology ; *Foot-and-Mouth Disease Virus/immunology/genetics ; Animals ; *Foot-and-Mouth Disease/prevention & control/immunology/virology ; *Vaccine Development/methods ; Epitopes, T-Lymphocyte/immunology/genetics ; *CRISPR-Cas Systems ; Epitopes, B-Lymphocyte/immunology/genetics ; *Viral Vaccines/immunology/genetics ; Nanovaccines ; Nanoparticles/chemistry ; Capsid Proteins/genetics/immunology ; Protein Subunit Vaccines ; },
abstract = {Peptide-based vaccines offer a safer alternative to inactivated vaccines. However, the immunogenicity of the peptides is usually poor, and therefore, adjuvants or delivery systems are required. Bacteriophage T4, with its intrinsic immunostimulatory properties, provides a promising platform for antigen display. Here, we used CRISPR-Cas genome editing to insert a gene encoding a foot-and-mouth disease virus (FMDV) B-cell epitope (VP1130-158) into the C-terminus of the soc gene in the T4 genome. The T4 phage self-assembly system enables the display of epitopes on the capsid surface in vivo, generating VP1130-158-T4 virus-like particles. To further enhance immune activation, CD4[[+]] T-cell epitopes FMDV 3A21-35 or tetanus toxoid P2830-844 were fused downstream of the VP1 epitope. These recombinant T4 phages provide proof-of-concept for the development of safe, epitope-based FMDV vaccines.},
}

*Bacteriophage T4/genetics/immunology
*Foot-and-Mouth Disease Virus/immunology/genetics
Animals
*Foot-and-Mouth Disease/prevention & control/immunology/virology
*Vaccine Development/methods
Epitopes, T-Lymphocyte/immunology/genetics
*CRISPR-Cas Systems
Epitopes, B-Lymphocyte/immunology/genetics
*Viral Vaccines/immunology/genetics
Nanovaccines
Nanoparticles/chemistry
Capsid Proteins/genetics/immunology
Protein Subunit Vaccines

@article {pmid42681180,
year = {2026},
author = {Wang, Y and Li, X and Huang, Y and Zhong, M and Yang, H},
title = {CRISPR-Cpf1-Mediated T4 Phage Genome Editing for One-Step In Vivo Display of Heterologous Protein.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {135-145},
pmid = {42681180},
issn = {1940-6029},
mesh = {*Bacteriophage T4/genetics ; *Capsid Proteins/genetics/metabolism ; *Gene Editing/methods ; Escherichia coli/genetics ; *Genome, Viral ; *CRISPR-Cas Systems ; Recombinant Proteins/genetics ; },
abstract = {The T4 phage is a robust vector for high-density heterologous protein display. It leverages two non-essential outer capsid proteins, i.e., Soc (~870 copies) and Hoc (~155 copies). These two proteins enable the efficient display of target proteins on the capsid of T4. Here, we detail the workflow for one-step in vivo display of a heterologous protein. Specifically, this method utilizes CRISPR-Cpf1-mediated gene editing technology to insert the sequence of interest (using mCherry as an example) downstream of the Soc encoding gene within the T4 phage genome, resulting in a Soc-fused recombinant protein. This engineering method allows for endogenous expression of the Soc-mCherry recombinant protein within Escherichia coli cells during phage replication, after which the recombinant protein spontaneously assembles onto the capsid of the engineered phage. By providing a universal framework suitable for in vivo display, this approach empowers researchers to readily construct tailored T4 nanoparticles for diverse biotechnological applications.},
}

*Bacteriophage T4/genetics
*Capsid Proteins/genetics/metabolism
*Gene Editing/methods
Escherichia coli/genetics
*Genome, Viral
*CRISPR-Cas Systems
Recombinant Proteins/genetics

@article {pmid42681181,
year = {2026},
author = {Mahdavi-Amiri, Y and Kim, SB},
title = {Methods for Applying Prime Editing and Inverse Prime Editing in Mammalian Cell Culture.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {147-161},
pmid = {42681181},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Animals ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Point Mutation ; Flow Cytometry ; Cell Culture Techniques/methods ; Moloney murine leukemia virus/genetics/enzymology ; High-Throughput Nucleotide Sequencing ; },
abstract = {Prime editing (PE) is a powerful method for introducing point mutations into the genomes of living organisms. PE utilizes a Cas9 nickase fused to an engineered Moloney Murine Leukemia Virus reverse transcriptase (MLV-RT), paired with an extended guide RNA known as pegRNA, which contains a primer binding site (PBS) and a reverse transcriptase template (RTT) complementary to the non-target strand DNA. Recently described inverse prime editing (iPE) also employs reverse transcriptase and pegRNAs; however, it utilizes an RNA template complementary to the target strand, resulting in the polymerization of target-strand DNA in the opposite direction. In this work, we provide a practical protocol for using either PE or iPE to introduce point mutation(s) or small-to-medium sized insertions and deletions in cell culture, and demonstrate how to assess editing efficiency via flow cytometry and next-generation sequencing. We include recommendations for prime editor selection and straightforward guidelines for pegRNA design, intended for researchers unfamiliar with genome editing technologies.},
}

*Gene Editing/methods
Animals
Humans
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Point Mutation
Flow Cytometry
Cell Culture Techniques/methods
Moloney murine leukemia virus/genetics/enzymology
High-Throughput Nucleotide Sequencing

@article {pmid42681182,
year = {2026},
author = {Golla, DA and Daniel, TC and Haugh, L and Gao, X},
title = {Constructing Drive-and-Process (DAP) CRISPR Guide RNA Arrays for Multiplexed Base- and Prime-Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3032},
number = {},
pages = {163-180},
pmid = {42681182},
issn = {1940-6029},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; HEK293 Cells ; *CRISPR-Cas Systems ; RNA, Transfer/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Advancements in base- and prime editing technologies in recent years have offered researchers a plethora of options for introducing precise insertions, deletions, or substitutions into targeted genomic loci. These precision editors' applications have rapidly expanded to the modeling and treatment of polygenic diseases, as well as into the growing field of functional genomics. This has created a need for compact, modular expression systems capable of producing multiple guide RNAs (gRNAs) from a single transcript while preserving high editing efficiency. To address this, we have developed the drive-and-process (DAP) array, a modular architecture composed of alternating gRNA and tRNA units. The DAP array design exploits the cell's endogenous tRNA processing machinery to cleave each tRNA from the array and release individual gRNAs, thereby enabling simultaneous editing at multiple loci following hybridization with Cas9. Here, we outline key design considerations and experimental steps required for the construction and deployment of DAP arrays as multiplex base- or prime editing tools in human cells (e.g., HEK293T). We also highlight how the DAP array can be leveraged to enable efficient processing of gRNAs along with other RNAs of similar size, such as shRNA, potentially broadening its usage in addressing complex biological questions and therapeutic applications that require coordinated genetic perturbation.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
HEK293 Cells
*CRISPR-Cas Systems
RNA, Transfer/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42240879,
year = {2026},
author = {Jeong, H and Kim, H and Cho, E and Lee, HK and Kim, DY and Keum, B and Jung, C},
title = {Enhancing Next-Generation Sequencing Sensitivity with High-Recovery Adapter Ligation and Cas9-Mediated Dimer Elimination.},
journal = {Clinical chemistry},
volume = {72},
number = {9},
pages = {973-983},
doi = {10.1093/clinchem/hvag052},
pmid = {42240879},
issn = {1530-8561},
support = {//Technology Innovation Program/ ; 20009356//Ministry of Trade, Industry & Energy/ ; //National Research Foundation of Korea/ ; 2021-NR061248//Korean government/ ; RS-2024-00440975//Korean government/ ; //Bio & Medical Technology Development Program/ ; //National Research Foundation/ ; RS-2022-NR067272//Ministry of Science & ICT/ ; RS-2023-00259824//Ministry of Science & ICT/ ; },
mesh = {Humans ; *High-Throughput Nucleotide Sequencing/methods ; *CRISPR-Cas Systems ; *Circulating Tumor DNA/genetics/blood ; Sensitivity and Specificity ; Gene Library ; Sequence Analysis, DNA/methods ; },
abstract = {BACKGROUND: Accurate detection of ultra-low-frequency variants is a major challenge in clinical liquid biopsy. In early cancer detection and minimal residual disease monitoring, Circulating tumor (ctDNA) may fall below 0.1% variant allele frequency, making sensitivity highly dependent on molecular recovery during library preparation. Losses at early steps, especially adapter ligation, permanently reduce analyzable molecules and cannot be rescued by deeper sequencing or bioinformatic refinement.

METHODS: We developed Powerful Recovery and Improved Dimer Elimination (PRIDE) next-generation sequencing NGS, a library preparation strategy that increases adapter ligation efficiency and removes adapter dimers via sequence-specific Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) cleanup. PRIDE NGS is compatible with standard clinical work flows and requires no added sequencing depth or changes to downstream bioinformatic pipelines. Performance was assessed by targeted sequencing of cell-free (cfDNA) reference standards and clinical plasma samples.

RESULTS: PRIDE NGS improved recovery and detection of low-frequency variants vs conventional preparation. In reference standards, it detected more variants at low allele frequencies, particularly below 0.1%. In clinical plasma samples, it similarly increased detection, including variants predicted to have moderate or high functional impact. These gains occurred at comparable or lower sequencing depth, indicating sensitivity improvements driven by enhanced molecular recovery.

CONCLUSIONS: By overcoming a key bottleneck in library preparation, PRIDE NGS lowers the practical detection threshold for ultra-low-frequency variants in liquid biopsy. This clinically applicable approach improves analytical sensitivity by lowering the detection limit without increasing the sequencing burden, supporting routine testing and longitudinal monitoring.},
}

Humans
*High-Throughput Nucleotide Sequencing/methods
*CRISPR-Cas Systems
*Circulating Tumor DNA/genetics/blood
Sensitivity and Specificity
Gene Library
Sequence Analysis, DNA/methods

@article {pmid42261770,
year = {2026},
author = {Zhang, L and Yang, C and Yao, Q and Du, X and Ding, S and Shi, Y and Sheng, C and Wang, M and Han, Y and Luo, H},
title = {Machine Learning-Enhanced Ultrasensitive Immuno-CRISPR Array Facilitates Early Diagnosis of Alzheimer's Disease by Detecting Multiple Plasma Biomarkers.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {49},
pages = {e75983},
pmid = {42261770},
issn = {2198-3844},
support = {82401859//National Natural Science Foundation of China/ ; XKTP2025B04//First-class Discipline Breakthrough Initiative of Hainan University/ ; 826MS0122//Hainan Provincial Natural Science Foundation of China/ ; 826QN0570//Hainan Provincial Natural Science Foundation of China/ ; 825QN284//Hainan Provincial Natural Science Foundation of China/ ; },
mesh = {*Alzheimer Disease/diagnosis/blood ; Humans ; *Biomarkers/blood ; *Machine Learning ; Early Diagnosis ; *Amyloid beta-Peptides/blood ; Sensitivity and Specificity ; tau Proteins/blood ; CRISPR-Cas Systems/genetics ; },
abstract = {Early and accurate diagnosis of Alzheimer's disease (AD) remains a significant challenge due to the multifactorial and dynamic nature of its pathology. Although plasma-based biomarkers such as amyloid-β (Aβ) and phosphorylated tau (p-tau) have shown promise as diagnostic indicators, current single-biomarker detection techniques lack the requisite sensitivity and specificity for early-stage diagnosis. Here, we present the development of an ultrasensitive CRISPR-based multi-protein detection array (UCMDA) capable of concurrently detecting six core AD biomarkers, including Aβ40, Aβ42, p-tau[181], p-tau[217], p-tau[231], and p-tau[396,404]. By integrating antibody pair-based multiplex recombinase polymerase amplification (RPA) with spatially encoded CRISPR-Cas12a detection, the UCMDA achieves a detection limit of 1 fg/mL, which is 10 000-fold more sensitive than conventional ELISA. Clinical validation in a cohort of 155 plasma samples demonstrated that logistic regression (LR)-based integration of the six biomarkers significantly enhanced diagnostic performance, with the multi-biomarker model substantially outperforming single-biomarker approaches in diagnosing AD-MCI and AD. This platform offers a scalable, cost-effective, and minimally invasive strategy for early detection and disease monitoring. This work highlights the potential of CRISPR-based multiplex protein detection technologies combined with machine learning-assisted analysis to enhance the precision of diagnosing neurodegenerative disorders.},
}

*Alzheimer Disease/diagnosis/blood
Humans
*Biomarkers/blood
*Machine Learning
Early Diagnosis
*Amyloid beta-Peptides/blood
Sensitivity and Specificity
tau Proteins/blood
CRISPR-Cas Systems/genetics

@article {pmid42295796,
year = {2026},
author = {Miller, MA and Vo, N and Utkarsh, and Sokirniy, I and Pritchard, J and Freedman, BS and Medina, SH},
title = {Ultrasound-Actuated Gene Editing in Human Kidney Organoids.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {49},
pages = {e20402},
pmid = {42295796},
issn = {2198-3844},
support = {R21DK128638/GF/NIH HHS/United States ; R35GM142902/GF/NIH HHS/United States ; U01DK127553/GF/NIH HHS/United States ; U01AI176460/GF/NIH HHS/United States ; R21DK128638/DK/NIDDK NIH HHS/United States ; R35GM142902/GM/NIGMS NIH HHS/United States ; U01DK127553/DK/NIDDK NIH HHS/United States ; U01AI176460/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Organoids/metabolism ; *Kidney/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics ; },
abstract = {Efficient delivery of gene editing ribonucleoproteins (RNPs) into the interior of solid tissues remains a key hurdle to the clinical translation of non-viral CRISPR-Cas9 technologies. Here, we report acoustically-actuated peptide nanoemulsions (NPeps) that can be spatiotemporally guided and activated by ultrasound to ballistically deliver RNPs into cells within the bulk of dense 3D cellular structures. Using human kidney organoids as a model, we demonstrate NPep vectors improve the spatial profile of gene editing in the organoid mass relative to commercial lipofection reagents, without disruption of tissue structure or qualitative viability features. This technologic paradigm is poised to advance imaging-guided, deep tissue RNP delivery modalities to expand the clinical diagnostic and therapeutic potential of CRISPR-Cas9 editing strategies.},
}

Humans
*Organoids/metabolism
*Kidney/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics

@article {pmid42360136,
year = {2026},
author = {Ma, L and Yao, P and Wu, S and Shi, Y and Qin, L and Li, B and Zhu, J and Huang, M and Zhu, Y and Song, Y and Pang, J and Guo, Z and Wu, G and Wang, C and Xu, K and Huang, R and Kuang, Q and Qu, L and Pan, C and Xie, X and Zhu, Q and Huang, J and Lin, Q},
title = {Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {49},
pages = {e75888},
pmid = {42360136},
issn = {2198-3844},
support = {2024YFC3408200//National Key R&D Program of China/ ; 2024YFF1000800//National Key R&D Program of China/ ; 2023ZD04074//STI 2030-Major Projects/ ; 20253BAC260005//Frontier Technology Program of Jiangxi Provincial Natural Science Foundation/ ; 2023-NJS-00-012//Invigorate the Seed Industry of Guangdong Province/ ; 32422050//National Natural Science Foundation of China/ ; 32401250//National Natural Science Foundation of China/ ; 2023ZT10N019//Young Elite Scientists Sponsorship Program of the China Association for Science and Technology, the Guangdong Provincial "Pearl River Talent Program" Innovation and Entrepreneurship Team Project/ ; AB24153006//Key R&D Program of Guangxi Province/ ; 2025A04J7124//Science and Technology Projects in Guangzhou/ ; 2025A04J3669//Science and Technology Projects in Guangzhou/ ; 2023B10564004//specific university discipline construction project/ ; },
mesh = {Animals ; *Gene Editing/methods ; Rats ; *RNA-Directed DNA Polymerase/genetics/metabolism ; *Endogenous Retroviruses/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {CRISPR-based prime editors (PEs) install precise edits into genomic DNA without generating double-strand breaks. Their editing efficiency is highly dependent on reverse transcriptases (RTs), but efficient RT candidates remain limited. Here, we identified 19 novel active RTs by screening 558 candidates. Among them, RERV-RT, derived from Rattus norvegicus, exhibited the highest activity. Through structure-guided engineering and deep mutational scanning, we developed an optimized variant, enRERV-RT, which outperforms conventional M-MLV-RT-based PE systems by 1.20-fold in mammalian and plant cells, and by 1.88-fold at hard-to-edit loci, while enabling precise multiplex editing of functionally relevant genes. Additionally, we developed a high-throughput platform, TRAP-seq-PE, to systematically evaluate prime editor performance. Across diverse mutation types, we found that PE systems based on enRERV-RT exhibited higher editing efficiencies than those based on M-MLV-RT. Collectively, our work establishes a versatile, high-efficiency PE system, thereby facilitating advances in clinical gene therapy and precise crop breeding.},
}

Animals
*Gene Editing/methods
Rats
*RNA-Directed DNA Polymerase/genetics/metabolism
*Endogenous Retroviruses/genetics/enzymology
*CRISPR-Cas Systems/genetics
Humans

@article {pmid42470685,
year = {2026},
author = {Hou, C and Yang, R and Guan, X and Zhang, J and Guo, C and Zhang, S and Pei, M and Schalper, KT and Schreiber, D and Liu, X and Liu, C},
title = {Wax-in-a-Tube: A Simple, Rapid, One-Pot Platform for Molecular Detection.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {6673-6681},
doi = {10.1021/acssensors.5c04476},
pmid = {42470685},
issn = {2379-3694},
support = {U01CA269147/CA/NCI NIH HHS/United States ; R01AI194917//National Institute of Allergy and Infectious Diseases/ ; R01EB023607/EB/NIBIB NIH HHS/United States ; },
mesh = {*DNA, Viral/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *Herpesvirus 2, Human/genetics/isolation & purification ; Humans ; Rapid Diagnostic Tests ; Recombinases/metabolism ; CRISPR-Cas Systems ; },
abstract = {CRISPR technology has emerged as a powerful platform for highly sensitive and specific nucleic acid detection, particularly when coupled with isothermal amplification. However, conventional two-step CRISPR assays still require manual operations, such as shaking, centrifugation, or vortexing, that complicate the workflow and increase the risk of aerosol contamination. Here, we present a wax-in-a-tube (WIAT) platform that enables simple, rapid, one-pot recombinase polymerase amplification (RPA) and CRISPR-based detection by leveraging the phase-change properties of a molded wax separator, thereby eliminating additional manual steps and minimizing contamination risks. Using a molding approach, we directly integrated the wax separator into the reaction tube to physically partition different reaction components. The WIAT platform achieved a detection sensitivity of 10 aM for HSV-2 DNA, comparable to that of standard two-step assays. We further validated its clinical performance using HSV-2 swab samples, demonstrating results comparable to those obtained with PCR. Together, these findings establish the WIAT platform as a simple, rapid, and highly sensitive one-pot RPA-CRISPR assay with strong potential for point-of-care infectious disease detection and early surveillance.},
}

*DNA, Viral/analysis/genetics
*Nucleic Acid Amplification Techniques/methods/instrumentation
*Herpesvirus 2, Human/genetics/isolation & purification
Humans
Rapid Diagnostic Tests
Recombinases/metabolism
CRISPR-Cas Systems

@article {pmid42486986,
year = {2026},
author = {Gao, Y and Ma, Y and Yu, K and Liu, Y and Gu, B and Tang, H and Yan, W and Yang, S and Su, J and Wang, X and Ma, X and Wang, X and Wang, F and Li, Q and Liu, M and Wang, H},
title = {Efficient and precise programmable DNA knock-in without double-strand breaks.},
journal = {Nature},
volume = {657},
number = {8130},
pages = {284-294},
pmid = {42486986},
issn = {1476-4687},
mesh = {*Gene Knock-In Techniques/methods ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Gene Editing/methods ; *DNA/genetics ; Deoxyribonuclease I/metabolism ; Receptors, Chimeric Antigen/genetics ; Mice ; Receptors, Antigen, T-Cell/genetics ; INDEL Mutation/genetics ; },
abstract = {Programmable gene knock-in holds substantial promise for treating genetic diseases and advancing cell therapies. However, achieving precise and efficient kilobase-scale DNA fragment integration remains challenging[1,2]. Here we report CRISPR kilobase-scale nickase-targeting (KNIT) editing for efficient, precise and programmable kilobase-scale DNA insertion without double-strand DNA cleavage, which is enabled through the coupling of a Cas9 nickase with a DNA donor recruiting system. KNIT editing facilitates programmable integration of DNA fragments from 0.7 kb to more than 10 kb and is effective across genomic loci and cell types. It achieves up to 89% efficiency and markedly reduces unintended insertion-deletion mutation (indels) rates, translocations and off-target editing. The system supports repeated insertion editing and multiloci gene knock-in with minimal translocations. Its enhanced version, KNIT editor 2, further improves efficiency via a single transfection. Moreover, in mutant cells with a pathological mutation, KNIT editing restores normal gene expression by inserting a therapeutic gene into a safe harbour locus or its native locus. Notably, KNIT editing enables non-viral and programmable chimeric antigen receptor T cell (CAR-T cell) engineering without double-strand breaks and with clinically relevant efficiencies. Moreover, the engineered CAR-T cells exhibit effective antitumour activity in vitro and in mouse models. Therefore, by achieving programmable and site-specific kilobase-scale DNA insertions without double-strand breaks while reducing unintended outcomes, KNIT editing provides a versatile platform for advancing personalized medicine.},
}

*Gene Knock-In Techniques/methods
*DNA Breaks, Double-Stranded
*CRISPR-Cas Systems/genetics
Humans
Animals
*Gene Editing/methods
*DNA/genetics
Deoxyribonuclease I/metabolism
Receptors, Chimeric Antigen/genetics
Mice
Receptors, Antigen, T-Cell/genetics
INDEL Mutation/genetics

@article {pmid42571623,
year = {2026},
author = {Zhou, M and Du, K and Jiang, M and Xu, X and Su, X and Xie, Y and Zhang, D and Sun, X and Peng, G and Xia, K and Hu, Z},
title = {FOCUS: A Dual-Mismatch crRNA Strategy Unlocks High-Fidelity One-Step SNV Detection with Cas12a.},
journal = {ACS sensors},
volume = {11},
number = {8},
pages = {7335-7348},
doi = {10.1021/acssensors.6c01689},
pmid = {42571623},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Nucleic Acid Amplification Techniques/methods ; Base Pair Mismatch ; DNA/genetics ; Rapid Diagnostic Tests ; },
abstract = {CRISPR/Cas12a has emerged as a powerful tool for nucleic acid detection; however, its clinical utility is severely hampered by intrinsic limitations in single-nucleotide variant (SNV) discrimination, reliance on pre-processed single-stranded DNA (ssDNA) templates, and cumbersome multi-step workflows. Here, we report a novel molecular design principle by engineering crRNAs with site-specific dual mismatches (positions 12 and 14 relative to the PAM), which we systematically demonstrate to drastically enhance the SNV discrimination capability of Cas12a. Leveraging this breakthrough, we developed FOCUS (Fast One-step CRISPR-based Universalizable SNV detection system), an all-in-one CRISPR sensing platform that enables isothermal detection of SNVs from double-stranded DNA (dsDNA) by integrating amplification and detection in a single reaction system. FOCUS achieved attomolar-level sensitivity (13.15 aM) and ultrafast readout (< 20 min) for distinguishing the highly homologous survival motor neuron 1 (SMN1) and SMN2 genes-the gold standard challenge for SNV genotyping in spinal muscular atrophy (SMA) diagnostics. To validate its clinical translatability, FOCUS was successfully adapted to a low-cost, equipment-free assay using lateral flow strips and UV visualization, facilitating point-of-care testing (POCT). In a comprehensive validation across 175 clinical samples, FOCUS exhibited 100% diagnostic concordance with gold-standard methods for SMA (21 samples), high-risk HPV 16/18 (27 samples), Staphylococcus aureus (20 samples), and SARS-CoV-2 (107 samples). Collectively, our study establishes a generalizable engineering strategy for Cas12a crRNAs and presents FOCUS as a robust, versatile, and field-deployable solution for precision SNV genotyping, underscoring the translational medicine value of FOCUS in molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
*Polymorphism, Single Nucleotide/genetics
*CRISPR-Associated Proteins/metabolism/genetics
Humans
*Endodeoxyribonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Nucleic Acid Amplification Techniques/methods
Base Pair Mismatch
DNA/genetics
Rapid Diagnostic Tests

@article {pmid42608059,
year = {2026},
author = {Chen, Z and Zhou, J and Galli, M and Iohannes, SD and Clark, T and Debernardi, JM and Dubcovsky, J and Jackson, D and Gallavotti, A},
title = {The combination of morphogenic regulators BABY BOOM and GRF-GIF improves maize transformation efficiency and promotes leaf regeneration.},
journal = {The New phytologist},
volume = {252},
number = {1},
pages = {260-275},
pmid = {42608059},
issn = {1469-8137},
support = {2424271//Division of Molecular and Cellular Biosciences/ ; 1916804//Division of Integrative Organismal Systems/ ; },
mesh = {*Zea mays/genetics/physiology/embryology ; Plants, Genetically Modified ; *Regeneration/genetics ; *Transformation, Genetic ; *Plant Leaves/physiology ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; *Transcription Factors/metabolism/genetics ; Triticum/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Transformation is an indispensable tool for plant genetics and functional genomics. Although stable transformation in maize is no longer a major obstacle, there remains a need for accessible and efficient methods for academic laboratories. Here, we present the GGB system, a rapid and efficient approach optimized for immature embryo transformation in B104 and other maize lines. This system combines two distinct morphogenetic regulators, the wheat GRF4-GIF1 chimera and the maize BABY BOOM (BBM) transcription factor (hence the name "GGB") with a modified QuickCorn protocol, enabling regeneration of transformed maize plantlets in c. 2 months with an efficiency 7-fold higher than when compared to either morphogenic factor used in isolation. Expression of both regulators did not significantly affect development, eliminating the need to excise them after regeneration. However, transmission of the transgenic GGB construct through pollen was significantly reduced, potentially aiding transgenic line containment. We show that the GGB system is adaptable for CRISPR-Cas9 editing and reporter line generation. Furthermore, stable GGB transformants exhibited high leaf regeneration capacity via somatic embryogenesis. RNA-seq time-course profiling of GGB leaf cultures identified additional factors that could promote regeneration and led to the discovery of asparagine and trehalose as additional media components that significantly enhanced leaf regeneration.},
}

*Zea mays/genetics/physiology/embryology
Plants, Genetically Modified
*Regeneration/genetics
*Transformation, Genetic
*Plant Leaves/physiology
*Plant Proteins/metabolism/genetics
Gene Expression Regulation, Plant
*Transcription Factors/metabolism/genetics
Triticum/genetics
CRISPR-Cas Systems/genetics

@article {pmid42669708,
year = {2026},
author = {Boggess, SC and Gandhi, V and Tsai, MC and Marzette, E and Teyssier, N and Chou, JY and Hu, X and Cramer, A and Yadanar, L and Shroff, K and Jeong, CG and Eidenschenk, C and Hanson, JE and Tian, R and Kampmann, M},
title = {A massively parallel CRISPR-based screening platform for modifiers of neuronal depolarization.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42669708},
issn = {2041-1723},
support = {U54 NS123746/NS/NINDS NIH HHS/United States ; 23AARF-1027616/ALZ/Alzheimer's Association/United States ; EDUC2-12730//California Institute for Regenerative Medicine (CIRM)/ ; U54 NS123746//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {Humans ; *Neurons/metabolism/physiology ; Induced Pluripotent Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Calcium/metabolism ; },
abstract = {Understanding the complex interplay between gene expression and neuronal activity is crucial for unraveling the molecular mechanisms underlying cognitive function and neurological disorders. Here, we developed pooled screens using CRISPR interference (CRISPRi) and the fluorescent calcium integrator CaMPARI2 to evaluate genetic modifiers of neuronal depolarization. Using this screening method, we evaluated 1343 genes for their effect on depolarization in a human iPSC-derived neuron model, revealing potential links to neurodegenerative and neurodevelopmental disorders. These genes include known regulators of neuronal excitability, such as TARPs and ion channels, as well as genes associated with autism spectrum disorder and Alzheimer's disease not previously described to affect neuronal depolarization. This CRISPRi-based screening platform offers a versatile tool to uncover molecular mechanisms controlling neuronal function in health and disease.},
}

Humans
*Neurons/metabolism/physiology
Induced Pluripotent Stem Cells/cytology/metabolism
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Calcium/metabolism

@article {pmid42669834,
year = {2026},
author = {},
title = {Correction to "Accurate Molecular Sensing based on a Modular and Customizable CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON)".},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e6219762},
doi = {10.1002/anie.6219762},
pmid = {42669834},
issn = {1521-3773},
}

@article {pmid42670258,
year = {2026},
author = {Ng, CF and Krishnamurthy, D and Dextre, A and Chorlay, A and Ott, M and Fletcher, DA},
title = {LUCas: Light-Uncaged Cas13a using photocleavable interfering guide RNAs.},
journal = {Nucleic acids research},
volume = {54},
number = {16},
pages = {},
pmid = {42670258},
issn = {1362-4962},
support = {//Schmidt Science Fellowship/ ; //Rhodes Trust/ ; //Burroughs Wellcome Career Award/ ; //European Molecular Biology Organization/ ; DBI-1548297//National Science Foundation/ ; //Wagner Foundation/ ; 4R33AI140465-04//National Institute of Allergy and Infectious Diseases/ ; //James B. Pendleton Charitable Trust/ ; //Gordon and Betty Moore Foundation/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Kinetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; Ultraviolet Rays ; Photolysis ; Light ; },
abstract = {CRISPR diagnostics enable sensitive detection of infectious diseases, with the RNA endonuclease Cas13a providing specific, amplification-free RNA detection through collateral trans-cleavage of fluorescent reporters. However, background cleavage from unbound enzyme, contaminating nucleases, and unsynchronized initiation of reactions limits assay sensitivity and interpretability. A strategy to precisely control the onset of Cas13a catalytic activity, essentially a molecular "starting gun," would address these challenges. Here, we introduce Light-Uncaged Cas13a (LUCas), a light-controllable system that directly blocks Cas13a trans-cleavage activity using a photocleavable interfering guide RNA, even in the presence of target RNA. Brief UV illumination releases this suppression, restoring full activity. Quantitative kinetic analysis reveals an ~100-fold suppression of trans-cleavage activity prior to photo-uncaging, including suppression of target-independent background activity. Using measured kinetic parameters, we predict and experimentally validate the limit of detection of the LUCas system for direct detection. We further demonstrate a multiplexed detection strategy termed "temporal barcoding," enabling quantitative detection of viral co-infections in a single bulk reaction. Finally, LUCas is shown to be compatible with one-pot isothermal amplification for enhanced sensitivity and direct detection of target RNA spiked into blood plasma. Together, these results establish LUCas as a general framework for mechanistically informed, light-based control of Cas13a activity.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
Kinetics
*CRISPR-Associated Proteins/genetics/metabolism
Humans
Ultraviolet Rays
Photolysis
Light

@article {pmid42670666,
year = {2026},
author = {Mirzaee, Z},
title = {Advancing antimicrobial peptides: Mechanisms, design, and applications in the post-antibiotic era.},
journal = {Protein and peptide letters},
volume = {34},
number = {1},
pages = {19-30},
doi = {10.1016/j.ppl.2026.07.002},
pmid = {42670666},
issn = {1875-5305},
mesh = {*Antimicrobial Peptides/chemistry/pharmacology/therapeutic use ; Humans ; *Drug Design ; Protein Engineering ; Animals ; Structure-Activity Relationship ; *Anti-Bacterial Agents/chemistry/pharmacology ; Bacteria/drug effects ; *Antimicrobial Cationic Peptides/chemistry/pharmacology ; },
abstract = {The rapid emergence of multidrug-resistant and extensively drug-resistant bacteria has intensified the need for alternative antimicrobial strategies in the post-antibiotic era. Antimicrobial peptides (AMPs), as evolutionarily conserved components of innate immunity, have attracted considerable attention due to their broad-spectrum antimicrobial activity, rapid mechanisms of action, and lower propensity for resistance development. This review summarizes the structural diversity, mechanisms of action, and structure-activity relationships (SAR) of AMPs that underpin their biological activity and guide the rational design of next-generation peptide therapeutics. It further discusses recent advances in peptide engineering, peptidomimetic design, machine learning-assisted discovery, innovative production platforms, and the application of CRISPR-Cas genome editing for production host optimization. In addition, the review highlights synergistic therapeutic strategies, current clinical progress, and the expanding applications of AMPs in medicine, food preservation, agriculture, and aquaculture. Despite these advances, challenges including limited stability, potential toxicity, manufacturing costs, and regulatory barriers continue to hinder widespread clinical translation of AMP-based therapeutics. By integrating recent experimental and computational advances with current translational challenges and future perspectives, this review provides a comprehensive overview of the field and highlights key directions for the rational development and clinical translation of next-generation antimicrobial peptides to combat antimicrobial resistance.},
}

*Antimicrobial Peptides/chemistry/pharmacology/therapeutic use
Humans
*Drug Design
Protein Engineering
Animals
Structure-Activity Relationship
*Anti-Bacterial Agents/chemistry/pharmacology
Bacteria/drug effects
*Antimicrobial Cationic Peptides/chemistry/pharmacology

@article {pmid42671221,
year = {2026},
author = {Xu, Y and He, X and Xu, T},
title = {LAMP-CRISPR Integrated Platforms for Rapid Detection of Microbial Pathogens: Principles, Applied Strategies, and the Road to Field Translation.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxag220},
pmid = {42671221},
issn = {1365-2672},
abstract = {Loop-mediated isothermal amplification (LAMP) integrated with CRISPR-Cas systems has emerged as a promising molecular diagnostic platform for the rapid detection of microbial pathogens. By combining the efficient nucleic acid amplification of LAMP with the sequence-specific recognition capability of CRISPR-Cas effectors, these platforms offer potential advantages in analytical sensitivity, specificity, operational simplicity, and field applicability. In this review, we summarize the principles, assay formats, and recent advances of LAMP-CRISPR technologies for detecting a broad spectrum of microbial pathogens, including bacterial, viral, fungal, and parasitic agents across clinical, veterinary, food safety, environmental, and agricultural applications. Representative studies are compared with attention to pathogen type, sample matrix, assay design, CRISPR-Cas system, readout format, analytical performance, and practical application. We further discuss major technical challenges that continue to hinder practical implementation, particularly complex sample pretreatment, workflow integration, carry-over contamination, reagent stability, multiplexing capability, and platform standardization. Attention is given to sample pretreatment and system-level integration, including current extraction and rapid lysis strategies, closed-tube reactions, portable readouts, and microfluidic or cartridge-based formats, which may support simplified "sample-in, answer-out" diagnostic workflows. Finally, we outline future directions for improving matrix-adapted sample processing, assay robustness, standardized validation, large-scale evaluation, and field deployment. This review provides a structured overview of current LAMP-CRISPR platforms and highlights key technological considerations for translating rapid microbial pathogen detection from laboratory research to real-world applications.},
}

@article {pmid42673787,
year = {2026},
author = {Lv, L and Zhang, Y and Fan, Y and Guo, B and Chen, Y},
title = {Bivalent aptamer-assisted CRISPR-Cas12a sensor for precise vancomycin therapeutic drug monitoring.},
journal = {Talanta},
volume = {312},
number = {Pt B},
pages = {130504},
doi = {10.1016/j.talanta.2026.130504},
pmid = {42673787},
issn = {1873-3573},
abstract = {Therapeutic drug monitoring (TDM) of vancomycin (VAN) is critical for maximizing efficacy and minimizing toxicity, but conventional methods are constrained by high costs, slow turnaround times, and operational complexity. To address these limitations, we developed a novel Bivalent Aptamer-assisted CRISPR-Cas12a Sensor (termed BACS) for rapid and precise VAN detection. Central to this platform is a high-affinity bivalent aptamer (2AP33), engineered via molecular docking-guided truncation and rational linker design, which exhibits significantly enhanced binding avidity compared to its monovalent counterpart. This aptamer was integrated into a CRISPR-Cas12a system based on a competitive binding mechanism, where target binding modulates Cas12a trans-cleavage activity. The optimized BACS achieved a wide linear detection range (1-50 μM) with a low limit of detection (0.64 μM) in clinical serum, fully covering the clinical therapeutic window. Notably, the assay is rapid (within 10 min), cost-effective, and simple. Critically, the clinical practicality and reliability of BACS were rigorously validated with 175 clinical serum samples, showing exceptional concordance with both the gold standard method and a classical method. This work not only provides a reliable tool for VAN TDM but also offers an adaptable strategy for developing high-performance CRISPR-powered biosensors for diverse clinical analytes through a streamlined molecular engineering pipeline.},
}

@article {pmid42674013,
year = {2026},
author = {Liu, H and Liu, Y and Xu, Y and Wang, Z and Yu, Y and Su, G and Qu, G},
title = {Inhibitor-Regulated Cas12a Activation Enables Highly Sensitive and One-Pot Detection of Drug-Resistant Genes in River Water.},
journal = {Analytical chemistry},
volume = {98},
number = {33},
pages = {24024-24034},
doi = {10.1021/acs.analchem.6c00793},
pmid = {42674013},
issn = {1520-6882},
support = {22325606//National Natural Science Foundation of China/ ; 22576049//National Natural Science Foundation of China/ ; 2024HIAS-V001//Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences/ ; 2025ZY01044//Central Guiding Local Science and Technology Development Fund Projects/ ; },
mesh = {*Rivers/microbiology/chemistry ; CRISPR-Cas Systems ; *Bacterial Proteins/genetics/metabolism ; *Drug Resistance, Bacterial/genetics ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {Monitoring environmental drug-resistance genes (DRGs) plays a pivotal role in preventing the transmission of antimicrobial resistance, thereby reducing public health risks. In this study, a one-pot recombinase polymerase amplification (RPA)/clustered regularly interspaced short palindromic repeat (CRISPR) assay was developed for monitoring DRGs in river water. To overcome compatibility challenges between RPA and CRISPR systems, four glycosaminoglycans (heparin sodium, nadroparin calcium, dalteparin sodium, and chondroitin sulfate A sodium salt) with different molecular weights or negative charge density were evaluated as Cas-enzyme activity modulators. Among them, heparin sodium with the high molecular weight and high strong negative charge density exhibited the best performance in the one-pot DRG detection assay. In the system, CRISPR-Cas12a activity was temporarily inhibited during the RPA amplification phase. When sufficient amplicons were accumulated, Cas12a was activated for signal readout, thereby achieving orderly coupling and precise control of both reactions. To further simplify and improve the reliability of environmental DRG monitoring, a pretreatment method that can eliminate nucleic acid extraction was developed and integrated with the inhibitor-controlled one-pot platform. This assay achieved high sensitivity and specificity when it was applied to river samples, matching the performance of qPCR. The developed assay is simple to operate, has high sensitivity, and is widely adaptable, providing a robust tool for rapid antimicrobial resistance surveillance and exhibiting promise for public health management applications.},
}

*Rivers/microbiology/chemistry
CRISPR-Cas Systems
*Bacterial Proteins/genetics/metabolism
*Drug Resistance, Bacterial/genetics
Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/metabolism/genetics

@article {pmid42674033,
year = {2026},
author = {Lin, S and Chen, X and Lang, Z and Lu, B and Jia, Y and Ju, H and Cao, H},
title = {Programmable Versatile Socket: A Tight-Locking and High-Gain CRISPR/Cas12a Molecular Circuit for Ultrasensitive Sensing of Diverse Targets.},
journal = {Analytical chemistry},
volume = {98},
number = {33},
pages = {24577-24592},
doi = {10.1021/acs.analchem.6c04985},
pmid = {42674033},
issn = {1520-6882},
support = {ZDYF2026SHFZ032//Key Research and Development Project of Hainan Province/ ; 2025DNJP0208//International Cooperative Research Project/ ; 22104027//National Natural Science Foundation of China (NSFC)/ ; 22364014//National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; *Aflatoxin B1/analysis ; *Biosensing Techniques/methods ; RNA/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/genetics ; },
abstract = {CRISPR/Cas12a has emerged as an important platform for nucleic acid analysis, yet limited catalytic turnover and intrinsic nucleic acid specificity constrain its sensitivity and analyte scope. Herein, a tight-locking and high-gain Cas12a-driven strand displacement amplification (CSDA) molecular circuit is developed as a versatile socket without preamplification for programmable sensing of nucleic acid and non-nucleic acid analytes. CSDA relies on an RNA-DNA three-strand hairpin (RD-TSH) switch containing a 2-nt mismatch. RD-TSH suppresses nonspecific amplification and unintended Cas12a self-activation to ensure tight locking. Screening the number of mismatched bases in RD-TSH and molecular-level mechanistic analyses reveal a DNA breathing-driven two-step unlocking mechanism. Only complete unlocking triggers autocatalytic CSDA, allowing high-gain amplification. The sequence-independent unlocking of RD-TSH confers high orthogonality to CSDA, enabling target-specific modules to be coupled to the CSDA socket as interchangeable plugs via programmable crRNA guides, thus achieving universal detection of both nucleic and non-nucleic analytes. Using Vibrio parahaemolyticus DNA, thermostable direct hemolysin, and aflatoxin B1 as representative targets, CSDA achieved ultrasensitive detection in complex matrices with sensitivity improvements of over 5 orders of magnitude, 42-fold, and 602-fold, respectively. This plug-and-play architecture establishes CSDA as a broadly adaptable and ultrasensitive CRISPR/Cas12a sensing socket, providing a general route toward programmable sensing of diverse analyte classes and a promising strategy for more accurate integrated multitarget analytical platforms.},
}

*CRISPR-Cas Systems/genetics
DNA/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/metabolism/genetics
*Aflatoxin B1/analysis
*Biosensing Techniques/methods
RNA/genetics/chemistry
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/genetics

@article {pmid42675078,
year = {2026},
author = {Tsui, W and Yang, Y and Wang, C and Li, D and Zhang, Y and Zhao, X and Wu, J and Guo, J and Wang, Y and Cheng, X and Li, X and Kotovskaya, O and Isaev, A and Ma, J and Wang, M},
title = {Dynamic balance of CRISPR-Cas immunity and resistance plasmid anti-immunity mediated by a bifunctional protein AcrIE10.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42675078},
issn = {2041-1723},
support = {W2512095, 81991531, and 32471347//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82402671//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22410710300//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; 25-44-02137//Russian Science Foundation (RSF)/ ; },
mesh = {*Klebsiella pneumoniae/genetics/immunology/drug effects/metabolism ; *CRISPR-Cas Systems/immunology/genetics ; *Plasmids/genetics/metabolism ; *Bacterial Proteins/metabolism/genetics/chemistry/immunology ; Promoter Regions, Genetic ; Drug Resistance, Bacterial/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {Despite targeting by CRISPR-Cas system, antimicrobial resistance plasmids are prevalent in clinical isolates of carbapenem-resistant Klebsiella pneumoniae which represent a major public health threat. A stable co-existence of plasmids and CRISPR-Cas systems is mediated by anti-CRISPR (Acr) proteins. Here, we report that previously identified AcrIE10 encoded by a resistance plasmid combines two functions: it inhibits CRISPR immunity by directly binding Cas7* subunit through its Acr domain, and acts as an Acr-associated (Aca) protein that self-represses the transcription of Acr locus. AcrIE10 is an example of an Aca protein that utilizes N-terminal ribbon-helix-helix (RHH) domain to specifically recognize the inverted repeat (IR) region in its own promoter. Crucially, a dimerization of AcrIE10 dimers is required for the effective binding to the IR and self-repression, while stoichiometry-dependent interaction with Cas7* facilitates transition to de-repressed state. These findings elucidate molecular mechanisms by which AcrIE10 operates as a dual functionAcr-Aca protein to achieve a delicate balance between host CRISPR-Cas immunity and plasmid anti-defense.},
}

*Klebsiella pneumoniae/genetics/immunology/drug effects/metabolism
*CRISPR-Cas Systems/immunology/genetics
*Plasmids/genetics/metabolism
*Bacterial Proteins/metabolism/genetics/chemistry/immunology
Promoter Regions, Genetic
Drug Resistance, Bacterial/genetics
Gene Expression Regulation, Bacterial

@article {pmid42675231,
year = {2026},
author = {Rathna, ARS and Ceasar, SA},
title = {Accelerating iron biofortification in millets: progress, challenges, and future prospects.},
journal = {Planta},
volume = {264},
number = {4},
pages = {},
pmid = {42675231},
issn = {1432-2048},
mesh = {*Biofortification/methods ; *Iron/metabolism ; *Millets/genetics/metabolism ; Gene Editing ; Plant Breeding ; CRISPR-Cas Systems ; },
abstract = {Integrating conventional breeding, omics, and CRISPR-based genome editing can overcome genetic and antinutrient constraints, enabling efficient iron biofortification of millets for sustainable and nutrition-secure food systems. Iron (Fe) deficiency remains one of the most widespread forms of micronutrient malnutrition. Biofortification of staple crops has emerged as a particularly sustainable and scalable strategy to combat this issue. Millets are nutrient-dense staple cereals with exceptional nutritional quality and climate resilience. However, genetic variations and the presence of antinutrients limit Fe content in millets, which highlights the necessity of advancing biofortification strategies. This review examines the present state of multi-dimensional strategies and discusses the future prospects for efficient iron biofortification in millets. We analyzed the efforts made for Fe biofortification in millets, ranging from conventional breeding practices to next-generation molecular approaches. Recent advances in omics have enhanced understanding of Fe uptake, transport, and storage in millets. Furthermore, CRISPR/Cas-based genome editing is discussed for the regulated expression of key Fe-transporter genes and targeted knockout of genes responsible for antinutrients. A multidisciplinary approach is essential to develop high-yielding and Fe-rich millet varieties that can contribute to sustainable nutrition security.},
}

*Biofortification/methods
*Iron/metabolism
*Millets/genetics/metabolism
Gene Editing
Plant Breeding
CRISPR-Cas Systems

@article {pmid42677405,
year = {2026},
author = {Zhang, WY and Pei, WK},
title = {[Cellular barcoding and next-generation lineage tracing: concepts and applications].},
journal = {Sheng li xue bao : [Acta physiologica Sinica]},
volume = {78},
number = {4},
pages = {731-740},
doi = {10.13294/j.aps.2026.0063},
pmid = {42677405},
issn = {0371-0874},
mesh = {*Cell Lineage/genetics ; Humans ; *DNA Barcoding, Taxonomic ; High-Throughput Nucleotide Sequencing ; CRISPR-Cas Systems ; Animals ; Single-Cell Analysis ; },
abstract = {Lineage tracing is a fundamental technique for dissecting cell fate decisions and development process. With recent advances in high-throughput sequencing and single-cell sequencing technologies, cellular barcoding-based lineage tracing strategies have transitioned from low-throughput labeling methods to high-resolution, multidimensional lineage reconstruction. In this review, we systematically summarize four major barcoding paradigms: viral integration-based random integration barcodes, transposon-based random integration barcodes, recombinase-mediated DNA rearrangement (e.g., Cre-loxP), and CRISPR-Cas9-based mutation recording systems. We describe their principles, representative studies, technical advantages, and limitations. Furthermore, we discuss the core bottlenecks in terms of editing precision, integration of spatiotemporal information, and non-invasive lineage tracing, with a focus on cutting-edge advancements such as prime editing, sequential recording systems, strategies for integrating spatial transcriptomics, and epigenetic tracing. Overall, single-cell lineage tracing is evolving from clonal labeling toward the multi-dimensional integration of lineage, state, and space. In the future, the deep integration of precise gene-editing tools with high-resolution spatial omics technologies is expected to enable dynamic and systematic analysis of cellular fate trajectories, thereby providing critical technical support for research in developmental biology and regenerative medicine.},
}

*Cell Lineage/genetics
Humans
*DNA Barcoding, Taxonomic
High-Throughput Nucleotide Sequencing
CRISPR-Cas Systems
Animals
Single-Cell Analysis

@article {pmid42678547,
year = {2026},
author = {Akhtar, MS and Amin, A},
title = {CRISPR-cas systems in pharmacology: functional pharmacogenomics, drug screening, resistance, and therapeutic translation.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42678547},
issn = {1438-7948},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Pharmacogenetics/methods ; Animals ; Precision Medicine ; *Drug Discovery/methods ; },
abstract = {CRISPR-Cas9 gene-editing technology has advanced pharmacological research by enabling targeted genetic modification for disease modeling, therapeutic development, and precision medicine. This review discusses the applications of CRISPR-Cas9 in drug discovery, personalized therapy, cancer drug resistance research, genetic disorders, and antimicrobial resistance. By editing disease-associated genes, CRISPR-Cas9 supports the development of patient-specific therapeutic strategies and more accurate preclinical models. In cancer, CRISPR-Cas9 is used to investigate the target genes involved in treatment resistance, while in genetic disorders, it offers potential mutation-correcting approaches, with the most robust clinical evidence currently seen in selected hemoglobinopathies. CRISPR-based strategies also hold promise for restoring antibiotic susceptibility by targeting genes that confer antibiotic resistance. Despite these advances, clinical translation remains limited by off-target effects, delivery challenges, immune responses, long-term safety concerns, and ethical and regulatory issues. Continued improvements in editing precision, delivery systems, and governance frameworks are essential for responsible clinical integration. Overall, CRISPR-Cas9 represents a vital platform for future pharmacological innovation, but its broad clinical use may require further validation of safety, efficacy, durability, and accessibility.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Pharmacogenetics/methods
Animals
Precision Medicine
*Drug Discovery/methods

@article {pmid41966922,
year = {2026},
author = {Cui, J and Zhang, L and Zhou, J and Shi, T and Wu, S and Dai, T and Hao, L and Pan, J and Lai, X and Lu, W and Huang, X and Li, Z and Lai, L and Wang, X},
title = {A universal light-controlled highly sensitive one-pot CRISPR/Cas12a diagnostic based on structure-engineered crRNA.},
journal = {Trends in biotechnology},
volume = {44},
number = {9},
pages = {2699-2721},
doi = {10.1016/j.tibtech.2026.03.018},
pmid = {41966922},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleic Acid Amplification Techniques/methods ; Human papillomavirus 16/genetics/isolation & purification ; Human papillomavirus 18/genetics/isolation & purification ; Polymorphism, Single Nucleotide ; *Molecular Diagnostic Techniques/methods ; CRISPR-Associated Proteins ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection has transformed molecular diagnostics through its speed and accuracy; however, one-pot formats are often limited by sensitivity and field suitability. Herein, we developed a universal light-controlled high-sensitivity one-pot CRISPR/Cas12a testing (ULTRAt) platform based on structure-engineered CRISPR RNA (crRNA) scaffolds. By incorporating photocaged 6-nitropiperonyloxymethyl groups into the crRNA stem-loop, Cas12a activity is transiently suppressed during isothermal amplification via structural modulation, enabling efficient target enrichment. Subsequent UV irradiation removes the protecting groups, restoring the native conformation and activating robust trans-cleavage. ULTRAt achieves a limit of detection of two copies of monkeypox virus per reaction with a 15-min time-to-result, representing a 100-fold sensitivity improvement over conventional assays. The platform further supports single-nucleotide polymorphism discrimination and human papillomavirus 16/18 genotyping. Analysis of 91 clinical samples demonstrates strong concordance between ULTRAt and reference qPCR and sequencing assays. Collectively, ULTRAt enables rapid, ultra-sensitive, and versatile one-pot detection, supporting near-patient diagnostics and genotyping.},
}

*CRISPR-Cas Systems/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Nucleic Acid Amplification Techniques/methods
Human papillomavirus 16/genetics/isolation & purification
Human papillomavirus 18/genetics/isolation & purification
Polymorphism, Single Nucleotide
*Molecular Diagnostic Techniques/methods
CRISPR-Associated Proteins
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42237538,
year = {2026},
author = {Sarko, LE and Givand, D and Rattin, B and Shepley, C and Tommasi, A and Attar, A and Taylor, R and Kutler, B and Traynor, RM and Upadhyaya, A and Mnuk, M and Gehrke, C and Murren, N and Ulland, TK and Capitini, CM and Kotanchek, T and Saha, K},
title = {Directing fratricide within T cell products using an anti-uPAR chimeric antigen receptor to drive the production of potent therapeutic cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {9},
pages = {5190-5206},
doi = {10.1016/j.ymthe.2026.05.029},
pmid = {42237538},
issn = {1525-0024},
support = {P30 CA014520/CA/NCI NIH HHS/United States ; T32 GM135119/GM/NIGMS NIH HHS/United States ; S10 RR025483/RR/NCRR NIH HHS/United States ; R35 GM119644/GM/NIGMS NIH HHS/United States ; R01 CA278051/CA/NCI NIH HHS/United States ; R01 AG083883/AG/NIA NIH HHS/United States ; },
mesh = {Humans ; *Receptors, Chimeric Antigen/genetics/metabolism ; *T-Lymphocytes/metabolism/immunology ; Gene Editing ; *Receptors, Urokinase Plasminogen Activator/antagonists & inhibitors/genetics/immunology/metabolism ; CRISPR-Cas Systems ; Lymphocyte Activation/immunology ; Immunotherapy, Adoptive/methods ; *Receptors, Antigen, T-Cell/genetics/metabolism ; Cell- and Tissue-Based Therapy/methods ; },
abstract = {Cell therapy manufacturing of primary T cells often results in heterogeneous cell populations in the final product, with many cells lacking desired receptor expression or exhausted and other dysfunctional phenotypes. Here, we design a novel cell-intrinsic strategy to genetically reprogram primary human T cells to autonomously detect and eliminate dysfunctional cells. This integrated detection and elimination process, known as directed fratricide, is programmed via non-viral CRISPR genome editing to eliminate the T cell receptor (TCR) alpha chain (TRAC gene knockout) and integrate a chimeric antigen receptor (CAR) against the urokinase-type plasminogen activator receptor (uPAR), also known as CD87. In these cell products, strong T cell stimulation or activation during manufacturing causes a small subset of cells to express uPAR, which triggers CAR-mediated killing within the product. This fratricide induces proliferation in the desired cells and destroys undesired cells, a process that could be modeled computationally and controlled robustly via supplements to the culture media. The strategy enabled enrichment of anti-uPAR and anti-disialoganglioside (GD2) CAR-T cell products up to ≥99% CAR+/TCR-, favoring a memory-like phenotype. Understanding growth dynamics among T cell subsets and reprogramming them via CRISPR could accelerate the biomanufacturing of potent cell products without extensive selection methods.},
}

Humans
*Receptors, Chimeric Antigen/genetics/metabolism
*T-Lymphocytes/metabolism/immunology
Gene Editing
*Receptors, Urokinase Plasminogen Activator/antagonists & inhibitors/genetics/immunology/metabolism
CRISPR-Cas Systems
Lymphocyte Activation/immunology
Immunotherapy, Adoptive/methods
*Receptors, Antigen, T-Cell/genetics/metabolism
Cell- and Tissue-Based Therapy/methods