@article {pmid41671414,
year = {2026},
author = {Yu, J and Park, JC and Uhm, H and Kim, YW and Im, HW and Bae, S},
title = {Evolution of Prime Editing: Enhancing Efficiency and Expanding Capacity.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e21015},
doi = {10.1002/advs.202521015},
pmid = {41671414},
issn = {2198-3844},
support = {RS-2024-00332601//Korean Fund for Regenerative Medicine/ ; 25202MFDS003//Ministry of Food and Drug Safety/ ; 25B-001-0700//SNUH Lee Kun-hee Child Cancer and Rare Disease/ ; 2021M3A9H3015389//National Research Foundation of Korea/ ; RS-2024-00451880//National Research Foundation of Korea/ ; RS-2024-00455559//National Research Foundation of Korea/ ; SRC-NRF2022R1A5A102641311//National Research Foundation of Korea/ ; RS-2024-00404132//Ministry of Health & Welfare/ ; },
abstract = {Genetic mutations cause approximately 80% of rare human diseases, highlighting the urgent need for precise genome editing. Since clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) nucleases were first used for genome editing in 2012, genome editing technologies have rapidly advanced. Base editors, derived from the CRISPR-Cas system, were developed to introduce specific point mutations without requiring DNA double-strand breaks, and subsequently, prime editing (PE) technology was created to enable insertions, deletions, and all types of point mutations. The precision and versatility of PE make it a promising tool for clinical applications. However, PE has potential limitations, including low editing efficiency and limited capacity for large-scale manipulation. To overcome these limitations, research has been continuously conducted to improve PE efficiency and expand its capabilities. Therefore, this review aims to highlight current efforts and future directions for developing and improving PE-related tools.},
}

@article {pmid41671402,
year = {2026},
author = {Shi, M and Ge, W and Li, C and Liu, B and Deng, X and Liu, C and Zheng, M and Zhang, P and Li, L and Guo, Y and Han, Y and Yang, Y and Yu, YV and Jin, YN},
title = {Versatile CRISPR-Cas Tools for Gene Regulation in Zebrafish via an Enhanced Q Binary System.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e11485},
doi = {10.1002/advs.202511485},
pmid = {41671402},
issn = {2198-3844},
support = {32070832//National Natural Science Foundation of China/ ; 32150610476//National Natural Science Foundation of China/ ; 2042022dx0003//Fundamental Research Funds for the Central Universities/ ; },
abstract = {CRISPR-Cas systems revolutionize gene regulation across diverse organisms, including zebrafish. However, most zebrafish studies still rely on transient delivery of CRISPR components, with limited use of transgenic models, primarily restricted to Cas9-mediated knockouts. This limitation arises from challenges in achieving sustained, tissue-specific, and efficient expression of transgenic CRISPR effectors. To address these challenges, we introduce CRISPR-Q, a transgenic system that combines the QFvpr/QUAS binary expression platform with CRISPR-Cas technologies. CRISPR-Q overcomes the drawbacks of transient mRNA or protein delivery and circumvents the toxicity and transgene silencing issues associated with other binary systems, such as Gal4/UAS. The system enables robust and spatiotemporal expression of CasRx or dCas9vpr, allowing precise transcript knockdown (CRISPR-QKD) or gene activation (CRISPR-Qa). Using CRISPR-QKD, we achieve effective knockdown of smn1 and simultaneous knockdown of tardbp and tardbpl, modeling spinal muscular atrophy and amyotrophic lateral sclerosis, respectively. CRISPR-Qa activates endogenous lin28a and sox9b, demonstrating its functional versatility. We further validate CRISPR-Q's tissue-specific applicability in heart-specific transgenic zebrafish. Together, CRISPR-Q represents a robust and versatile platform for studying gene function and modeling human diseases in zebrafish, with broad potential for adaptation in other model organisms.},
}

@article {pmid41670257,
year = {2026},
author = {Vollmer, SK and Stetter, MG and Hensel, G},
title = {First Successful Targeted Mutagenesis Using CRISPR/Cas9 in Stably Transformed Grain Amaranth Tissue.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70590},
pmid = {41670257},
issn = {1467-7652},
support = {EXC-2048/1 project ID 390686111//Deutsche Forschungsgemeinschaft/ ; STE 2654/4//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Grain amaranth is a nutritionally rich, stress-tolerant C4 dicot with considerable potential for climate-resilient agriculture; however, efficient and reproducible protocols for stable transformation, regeneration, and CRISPR/Cas9-mediated editing have not yet been established. CRISPR/Cas-based genome editing is a cornerstone technology for accelerating the development of climate-resilient, high-yielding crops. Its effective application depends on robust, stable transformation procedures and CRISPR/Cas systems optimised for the target species. The absence of such tools remains a critical constraint for the genetic improvement of many promising yet underexplored crops. In this study, we edited key genes of the betalain biosynthesis pathway in grain amaranth (Amaranthus hypochondriacus L.) using the CasCADE modular cloning system, thereby demonstrating the feasibility of targeted mutagenesis in an orphan crop. We observed successful edits in up to 49% of transformed calli, resulting in deletions or insertions in the target genes. Our CRISPR/Cas9-mediated editing paves the way for targeted molecular research and breeding of grain amaranth.},
}

@article {pmid41669806,
year = {2026},
author = {Wang, ZH and Yang, ZQ},
title = {Advances in site-specific knock-in techniques for gene editing.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {2},
pages = {128-141},
doi = {10.16288/j.yczz.25-076},
pmid = {41669806},
issn = {0253-9772},
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Animals ; DNA Nucleotidyltransferases ; },
abstract = {Gene-targeted knock-in technology serves as a cornerstone tool in genetic engineering and gene therapy, designed to circumvent the unpredictability and heterogeneityassociated with conventional random integration methods. However, its practical application has long been constrained by off-target activity and low efficiency during the editing process. Recent advances in site-specific recombinase systems (e.g., Bxb1 integrase) and programmable nuclease systems (e.g., CRISPR/Cas9) have significantly enhanced the precision and efficiency of gene knock-in. Notably, the Cas9-Bxb1 integrase system enables targeted integration of large DNA fragments (5-43 kb) into genomic safe harbor (GSH) sites, offering a transformative platform for disease modeling, functional genomics, and clinical therapeutics. This review systematically summarizes the progress of site-specific recombinase and nuclease systems, discusses GSH screening strategies and the role of multi-omics data in optimizing predictive models, and compares the strengths and limitations of twinPE+Bxb1 and PASTE systems. Future research should focus on developing novel integrases with low off-target activity, refining DSB-free editing technologies, and establishing cross-species GSH databases to advance applications in precision medicine and synthetic biology.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
Animals
DNA Nucleotidyltransferases

@article {pmid41669364,
year = {2025},
author = {Elkonin, LA and Gerashchenkov, GA and Borisenko, NV and Sarsenova, SK and Panin, VM},
title = {Study of the progeny of sorghum mutants obtained using the CRISPR/Cas9 genetic construct directed at inducing mutations in the α-kafirin k1C5 gene.},
journal = {Vavilovskii zhurnal genetiki i selektsii},
volume = {29},
number = {8},
pages = {1161-1168},
doi = {10.18699/vjgb-25-122},
pmid = {41669364},
issn = {2500-0462},
abstract = {Site-directed mutagenesis using genetic constructs carrying the CRISPR/Cas system is an effective technology that is actively used to solve a variety of problems in plant genetics and breeding. One of these problems is to improve the nutritional value of grain sorghum, a high-yielding heat- and drought-tolerant cereal crop that is becoming increasingly important in the conditions of climate aridization. The main reason for the relatively low nutritional value of sorghum grain is the resistance of its storage proteins, kafirins, to proteolytic digestion. We have previously obtained mutants with improved kafirin in vitro digestibility using the CRISPR/Cas technology in grain sorghum variety Avance. The nucleotide sequence of one of the genes (k1C5) of the gene family encoding the signal polypeptide of 22 kDa α-kafirin was used as a target. The aim of this study was to investigate the manifestation of the main agronomically-important traits in the progeny of these mutants and inheritance of high in vitro protein digestibility, and also sequencing nucleotide sequences encoding the 22 kDa α-kafirin signal polypeptide in a number of plants from the T0 generation and their T1 progeny. It was revealed that four of the six studied T0 plants, as well as their progeny, had the same mutation: a T→C substitution in the 23rd position of the nucleotide sequence of the k1C5 gene encoding the signal polypeptide, which led to a substitution of the coding triplet CTC→CCC (Leu→Pro). This mutation is located off-target, 3' from the PAM sequence. It is suggested that this mutation may have arisen as a result of Cas9 nuclease errors caused by the presence of multiple PAM sequences located close to each other. It was found that the progeny of two of the three studied mutants (T2 and T3 families), grown in the experimental field conditions, differed from the original variety by a reduced plant height (by 12.4-15.5 %). The peduncle length, 1,000-grain mass, and grain mass per panicle did not differ from the original variety, with the exception of the progeny of the 2C-1.2.5b mutant, which had a reduced grain yield per panicle. Unlike the original variety, plants from the T2 and T3 generations had kernels with a modified type of endosperm (completely floury, or floury with inclusions of vitreous endosperm, or with a thin vitreous layer). The level of grain protein digestibility in the progeny of mutants 2C-2.1.1 #13 and 2C-1.2.5a #14 varied from 77 to 84 %, significantly exceeding the original variety (63.4 ± 2.3 %, p < 0.05). The level of protein digestibility from kernels with modified endosperm was higher than that of kernels with normal vitreous endosperm (84-93 %, p <0.05). The reasons for the variation in endosperm texture in the progeny of the mutants and its relationship with the high digestibility of kafirins are discussed.},
}

@article {pmid41666229,
year = {2026},
author = {Fan, X and Lyu, S and Fan, W and Shu, J and Cheng, X},
title = {Precision targeting: The dawn of artificially customized disease resistance.},
journal = {PLoS pathogens},
volume = {22},
number = {2},
pages = {e1013942},
pmid = {41666229},
issn = {1553-7374},
mesh = {*Plant Diseases/immunology/prevention & control/genetics ; *Disease Resistance/genetics/immunology ; *Plant Immunity/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics/immunology ; *Crops, Agricultural/genetics/immunology ; },
abstract = {Advanced plant disease management strategies are essential to sustainable agriculture and global food security. Advances in plant immunity have given rise to a variety of innovative disease control strategies, such as NLR gene transfer, RNA silencing technology, and CRISPR/Cas9-based gene disruption, as well as the use of immunity inducers. Recently, several novel resistance strategies, including the bioengineering of immunoreceptors, protease-triggered resistance design, and the sentinel approach, have enabled the customized development of disease resistance traits. These new approaches envisage a new paradigm of precision-targeted, artificially engineered resistance to enhance crop protection.},
}

*Plant Diseases/immunology/prevention & control/genetics
*Disease Resistance/genetics/immunology
*Plant Immunity/genetics
CRISPR-Cas Systems
Plants, Genetically Modified/genetics/immunology
*Crops, Agricultural/genetics/immunology

@article {pmid41665762,
year = {2026},
author = {Farooq, M and Khan, A and Hassan, A and Shah, MM},
title = {Advances in CRISPR/Cas systems for engineering abiotic stress tolerance in plants: mechanisms and future prospects.},
journal = {Planta},
volume = {263},
number = {3},
pages = {72},
pmid = {41665762},
issn = {1432-2048},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Stress, Physiological/genetics ; *Crops, Agricultural/genetics/physiology ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; Droughts ; },
abstract = {Abiotic stress factors such as drought, salinity, extreme temperatures, and oxidative stress significantly limit crop productivity and threaten global food security. Traditional breeding and transgenic approaches have been employed to enhance stress tolerance, but they are often time-consuming and face regulatory hurdles. The advent of CRISPR/Cas genome editing technology has revolutionized plant genetic engineering by enabling precise modifications to stress-responsive genes. This review explores recent advancements in CRISPR/Cas-based genome editing for improving abiotic stress resilience in crops. We discuss the mechanisms of CRISPR/Cas systems, their applications in stress tolerance, and emerging approaches such as multiplex genome editing, base editing, and AI-assisted CRISPR. Furthermore, we highlight challenges, ethical considerations, and future directions for integrating CRISPR into agricultural biotechnology. This review underscores the potential of CRISPR-based strategies in developing climate-resilient crops to ensure sustainable food production in the face of global climate change.},
}

*CRISPR-Cas Systems/genetics
Gene Editing/methods
*Stress, Physiological/genetics
*Crops, Agricultural/genetics/physiology
Plants, Genetically Modified/genetics
Genetic Engineering/methods
Droughts

@article {pmid41664621,
year = {2026},
author = {Ullah, Q and Haider, W and Zeshan, M and Waqar, M and Arshad, MT and Parveen, H and Mukhtar, S and Zahir, A},
title = {Advancing climate adaptation in saffron through CRISPR-based modulation of stress tolerance and photoperiodic flowering control.},
journal = {GM crops & food},
volume = {17},
number = {1},
pages = {2626180},
pmid = {41664621},
issn = {2164-5701},
mesh = {*Flowers/genetics/physiology/growth & development ; *Crocus/genetics/physiology/growth & development ; Gene Editing ; *CRISPR-Cas Systems ; Photoperiod ; Stress, Physiological ; Plants, Genetically Modified/genetics ; Adaptation, Physiological ; Droughts ; Climate Change ; },
abstract = {Saffron (Crocus sativus L.) is a high-value crop known for its intricate harvesting process and limited production due to factors like triploid sterility and specific climatic needs. This review discusses biotechnological methods, particularly CRISPR/Cas9 genome editing, aimed at improving heat and drought tolerance and achieving year-round flowering. Such genetic edits as evidenced with experimental CRISPR/Cas9 systems that reach up to 70% callus initiation in saffron. Cultivation efficiency and quality are increased in Hydroponic systems and synthetic bioreactors, which have been proven in trials in non-traditional areas such as North Bengal, India. Nonetheless, internationalization threatens the market value and cultural integrity of saffron, and such measures as fair-trade labels, GI laws, and cooperatives of stakeholders must be implemented fairly and equally.},
}

*Flowers/genetics/physiology/growth & development
*Crocus/genetics/physiology/growth & development
Gene Editing
*CRISPR-Cas Systems
Photoperiod
Stress, Physiological
Plants, Genetically Modified/genetics
Adaptation, Physiological
Droughts
Climate Change

@article {pmid41663678,
year = {2026},
author = {Zhu, Y and Zhang, J and Ruan, Y and Lei, T and Li, S and Cao, L and Zou, X and He, Y and Li, Q and Chen, S and Peng, A},
title = {Application of compact CRISPR/Cas nucleases for citrus genome editing.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {7},
pmid = {41663678},
issn = {1573-9368},
support = {CSTB2024TIAD-CYKJCXX0021//Sichuan-Chongqing Science and Technology Innovation Cooperation Program Project/ ; CSTB2023TIAD-KPX0044//the Technology Innovation and Application Development Key Project of Chongqing/ ; CARS-26//Earmarked Fund for China Agriculture Research System/ ; CSTB2023NSCQ-MSX1085//Chongqing Natural Science Foundation Project/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Citrus/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Gene editing technology continues to advance, and the range of available editing tools is steadily expanding. Recently, several compact and ultracompact systems have been developed, gaining considerable attention because their components can be efficiently packaged into viral vectors. To identify compact tools suitable for efficient genome editing in citrus, Casπ, CoCas9, along with their respective single guide RNAs, were synthesized, and CRISPR/Casπ and CRISPR/CoCas9 constructs were designed to assess their editing efficiency in 'Wanjincheng' orange (Citrus sinensis Osbeck). The Casπ was able to mediate genome editing in the citrus genome, although with low efficiency. In comparison, CoCas9 showed a transformation efficiency three times higher than that of the widely used SpCas9. Moreover, while the gene editing efficiency of CoCas9 was comparable to that of SpCas9, the significantly elevated transformation efficiency resulted in a significantly higher overall editing efficiency for CoCas9 relative to SpCas9. Mutation profiles generated by CoCas9 and SpCas9 were highly similar, and both nucleases displayed comparable target specificity at three potential off-target sites. These results indicate that Casπ is not suitable for application in citrus genome editing, whereas CoCas9 represents a promising alternative to SpCas9 for efficient and precise genome modification in citrus.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Citrus/genetics
*Genome, Plant
Plants, Genetically Modified/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics

@article {pmid41663312,
year = {2026},
author = {Eraña, H and Vidal, E and Fernández-Borges, N and Charco, JM and Díaz-Domínguez, CM and Sampedro-Torres-Quevedo, C and Galarza-Ahumada, J and Fernández-Muñoz, E and San-Juan-Ansoleaga, M and Pérez-Castro, MÁ and Gonçalves-Anjo, N and Piñeiro, P and Giler, S and González-Martín, N and Lorenzo, NL and Manero-Azua, A and Perez de Nanclares, G and Geijo, M and Sánchez-Martín, MA and Requena, JR and Castilla, J},
title = {The L108I polymorphism in mouse prion protein drives spontaneous disease and enhances transmission of atypical and classical prion strains.},
journal = {Brain pathology (Zurich, Switzerland)},
volume = {},
number = {},
pages = {e70083},
doi = {10.1111/bpa.70083},
pmid = {41663312},
issn = {1750-3639},
support = {PID2024-160022OB-I00//Agencia Estatal de Investigación/ ; PID2021-122201OB-C21//Agencia Estatal de Investigación/ ; PID2020-117465GB-I00//Agencia Estatal de Investigación/ ; PID2021-1222010B-C22//Agencia Estatal de Investigación/ ; CEX2021-001136-S//Ministerio de Ciencia e Innovación/ ; EFA031/01 NEURO-COOP//Interreg/ ; PT23/00123//Instituto de Salud Carlos III/ ; BN661-FTPGB-2023//Fundación Tatiana Pérez de Guzmán el Bueno/ ; //Creutzfeldt-Jakob Disease Foundation-2022/ ; },
abstract = {Prion diseases are fatal neurodegenerative disorders that can be idiopathic, associated with genetic mutations, or acquired by infection with misfolded prion protein. We developed two complementary transgenic mouse models to investigate how the L108I substitution in mouse prion protein (PrP) influences spontaneous prion formation and transmission characteristics. The transgenic mouse model overexpressing the variant at approximately three times wild-type (WT) PrP levels (TgMo(L108I)3x) consistently developed a spontaneous neurodegenerative disorder between 219 and 536 days of age with 100% penetrance. This spontaneous disease exhibited biochemical and neuropathological characteristics of atypical prion disorders, featuring a distinctive 7-10 kDa protease-resistant PrP fragment and pathology comparable to small ruminants' atypical scrapie and certain forms of Gerstmann-Sträussler-Scheinker syndrome (GSS). In contrast, the knock-in model expressing the same variant at physiological levels (TgMo(L108I)1x) showed no spontaneous disease beyond 600 days, demonstrating that both the specific amino acid substitution and elevated expression levels are necessary for spontaneous prion formation. The spontaneously generated prions transmitted efficiently to models expressing the I108 variant and to Tga20 mice overexpressing WT PrP but encountered a robust transmission barrier toward WT mice, indicating strain-specific replication requirements. The TgMo(L108I)3x model demonstrated exceptional versatility as a universal acceptor for heterogeneous prion isolates, demonstrating superior efficiency in propagating atypical variants like GSS A117V (57 ± 0.6 days) and rapid propagation of classical scrapie-derived mouse prion strains, including Rocky Mountains Laboratory mouse prion strain (RML) (85 ± 3.8 days) and 22L (95 ± 1 days). Comparative analysis revealed that the L108I substitution differentially impacts strain propagation, with greater acceleration of RML (~33% shorter incubation) than 22L (~0.5% shorter) compared to WT mice. These complementary systems offer powerful experimental platforms for investigating the molecular determinants of spontaneous prion formation, strain selection and transmission barriers, providing insights into idiopathic prion pathogenesis and developing therapeutic interventions.},
}

@article {pmid41663226,
year = {2026},
author = {Yin, X and Zhang, Z and Luo, H and Qin, X and Chen, Y and Chen, W and Zheng, H},
title = {Amplification-free one-pot RNA detection by pairing CRISPR-Cas13a with cascade amplification circuit-driven DNAzyme (RAPID).},
journal = {Analytica chimica acta},
volume = {1391},
number = {},
pages = {345138},
doi = {10.1016/j.aca.2026.345138},
pmid = {41663226},
issn = {1873-4324},
mesh = {*DNA, Catalytic/metabolism/chemistry/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Neisseria gonorrhoeae/genetics/isolation & purification ; *RNA, Bacterial/analysis/genetics ; Humans ; },
abstract = {RNA has become a versatile target for diagnosing a wide range of pathogens. The demand for rapid and accurate diagnostics in point-of-care (POC) or resource-limited settings is growing. However, most RNA-based assays depend on reverse transcription and complex instruments (e.g., RT-qPCR), restricting their use in these settings. Isothermal amplification methods provide a simpler alternative with reduced instrumentation requirements, but their high amplification efficiency raises concerns about nucleic acid carry-over contamination. To address these challenges, we developed RAPID (CRISPR-Cas13a with a cascade amplification circuit-driven DNAzyme), an isothermal, one-pot RNA detection biosensing platform that eliminates the need for sample pre-amplification. RAPID integrates the precise target recognition by CRISPR-Cas13a with robust signal amplification by a toehold-mediated strand-displacement DNA circuit, eliminating the need for reverse transcription and thermal cycling. This platform enables quantitative RNA detection within 30 min at 37 °C. By reprogramming RAPID crRNAs, we successfully detected both bacterial (e.g., Treponema pallidum and Neisseria gonorrhoeae) and viral (e.g., herpes simplex virus) targets. The RAPID platform is designed for versatile detection, being compatible with both fluorescence-based (RAPID-Flu) and lateral flow assay (RAPID-LFA) readouts. The RAPID-Flu and RAPID-LFA both demonstrated a sensitivity of 5 fM per reaction, exhibiting comparable detection limits. Both methods showed excellent specificity and high concordance with clinical diagnoses of Neisseria gonorrhoeae. In summary, the RAPID platform provides rapid, programmable, and visually interpretable solutions with strong potential for POC diagnostics. Its flexibility and portability make it particularly suitable for early diagnosis and on-site monitoring of diverse infectious pathogens.},
}

*DNA, Catalytic/metabolism/chemistry/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
Neisseria gonorrhoeae/genetics/isolation & purification
*RNA, Bacterial/analysis/genetics
Humans

@article {pmid41663224,
year = {2026},
author = {Li, Y and Chen, X and Yang, Z and Wang, Z and Wang, R},
title = {CRISPR/Cas12a empowered electrochemical biosensor for ultrasensitive detection of Vibrio parahaemolyticus in seafood samples.},
journal = {Analytica chimica acta},
volume = {1391},
number = {},
pages = {345158},
doi = {10.1016/j.aca.2026.345158},
pmid = {41663224},
issn = {1873-4324},
mesh = {*Vibrio parahaemolyticus/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Seafood/microbiology/analysis ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Food Contamination/analysis ; Animals ; DNA, Bacterial/genetics/analysis ; },
abstract = {Rapid and ultrasensitive detection of Vibrio parahaemolyticus (V. parahaemolyticus, Vp) is of great significance for the early prevention of foodborne disease. Traditional methods for detecting Vp are time-consuming, exhibiting low sensitivity and specificity. In this study, CRISPR/Cas12a system is integrated with electrochemical sensing and polymerase chain reaction (PCR) to establish a PCR-based E-CRISPR biosensor for Vp detection. The target DNA extracted from Vp is amplified by PCR, then activate CRISPR/Cas12a system to cleave methylene blue (MB)-labeled hairpin DNA probes on electrode, resulting in great changes in current. The employment of hairpin DNA probes reduces the steric hindrance for Cas12a trans-cleavage, acquiring a better cleavage efficiency and sensing performance. Under optimal conditions, the limit of detection reaches 1.17 copies/μL (genomic DNA), 1.23 CFU/mL (standard bacteria), and 12.3 CFU/g (artificially contaminated shrimp samples) respectively. Moreover, the PCR-based E-CRISPR biosensor demonstrates superior reproducibility and specificity. Most importantly, the E-CRISPR biosensor were in 100 % agreement with real time quantitative PCR for the detection of 18 seafood samples, which confirms the biosensor's broad applicability for monitoring Vp in complex food matrix. Our developed E-CRISPR biosensor demonstrates to be a simple, rapid and ultrasensitive method for Vp detection in the food supply chain, and can be extended to other foodborne pathogens.},
}

*Vibrio parahaemolyticus/isolation & purification/genetics
*Biosensing Techniques/methods
*Seafood/microbiology/analysis
*Electrochemical Techniques/methods
*CRISPR-Cas Systems/genetics
Limit of Detection
Food Contamination/analysis
Animals
DNA, Bacterial/genetics/analysis

@article {pmid41662274,
year = {2026},
author = {Tek, MI and Budak Tek, K and Sarikaya, P and Ahmed, AR and Fidan, H},
title = {Choosing the best route: Comparative optimization of wheat transformation methods for improving yield by targeting TaARE1-D with CRISPR/Cas9.},
journal = {PloS one},
volume = {21},
number = {2},
pages = {e0342491},
pmid = {41662274},
issn = {1932-6203},
mesh = {*Triticum/genetics/growth & development ; *CRISPR-Cas Systems ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Plant Proteins/genetics ; Plant Breeding/methods ; },
abstract = {Wheat (Triticum aestivum L.) is one of the most important crops worldwide, supplying a major share of calories and protein for the global population. Incorporating gene editing into breeding programs is critical to improve yield and stress tolerance, yet wheat remains difficult to transform and regenerate efficiently. These bottlenecks limit the full application of CRISPR/Cas9 for improvement yield in wheat. To address this, transformation parameters were optimized for three methods: immature embryo transformation, callus transformation, and injection-based in planta transformation. Systematic optimization of Agrobacterium strain, bacterial density, acetosyringone concentration, and incubation conditions resulted in substantially improved transformation success. Efficiencies of 66.84% for immature embryos, 55.44% for callus, and 33.33% for in planta transformation were achieved, representing more than tenfold increase compared with previously reported rate of ~3%. A key innovation was the shortening of the callus induction stage for immature embryos, reducing the time required for plant regeneration by approximately one month while maintaining high transformation efficiency. The protocols were validated through CRISPR/Cas9-mediated knockout of TaARE1-D, a negative regulator of nitrogen uptake and yield. Generated mutants exhibited increased grain number, spike length, grain length, and thousand-grain weight, as well as the characteristic stay-green phenotype associated with loss of TaARE1-D function. The optimized protocols provide robust platforms to accelerate gene-editing in wheat to increase yield and stress-tolerance.},
}

*Triticum/genetics/growth & development
*CRISPR-Cas Systems
Gene Editing/methods
Plants, Genetically Modified/genetics
*Transformation, Genetic
*Plant Proteins/genetics
Plant Breeding/methods

@article {pmid41661420,
year = {2026},
author = {Zhang, H and Feng, G and Feng, Y},
title = {Quinoa as a naturally stress-resistant crop: current status and future promises.},
journal = {Stress biology},
volume = {6},
number = {1},
pages = {12},
pmid = {41661420},
issn = {2731-0450},
support = {2022B02010-1//Science and Technology Department of Xinjiang Uygur Autonomous Region/ ; 2022YFF1003403-4//Key Technologies Research and Development Program/ ; 2021YFA1300401//Key Technologies Research and Development Program/ ; 32441015//National Natural Science Foundation of China/ ; },
abstract = {Quinoa (Chenopodium quinoa Willd.), a semi-domesticated halophyte originating in the Andean region, has emerged as a promising crop for exploiting marginal lands, valued for its exceptional nutritional profile and remarkable resilience to high salinity and drought. This review analyzes the current status and future potential of quinoa as a model halophytic crop. We begin by examining the physiological mechanisms that enable quinoa to thrive in marginal environments, which have been the subject of extensive study. Thanks to the advancement in high-throughput sequencing technology, genomic resources - including the recent development of high-quality reference genomes and a Chenopodium pangenome - are rapidly expanding. Sequence-based genetic mapping techniques hold the promise to dissect the molecular basis of complex traits in combination with the utility of functional genomics tools such as virus-induced gene silencing (VIGS) and stable genetic transformation. Ultimately, the application of modern breeding technologies, such as phenomics, genomic selection (GS), and CRISPR/Cas, will expedite the development of locally adapted, climate-resilient quinoa cultivars worldwide.},
}

@article {pmid41661284,
year = {2026},
author = {Li, Z and Chen, L and Luo, J and Lu, Y and Zhang, H and Zhao, P},
title = {One-pot recombinase polymerase amplification and CRISPR/Cas12a assay for Cryptococcus neoformans.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {61},
pmid = {41661284},
issn = {1432-0614},
mesh = {*Cryptococcus neoformans/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Recombinases/genetics/metabolism ; *Cryptococcosis/diagnosis/microbiology ; Humans ; Real-Time Polymerase Chain Reaction/methods ; Point-of-Care Testing ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Cryptococcus neoformans, an opportunistic fungal pathogen, can induce central nervous system infections, posing a life-threatening risk and imposing substantial global health challenges and economic burdens. Given the significant reduction in mortality achieved through early monitoring of C. neoformans, there is an urgent demand for a rapid detection method for this pathogen. Herein, we developed a rapid, sensitive, and specific assay for the detection of C. neoformans based on a one-pot recombinase polymerase amplification (RPA) and CRISPR/Cas12a system, which can be read using a real-time fluorescent PCR instrument or lateral flow strips. This assay exhibits high sensitivity, with a detection limit of 1 copy/µL for C. neoformans, and no cross-reactivity was observed across different fungal strains. Notably, the assay can be performed in harsh environments without reliance on complex equipment, making it suitable for point-of-care testing (POCT). Collectively, this method not only provides a robust alternative for C. neoformans detection but also offers valuable insights for the identification of other fungal pathogens. KEY POINTS: • We developed a one-pot RPA and CRISPR/Cas12a assay to detect Cryptococcus neoformans. • This assay exhibits high sensitivity and specificity. • Detection results can be obtained by three ways which is suitable for POCT.},
}

*Cryptococcus neoformans/genetics/isolation & purification
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Recombinases/genetics/metabolism
*Cryptococcosis/diagnosis/microbiology
Humans
Real-Time Polymerase Chain Reaction/methods
Point-of-Care Testing
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41660305,
year = {2026},
author = {Zhang, W and Jiang, A and Jia, BK and Jin, Y and Chen, Y and Li, Z and Liao, Y and Zhang, H and Lin, Z and Fang, X and Wang, L},
title = {Progress in RNA-Targeted Therapeutics for Human Diseases.},
journal = {MedComm},
volume = {7},
number = {2},
pages = {e70607},
pmid = {41660305},
issn = {2688-2663},
abstract = {RNA-targeted therapy is reshaping molecular medicine by shifting the traditional "protein-centric" view toward an "RNA-regulatory network" paradigm. Beyond carrying genetic information, RNA plays essential roles in posttranscriptional regulation, signaling pathways, and epigenetic modulation. Advances in high-throughput sequencing, structural biology, and delivery technologies have accelerated the development of diverse RNA therapeutics, including antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA) modulators, messenger RNA (mRNA) therapeutics, aptamers, short hairpin RNA, and CRISPR/Cas-guided single-guide RNAs. However, a concise comparison of these major RNA modalities and the translational barriers that limit their broader clinical application is still lacking. This review outlines the mechanisms and representative applications of these RNA-based strategies in gene silencing, editing, protein replacement, immune activation, and targeted drug delivery. Special emphasis is placed on ASOs and siRNAs for neurological, metabolic, and infectious diseases, as well as mRNA therapeutics that are transforming vaccine development. Common challenges-such as in vivo stability, delivery efficiency, and immune activation-are also discussed. Finally, we highlight how chemical modification, nanotechnology, and artificial intelligence-assisted design are enhancing the specificity, stability, and safety of RNA therapeutics, providing a framework for advancing next-generation precision RNA medicine.},
}

@article {pmid41616513,
year = {2026},
author = {Xu, L and Jin, J and Lyu, W and Liang, X and Wang, Q and Zhang, J and Luo, Y and Chen, J and Lu, H and Li, X and Shen, F},
title = {Point-of-care profiling of H. pylori virulence and antibiotic resistance from endoscopic biopsies using an integrated restriction enzyme-CRISPR microfluidic platform.},
journal = {Biosensors & bioelectronics},
volume = {299},
number = {},
pages = {118438},
doi = {10.1016/j.bios.2026.118438},
pmid = {41616513},
issn = {1873-4235},
mesh = {*Helicobacter pylori/genetics/pathogenicity/drug effects/isolation & purification ; Humans ; *Helicobacter Infections/microbiology/diagnosis/drug therapy ; *Drug Resistance, Bacterial/genetics ; Point-of-Care Systems ; Anti-Bacterial Agents/pharmacology ; Virulence/genetics ; CRISPR-Cas Systems ; Lab-On-A-Chip Devices ; Biopsy ; Biosensing Techniques ; Clarithromycin/pharmacology ; Levofloxacin/pharmacology ; Polymorphism, Single Nucleotide ; Gastric Mucosa/microbiology/pathology ; },
abstract = {Endoscopically obtained gastric mucosal biopsies contain abundant molecular information that could inform Helicobacter pylori (H. pylori) eradication therapy. However, the lack of point-of-care tools for processing solid tissue limits rapid, on-site genotyping, leading to empirical therapy and higher eradication failure rates. Here, we present the SlipChip-based On-site and User-friendly Testing (SCOUT) together with the restriction enzyme-assisted CRISPR/Cas12a (RCut) method for point-of-care genotyping from raw endoscopic gastric biopsies. This fully automated platform enables analysis of key virulence genes and resistance-associated single-nucleotide variants (SNVs) at mutant allele frequencies down to 0.1 %. Results were available within 1 h, enabling actionable molecular profiling during the same endoscopic encounter. In 159 clinical validations, SCOUT showed concordant virulence profiling with quantitative PCR (κ = 0.978) and accurately detected clarithromycin and levofloxacin resistance mutations with up to 100 % sensitivity and specificity. These results demonstrate that SCOUT bridges the gap between diagnostic sampling and informed eradication strategies, providing a scalable framework for point-of-care molecular genotyping.},
}

*Helicobacter pylori/genetics/pathogenicity/drug effects/isolation & purification
Humans
*Helicobacter Infections/microbiology/diagnosis/drug therapy
*Drug Resistance, Bacterial/genetics
Point-of-Care Systems
Anti-Bacterial Agents/pharmacology
Virulence/genetics
CRISPR-Cas Systems
Lab-On-A-Chip Devices
Biopsy
Biosensing Techniques
Clarithromycin/pharmacology
Levofloxacin/pharmacology
Polymorphism, Single Nucleotide
Gastric Mucosa/microbiology/pathology

@article {pmid41607132,
year = {2026},
author = {Yu, K and Li, H and Hu, Y and Yu, Y and Deng, S and Yang, Y and Guo, M and Li, M and Zhe, M and He, H and Fan, C},
title = {Integrating transcriptomics and high-throughput gene editing uncovers shoot apical meristem regulators in Brassica napus.},
journal = {Plant physiology},
volume = {200},
number = {2},
pages = {},
doi = {10.1093/plphys/kiag032},
pmid = {41607132},
issn = {1532-2548},
support = {2022YFD1200400//National Key Research and Development Program of China/ ; 2023YFF1000700//National Key Research and Development Program of China/ ; 31671279//National Natural Science Foundation of China/ ; 2024BBA001//Agricultural Gene Editing Platform Technology and Breeding Research & Development/ ; },
mesh = {*Brassica napus/genetics/growth & development ; *Meristem/genetics/growth & development ; *Gene Editing/methods ; *Transcriptome ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Plant Shoots/genetics/growth & development ; Plant Proteins/genetics/metabolism ; Mutation ; },
abstract = {The shoot apical meristem (SAM) determines plant architecture, but the key components of its regulatory network remain elusive in rapeseed (Brassica napus L.). Here, we integrated transcriptomic profiling of 3 multilocular silique mutants (Bnaclv1, Bnaclv2, and Bnaclv3) across key SAM development stages (IM, stage6, and stage8) with large-scale CRISPR/Cas9 functional screening to identify regulators of SAM maintenance. Differential gene expression and GO enrichment highlighted genes significantly associated with meristem development processes. Weighted gene co-expression network analysis of stage-specific transcriptomes identified 42 candidate genes potentially related to SAM development. To enable systematic functional screening, we established a high-throughput multiplex CRISPR/Cas9 pipeline, simultaneously targeting 198 sites across 42 candidate genes through optimized sgRNA design and pooled transformation. We successfully obtained mutants for 25 genes with homozygous mutants for 9 genes. Phenotypic analysis demonstrated that mutants of BnaSCPL family genes (SCPL29, SCPL44, and SCPL45) exhibited a multi-stem phenotype and disrupted SAM organization. Mechanistic studies revealed that BnaSCPL mutations disrupt the canonical CLV3/WUS feedback loop, uncovering their roles in SAM homeostasis. Additionally, knockout of BnaLFY homologs caused permanent vegetative state and sterility, demonstrating their conserved role in floral meristem identity in Brassica napus. Collectively, our study not only elucidates the critical function of BnaSCPLs in SAM maintenance but also establishes a regulatory framework for understanding meristem phase transitions in B. napus, providing potential targets for crop architecture improvement.},
}

*Brassica napus/genetics/growth & development
*Meristem/genetics/growth & development
*Gene Editing/methods
*Transcriptome
Gene Expression Profiling
Gene Expression Regulation, Plant
CRISPR-Cas Systems
Plant Shoots/genetics/growth & development
Plant Proteins/genetics/metabolism
Mutation

@article {pmid41591771,
year = {2026},
author = {Chen, D and Zhu, B and Zhou, Y and Fang, Z and Zhu, Z and Chen, C and Shen, T},
title = {Dual-mode CRISPR/Cas12a-mediated alkaline phosphatase detection (CAD) biosensor.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {6},
pages = {1239-1247},
doi = {10.1039/d5ay01195g},
pmid = {41591771},
issn = {1759-9679},
mesh = {*Alkaline Phosphatase/analysis/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Immunoassay/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Alkaline phosphatase (ALP), a crucial biomarker for hepatobiliary disorders, bone diseases, and cancer progression, requires ultrasensitive detection methods to meet clinical diagnostic requirements. Current methodologies predominantly depend on single-readout mechanisms that fail to address the growing requirements of sensitivity, operational simplicity, and adaptability to resource-limited settings. Herein, we present a CRISPR/Cas12a-mediated ALP detection (CAD) isothermal amplification system that overcomes these challenges through a novel dual-signal (fluorescence and lateral flow immunoassay (LFIA)) readout mechanism. The system features a rationally engineered hairpin DNA probe (HPP) that initiates Klenow (exo-)-driven polymerase elongation upon ALP recognition, subsequently activating Cas12a's trans-cleavage activity for exponential signal amplification. With fluorescence readout, this cascade amplification strategy achieves unprecedented sensitivity with a detection limit of 0.1 U L[-1] and a wide linear range (0.1-10 U L[-1]), outperforming conventional colorimetric methods by one order of magnitude while maintaining exceptional specificity against biological interferents. Furthermore, the LFIA adaptation of the readout bridges the gap between laboratory-based detection and point-of-care applications. This user-friendly adaptation enables instrument-free visual detection with a clear cut-off value of about 7 U L[-1], offering the potential to effectively differentiate pathological samples from normal physiological levels with an appropriate dilution factor for clinical samples. Our dual-mode biosensing strategy not only enables high-precision quantitative analysis in clinical settings but also facilitates rapid qualitative detection under resource-limited conditions, thus offering significant potential for early disease diagnosis and long-term therapeutic monitoring.},
}

*Alkaline Phosphatase/analysis/blood
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Humans
Nucleic Acid Amplification Techniques/methods
Limit of Detection
Immunoassay/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41590404,
year = {2026},
author = {Li, W and Wang, M and Wang, S},
title = {A label-free and universal CRISPR/Cas12a platform for the detection of hazardous substances in food.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {6},
pages = {1216-1223},
doi = {10.1039/d5ay01887k},
pmid = {41590404},
issn = {1759-9679},
mesh = {*CRISPR-Cas Systems/genetics ; *Food Contamination/analysis ; *Hazardous Substances/analysis ; *Food Analysis/methods ; Limit of Detection ; },
abstract = {The present study proposes a label-free and universal CRISPR/Cas12a-based platform for the detection of hazardous substances in food. A label-free reporter, termed the G4 reporter, was carefully engineered. In the absence of the target analyte, the catalytic hairpin assembly (CHA) remains inactive, thereby preventing activation of the CRISPR/Cas12a system and preserving the cleavage of the G4 reporter, which emits a label-free fluorescence signal. Conversely, in the presence of the target analyte, the CHA process is triggered, activating the CRISPR/Cas12a system, which cleaves the G4 reporter into fragments, leading to a decrease in the label-free fluorescence signal. This detection strategy follows a negative response model, wherein quantification is based on the net fluorescence difference between positive and negative controls. The developed label-free signal output modality for the CRISPR/Cas12a system offers advantages of simplicity and cost-effectiveness. Moreover, the proposed method incorporates a CHA process to facilitate signal transduction and activate the subsequent CRISPR system. The system upon integration of CHA with CRISPR functions in a dual-amplification mode, resulting in enhanced signal amplification efficiency and superior sensitivity compared to the standalone CRISPR system. Thus, the platform achieves highly sensitive detection of hazardous substances, with limits of detection (LODs) of 3.0 fg mL[-1] for aflatoxin B1 (AFB1) and 0.02 pg mL[-1] for acetamiprid (ACE). Moreover, trace amounts of AFB1 and ACE were successfully identified in real food samples. By altering the sequences of the detection components, this platform can be readily adapted for the detection of other hazardous substances in food matrices. Therefore, this work introduces a novel, label-free, universal, and sensitive CRISPR/Cas12a-based detection platform, demonstrating considerable potential for applications in food safety surveillance and public health protection.},
}

*CRISPR-Cas Systems/genetics
*Food Contamination/analysis
*Hazardous Substances/analysis
*Food Analysis/methods
Limit of Detection

@article {pmid41554880,
year = {2026},
author = {Bot, JF and Zhao, Z and Li, M and Kammeron, D and Shang, P and Geijsen, N},
title = {Temporal dynamics of collateral RNA cleavage by LbuCas13a in human cells.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {233},
pmid = {41554880},
issn = {2399-3642},
support = {NNF21CC0073729//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; W.OR18-11//Prinses Beatrix Spierfonds/ ; 15804//Netherlands Organisation for Scientific Research | Stichting voor de Technische Wetenschappen (Technology Foundation STW)/ ; 201706890022//China Scholarship Council (CSC)/ ; 202206300033//China Scholarship Council (CSC)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA Cleavage ; *CRISPR-Associated Proteins/metabolism/genetics ; *RNA/metabolism/genetics ; HEK293 Cells ; Ribonucleoproteins/metabolism/genetics ; Apoptosis/genetics ; },
abstract = {CRISPR-Cas13 exclusively targets RNA. In prokaryotes, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA, but in eukaryotic cells collateral cleavage activity has been limited. Here we report that LbuCas13a exhibits strong collateral RNA cleavage activity in human cells when delivered as ribonucleoprotein, independent of cell line and targeting both exogenous and endogenous transcripts. Collateral RNA cleavage starts within 50 minutes of ribonucleoprotein delivery resulting in major alterations to the total RNA profile. In response to the collateral RNA cleavage, cells upregulate genes associated with the stress and innate immune response, ultimately leading to apoptotic cell death. This enables us to use LbuCas13a as a flexible and repeatable target-RNA-specific cell elimination tool. Finally, using both total RNA sequencing and Nanopore sequencing, we find that LbuCas13a activation leads to rapid and near-global depletion of cytoplasmic RNAs, and that cleavage occurs at specific nucleotide positions.},
}

Humans
*CRISPR-Cas Systems
*RNA Cleavage
*CRISPR-Associated Proteins/metabolism/genetics
*RNA/metabolism/genetics
HEK293 Cells
Ribonucleoproteins/metabolism/genetics
Apoptosis/genetics

@article {pmid41459814,
year = {2026},
author = {Celle, M and Aniorte, S and Issa, AR and Falabregue, M and Jin, H and Sanchez-Mirasierra, I and Ding, S and Soukup, SF and Seugnet, L and Liao, L and Lesca, G and Walter, L and Mollereau, B},
title = {A dwdr45 knock-out drosophila model to decipher the role of autophagy in BPAN.},
journal = {Human molecular genetics},
volume = {35},
number = {3},
pages = {},
doi = {10.1093/hmg/ddaf198},
pmid = {41459814},
issn = {1460-2083},
support = {//French Ministry of Higher Education and Research/ ; 101067877//Marie Sklodowska-Curie Action fellowship/ ; //China Scholarship Council/ ; },
mesh = {Animals ; *Autophagy/genetics ; Disease Models, Animal ; *Drosophila Proteins/genetics/metabolism ; Gene Knockout Techniques ; Drosophila melanogaster/genetics ; CRISPR-Cas Systems ; *Neurodegenerative Diseases/genetics/pathology/metabolism ; *Carrier Proteins/genetics/metabolism ; Phenotype ; Drosophila/genetics ; Humans ; },
abstract = {Beta-propeller protein-associated neurodegeneration (BPAN) is a rare neurological disorder characterized by severe cognitive and motor impairments. BPAN is caused by de novo pathogenic variants in the WDR45 gene on the X chromosome. WDR45 gene encodes the protein WDR45/WIPI4, a known regulator of autophagy. A defective autophagy has been observed in cellular models of BPAN disease and is associated with neurological dysfunctions in wdr45 knockout (KO) mice. However, it remains unclear whether the autophagic defect directly contributes to all WDR45 loss-induced phenotypes or whether other WDR45-dependent cellular functions are involved. To investigate this, we generated a CRISPR/Cas9-mediated KO of CG11975 (dwdr45 KO), the Drosophila homolog of WDR45. Our analysis revealed that dwdr45 KO flies display BPAN-like phenotypes, including impaired locomotor function, seizure-like behavior, autophagy dysregulation and iron dyshomeostasis. Additionally, dwdr45 KO flies exhibit shortened lifespan compared to control flies. These findings demonstrate that dwdr45 KO fly is a relevant in-vivo model for investigating the key cellular and molecular mechanisms underlying BPAN-associated phenotypes. Here we showed that induction of autophagy in dwdr45 KO flies improved both the shortened lifespan and the seizure-like behavior, but did not restore locomotor function. This suggests that defective autophagy contributes to some, but not all, aspects of the phenotypes resulting from loss of dWdr45 function.},
}

Animals
*Autophagy/genetics
Disease Models, Animal
*Drosophila Proteins/genetics/metabolism
Gene Knockout Techniques
Drosophila melanogaster/genetics
CRISPR-Cas Systems
*Neurodegenerative Diseases/genetics/pathology/metabolism
*Carrier Proteins/genetics/metabolism
Phenotype
Drosophila/genetics
Humans

@article {pmid41330380,
year = {2026},
author = {Feng, C and Peets, EM and Zhou, Y and Crepaldi, L and Usluer, S and Dunham, A and Braunger, JM and Su, J and Strauss, ME and Muraro, D and Xian Cheam, KA and Bonder, MJ and Nogales, EG and Cooper, S and Bassett, A and Leonard, S and Gu, Y and Fussing, B and Burke, D and Parts, L and Stegle, O and Velten, B},
title = {A genome-scale single-cell CRISPRi map of trans gene regulation across human pluripotent stem cell lines.},
journal = {Cell genomics},
volume = {6},
number = {2},
pages = {101076},
doi = {10.1016/j.xgen.2025.101076},
pmid = {41330380},
issn = {2666-979X},
mesh = {Humans ; *Single-Cell Analysis/methods ; *Pluripotent Stem Cells/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation/genetics ; *Genome, Human/genetics ; Quantitative Trait Loci/genetics ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Population-scale resources of genetic, molecular, and cellular information form the basis for understanding human genomes, charting the heritable basis of disease and tracing the effects of mutations. Pooled perturbation assays, probing the effect of many perturbations coupled with single-cell RNA sequencing (scRNA-seq) readout, are especially potent references for interpreting disease-linked mutations or gene-expression changes. However, the utility of existing maps has been limited by the comprehensiveness of perturbations conducted and the relevance of their cell-line context. Here, we present a genome-scale CRISPR interference perturbation map with scRNA-seq readout across many genetic backgrounds in human pluripotent cells. We map trans expression changes induced by knockdowns and characterize their variation across donors, with expression quantitative trait loci linked to higher genetic modulation of perturbation effects. This study pioneers population-scale CRISPR perturbations with high-dimensional readouts, which will fuel the future of effective modulation of cellular disease phenotypes.},
}

Humans
*Single-Cell Analysis/methods
*Pluripotent Stem Cells/metabolism
*CRISPR-Cas Systems/genetics
*Gene Expression Regulation/genetics
*Genome, Human/genetics
Quantitative Trait Loci/genetics
Cell Line
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41659011,
year = {2025},
author = {Wang, B and Lu, J and Zhang, X and Hu, R and Ma, H},
title = {Advances in nanomaterial-mediated CRISPR/Cas delivery: from lipid nanoparticles to vesicle-derived systems.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {13},
number = {},
pages = {1669104},
pmid = {41659011},
issn = {2296-4185},
abstract = {Gene and genome editing therapies are increasingly connected with nanomaterials, which protect and transport fragile nucleic acids and CRISPR/Cas systems through biological barriers safely and accurately. This review discusses how different nanocarriers, including lipid-based, polymeric, inorganic, and vesicle-derived systems, can improve delivery efficiency, cell targeting, endosomal escape, and intracellular movement for gene and genome editing. It summarizes findings from early clinical and preclinical studies, comparing several carrier types such as ionizable lipid nanoparticles, polymeric nanoparticles, micelles, gold and silica nanostructures, and engineered extracellular vesicles. The review also explains how specific design factors, such as surface ligands, charge modification, PEGylation, and stimulus-responsive behaviors, influence biodistribution, and improve on-target efficiency while lowering immune responses and off-target effects. Ethical and regulatory concerns for in vivo editing are highlighted, along with current methods used to study nano-bio interactions. Among these carriers, ionizable lipid nanoparticles show the most advanced performance for delivering nucleic acids and CRISPR systems. However, new polymer-based and exosome-inspired carriers are progressing rapidly for repeated and targeted applications. Hybrid and responsive systems may also enable better spatial and temporal control of editing. Future research should focus on stronger in vivo potency testing, improved biocompatibility evaluation, and standardized manufacturing to ensure clinical safety and reliability.},
}

@article {pmid41658853,
year = {2026},
author = {Rahimian, M and Aghazadeh-Soltan-Ahmadi, M and Panahi, B},
title = {In silico exploration of the genomic repertoire of Iranian aquatic bacteria: Prophage carriage, bioactive compound potential, CRISPR-Cas immunity, and integrated defensive-metabolic islands.},
journal = {Biochemistry and biophysics reports},
volume = {45},
number = {},
pages = {102452},
pmid = {41658853},
issn = {2405-5808},
abstract = {The unique and underexplored aquatic ecosystems of Iran represent a significant reservoir of microbial diversity. This study presents the first comprehensive genomic survey of 38 native Iranian bacterial strains from hypersaline lakes and wetlands, integrating in silico analyses of their secondary metabolome, bacteriocin potential, resident prophages, and genomic architecture. Our genome mining revealed a prolific capacity for secondary metabolite production, identifying dozens of biosynthetic gene clusters (BGCs). Ectoine biosynthesis was ubiquitous, underscoring its role as a key osmoprotectant, while diverse BGCs for terpenes, polyketides, and hybrid metabolites were also prevalent. Concurrently, we identified a wide array of ribosomally synthesized and post-translationally modified peptides (RiPPs), including known bacteriocins. Furthermore, we characterized eight high-quality prophages integrated within these genomes, encoding auxiliary genes such as carbohydrate-active enzymes (CAZymes) and putative anti-CRISPR (ACR) proteins. The bacterial hosts themselves were equipped with robust defense systems, with CRISPR-Cas loci, predominantly Type I, detected in most strains. Crucially, we identified multi-functional genomic islands that physically link BGCs with defense systems (e.g., CRISPR-Cas, restriction-modification) and prophage regions. We propose the "Fortress Hypothesis" to explain this architecture, wherein the co-localization of metabolite production and defense machinery protects metabolic investment against phage predation and genetic loss. This integrative genomic arrangement highlights a sophisticated co-evolutionary strategy for survival in extreme environments. Our findings position these indigenous bacteria as a promising genetic repository for discovering novel bioactive compounds, enzymes, and biotechnological tools, with implications for antibiotic discovery, CRISPR modulation, and understanding adaptive microbial genomics in extreme niches.},
}

@article {pmid41658434,
year = {2026},
author = {Han, Y and Chen, R and Shentu, X},
title = {Advances and challenges of CRISPR/Cas gene editing for corneal diseases.},
journal = {Advances in ophthalmology practice and research},
volume = {6},
number = {1},
pages = {68-79},
pmid = {41658434},
issn = {2667-3762},
abstract = {BACKGROUND: Corneal diseases are a major cause of global visual impairment, and current treatments remain inadequate for severe or refractory cases. The CRISPR/Cas system offers robust and precise gene-editing capabilities, yet its therapeutic potential for corneal disorders remains largely unexplored.

MAIN TEXT: This narrative review introduces the CRISPR/Cas system and summarizes its recent advances in treating various corneal diseases, including inherited corneal dystrophies, infectious keratitis, corneal injury, and pathological neovascularization. We outline emerging preclinical and clinical studies, and analyze key issues that should be addressed for translation, including administration strategies, vector platform optimization and the mitigation of off-target toxicity.

CONCLUSIONS: This review provides a comprehensive and integrated overview of the current translational directions and challenges of CRISPR/Cas technology in corneal diseases from a novel perspective. It offers valuable guidance for future research and may accelerate the development of gene-editing therapies toward clinical application.},
}

@article {pmid41656481,
year = {2026},
author = {Laxmi, V and Verma, S and Kumar, M and Venkatesh, V and Mohit, and Maury, J and Mohd, S and Tripathi, S},
title = {Lactic Acid Bacteria-derived Bacteriocins: A Promising Antimicrobial Strategy against Multidrug-resistant for Neonatal Sepsis Pathogens.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41656481},
issn = {1867-1314},
abstract = {Neonatal sepsis continues to pose rising critical challenges to global health, particularly in low and middle-income countries, where it accounts for a substantial proportion of neonatal morbidity and mortality. The increasing prevalence of multidrug-resistant (MDR) pathogens in neonatal sepsis has significantly weakened the efficacy of conventional antibiotics, necessitating urgent exploration of alternative antimicrobial therapies for better clinical outcomes. Emerging research interest is growing to develop the microbial-derived peptides as novel antimicrobial agents, particularly "bacteriocins." In comparison to traditional antibiotics, many bacteriocins exhibit narrow-spectrum action, enabling them to inhibit specific pathogens without disrupting the host microbiota. Recent studies have highlighted the remarkable potential of lactic acid bacteria (LAB) derived bacteriocins in combating MDR pathogens responsible for neonatal sepsis. In this review, we compile current literature on the in vitro antimicrobial activity of LAB-derived bacteriocins, molecular diversity, mechanisms of action and clinical potential. Special attention is given to bacteriocins produced by LAB associated with the milk-derived microbiota, whose natural protective functions could be particularly beneficial for immunocompromised newborns. Additionally, we discuss the physicochemical properties of microbial peptides, including thermal stability, enzymatic resistance, and pH tolerance, which support their suitability for pharmaceutical applications. Overall, LAB-derived bacteriocins represent a novel, biocompatible, and complementary antimicrobial strategy integrated with conventional antibiotics to fight against MDR in neonatal sepsis. Further research and multicentric clinical trials are necessary to fully explore its compliance and efficacy as a future antimicrobial agent in neonatal medicine to underscore their potential as adjunct or preventive biotherapeutics in NICU settings.},
}

@article {pmid41656299,
year = {2026},
author = {Fang, T and Bogensperger, L and Feer, L and Allam, A and Bezshapkin, V and Balázs, Z and von Mering, C and Sunagawa, S and Krauthammer, M and Schwank, G},
title = {Uncovering Cas9 PAM diversity through metagenomic mining and machine learning.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-69098-5},
pmid = {41656299},
issn = {2041-1723},
abstract = {Recognition of protospacer adjacent motifs (PAMs) is crucial for target site recognition by CRISPR-Cas systems. In genome editing applications, the requirement for specific PAM sequences at the target locus imposes substantial constraints, driving efforts to search for novel Cas9 orthologs with extended or alternative PAM compatibilities. Here, we present CRISPR-PAMdb, a comprehensive and publicly accessible database compiling Cas9 protein sequences from 3.8 million bacterial and archaeal genomes and PAM profiles from 7.4 million phage and plasmid sequences. Through spacer-protospacer alignment, we infer consensus PAM preferences for 8003 unique Cas9 clusters. To extend PAM discovery beyond traditional alignment-based approaches, we develop CICERO, a machine learning model predicting PAM preferences directly from Cas9 protein sequences. Built on the ESM2 protein language model and trained on the CRISPR-PAMdb database, CICERO achieves an average cosine similarity of 0.69 on test data and 0.75 on experimentally validated Cas9 orthologs. For Cas9 clusters where alignment-based predictions are infeasible, CICERO generates PAM profiles for an additional 50,308 Cas9 proteins, including 17,453 high-confidence predictions with CICERO confidence scores above 0.8. Together, CRISPR-PAMdb and CICERO enable large-scale exploration of PAM diversity across Cas9 proteins, accelerating design of next-generation CRISPR-Cas9 tools for precise genome engineering.},
}

@article {pmid41656257,
year = {2026},
author = {Ruis, BL and Ward, H and Myers, CL and Bielinsky, AK and Hendrickson, EA},
title = {SETDB1/ATF7IP regulate the precise genome engineering of HUSH-regulated genes.},
journal = {Epigenetics & chromatin},
volume = {19},
number = {1},
pages = {10},
pmid = {41656257},
issn = {1756-8935},
support = {AG077174//National Institute of Aging/ ; AG077174//National Institute of Aging/ ; R35GM141805//National Institutes of General Medical Sciences/ ; GM088351/GM/NIGMS NIH HHS/United States ; CA154461/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Histone-Lysine N-Methyltransferase/metabolism/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Recombinational DNA Repair ; DNA Breaks, Double-Stranded ; HEK293 Cells ; },
abstract = {BACKGROUND: The use of programmable nucleases has transformed genome editing and functional genomics. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) was developed such that targeted genomic lesions [usually DNA double-stranded breaks (DSBs)] could be introduced in vivo with ease and precision. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. Because DSBs can lead to genomic instability, however, a large amount of effort has been invested in methodologies (e.g., base editors) that only require nicking the chromosomal DNA on one strand. Indeed, we have demonstrated in human cells that oligodeoxynucleotide (ODN)-mediated PGE using nickase variants of Cas9 can proceed by at least two HDR subpathways termed synthesis-dependent strand annealing (SDSA) and single-stranded DNA incorporation (ssDI). Which pathway is utilized is determined by which chromosomal strand (sense or anti-sense/Watson or Crick) is nicked and by the strandedness (sense or anti-sense/Watson or Crick) of the donor ODN.

RESULTS: While the mechanism of mammalian SDSA is moderately well understood, that of ssDI is not. To gain genetic insight into ssDI, we carried out a genome-wide CRISPR knockout screen to identify those genes which, when absent, enable increased ssDI. This screen identified the protein lysine methyl transferase (PKMT) Su(var)3-9, enhancer-of-zeste and trithorax (SET) domain bifurcated histone lysine methyltransferase 1 (SETDB1):activating transcription factor 7-interacting protein (ATF7IP) heterodimer and the downstream human silencing hub (HUSH) complex as strong negative regulators of ssDI. Consistent with their well-known biological effects, the negative regulation of ssDI by SETDB1/ATF7IP and HUSH was specific for transgenic reporters and for a HUSH-regulated single-copy gene, but was not observed at other (non-HUSH regulated) single-copy endogenous loci.

CONCLUSIONS: In toto, these experiments underscore the profound impact that chromatin modifiers - and by extension, chromatin structure - have on PGE outcomes. Specifically, we have identified SETDB1/ATF7IP and the HUSH complex as major negative regulators of the HDR subpathway, ssDI, when the target is a transgene. These experiments are a proof-of-principle that chromatin can act as a potent barrier to genetic recombination and they strongly support the feasibility of extending similar chromatin modulating strategies to enhance PGE efficiency at endogenous single-copy loci.},
}

Humans
*Histone-Lysine N-Methyltransferase/metabolism/genetics
*Gene Editing/methods
CRISPR-Cas Systems
Recombinational DNA Repair
DNA Breaks, Double-Stranded
HEK293 Cells

@article {pmid41655694,
year = {2026},
author = {Saito, A and Tankou, S and Ishii, K and Sakao-Suzuki, M and Oh, EC and Murdoch, H and Namkung, H and Adelakun, S and Furukori, K and Fujimuro, M and Salomoni, P and Maul, GG and Hayward, GS and Tang, Q and Yolken, RH and Houslay, MD and Katsanis, N and Kosugi, I and Yang, K and Kamiya, A and Ishizuka, K and Sawa, A},
title = {Cytomegalovirus (CMV)-encoded immediate early 1 (IE1) protein perturbs neural progenitor proliferation via interfering with host PML-DISC1 interaction.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {111269},
doi = {10.1016/j.jbc.2026.111269},
pmid = {41655694},
issn = {1083-351X},
abstract = {Congenital CMV infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remains unknown. Here we discovered the molecular interplay of key host (DISC1 and PML) and viral (IE1) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation in congenital CMV infection. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. We further observed that CMV-encoded IE1 protein interferes with host PML-DISC1 interaction, resulting in disturbance of the Notch pathway in vitro and in embryonic brains. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.},
}

@article {pmid41654278,
year = {2026},
author = {Wang, X and You, J and Li, X and Xu, Y and Li, Z and Wang, L},
title = {Limitations of traditional mycotoxin control and biotechnological advances toward sustainable solutions.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108836},
doi = {10.1016/j.biotechadv.2026.108836},
pmid = {41654278},
issn = {1873-1899},
abstract = {Mycotoxins are harmful fungal metabolites that contaminate food and feed, posing serious health and economic risks worldwide. Traditional control methods often fall short due to inefficiency and safety concerns, prompting the development of innovative biotechnological approaches. This review explores recent advances in mycotoxin management, focusing on engineered microbes for targeted degradation, nanotechnology-based detection and removal systems, phage therapy targeting toxin-producing fungi, CRISPR-Cas gene editing of mycotoxin biosynthesis pathways, and plant-microbe interactions that suppress fungal growth. Additionally, enzyme immobilization strategies are highlighted for improving enzyme stability and reusability in detoxification processes. These integrated biotechnological tools offer promising, sustainable solutions to mitigate mycotoxin contamination, enhancing food safety and agricultural productivity. The review also discusses current challenges and future directions for translating these advances into practical applications. Highlighting that biotechnological tools are technically feasible and increasingly close to industrial deployment in food and feed chains.},
}

@article {pmid41621146,
year = {2026},
author = {Kundu, A and Jerala, R},
title = {Small RNAs, big potential: Engineering microRNA-based synthetic gene circuits.},
journal = {Current opinion in chemical biology},
volume = {90},
number = {},
pages = {102652},
doi = {10.1016/j.cbpa.2026.102652},
pmid = {41621146},
issn = {1879-0402},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Gene Regulatory Networks ; *Synthetic Biology/methods ; Animals ; *Genetic Engineering/methods ; CRISPR-Cas Systems ; },
abstract = {MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression. Their dysregulation is closely associated with various diseases, positioning them as biomarkers of cellular state. Synthetic biology has leveraged these properties to engineer miR-based genetic circuits capable of sensing and interpreting endogenous miR levels. Early miR-OFF systems relied on reporter gene repression but were limited by ambiguous signal interpretation. Subsequent advances introduced miR-ON architectures, logic-based classifiers integrating multiple miRs, and layered regulatory strategies combining transcriptional, translational, and cleavage-based modules to enhance sensitivity and specificity. Recent innovations include CRISPR-associated miR-responsive systems and incoherent feed-forward loop (iFFL) architectures that stabilize gene expression amid cellular variability, shifting applications from passive sensing to therapeutic intervention. Despite challenges such as leakage, cellular resource resources, and delivery, progress in orthogonal miR toolkits, computational modeling, and RNA-based delivery platforms is rapidly driving miR-based circuits toward diagnostic and therapeutic applications.},
}

*MicroRNAs/genetics/metabolism
Humans
*Gene Regulatory Networks
*Synthetic Biology/methods
Animals
*Genetic Engineering/methods
CRISPR-Cas Systems

@article {pmid41577003,
year = {2026},
author = {Zade, NH and Jain, M and Garg, M and Checker, R and Ghosh, A and Khattar, E},
title = {Proteomics method for identifying POT1-associated complexes at telomeres using ChIP-Mass spectrometry.},
journal = {Methods (San Diego, Calif.)},
volume = {247},
number = {},
pages = {161-174},
doi = {10.1016/j.ymeth.2026.01.007},
pmid = {41577003},
issn = {1095-9130},
mesh = {Shelterin Complex ; *Telomere/metabolism/genetics/chemistry ; *Proteomics/methods ; Humans ; *Telomere-Binding Proteins/metabolism/genetics ; Mass Spectrometry/methods ; CRISPR-Cas Systems ; *Chromatin Immunoprecipitation/methods ; Protein Binding ; Gene Editing ; Tripeptidyl-Peptidase 1 ; },
abstract = {POT1 is the only single stranded telomere binding protein in the shelterin complex. Together with TPP1, POT1 plays a crucial role in regulating telomere length and protecting telomeres from DNA damage repair proteins. The activation of DNA damage repair proteins at telomeres can be detrimental to cells, so their activity must be suppressed. POT1 interacts with other telomeric proteins (TRF2, TRF1, TIN2 and RAP1) via its association with TPP1. These proteins function together to protect and maintain the telomeres. Despite extensive knowledge of POT1's role within the shelterin complex, the full spectrum of its interactors at the single-stranded telomeric overhang remains poorly defined. To study these interactions, we generated an endogenous Flag-tag knock-in of POT1 using the CRISPR-Cas9 gene editing system. To address the risk of unintended gene disruption associated with this technique, we conducted an in-depth characterization of the endogenously Flag-tagged POT1 clone to ensure that its telomere and TPP1 binding functions remained intact. Further, we performed proteomic profiling of the Flag-tagged POT1 within the chromatin fraction using ChIP-MS to explore its proteome. Our analysis uncovered a novel set of POT1-associated proteins at the extremes of telomeres. Given that POT1 exclusively binds to the single-stranded 3' overhang of telomeres, the proteomic data obtained indicates POT1 interactions occurring at the extreme ends of telomeres. In conclusion, our study reveals previously uncharacterized POT1 associated proteins using ChIP mass spectrometric approach, paving the way for further investigations into telomere biology and potential therapies targeting telomere regulation.},
}

Shelterin Complex
*Telomere/metabolism/genetics/chemistry
*Proteomics/methods
Humans
*Telomere-Binding Proteins/metabolism/genetics
Mass Spectrometry/methods
CRISPR-Cas Systems
*Chromatin Immunoprecipitation/methods
Protein Binding
Gene Editing
Tripeptidyl-Peptidase 1

@article {pmid41576935,
year = {2026},
author = {Hommersom, MP and Puvogel, S and Scheefhals, N and Carpentiero, E and Bouma, M and van Beusekom, E and Dillen, L and van de Warrenburg, BPC and Nadif Kasri, N and van Bokhoven, H},
title = {Human neuronal networks on micro-electrode arrays as a tool to assess genotype-phenotype correlation in CACNA1A-related disorders.},
journal = {Stem cell reports},
volume = {21},
number = {2},
pages = {102783},
doi = {10.1016/j.stemcr.2025.102783},
pmid = {41576935},
issn = {2213-6711},
mesh = {Humans ; *Genetic Association Studies/methods ; *Nerve Net/metabolism ; *Neurons/metabolism ; *Calcium Channels/genetics ; Phenotype ; CRISPR-Cas Systems/genetics ; Electrodes ; Genotype ; Calcium Channels, N-Type ; },
abstract = {CACNA1A-related disorders constitute a diverse group of neurological conditions, including ataxia, migraine, and epilepsy. Despite extensive genetic studies, clear genotype-phenotype correlations remain elusive. Moreover, next-generation sequencing has identified many variants of uncertain significance (VUS). Here, we leveraged patient-derived and CRISPR-Cas9-engineered human neuronal networks to explore relationships between CACNA1A variants and neurophysiological activity. CACNA1A haploinsufficiency induced subtle alterations in glutamatergic network activity, whereas missense variants had a more pronounced effect on overall network function. Network fingerprints were most affected from patients where ataxia co-occurred with migraine or epilepsy. Furthermore, we analyzed the impact of CRISPR-Cas9-induced VUS on network developmental trajectories. Although functional changes could not be directly linked to clinical phenotypes, all tested variants induced measurable alterations in neuronal network function, supporting their classification as likely pathogenic. These findings highlight the potential of human neuronal networks as a translational model for evaluating CACNA1A variant effects and improving clinical variant interpretation.},
}

Humans
*Genetic Association Studies/methods
*Nerve Net/metabolism
*Neurons/metabolism
*Calcium Channels/genetics
Phenotype
CRISPR-Cas Systems/genetics
Electrodes
Genotype
Calcium Channels, N-Type

@article {pmid41565761,
year = {2026},
author = {Beauchemin, KS and Supattapone, S},
title = {Genome-wide screens identify core regulators of cell surface prion protein expression.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {5895},
pmid = {41565761},
issn = {2045-2322},
support = {1R37NS125431/NS/NINDS NIH HHS/United States ; 5T32AI007519/NH/NIH HHS/United States ; 1R37NS125431/NS/NINDS NIH HHS/United States ; 1R37NS125431/NS/NINDS NIH HHS/United States ; 5T32AI007519/NH/NIH HHS/United States ; },
mesh = {*Neurons/metabolism/cytology ; Humans ; CRISPR-Cas Systems ; *Prion Proteins/genetics/metabolism ; Cell Differentiation/genetics ; *Gene Expression Regulation ; *PrPC Proteins/genetics/metabolism ; Animals ; Cell Line ; Cell Membrane/metabolism ; },
abstract = {Expression of the cellular prion protein, PrP[C], on the surface of neurons plays an important role in the pathogenesis of prion disease. We performed genome-wide CRISPR/Cas9 knockout screens in prion-infectible cells of neuronal origin (CAD5) to identify regulators of cell surface PrP[C] expression. We identified and validated 46 positive and 21 negative regulators of cell surface PrP[C] expression in undifferentiated CAD5 cells. Pathway analysis of the screening dataset showed that genes involved in the glycophosphatidylinositol (GPI) anchor and N-glycosylation biosynthetic pathways were overrepresented as positive regulators of cell surface PrP[C]. We also sought to determine whether the same or different genes regulate cell surface PrP[C] in CAD5 cells that have been differentiated to a more neuronal state and validated 41 positive and 13 negative regulators of CAD5 cell surface PrP[C] expression in the differentiated state. We identified 23 core genes as shared between the undifferentiated and differentiated cell states, including many positive regulators involved in GPI anchor biosynthesis. Intriguingly, unique regulators were also identified in the undifferentiated and differentiated cell states, suggesting that some mechanisms regulating cell surface PrP[C] expression in CAD5 cells are dependent on cell state. This list of core genes involved in regulating cell surface PrP[C] expression in a prion-susceptible, neuron-like cell type offers a valuable guide for future research and may help identify potential therapeutic targets for prion disease and other neurodegenerative diseases.},
}

*Neurons/metabolism/cytology
Humans
CRISPR-Cas Systems
*Prion Proteins/genetics/metabolism
Cell Differentiation/genetics
*Gene Expression Regulation
*PrPC Proteins/genetics/metabolism
Animals
Cell Line
Cell Membrane/metabolism

@article {pmid41524400,
year = {2026},
author = {Clarke, JE and Faulkner, TR and Seipke, RF},
title = {A platform for CRISPRi-seq in Streptomyces albidoflavus.},
journal = {mBio},
volume = {17},
number = {2},
pages = {e0306525},
doi = {10.1128/mbio.03065-25},
pmid = {41524400},
issn = {2150-7511},
support = {BB/T014962/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Streptomyces/genetics ; Operon ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Bacterial ; *CRISPR-Cas Systems ; Genomics/methods ; },
abstract = {UNLABELLED: Streptomyces produce a multitude of secondary metabolites, which have been exploited in drug discovery campaigns for more than three-quarters of a century. Our understanding of microbial physiology has been revolutionized by genome sequencing and large-scale functional studies. Technology for genome-wide investigations in Streptomyces species, however, has lagged behind that for other bacterial systems, hindering exploitation of unprecedented quantities of genomic data. Here, we develop a platform for en masse clustered regularly interspaced short palindromic repeats interference sequencing (CRISPRi-seq) for Streptomyces spp. By performing CRISPRi-seq with 2,160 unique sgRNAs targeting all operons (432 operons) encoding membrane transporters (629 genes) representing 1.1Mb of the 6.8Mb genome for S. albidoflavus, combined with hit validation, we discovered that only a small proportion (13 of 432 operons, 25 kb) contribute positively to fitness. Our work provides both a first-in-class platform for high-throughput functional genomics and a generalized blueprint for en masse screens in Streptomyces species.

IMPORTANCE: Streptomyces bacteria are prolific producers of clinically essential natural products, yet high-throughput tools to systematically interrogate their genomes remain underdeveloped. By establishing a robust CRISPRi-seq platform for en masse functional screening in Streptomyces albidoflavus, our work closes a critical technological gap in Streptomyces functional genomics. Our study not only identifies a small subset of transporter operons essential for fitness but also introduces a scalable, generalizable approach for dissecting gene function. This platform will accelerate systems-level understanding of an industrially and medically important genus.},
}

*Streptomyces/genetics
Operon
*Clustered Regularly Interspaced Short Palindromic Repeats
Genome, Bacterial
*CRISPR-Cas Systems
Genomics/methods

@article {pmid41467797,
year = {2026},
author = {Ji, HJ and Jang, A-Y and Han, SH and Kim, M-K and Lamien, CE and Wijewardana, V and Ahn, KB and Kim, K-H and Song, JY and Seo, HS},
title = {Accurate serotype identification of Streptococcus pneumoniae using nanopore Cas9-targeted serotype identification (nCATSerotyping).},
journal = {Journal of clinical microbiology},
volume = {64},
number = {2},
pages = {e0098425},
doi = {10.1128/jcm.00984-25},
pmid = {41467797},
issn = {1098-660X},
support = {22202MFDS171//Ministry of Food and Drug Safety/ ; 2018E240600//Korea Disease Control and Prevention Agency/ ; 523140-26//Korea Atomic Energy Research Institute/ ; RS-2022-00164721//National Research Foundation of Korea/ ; CRP D32039//International Atomic Energy Agency/ ; },
mesh = {*Streptococcus pneumoniae/classification/genetics/isolation & purification ; Humans ; *Serotyping/methods ; *Pneumococcal Infections/microbiology ; Serogroup ; *Nanopore Sequencing/methods ; Republic of Korea ; CRISPR-Cas Systems ; Pneumococcal Vaccines ; },
abstract = {Streptococcus pneumoniae (pneumococcus) is a leading cause of community-acquired pneumonia and invasive diseases, particularly among children and the elderly. The introduction of pneumococcal conjugate vaccines has significantly reduced invasive pneumococcal disease, but the prevalence of non-vaccine serotypes and newly emerging serotypes is increasing globally. Thus, accurate serotyping is essential for epidemiological surveillance and the development of next-generation multivalent pneumococcal vaccines. Conventional serotyping methods, including multiplex polymerase chain reaction (mPCR), monoclonal antibody (mAb) assays, and Quellung reaction using rabbit antisera, are limited by serotype coverage and cross-reactivity, making the detection of new or emerging serotypes challenging. In this study, we developed a nanopore Cas9-targeted serotyping (nCATSerotyping) platform, which employs Cas9-mediated enrichment of the capsular polysaccharide synthesis locus followed by Oxford Nanopore sequencing. Applying this method to 276 clinical pneumococcal isolates collected in South Korea (2018-2020), we achieved a serotyping success rate of 97.10% (268/276), significantly outperforming conventional methods such as mAb and mPCR, which identified only 76.45% (211/276) of isolates. Whole-genome sequencing of the remaining eight non-typeable isolates revealed them to be non-pneumococcal (oral streptococci), confirming 100% accuracy for S. pneumoniae serotyping. Importantly, our method identified emerging and underrepresented serotypes, including serotype 13 and null capsule clade strains. nCATSerotyping offers a rapid, accurate, and comprehensive solution for pneumococcal serotyping, with significant advantages in identifying novel and non-typeable strains. This scalable platform will be a valuable tool for global serotype surveillance and next-generation multivalent pneumococcal vaccine development.IMPORTANCEAccurate pneumococcal serotyping is critical for vaccine development and epidemiological surveillance, particularly as non-vaccine serotypes emerge following widespread pneumococcal conjugate vaccine implementation. Current serotyping methods face significant limitations in coverage and accuracy, identifying around 76% of pneumococcal isolates and failing to detect emerging serotypes like serotype 13 and null capsule clades. The nanopore Cas9-targeted serotyping platform addresses these critical gaps by achieving 100% serotyping accuracy for confirmed Streptococcus pneumoniae isolates while identifying previously undetectable strains that conventional methods missed. This comprehensive approach is essential for monitoring vaccine effectiveness, understanding serotype replacement patterns, and informing next-generation vaccine development strategies. Furthermore, the identification of misclassified oral streptococci highlights the diagnostic precision needed for accurate pneumococcal surveillance, ensuring that epidemiological data accurately reflect true pneumococcal disease burden and serotype distribution patterns.},
}

*Streptococcus pneumoniae/classification/genetics/isolation & purification
Humans
*Serotyping/methods
*Pneumococcal Infections/microbiology
Serogroup
*Nanopore Sequencing/methods
Republic of Korea
CRISPR-Cas Systems
Pneumococcal Vaccines

@article {pmid41654255,
year = {2026},
author = {Saini, H and Yadav, J and Pandey, S and Kumar, A and Nanda, D and Sachan, S and Kaushik, JJ},
title = {Field-deployable CRISPR-cas variants for rapid on-site detection of plant pathogens.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {365},
number = {},
pages = {113028},
doi = {10.1016/j.plantsci.2026.113028},
pmid = {41654255},
issn = {1873-2259},
abstract = {Rapid, field-deployable diagnostics are essential for effective plant disease management. Although CRISPR-Cas systems offer high sensitivity and programmability, their use in on-site plant pathogen detection has been hindered by the lack of standardized, practical workflows. Here we present implementable CRISPR-Cas diagnostic protocols using Cas12a, Cas13a, and miniature Cas variants for rapid detection of major plant pathogens. Three field-ready assays are described: (i) an RPA-Cas12a lateral-flow test for DNA pathogens, (ii) a Cas13a RT-RPA assay for RNA viruses, and (iii) an amplification-free Cas12a electrochemical biosensor suited for portable laboratories. Each protocol includes sample preparation steps, reagent formulations, incubation conditions, and troubleshooting guidance. Across platforms, detection limits of 1-100 copies µL[-1] were achieved within 20-45 min, demonstrating analytical sensitivity comparable to conventional PCR-based diagnostics while offering substantially reduced assay time and improved field deployability. We also address practical constraints including sample inhibitors, reagent stability, and biosafety and propose solutions for field implementation. These standardized workflows translate recent advances in CRISPR diagnostics into reproducible, field-deployable tools for plant health surveillance and rapid disease detection.},
}

@article {pmid41653672,
year = {2026},
author = {Bai, T and Qu, X and Pan, J and Tang, Y and Wang, L and Zhou, P and Hu, Z and Guo, Z and Zhu, Y and Zhang, Y},
title = {CRISPR-initiated exponential amplification on fluorescently-barcoded microspheres for deep learning-assisted multiplexed HPV detection.},
journal = {Biosensors & bioelectronics},
volume = {300},
number = {},
pages = {118488},
doi = {10.1016/j.bios.2026.118488},
pmid = {41653672},
issn = {1873-4235},
abstract = {Rapid, low-cost, and multiplexed nucleic acid testing is essential for human health but remains challenging. Although CRISPR-Cas systems offer high specificity, their integration into Multiplexed platforms suitable for near-patient testing has been limited. Here, we introduce a biosensing platform that combines CRISPR-initiated enzymatic amplification with quantum-dot encoded microbeads and deep-learning based image analysis for Multiplexed detection of human papillomavirus (HPV) DNA. The high specificity of CRISPR/Cas9 first triggers an exponential amplification. The products are then specifically captured on the microbead surface for localized fluorescent readout. A custom deep-learning algorithm automatically quantifies the bead fluorescence, enabling robust and automated analysis. The integrated approach achieves simultaneous detection of HPV16, HPV18, and HPV33 with detection limits as low as 0.2 pM. By using recognition-triggered amplification and a simple deep-learning assisted fluorescence readout, the workflow is significantly simplified. The platform establishes a universal and practical strategy for molecular diagnostics, demonstrating strong potential for near-patient testing.},
}

@article {pmid41617656,
year = {2026},
author = {He, M and Chen, J and Chao, M and Yin, F and Pan, H and Wang, Q and Cheng, W and Wang, Z and Xiang, Y},
title = {Novel One-Pot Detection of Tumor mRNA Vaccine by the Target Serving as crRNA of the Split Cas12a System and Synergistically Triggering an Entropy-Driven Reaction.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {4134-4142},
doi = {10.1021/acs.analchem.5c06932},
pmid = {41617656},
issn = {1520-6882},
mesh = {*Entropy ; *Cancer Vaccines/analysis/genetics ; *RNA, Messenger/genetics/analysis ; Humans ; *mRNA Vaccines/analysis ; *Endodeoxyribonucleases/metabolism/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {mRNA vaccines, as a new form of vaccine, are gradually revealing their potential in the field of tumor treatment. With the development of mRNA vaccines, monitoring their levels has become particularly important. In this work, a novel and innovative strategy was proposed for the first time, which directly used the target as crRNA and simultaneously triggered an entropy-driven split Cas12a system for triple signal amplification for the high-sensitivity detection of mRNA vaccines. This study broke the traditional full-length crRNA and innovatively designed a split crRNA structure, directly using the target mRNA as the variable sequence of the crRNA. This design avoided the need for additional introduction of variable sequences, thereby eliminating nonspecific signal leakage and multicomponent interference from the source. At the same time, the target molecule served as the initiating chain for the EDC reaction, allowing the split Cas12a system to be coupled with the entire entropy-driven reaction process, achieving efficient collaboration and triple signal amplification and significantly improving the detection sensitivity and specificity. Moreover, the entire detection process required only one reaction step, which was simple, fast, and efficient. The aim was to provide strategies for screening and pharmacokinetic studies of mRNA vaccines.},
}

*Entropy
*Cancer Vaccines/analysis/genetics
*RNA, Messenger/genetics/analysis
Humans
*mRNA Vaccines/analysis
*Endodeoxyribonucleases/metabolism/genetics
CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Nucleic Acid Amplification Techniques/methods

@article {pmid41587498,
year = {2026},
author = {Marpaung, DSS and Yap Sinaga, AO},
title = {Toehold-mediated strand displacement in CRISPR/Cas12a reactions: Advances in programmable and universal biosensing strategies.},
journal = {Talanta},
volume = {302},
number = {},
pages = {129442},
doi = {10.1016/j.talanta.2026.129442},
pmid = {41587498},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; DNA/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a-based biosensors have emerged as powerful tools for nucleic acid detection due to their programmability, high sensitivity, and versatility. However, challenges such as PAM dependence, limited mismatch discrimination, and difficulty in detecting non-nucleic acid analytes constrain their universality. Toehold-mediated strand displacement (TMSD) offers a programmable mechanism to overcome these limitations by dynamically regulating hybridization kinetics and molecular interactions. This review systematically summarizes recent advances integrating TMSD into CRISPR/Cas12a systems, including crRNA release, crRNA-DNA activation, activator generation, and reporter signal modulation. By coupling TMSD's precise strand exchange capabilities with Cas12a's trans-cleavage activity, these hybrid biosensors achieve improved specificity, tunable kinetics, and multi-analyte adaptability. The review further discusses design principles, thermodynamic foundations, and application examples across biomedical, environmental, and food diagnostics. Collectively, TMSD-assisted CRISPR/Cas12a biosensing provides a universal, programmable framework for next-generation molecular diagnostics with enhanced control, sensitivity, and functional diversity.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
DNA/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41586479,
year = {2026},
author = {Huang, C and Yang, Y and Yin, H and Chang, H and Gong, R and Jiang, Z and Zhang, XE and Chen, Y},
title = {Ultrasensitive CRISPR Platform Enables Rapid and Extraction-Free Detection of Viral Nucleic Acid.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {4028-4035},
doi = {10.1021/acs.analchem.5c06518},
pmid = {41586479},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; Humans ; *RNA, Viral/analysis/genetics ; Nucleic Acid Amplification Techniques/methods ; *COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *COVID-19 Nucleic Acid Testing/methods ; Nasopharynx/virology ; },
abstract = {CRISPR-based molecular diagnostics offers great potential for the rapid identification of pathogens. However, existing one-pot detection systems remain constrained by their restricted versatility and operational complexity. Herein, we report a novel strategy termed interspaced phosphorothioate primer-mediated one-pot detection (iPSOT), which substitutes conventional phosphodiester primers with interspaced phosphorothioate (iPS) primers. The iPS primers demonstrate strong compatibility with AapCas12b during loop-mediated isothermal amplification, enabling robust fluorescence signal generation within 15 min. The iPSOT system achieves highly specific nucleic acid detection and reliably identifies low-copy ribonucleic acid targets at concentrations of as low as 0.5 aM (0.3 copies/μL). Clinical validation further demonstrated that iPSOT enables the direct detection of SARS-CoV-2 from nasopharyngeal swabs without RNA extraction. This method reduces reagent cost and shortens assay time, achieving sample-to-result under 20 min. Overall, iPSOT enhances both sensitivity and specificity in one-pot detection and offers a promising platform for rapid, reliable point-of-care testing and large-scale pathogen surveillance.},
}

*SARS-CoV-2/genetics/isolation & purification
Humans
*RNA, Viral/analysis/genetics
Nucleic Acid Amplification Techniques/methods
*COVID-19/diagnosis/virology
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Limit of Detection
*Clustered Regularly Interspaced Short Palindromic Repeats
*COVID-19 Nucleic Acid Testing/methods
Nasopharynx/virology

@article {pmid41582525,
year = {2026},
author = {Ding, S and Li, Y and Wang, F and Liu, Q and Liu, L and Li, J and Wu, Y and Liu, X and Jiang, D and Xu, X and Gao, X and Huang, C},
title = {DNA Flap-Controlled CRISPR/Cas12a Trans-Cleavage Enables Mix-and-Read FEN1 Activity Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {3998-4007},
doi = {10.1021/acs.analchem.5c06447},
pmid = {41582525},
issn = {1520-6882},
mesh = {*Flap Endonucleases/metabolism/analysis ; Humans ; *CRISPR-Cas Systems ; *DNA/chemistry/metabolism/genetics ; *Biosensing Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a has emerged as a powerful biosensing tool, owing to its exceptional specificity and trans-cleavage-mediated signal amplification capability, for detecting flap endonuclease 1 (FEN1) activity. However, its reliance on additional preligation or replication steps increases operational complexity and raises the risk of false signals. Herein, we report a DNA flap-controlled CRISPR/Cas12a trans-cleavage (FCT-CRISPR) strategy that enables sensitive, accurate, and mix-and-read detection for FEN1 activity. In FCT-CRISPR, a flap-structured dumbbell DNA probe was ingeniously designed, in which the flap domain serves as a split activator, and the dumbbell scaffold acts as a steric hindrance unit. Upon FEN1 recognition and cleavage, the split activator is liberated from its constrained configuration in the dumbbell scaffold, thereby activating CRISPR/Cas12a trans-cleavage activity. FCT-CRISPR strategy avoids the reliance on exogenous DNA ligation or replication processes, allowing mix-and-read detection of FEN1 activity with a detection limit as low as 0.2 mU and excellent specificity against nontarget enzymes. In addition, the successful detection application in lysates of cancer cells demonstrates the potential of FCT-CRISPR for clinical use. This work establishes a sensitive, accurate, and mix-and-read platform for monitoring FEN1 activity and offers a promising tool for the early diagnosis of FEN1-related diseases.},
}

*Flap Endonucleases/metabolism/analysis
Humans
*CRISPR-Cas Systems
*DNA/chemistry/metabolism/genetics
*Biosensing Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41496452,
year = {2026},
author = {Liu, J and Liu, Z and Qin, D and Qi, X and Chen, M and Cheng, D and Guo, S and Zhang, X and Zhang, L and Yan, T and Li, F and He, W and Lu, Z and Wang, H and Li, J and Yang, X and Shi, Y and Zhang, S and Zhao, H and Liu, C and Chen, S and Zhong, Y},
title = {An all-in-one visual selection system for male-sterile line production in maize and rice.},
journal = {Plant communications},
volume = {7},
number = {2},
pages = {101693},
doi = {10.1016/j.xplc.2026.101693},
pmid = {41496452},
issn = {2590-3462},
mesh = {*Zea mays/genetics/physiology ; *Oryza/genetics/physiology ; *Plant Infertility/genetics ; Seeds/genetics/growth & development ; *Plant Breeding/methods ; CRISPR-Cas Systems ; Hybrid Vigor/genetics ; Plants, Genetically Modified ; },
abstract = {Heterosis has significantly improved crop yields, yet hybrid seed production remains hindered by labor-intensive manual emasculation. Although current male-sterility systems, such as cytoplasmic male sterility and environment-sensitive genic male sterility, have improved the efficiency of hybrid seed production, their limited genetic adaptability and high environmental dependence remain major challenges. Here, we report an all-in-one seed production technology (ASPT) that integrates CRISPR-Cas9, RUBY, and key seed production technology (SPT) components into a single vector, enabling efficient generation and propagation of male-sterile lines in both maize and rice. The engineered RUBY marker enables visual identification of male-sterile and maintainer lines, with an accuracy of 99.81% in automated seed sorting and 100% in secondary field screening. Notably, ASPT was successfully introduced into 21 genetically diverse elite maize inbred lines, demonstrating broad compatibility. ASPT enables scalable and precise propagation of male-sterile lines in both maize and rice, providing a broadly applicable strategy to advance hybrid seed production in crops.},
}

*Zea mays/genetics/physiology
*Oryza/genetics/physiology
*Plant Infertility/genetics
Seeds/genetics/growth & development
*Plant Breeding/methods
CRISPR-Cas Systems
Hybrid Vigor/genetics
Plants, Genetically Modified

@article {pmid40579542,
year = {2026},
author = {Ullah, HMA and Huang, Q and Chiola, S and Wang, Y and Shcheglovitov, A},
title = {Generating and characterizing human telencephalic brain organoids from stem cell-derived single neural rosettes.},
journal = {Nature protocols},
volume = {21},
number = {2},
pages = {718-748},
pmid = {40579542},
issn = {1750-2799},
support = {R01 MH113670/MH/NIMH NIH HHS/United States ; T32HG008962//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01NS123849//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R01MH113670//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R01 NS123849/NS/NINDS NIH HHS/United States ; R21NS104963//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R21 CA279773/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Organoids/cytology ; *Telencephalon/cytology ; *Neural Stem Cells/cytology ; Nerve Tissue Proteins/genetics ; CRISPR-Cas Systems ; Neurons/cytology ; *Cell Culture Techniques/methods ; },
abstract = {We have developed a method for generating human telencephalic organoids from stem cell-derived isolated single neural rosettes. The use of single neural rosettes for generating organoids offers several important advantages. First, it mimics the development of neural tissue from a singular neural tube in vivo. Second, single neural rosette-derived organoids exhibit a relatively consistent and reproducible composition of telencephalic neural cells. Finally, single neural rosette-derived organoids demonstrate predictable organization of the identified neural cells around a single neural rosette-derived lumen and contain a large proportion of functionally mature neurons that generate action potentials and receive both excitatory and inhibitory synaptic inputs. These unique features of our protocol enable the study of the specification and organization of different neural cells in the developing human telencephalon, as well as modeling of neurodevelopmental disorders associated with disrupted neural networks. Here, we describe our protocols for generating CRISPR-Cas9-engineered human stem cells with a hemizygous SHANK3 deletion, stem cell-derived single neural rosettes and telencephalic brain organoids. We also offer insights on how to conduct single-cell RNA sequencing, immunohistochemistry and slice patch-clamp electrophysiology on these organoids. Completion of the protocols takes 5-6 months and requires experience working with cultured cells. We expect this protocol will prove useful for studies of human brain development and disease, as well as for advancing the development of new organoid-based biocomputers.},
}

Humans
*Organoids/cytology
*Telencephalon/cytology
*Neural Stem Cells/cytology
Nerve Tissue Proteins/genetics
CRISPR-Cas Systems
Neurons/cytology
*Cell Culture Techniques/methods

@article {pmid41652748,
year = {2026},
author = {Lei, Y and Ma, W and Wang, L and Chen, Y and Chen, X},
title = {Genomic insights into prophage and CRISPR-Cas system present in Lactobacillus delbrueckii subsp. bulgaricus strains.},
journal = {Food research international (Ottawa, Ont.)},
volume = {227},
number = {},
pages = {118213},
doi = {10.1016/j.foodres.2025.118213},
pmid = {41652748},
issn = {1873-7145},
mesh = {*Prophages/genetics ; *Lactobacillus delbrueckii/virology/genetics ; *CRISPR-Cas Systems/genetics ; Genomics ; Yogurt/microbiology ; Base Composition ; Genome, Bacterial ; Genome, Viral ; },
abstract = {As a key starter culture for yogurt fermentation, Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) was subjected to bioinformatics analysis to investigate the distribution of prophages and the structure of the CRISPR-Cas system in 119 L. bulgaricus strains, and explored the targeting relationship between them and annotated the functional genes of targeted prophages. A total of 1704 prophage fragments were identified, of which 8.74 % (149/1704) were classified as complete prophages, none of them carried virulence factor genes and antibiotic resistance genes. Among all complete prophage genomes, the GC content was significantly affected by geographical sources (Asia, Europe, and North America) of host bacteria (P < 0.01). The 123 CRISPR-Cas systems detected in L. bulgaricus, type I-E (69) and type II-A (40) systems were predominant. Spacers-prophages targeting analysis revealed a negative correlation (P < 0.05), with 23.43 % (1055/4503) spacers showing homology to prophages. Functional gene annotation revealed that the diversity of prophage functional genes showed significant differences in under different grouping conditions (P < 0.05). Nevertheless, in-depth analysis of targeted prophage genomes revealed a conserved functional gene--ABC transporter. This study provides data support for exploring the genomic diversity and adaptive evolution mechanism of prophages in L. bulgaricus, and providing insights for screening phage-resistant strains and developing novel anti-phage strategies in dairy industry.},
}

*Prophages/genetics
*Lactobacillus delbrueckii/virology/genetics
*CRISPR-Cas Systems/genetics
Genomics
Yogurt/microbiology
Base Composition
Genome, Bacterial
Genome, Viral

@article {pmid41652223,
year = {2026},
author = {Zhang, J and Zhang, J and Tang, S and Liu, C and Cai, Y and Zeng, H and Meng, X and Liu, B and Zhang, Y and Wang, Y},
title = {Single-round evolution of RNA aptamers with GRAPE-LM.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {41652223},
issn = {1546-1696},
support = {62302311//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82273967//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M742397//China Postdoctoral Science Foundation/ ; 2024T170585//China Postdoctoral Science Foundation/ ; },
abstract = {The directed evolution of biomolecules is an iterative process. Although advancements in language models have expedited protein evolution, effectively evolving RNA remains a challenge. RNA aptamers, selected for their binding properties, provide an ideal system to address this challenge, yet traditional aptamer discovery still relies on labor-intensive, multi-round screening. Here we introduce GRAPE-LM (generator of RNA aptamers powered by activity-guided evolution and language model), a generative artificial intelligence framework designed for the one-round evolution of RNA aptamers. GRAPE-LM integrates a transformer-based conditional autoencoder with nucleic acid language models and is guided by CRISPR-Cas-based aptamer screening data derived from intracellular environments. We validate GRAPE-LM on three disparate targets: the human T cell receptor CD3ε, the receptor-binding domain of the SARS-CoV-2 spike protein and the human oncogenic transcription factor c-Myc (an intracellular disordered protein). GRAPE-LM, informed with only a single round of CRISPR-Cas-based screening, successfully obtains RNA aptamers that outperform those driven from multiple rounds of human selection and optimization.},
}

@article {pmid41652115,
year = {2026},
author = {Omelchenko, DO and Barkovskaya, AM and Omelchenko, LV and Klepikova, AV and Penin, AA and Logacheva, MD},
title = {Optimization of functional genetics tools for a model tetraploid Capsella bursa-pastoris, with focus on homoeolog-aware gene editing.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {6},
pmid = {41652115},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *Capsella/genetics/growth & development ; Tetraploidy ; *Plants, Genetically Modified/genetics/growth & development ; Genome, Plant/genetics ; Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems ; },
abstract = {Capsella bursa-pastoris is a recent allotetraploid and a promising model for studying early consequences of polyploidy. One of the intriguing questions in polyploid research is how new functions arise from initially identical or nearly identical homoeologous genes. Functional genetics tools, including genetic editing, can help to understand this process, but they have not been developed for C. bursa-pastoris yet. We present here the results of our study aimed at filling this gap. In particular, we compared the efficiency of floral dip transformation in six accessions of C. bursa-pastoris representing distant populations. The Asian clade accession PGL0025 had the highest efficiency of transformation (~ 1.1%). Comparison of Agrobacterium tumefaciens strains EHA105 and GV3101 (pMP90) showed that the latter is more effective. Also, we created a genome-wide gRNA database for all pairs of homoeologs of the PGL0001 accession of C. bursa-pastoris and integrated it into publicly available genome browser: https://t2e.online/igv_capsella_bursa-pastoris/ . We assessed the possibility of differential editing for two pairs of homoeologous genes with high sequence similarity (> 90%) both in vitro and in silico. Despite the test results that indicated off-target activity, we have succeeded in obtaining lines of plants with homozygous frameshift mutations in each of the homoeologs separately in vivo. We expect that these findings and resources will promote the use of C. bursa-pastoris as a model in functional genetics experiments, in particular, the studies of the fate of duplicated gene after polyploidization event.},
}

*Gene Editing/methods
*Capsella/genetics/growth & development
Tetraploidy
*Plants, Genetically Modified/genetics/growth & development
Genome, Plant/genetics
Agrobacterium tumefaciens/genetics
CRISPR-Cas Systems

@article {pmid41652090,
year = {2026},
author = {Pan, X and Yildiz, U and Armstrong, SK and Bissonnette, K},
title = {Status and advancement of root-knot nematode management strategies and the emerging CRISPR/Cas biotechnology application.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {38},
pmid = {41652090},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; Animals ; *Plant Diseases/parasitology/genetics/prevention & control ; Gene Editing ; *Tylenchoidea/pathogenicity/genetics ; Crops, Agricultural/genetics/parasitology ; Disease Resistance/genetics ; Plant Roots/parasitology/genetics ; Gossypium/parasitology/genetics ; },
abstract = {Root-knot nematodes (RKNs), Meloidogyne spp., exhibit a broad host range, threatening more than 3000 species of plants, including agriculturally important crops such as cotton (Gossypium hirsutum), tomato (Lycopersicon esculentum) and rice (Oryza sativa). Among the over 90 RKN species, the four most prevalent are M. incognita, M. arenaria, M. javanica, and M. hapla, with M. incognita being the most damaging. This paper reviewed the current RKN management strategies, including chemical nematicides, biological control, crop rotation, and resistant varieties, with a focus on the application of the revolutionary CRISPR/Cas genome editing tool in developing RKN resistance in plants. CRISPR/Cas has been widely utilized for improving crop traits due to its specificity, streamline, and inheritability. Recent progress has demonstrated the simplicity and robustness of CRISPR/Cas technology in improving plant traits. Among these, the development of nematode resistance by CRISPR/Cas knocking out of plant compatibility factors in model and commercial plants, has achieved significant progress. This review summarizes the RKN parasitism mechanisms and plant compatibility factors that would be promising CRISPR/Cas targets. The fundamentals and key aspects of CRISPR/Cas genome editing technology are addressed and discussed, and an example experimental pipeline for developing nematode resistance in cotton is described.},
}

*CRISPR-Cas Systems
Animals
*Plant Diseases/parasitology/genetics/prevention & control
Gene Editing
*Tylenchoidea/pathogenicity/genetics
Crops, Agricultural/genetics/parasitology
Disease Resistance/genetics
Plant Roots/parasitology/genetics
Gossypium/parasitology/genetics

@article {pmid41651998,
year = {2026},
author = {Natsagdorj, ME and Hara, H and Uosaki, H and Nakahara, F and Inoue, M and Hanazono, Y},
title = {ATM Inhibition Enhances Knock-in Efficiency by Suppressing AAV-Induced Activation of Apoptotic Pathways.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {177},
pmid = {41651998},
issn = {2399-3642},
support = {JP23bm1123020//Japan Agency for Medical Research and Development (AMED)/ ; JP22bm0804018//Japan Agency for Medical Research and Development (AMED)/ ; JP23bm1123020//Japan Agency for Medical Research and Development (AMED)/ ; JP18am0301002//Japan Agency for Medical Research and Development (AMED)/ ; JP22ae0201007//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {*Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors/genetics/metabolism ; *Dependovirus/genetics ; Animals ; Mice ; *Apoptosis/genetics ; *Gene Knock-In Techniques/methods ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Morpholines/pharmacology ; Gene Editing/methods ; Mouse Embryonic Stem Cells/metabolism ; Thioxanthenes ; },
abstract = {CRISPR/Cas9-mediated genome editing has expanded the possibilities for precise gene modifications; however, the efficiency of targeted insertion remains suboptimal. In this study, we describe a triple-reporter system in mouse embryonic stem cells that simultaneously tracks double-strand break (DSB) induction, homology-directed repair (knock-in), and end-joining-mediated targeted insertion (EJ-TI). Using both plasmid and adeno-associated virus (AAV) donor vectors, our results demonstrate that ataxia telangiectasia and Rad3-related kinase (ATR) activity is essential for knock-in regardless of the donor type, whereas ataxia telangiectasia mutated (ATM) inhibition exhibits a donor-dependent role. In cells receiving circular plasmid donors, ATM inhibition with AZD1390 markedly reduced the knock-in and EJ-TI efficiencies, consistent with its canonical role in DSB repair. In contrast, with linear AAV donors, ATM inhibition enhanced the knock-in efficiency by suppressing the overactivation of the ATM-p53-caspase 3 apoptotic pathway and partially suppressing classical non-homologous end-joining. These findings highlight the critical influence of donor DNA configuration on DNA damage response signaling and provide a strategy for optimizing genome editing efficiency by selectively modulating the ATM pathways, an approach that may have significant implications for gene therapy, cell engineering, and other applications.},
}

*Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors/genetics/metabolism
*Dependovirus/genetics
Animals
Mice
*Apoptosis/genetics
*Gene Knock-In Techniques/methods
DNA Breaks, Double-Stranded
CRISPR-Cas Systems
Morpholines/pharmacology
Gene Editing/methods
Mouse Embryonic Stem Cells/metabolism
Thioxanthenes

@article {pmid41651087,
year = {2026},
author = {Cao, W and Lu, J and Jiang, Y and Yuan, M and Wang, M and Mu, X and Gao, Z and Yang, S and Li, T and Wei, X and Guo, L},
title = {Recent advances in metabolic engineering of purple non‑sulfur photosynthetic bacteria for enhanced biohydrogen production.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108832},
doi = {10.1016/j.biotechadv.2026.108832},
pmid = {41651087},
issn = {1873-1899},
abstract = {Photo-fermentative hydrogen production from lignocellulosic biomass offers a sustainable and carbon-neutral route for bioenergy conversion, providing an effective strategy to mitigate fossil resource depletion and greenhouse gas emissions. Among diverse microbial candidates, purple non‑sulfur bacteria (PNSB) have emerged as promising photosynthetic platforms owing to their broad substrate utilization, intrinsic tolerance to lignocellulose-derived inhibitors, and high hydrogen yields under light-anaerobic conditions. Their metabolic versatility allows dynamic redistribution of carbon and electron fluxes, facilitating efficient energy conversion. Recent progress in metabolic engineering has substantially advanced the hydrogen-producing capacity of PNSB through targeted manipulation of photosynthetic metabolism, redox balance and stress response pathways. Engineering strategies have focused on optimizing pigment biosynthesis to enhance light harvesting, reinforcing redox homeostasis and adenosine triphosphate (ATP) generation, and improving tolerance to environmental stresses such as ammonia, pH, and temperature fluctuations. These efforts have led to engineered strains exhibiting extraordinary improvement in hydrogen yield, stability and robustness. This review provides an overview of the fundamental mechanisms underlying photo-fermentative hydrogen metabolism in PNSB, summarizes recent advances in the metabolic and systems-level engineering strategies, and outlines the prospects of developing strains capable of approaching the theoretical limit of hydrogen yield through integrated engineering strategies, advanced tools such as CRISPR-Cas, and adaptive laboratory evolution methods.},
}

@article {pmid41648912,
year = {2026},
author = {Wang, R and Li, J and Zheng, Y and Wei, N and Li, W},
title = {CRISPR/Cas12a platform activated by a protospacer adjacent motif-engineered DNA circuit for specific target sensing.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay02079d},
pmid = {41648912},
issn = {1759-9679},
abstract = {CRISPR/Cas systems have found extensive applications in nucleic acid diagnostics. However, the generalizability of this approach, particularly for the sensing of non-nucleic acid targets, remains a challenge. This study presents the development of a CRISPR/Cas12a platform activated by a protospacer adjacent motif (PAM)-engineered DNA circuit. Initially, the influence of the presence or absence of the PAM on the DNA circuit and the subsequent CRISPR/Cas12a system was investigated, demonstrating that a PAM-engineered DNA circuit functions as an effective activator of Cas12a, whereas a DNA circuit lacking the PAM does not induce activation. Subsequently, through the strategic design of recognition elements, sensitive and selective detection of specific targets is achieved, with limits of detection (LODs) of 0.023 fM for circulating tumor DNA (ctDNA), 0.00004 U mL[-1] for uracil-DNA glycosylase (UDG), and 0.12 fM for acetamiprid (ACE). This approach exemplifies a two-stage signal amplification mechanism, achieving improved sensitivity relative to either the CRISPR/Cas12a system or the DNA circuits alone. Moreover, quantitative assays for these targets were successfully conducted in real samples, suggesting the practical applicability of the proposed method. This research establishes a versatile sensing platform for various targets, which holds significant promise for advancements in molecular diagnostics, food safety assessment, and environmental monitoring.},
}

@article {pmid41648761,
year = {2025},
author = {Lu, Z and Bukhari, STH and Azeem, M and Tariq, N and Shabbir, MAB},
title = {A narrative review of precision and ethical considerations in cardiovascular health: CRISPR-Cas9, telemedicine, and lifestyle interventions.},
journal = {Frontiers in public health},
volume = {13},
number = {},
pages = {1737251},
pmid = {41648761},
issn = {2296-2565},
mesh = {Humans ; *Telemedicine/ethics ; *Cardiovascular Diseases/prevention & control/therapy ; *CRISPR-Cas Systems ; *Life Style ; *Precision Medicine ; Gene Editing ; },
abstract = {BACKGROUND: Cardiovascular diseases (CVDs) remain the leading cause of global morbidity and mortality, influenced by lifestyle, socioeconomic status, and genetic factors. Emerging innovations, including wearable health technologies, telemedicine, and CRISPR-Cas9 gene editing, provide new possibilities for rapid prevention and personalized management.

METHODS: This narrative review collected evidence from Scopus, PubMed, and Google Scholar, using keywords such as cardiovascular (CV) prevention, lifestyle determinants, digital health, telemedicine, CRISPR-Cas9, and public health ethics. Eligible peer-reviewed studies, clinical guidelines, and policy documents were included to assess behavioral, technological, and genomic strategies for CVD care.

RESULTS: Modifications in lifestyle, such as quitting smoking, regular physical activity, following a heart-healthy diet plan, and getting adequate sleep, can significantly reduce the risk of CVD. Additionally, telemedicine and wearable devices facilitate early detection, better self-management, and treatment adherence, especially in underserved communities. CRISPR-Cas9 holds a significant potential for correcting genetic variants related to lipid disorders and inherited cardiomyopathies, but its clinical translation remains in early stages. However, existing evidence is limited by heterogeneity in study design, brief follow-up, particularly for digital health and CRISPR applications. Additional challenges, such as health inequities, digital access, data privacy, and ethical oversight, further influence their real-world implementation.

CONCLUSION: Effective integration of behavioral, digital and genomic innovations requires policy frameworks that ensure equity, ethical governance, and long-term sustainability. Combining precision medicine with efforts to address social determinants of health will be crucial in reducing the global burden of CVD and shaping the future of CV care.},
}

Humans
*Telemedicine/ethics
*Cardiovascular Diseases/prevention & control/therapy
*CRISPR-Cas Systems
*Life Style
*Precision Medicine
Gene Editing

@article {pmid41648339,
year = {2026},
author = {Phan, PT and Ozturk, M and Dougherty, EM and Ravishankar, J and Xue, C and Sashital, DG},
title = {Mismatch type impacts interference and priming activities in the type I-E CRISPR-Cas system.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.01.24.701482},
pmid = {41648339},
issn = {2692-8205},
abstract = {Type I-E CRISPR-Cas systems direct RNA-guided interference against foreign nucleic acids using the CRISPR RNA (crRNA)-guided Cascade complex and Cas3 helicase-nuclease. DNA targeting by Cascade-Cas3 promotes priming, a mechanism that allows for rapid acquisition of new spacers within the CRISPR array. Target mutations in the PAM and PAM-proximal seed region can block interference but may still allow priming. Previous studies have suggested that target mutations to T and A are tolerated, but that C and G substitutions are deleterious to interference and priming, respectively. However, the contributions of the crRNA spacer sequence to mutational tolerance remain unclear. Here, we systematically tested the effects of crRNA seed sequences on mutational tolerance. We engineered four E. coli strains with variable spacer sequences and tested CRISPR interference and priming against a plasmid library for each strain. Consistent with prior studies, we observe that mutations to C or G in the seed can be highly deleterious, especially at positions 1, 2 and 4. However, the corresponding crRNA sequence also strongly impacts the level of defect, with rC-dC and rA/G-dG causing the largest defects in our plasmid library experiments. Using in vitro biochemistry, we observe that mismatch type at the first position of the seed affects Cas8 conformation, and results in reduction in the rates of both Cascade-target binding and Cas3 recruitment. Overall, our results reveal that although nucleotide identity of target mutations is an important determinant of type I-E CRISPR immunity, the crRNA sequence also strongly impacts immune outcomes upon target mutation.},
}

@article {pmid41637875,
year = {2026},
author = {Wu, H and Merényi, Z and Virágh, M and Liu, XB and Hegedüs, B and Hou, Z and Ábrahám, E and Fürtön, A and Kristóffy, Z and Lipinszki, Z and Nagy, LG},
title = {Functional characterization of a Con7-related transcription factor in Coprinopsis cinerea indicates evolutionary conservation of morphogenetic roles.},
journal = {Microbiological research},
volume = {306},
number = {},
pages = {128462},
doi = {10.1016/j.micres.2026.128462},
pmid = {41637875},
issn = {1618-0623},
mesh = {*Transcription Factors/genetics/metabolism ; Fruiting Bodies, Fungal/growth & development/genetics ; *Fungal Proteins/genetics/metabolism ; Gene Expression Regulation, Fungal ; *Agaricales/genetics/growth & development ; Morphogenesis/genetics ; CRISPR-Cas Systems ; Phylogeny ; Gene Expression Profiling ; Gene Deletion ; Evolution, Molecular ; Zinc Fingers ; },
abstract = {Fruiting bodies of mushroom-forming fungi (Agaricomycetes) exhibit the highest degree of multicellular complexity in fungi, yet the molecular underpinnings of their developmental programs remain incompletely understood. Here, we characterize gcd1, a gene encoding a transcription factor in the Con7 subfamily of C2H2-type zinc finger proteins. This subfamily has previously been implicated in pathogenic morphogenesis in Ascomycota, but its role in Agaricomycetes has not previously been addressed. In Coprinopsis cinerea, CRISPR/Cas9-mediated deletion of gcd1 resulted in strains with severely impaired fruiting body morphogenesis, with malformed cap, stipe, and gill tissues. Gcd1 deletion strains lacked universal veil, resembling species with open (gymnocarpous) development. We find that GCD1/Con7 homologs are widely distributed in most Dikarya species and are mostly encoded by a single gene in each species' genome. Transcriptome analyses identified several misregulated genes in the Δgcd1 mutant, which pinpoint potential mechanisms underlying its developmental defects as well as provided insights into the morphogenesis of mushroom fruiting bodies. These findings establish GCD1 as a key regulator of multicellular development in C. cinerea and broaden the known functions of Con7-like transcription factors to include fruiting body morphogenesis in Agaricomycetes. Overall, our results and the morphogenetic role of Con7-like transcription factors of Ascomycota suggest functional conservation over half a billion years of evolution.},
}

*Transcription Factors/genetics/metabolism
Fruiting Bodies, Fungal/growth & development/genetics
*Fungal Proteins/genetics/metabolism
Gene Expression Regulation, Fungal
*Agaricales/genetics/growth & development
Morphogenesis/genetics
CRISPR-Cas Systems
Phylogeny
Gene Expression Profiling
Gene Deletion
Evolution, Molecular
Zinc Fingers

@article {pmid41553775,
year = {2026},
author = {Zheng, I and Learn, B and Bailey, S},
title = {Structural basis for inhibition of SpyCas9 by the anti-CRISPR protein AcrIIA26.},
journal = {The Biochemical journal},
volume = {483},
number = {3},
pages = {289-300},
doi = {10.1042/BCJ20250364},
pmid = {41553775},
issn = {1470-8728},
support = {GM097330//HHS | National Institutes of Health (NIH)/ ; },
mesh = {*Streptococcus pyogenes/genetics/enzymology/metabolism ; *CRISPR-Associated Protein 9/chemistry/metabolism/antagonists & inhibitors/genetics ; *CRISPR-Cas Systems ; *Viral Proteins/chemistry/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; Cryoelectron Microscopy ; *Bacteriophages/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Models, Molecular ; },
abstract = {CRISPR-Cas9 systems provide adaptive immunity in prokaryotes by targeting and cleaving invading phage DNA. In response, phages have evolved anti-CRISPR (Acr) proteins to inhibit Cas9 and evade this immune response. AcrIIA26 is a type II-A anti-CRISPR protein that inhibits Streptococcus pyogenes Cas9 (SpyCas9) DNA binding, but its molecular mechanism remains unclear. Here, we determined the 3.0 Å resolution cryo-EM structure of AcrIIA26 in complex with SpyCas9-single-guide RNA, revealing a dual inhibition mechanism. AcrIIA26 adopts a novel fold comprising a central β-sheet flanked by two α-helical bundles. The 5-helix bundle, which features a negatively charged surface whose shape mimics duplex DNA, occupies the same position as the protospacer adjacent motif (PAM) duplex in target-bound Cas9. This directly blocks PAM recognition by burying critical residues R1333 and R1335 in the PAM-interacting domain. Mutagenesis confirmed that residues E49 and D50 in AcrIIA26 are essential for this interaction. Simultaneously, the 4-helix bundle binds the Cas9 REC lobe and sterically prevents the conformational changes required for Cas9 activation, with mutation of AcrIIA26 F121 completely eliminating inhibitory activity. Structural comparisons reveal that despite diverse folds, multiple anti-CRISPRs convergently evolved to block PAM recognition, highlighting this as a critical vulnerability in Cas9 function. Our findings provide mechanistic insights into AcrIIA26 inhibition and offer a foundation for engineering improved Cas9 off-switches for genome editing applications.},
}

*Streptococcus pyogenes/genetics/enzymology/metabolism
*CRISPR-Associated Protein 9/chemistry/metabolism/antagonists & inhibitors/genetics
*CRISPR-Cas Systems
*Viral Proteins/chemistry/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics
Cryoelectron Microscopy
*Bacteriophages/metabolism/genetics
*Bacterial Proteins/chemistry/metabolism/genetics
Models, Molecular

@article {pmid41507205,
year = {2026},
author = {Wan, H and Kong, D and Yan, T and Zhou, Y and Liu, M and Ma, X and Zhao, T and Zhou, W and Liu, X and Yin, J and Guan, N and Ye, H},
title = {A compact and inducible dCas12f-based CRISPRa platform for programmable in vivo gene activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1447},
pmid = {41507205},
issn = {2041-1723},
support = {25J22800100//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; 32250010//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Dependovirus/genetics ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Transcriptional Activation/genetics ; HEK293 Cells ; *Gene Editing/methods ; Genetic Therapy/methods ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Precise activation of endogenous genes is a powerful strategy for functional genomics and therapeutic development, but current CRISPR-based transcriptional activation (CRISPRa) systems are limited by the large size of Cas proteins for adeno-associated virus (AAV) delivery. Here, we present a high-efficiency dCas12f-based transcriptional activation system (HEAL), which recruits transactivators through MS2 coat protein binding to MS2 aptamers embedded within the sgRNA scaffold. Engineered to enhance DNA binding, nuclear localization, and transactivator recruitment, HEAL induces over 100,000-fold activation of endogenous genes and outperforms existing CRISPRa systems in vitro and in vivo. We further develop red-light-inducible OptoHEAL and small-molecule-inducible ChemHEAL for remote and precise transcriptional control. AAV-delivered HEAL targeting interleukin 10 alleviates acute kidney injury in mice, while ChemHEAL-mediated activation of thymic stromal lymphopoietin reduces body weight in obese mice. HEAL provides a modular, compact, and controllable platform for endogenous gene activation with strong potential for fundamental research and gene therapy.},
}

Animals
Dependovirus/genetics
Mice
*CRISPR-Cas Systems/genetics
Humans
*Transcriptional Activation/genetics
HEK293 Cells
*Gene Editing/methods
Genetic Therapy/methods
Mice, Inbred C57BL
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41370233,
year = {2026},
author = {Nomura, Y and Nomura, J and Tamada, K and Eguchi, N and Torigata, K and Tokumoto, S and Nemoto, A and Shirafuji, T and Yamamoto, K and Hishimoto, A and Nagase, H and Nishikawa, T and Takumi, T},
title = {Isogenic modeling of 1q21.1 reciprocal CNVs in human ES cells reveals divergent neurodevelopmental trajectories.},
journal = {Human molecular genetics},
volume = {35},
number = {2},
pages = {},
doi = {10.1093/hmg/ddaf184},
pmid = {41370233},
issn = {1460-2083},
support = {21 K07820//KAKENHI/ ; 24 K10078//KAKENHI/ ; 22 K15750//KAKENHI/ ; 16H06316//KAKENHI/ ; 16H06463//KAKENHI/ ; 23KK0132//KAKENHI/ ; 24H00620//KAKENHI/ ; 24H01241//KAKENHI/ ; 23H04233//KAKENHI/ ; 24 K22036//KAKENHI/ ; //Japan Society for the Promotion of Science and Ministry of Education, Culture, Sports, Science, and Technology/ ; JP21wm0425011//Japan Agency for Medical Research and Development/ ; JPMJPF2018//Japan Science and Technology Agency/ ; JPMJMS2299//Japan Science and Technology Agency/ ; JPMJMS229B//Japan Science and Technology Agency/ ; //Takeda Science Foundation/ ; //Smoking Research Foundation/ ; //Taiju Life Social Welfare Foundation/ ; },
mesh = {Humans ; *DNA Copy Number Variations/genetics ; *Chromosomes, Human, Pair 1/genetics ; Neurogenesis/genetics ; *Neurodevelopmental Disorders/genetics/pathology ; *Human Embryonic Stem Cells/metabolism ; Chromosome Deletion ; Neural Stem Cells/metabolism ; Cell Differentiation/genetics ; Neurons/metabolism ; Chromosome Duplication ; CRISPR-Cas Systems ; Intellectual Disability/genetics ; Cell Line ; Schizophrenia/genetics ; },
abstract = {Copy number variations (CNVs) in the distal 1q21.1 region, both deletion (1q del) and duplication (1q dup) are associated with various neurodevelopmental and neuropsychiatric disorders such as autism spectrum disorder, intellectual disability, epilepsy, and schizophrenia. Besides common phenotypes, 1q del and 1q dup manifest opposite clinical phenotypes, e.g. microcephaly in 1q del and macrocephaly in 1q dup. However, molecular and cellular mechanisms underlying these phenotypes are still elusive. Here, to identify molecular mechanisms associated with neurodevelopmental phenotypes from the viewpoint of neurogenesis and neurodevelopment, we generate isogenic human ES cell (hESC) lines with reciprocal 1q21.1 CNVs using CRISPR/Cas9 system and differentiate them into 2-dimensional (2-D) neurons and neural progenitor cell (NPC) spheroids. Our study recapitulates reciprocal brain size in the NPC spheroids and shows dosage-dependent differentiation changes i.e. more GABAergic components in 1q del and more proliferative state in 1q dup. These results demonstrate that 1q21.1 CNVs dramatically affect cell fate in the early neurodevelopmental periods. This is the first isogenic cell model of human 1q21.1 CNVs, and our findings provide new insights into the underlying mechanisms of neurodevelopmental disorders.},
}

Humans
*DNA Copy Number Variations/genetics
*Chromosomes, Human, Pair 1/genetics
Neurogenesis/genetics
*Neurodevelopmental Disorders/genetics/pathology
*Human Embryonic Stem Cells/metabolism
Chromosome Deletion
Neural Stem Cells/metabolism
Cell Differentiation/genetics
Neurons/metabolism
Chromosome Duplication
CRISPR-Cas Systems
Intellectual Disability/genetics
Cell Line
Schizophrenia/genetics

@article {pmid41276946,
year = {2026},
author = {Pal, T and Liu, Z and Nair, MG and Chen, J},
title = {CRISPR in MOF Formulation with Enhanced Stability, Activity, and Altered PAM Specificity for Broad-Spectrum Diagnosis of Bacterial Sepsis.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {8},
pages = {e13439},
doi = {10.1002/advs.202513439},
pmid = {41276946},
issn = {2198-3844},
support = {R35GM147069/GM/NIGMS NIH HHS/United States ; R35GM147069/GM/NIGMS NIH HHS/United States ; },
mesh = {*Sepsis/diagnosis/microbiology/genetics ; Humans ; *Metal-Organic Frameworks/chemistry ; *CRISPR-Cas Systems/genetics ; RNA, Ribosomal, 16S/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Sepsis is a life-threatening condition caused by polymicrobial infections and remains a global health emergency that requires rapid and broad-spectrum diagnostics. Existing CRISPR-based assays face two major limitations that restrict their application for sepsis: narrow protospacer adjacent motif (PAM) site compatibility and poor enzyme stability under clinical and environmental stresses. A modular diagnostic platform is presented, CRISPR-FLEXMO (CRISPR with flexible PAM in metal-organic framework encapsulation, MOF), which integrates a PAM-relaxed Cas12a variant (K607R) with a manganese-coordinated MOF (Mn-MOF) for stable and specific detection of sepsis-causing bacteria. The system targets a conserved region upstream of the Shine-Dalgarno sequence in the 16S rRNA gene containing a universal TTCC PAM, enabling broad-spectrum detection with a single universal primer pair across Gram-negative and Gram-positive pathogens. The K607R variant shows enhanced cis- and trans-cleavage activity, while Mn-MOF encapsulation maintains enzyme functionality under ambient, thermal, and chaotropic stress. The assay detects as low as 10 CFU mL[-1] in bacterial lysates following amplification and achieves 100% sensitivity and specificity in serum samples from 15 sepsis patients and 3 healthy individuals, with no cross-reactivity to six respiratory viruses. The platform retains over 78% activity after 12 weeks of room-temperature storage, offering a field-deployable CRISPR diagnostic solution for next-generation infectious disease detection.},
}

*Sepsis/diagnosis/microbiology/genetics
Humans
*Metal-Organic Frameworks/chemistry
*CRISPR-Cas Systems/genetics
RNA, Ribosomal, 16S/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41276924,
year = {2026},
author = {Yun, S and Kim, S and Kim, S and Noh, M and Kim, DK and Lee, EJ and Lee, H},
title = {Parallel Genome-Wide CRISPR Screens Reveal SORL1 and ZFYVE19 as Sequential Host Determinants of Salmonella Infection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {8},
pages = {e15042},
doi = {10.1002/advs.202515042},
pmid = {41276924},
issn = {2198-3844},
support = {NRF-2022R1A2B5B02002256//National Research Foundation of Korea/ ; NRF-2022R1A4A1025913//National Research Foundation of Korea/ ; NRF-RS-2025-00561488//National Research Foundation of Korea/ ; NRF-RS-2024-00350131//National Research Foundation of Korea/ ; NRF-RS-2025-02219421//National Research Foundation of Korea/ ; RS-2025-16652968//MSIT/ ; },
mesh = {Humans ; *Salmonella Infections/genetics/microbiology/metabolism ; *Host-Pathogen Interactions/genetics ; *Membrane Transport Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Salmonella enterica/pathogenicity/genetics ; CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Salmonella enterica, a major cause of gastroenteritis and typhoid fever, hijacks host machinery to invade cells, and replicate within a specialized niche. While some host factors are known, a comprehensive, temporally-resolved understanding of the host-pathogen interface has been hindered by a lack of suitable genome-wide methodologies. To address this, a parallel CRISPR screening platform is developed to identify host determinants for distinct infection stages. An invasion screen captured factors for bacterial entry, while a fitness screen identified factors governing long-term survival. The screens reveal a temporal switch in host dependency, from endosomal trafficking in early infection to cell cycle and DNA damage response pathways governing host cell fitness in long-term infection. Notably, the approach uncovers two novel host factors with stage-specific roles, SORL1 as a mediator of bacterial invasion and ZFYVE19 as a factor supporting intracellular proliferation. Genetic disruption of SORL1 or ZFYVE19 validate these roles, leading to impaired invasion or replication, respectively. Importantly, antibody-mediated blockade of SORL1 effectively prevented Salmonella entry, highlighting it as a novel host-directed therapeutic target. Together, the screening strategy provides a powerful framework for the temporal dissection of host-pathogen interactions, revealing novel biology and promising therapeutic targets.},
}

Humans
*Salmonella Infections/genetics/microbiology/metabolism
*Host-Pathogen Interactions/genetics
*Membrane Transport Proteins/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Salmonella enterica/pathogenicity/genetics
CRISPR-Cas Systems/genetics
Animals

@article {pmid41276909,
year = {2026},
author = {Fokin, AI and Lin, Y and Guschin, DY and Chen, HY and James, J and Yan, J and Silberzan, P and Gautreau, AM},
title = {Identification of PKN2 and MOB4 as Coordinators of Collective Cell Migration.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {8},
pages = {e02907},
doi = {10.1002/advs.202502907},
pmid = {41276909},
issn = {2198-3844},
support = {ANR-20-CE13-0016//Agence Nationale de la Recherche/ ; ANR-22-CE13-0041//Agence Nationale de la Recherche/ ; ANR-24-CE44-4957//Agence Nationale de la Recherche/ ; 2021 060003815//Fondation ARC pour la Recherche sur le Cancer/ ; INCA_16712//Institut National du Cancer/ ; ANR-11-LABX-0038//Labex Cell(n)Scales/ ; ANR-10-IDEX-0001-02//Labex Cell(n)Scales/ ; //"Institut Pierre-Gilles de Gennes" laboratoire d'excellence/ ; ANR-10-IDEX-0001-02 PSL//"Investissements d'avenir" program/ ; ANR-10-LABX-31//"Investissements d'avenir" program/ ; //French National Cancer Institute/ ; NRF-MSG-2023-0001//National Research Foundation Singapore/ ; },
mesh = {*Cell Movement/physiology/genetics ; Humans ; *Adaptor Proteins, Signal Transducing/metabolism/genetics ; Adherens Junctions/metabolism ; Wound Healing ; CRISPR-Cas Systems ; Protein Kinase C ; },
abstract = {In animals, collective cell migration is critical during development and adult life for repairing organs. It remains, however, poorly understood compared with single-cell migration. The polymerization of branched actin by the RAC1-WAVE-Arp2/3 pathway is established to power membrane protrusions at the front of migrating cells, but also to maintain cell junctions in epithelial monolayers. Here, novel regulators of collective cell migration are identified using a two-pronged approach: candidates are extracted from publicly available RAC1-WAVE-Arp2/3 dependency maps and screened in a second step using CRISPR/Cas9 genetic inactivation. In a wound healing assay, PKN2 knockout (KO) MCF10A cells display decreased collective migration due to destabilization of adherens junctions, whereas MOB4 KO cells display increased collective migration with a loss of migration orientation. Upon wound healing, PKN2 relocalizes to lateral junctions and maintains coordinated migration in the monolayer, whereas MOB4 relocalizes to the front edge of leader and follower cells collectively migrating toward the wound. The role of MOB4 in controlling collective migration requires YAP1, since MOB4 KO cells fail to activate YAP1, and their phenotype is rescued by constitutively active YAP1. Together, these findings reveal two complementary activities required for coordinating cells in collective migration.},
}

*Cell Movement/physiology/genetics
Humans
*Adaptor Proteins, Signal Transducing/metabolism/genetics
Adherens Junctions/metabolism
Wound Healing
CRISPR-Cas Systems
Protein Kinase C

@article {pmid41230595,
year = {2026},
author = {Kim, K and Kachiprath, B and Yeo, IC and Shim, KY and Kwak, IS and Jeong, CB},
title = {Environmental DNA-based RPA-CRISPR/Cas12a assay for on-site detection of chironomid larvae in aquatic environments.},
journal = {Pest management science},
volume = {82},
number = {3},
pages = {2458-2467},
doi = {10.1002/ps.70382},
pmid = {41230595},
issn = {1526-4998},
support = {//Incheon National University Research Grant in 2021 (No. 2021-0447)/ ; },
mesh = {*Chironomidae/growth & development/genetics ; Animals ; *DNA, Environmental/analysis ; Larva/growth & development/genetics ; *CRISPR-Cas Systems ; *Environmental Monitoring/methods ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {BACKGROUND: Freshwater chironomid midges pose a significant threat to drinking water quality and freshwater ecosystems in South Korea by disrupting ecological balance and causing biofouling in drinking water treatment systems. To enable rapid detection and provide early warnings of chironomid presence, we developed a highly sensitive and accurate diagnostic assay based on environmental DNA (eDNA) analysis. This method integrates recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated cleavage, and visualization through either fluorescence-based detection or a lateral flow assay (LFA).

RESULTS: The RPA-CRISPR/Cas12a assays demonstrated remarkable specificity for chironomid eDNA, as confirmed through field surveys and microscopic observations. Although both fluorescence-based and LFA-based RPA-CRISPR/Cas12a assays exhibited comparable detection limits of 10 copies/μL, the LFA method proved more convenient for on-site applications due to its ease of deployment in the field.

CONCLUSION: Our eDNA-based RPA-CRISPR/Cas12a assay addresses the limitations of traditional biomonitoring by requiring only a small sampling volume, thereby minimizing habitat disturbance, while offering much higher sensitivity. This assay represents a valuable tool for on-site detection of invasive species and for monitoring environmental and public health threats (e.g., chironomids). © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.},
}

*Chironomidae/growth & development/genetics
Animals
*DNA, Environmental/analysis
Larva/growth & development/genetics
*CRISPR-Cas Systems
*Environmental Monitoring/methods
*Nucleic Acid Amplification Techniques/methods

@article {pmid40675140,
year = {2026},
author = {Karimi, MA and Paryan, M and Behrouzian Fard, G and Sadeghian, H and Zarrinfar, H and Hosseini Bafghi, M},
title = {Challenges and Opportunities in the Application of CRISPR-Cas9: A Review on Genomic Editing and Therapeutic Potentials.},
journal = {Medical principles and practice : international journal of the Kuwait University, Health Science Centre},
volume = {35},
number = {1},
pages = {1-17},
pmid = {40675140},
issn = {1423-0151},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Genetic Therapy/methods ; },
abstract = {

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) constitute a revolutionary gene-editing technology, allowing precise DNA modifications with vast potential for disease treatment and the creation of genetically modified organisms. This system consists of various components designed to target specific genes, requiring efficient nuclear access within target cells through diverse delivery methods, including physical techniques and carrier-based approaches. Despite its transformative promise, CRISPR faces several challenges, including efficient cellular delivery, off-target effects, immune responses, optimizing editing efficiency, and ethical concerns. Overcoming these hurdles is crucial for fully harnessing its applications. However, CRISPR-Cas9 offers remarkable opportunities for pioneering gene therapies across various disorders, including cancer, and could revolutionize agriculture by engineering disease-resistant crops. A key strength of CRIScPR lies in its adaptability to a broad spectrum of genes, significantly enhancing its versatility. The technology's potential further expands through emerging interdisciplinary integrations, such as artificial intelligence, machine learning, and biological imaging. These advancements can refine CRISPR's precision, improve efficiency, and mitigate existing limitations, positioning it as an indispensable tool in future genetic research. Overall, CRISPR-Cas9 promises transformative progress in healthcare and agriculture, solidifying its role as a cornerstone in the evolution of genetic engineering.

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

@article {pmid41649714,
year = {2024},
author = {Dong, G and Fan, Z},
title = {CRISPR/Cas-mediated germplasm improvement and new strategies for crop protection.},
journal = {Crop health},
volume = {2},
number = {1},
pages = {2},
pmid = {41649714},
issn = {2948-1945},
support = {SYND-2021-03//Administration Bureau of Sanya Yazhou Bay Science and Technology City/ ; CARS-02//China Agricultural Research System of MOF and MARA/ ; },
abstract = {Global agriculture and food security are encountering unprecedented challenges from both the ever-growing population and rapidly changing climate conditions. CRISPR/Cas-mediated genome editing technology has revolutionized plant functional genetic research and precision crop breeding with robustness, high target specificity and programmability. Furthermore, numerous emerging biotechnologies based on the CRISPR/Cas platform provide the opportunity to create new crop germplasms with durable resistance against disease or insect pests, herbicide tolerance, and other stress-tolerant improvements, reshaping crop protection to increase agricultural resilience and sustainability. In this review, we briefly describe the CRISPR/Cas toolbox, including base editing, prime editing, compact genome manipulation, transcriptional regulation and epigenetic editing, and then overview the most important applications of CRISPR/Cas-mediated crop genetic improvement, highlighting crop protection-based stress resistance engineering. In addition, we enumerate global regulations on genome-edited crops. Finally, we discuss some bottlenecks facing this cutting-edge technology and infinite possibilities for the future.},
}

@article {pmid41642758,
year = {2026},
author = {Sun, L and Liu, Z and Dong, C and Chi, T and Chen, J and Cheng, D and Zhu, L and Hu, B},
title = {Method Development and Sensitivity Optimization Paradigm for One-Step RPA-CRISPR/Cas12a Rapid Detection of the Hard-to-Amplify Gene blaNDM.},
journal = {Environmental science & technology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.est.5c10280},
pmid = {41642758},
issn = {1520-5851},
abstract = {The presence and spread of antibiotic resistance genes (ARGs) across various habitats have increased the risks of antibiotic resistance, highlighting the urgent need for effective monitoring methods. One key challenge in method development lies in balancing sensitivity, speed, and portability. To address it, a one-step assay targeting the carbapenem resistance gene blaNDM was developed based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a. A sensitivity optimization paradigm─MOSAIC (multistrategy optimized sensitive assay via integrated CRISPR/Cas12a)─was proposed, incorporating component optimization, suboptimal-PAM-mediated CRISPR inhibition, and glycerol-assisted phase separation. The glycerol-assisted strategy exhibited the largest enhancement, followed by the suboptimal-PAM strategy and component optimization. When combined, these strategies demonstrated a synergistic effect, yielding greater improvement (10 000-fold) than a single strategy alone. MOSAIC reached a limit of detection (LOD) of 260 copies/μL, comparable to that of qPCR, and enabled faster quantification of blaNDM at 37 °C within 1 h on a standard plate reader. It achieved 100% diagnostic sensitivity and 95.45% specificity in clinical isolates, and 77.41-99.73% accuracy in environmental matrix-spiked samples, comparable to that of qPCR. It provides a technological foundation for on-site detection of blaNDM and offers an optimization paradigm and new insights for the development of one-step RPA-CRISPR/Cas12a assays targeting various genes.},
}

@article {pmid41642528,
year = {2026},
author = {Shafique, MS and Liu, Y and Ji, Z},
title = {Genome Editing for Developing Disease-Resistant Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3012},
number = {},
pages = {253-264},
pmid = {41642528},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Disease Resistance/genetics ; CRISPR-Cas Systems/genetics ; *Genome, Plant ; *Plant Diseases/genetics ; Plants, Genetically Modified/genetics ; Oryza/genetics ; Plant Breeding/methods ; Crops, Agricultural/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; },
abstract = {Genome editing tools, most notably the CRISPR-Cas9 system, have transformed modern crop breeding by allowing precise and efficient modification of plant genomes, particularly for enhancing resistance to disease. In this chapter, we present two major CRISPR-based approaches that have been successfully applied to develop disease-resistant crops: (1) gene knockout of host susceptibility (S) genes to block pathogen compatibility, and (2) targeted insertion of resistance alleles or synthetic genes using the homology-directed repair (HDR) pathway. Both strategies utilize the specificity and efficiency of CRISPR-Cas9 to introduce defined edits in crops such as rice. This chapter outlines comprehensive step-by-step protocols covering key elements of the process, including target site selection, construction of sgRNA and Cas9 vectors, plant transformation methods, and mutation validation techniques. Together, these methods provide a powerful platform for engineering plant immune responses and offer practical tools to accelerate the breeding of disease-resistant cultivars.},
}

*Gene Editing/methods
*Disease Resistance/genetics
CRISPR-Cas Systems/genetics
*Genome, Plant
*Plant Diseases/genetics
Plants, Genetically Modified/genetics
Oryza/genetics
Plant Breeding/methods
Crops, Agricultural/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Knockout Techniques

@article {pmid41641699,
year = {2026},
author = {Fu, M and Wang, J and Li, J and Zhou, Y and Huang, X and Jia, Z and Luo, Y and Tan, X and Gao, Y and Yu, B and Duan, Y and Bu, Q and Li, X and Wang, Y and Takaya, N and Zhou, S},
title = {Dual-single-guide RNA strategy improves CRISPR-mediated homology-directed repair in Aspergillus.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41641699},
issn = {1362-4962},
support = {22077032//National Natural Science Foundation of China/ ; 21672065//National Natural Science Foundation of China/ ; K202415//State Key Laboratory of Natural and Biomimetic Drugs/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Aspergillus nidulans/genetics ; *CRISPR-Cas Systems ; *Recombinational DNA Repair ; Gene Editing/methods ; DNA Breaks, Double-Stranded ; Fungal Proteins/genetics/metabolism ; DNA, Fungal/genetics ; },
abstract = {CRISPR-Cas9 knock-in efficiency is often limited by geometric misalignment between donor DNA and the endogenous strand-invasion path. In Aspergillus nidulans, we found that integration drops sharply when the insertion site is offset from the invasion entry point, producing premature annealing or unsupported 3' ends that stall DNA synthesis. Chromatin immunoprecipitation-based profiling shows directional loading of the RAD51 homolog UvsC around Cas9-induced double-strand breaks, thereby defining the spatial origin of strand invasion. Guided by this insight, we introduce a dual-single-guide RNA design that places two cuts flanking the insertion site to create a geometry-matched strand-invasion window. This alignment consistently and markedly increases homology-directed-repair-mediated integration across insert sizes and editing tasks-including C-terminal tagging, bidirectional promoter rewiring, and long-distance dual-site mutagenesis-and generalizes across multiple fungal species. We propose a structural-docking model in which pairing fidelity between the resected chromosomal strand and donor homology arms governs knock-in outcomes, providing a practical design principle for efficient and precise genome engineering at structurally constrained loci.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
*Aspergillus nidulans/genetics
*CRISPR-Cas Systems
*Recombinational DNA Repair
Gene Editing/methods
DNA Breaks, Double-Stranded
Fungal Proteins/genetics/metabolism
DNA, Fungal/genetics

@article {pmid41640776,
year = {2025},
author = {Di Pasquale, G and Ottaviani, L and Camardo Leggieri, M and Giorni, P and Marocco, A and Battilani, P and Lanubile, A},
title = {Plant oxylipins: adaptation to environmental stresses and impact on mycotoxin contamination.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1739321},
pmid = {41640776},
issn = {1664-462X},
abstract = {Due to increasingly frequent changes in climatic conditions and global warming, plants consistently deal with severe weather events including extreme temperature variations, floods and drought. These abiotic stressors resulting from climate change weaken host crop resistance, making them more exposed to fungal disease insurgences and mycotoxin contamination. Oxylipins are major players in the plant-environment interaction. Their synthesis begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases (LOXs) to generate fatty acid hydroperoxides that in turn are converted into a huge assortment of bioactive compounds by specialized cytochrome P450 enzymes, known as CYP74. In the present review we focus on recent advances concerning oxylipin biosynthesis and the phylogenetic relationships among the main key enzymes of the oxylipin pathway considering five monocot and dicot plant species. Moreover, new information regarding the role of these signaling molecules on the plant physiology in response to abiotic stress and mycotoxin occurrence are provided along with the application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) (CRISPR/Cas)-based tools. Here, we report the intervention of LOX, allene oxide synthase, OPDA reductase, JASMONATE (JA) resistant and JA ZIM domain genes along with the accumulation of JA and its conjugates, 12-OPDA, ketols and green leaf volatiles in response to abiotic stress. The modulation of LOX genes and the production of several fatty acids, oxylipins and sphingolipids is also required against mycotoxin contamination.},
}

@article {pmid41575301,
year = {2026},
author = {Park, H and Bae, SM and Hong, T and Song, G and Lim, W},
title = {CRISPR/Cas9-mediated SLC2A1 gene knockout changes in energy metabolism and cellular behavior in human trophoblasts.},
journal = {Reproduction (Cambridge, England)},
volume = {171},
number = {2},
pages = {},
doi = {10.1093/reprod/xaag006},
pmid = {41575301},
issn = {1741-7899},
support = {//National Research Foundation of Korea/ ; RS-2024-00453204//Korea government/ ; RS-2025-02216962//Korea government/ ; },
mesh = {Humans ; *Trophoblasts/metabolism/cytology ; *CRISPR-Cas Systems ; *Glucose Transporter Type 1/genetics/metabolism ; *Energy Metabolism/genetics ; Endoplasmic Reticulum Chaperone BiP ; Female ; Pregnancy ; Gene Knockout Techniques ; Endoplasmic Reticulum Stress ; Mitochondria/metabolism ; Reactive Oxygen Species/metabolism ; Signal Transduction ; Autophagy ; },
abstract = {Glucose transport across the placenta is essential for fetal growth and development. Glucose transporter 1, encoded by the SLC2A1 gene, plays a central role in mediating maternal-fetal glucose exchange. Dysregulation of placental glucose transport is implicated in pregnancy-related complications, such as preeclampsia and fetal growth restriction; however, the mechanistic role of SLC2A1 in trophoblast function remains poorly defined. To functionally validate the role of SLC2A1 in human trophoblasts, we used clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-mediated knockout of the SLC2A1 gene, enabling complete and permanent loss of SLC2A1 expression. In the resulting SLC2A1 knockout human trophoblast HTR8/SVneo cells, SLC2A1 depletion induced a metabolic shift from glycolysis to oxidative phosphorylation, leading to increased mitochondrial respiration, ATP production, mitochondrial calcium overload, and elevated mitochondrial reactive oxygen species generation. These changes were accompanied by enhanced endoplasmic reticulum stress, as shown by the upregulation of p-PERK, IRE1α, and GRP78, as well as increased autophagic activity indicated by LC3B-II and p62 accumulation. Notably, mTOR signaling was also upregulated, suggesting a feedback loop that regulates autophagy. The loss of SLC2A1 impaired the PI3K/AKT pathway, reduced trophoblast migration and 3D spheroid formation, and disrupted epithelial-mesenchymal transition-like properties. These findings demonstrate that SLC2A1 is essential for maintaining trophoblast energy homeostasis, redox balance, and invasive capacity; its deficiency triggers mitochondrial and endoplasmic reticulum stress responses that may contribute to placental dysfunction during early pregnancy.},
}

Humans
*Trophoblasts/metabolism/cytology
*CRISPR-Cas Systems
*Glucose Transporter Type 1/genetics/metabolism
*Energy Metabolism/genetics
Endoplasmic Reticulum Chaperone BiP
Female
Pregnancy
Gene Knockout Techniques
Endoplasmic Reticulum Stress
Mitochondria/metabolism
Reactive Oxygen Species/metabolism
Signal Transduction
Autophagy

@article {pmid41640466,
year = {2026},
author = {Bharti, N and Modi, U and Bhatia, D and Solanki, R},
title = {Engineering delivery platforms for CRISPR-Cas and their applications in healthcare, agriculture and beyond.},
journal = {Nanoscale advances},
volume = {},
number = {},
pages = {},
pmid = {41640466},
issn = {2516-0230},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have transformed genome editing through unprecedented precision, and next-generation variants (base and prime editors) further enhance specificity by enabling targeted nucleotide changes without introducing double-strand DNA breaks. These technologies have unlocked broad applications in therapeutic gene correction, functional genomics, infectious disease management, diagnostics, agricultural engineering, environmental biotechnology, and synthetic biology. However, the targeted delivery of these systems remains a major challenge due to the large and chemically distinct nature of their components, including Cas protein or its base/prime editor fusions, guide RNA, and in some cases, DNA repair templates-which complicate packaging, stability, and cellular uptake. Additional hurdles arise from tissue and cell-type specificity, differential intracellular environments, variable editing efficiencies, and the persistent risk of off-target genome modifications. This review outlines the key challenges in the delivery of CRISPR technologies as well provides a comprehensive overview of both current and emerging delivery strategies, including viral vectors (adenovirus, adeno-associated virus, and lentivirus), non-viral physical approaches (microinjection, electroporation, ultrasound, and hydrodynamic tail-vein injection), and nanoparticle-based modalities (lipid and polymeric nanoparticles, gold nanoparticles, DNA nanostructures, and extracellular vesicles). We also discussed the diverse applications of CRISPR-Cas9 in gene therapy, immune cell engineering for cancer therapies, and agricultural innovation.},
}

@article {pmid41638451,
year = {2026},
author = {Asemoloye, MD},
title = {The fungal cure: Harnessing mycelial approach as sustainable green solution for industrial waste treatment.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108834},
doi = {10.1016/j.biotechadv.2026.108834},
pmid = {41638451},
issn = {1873-1899},
abstract = {Industrialization has intensified releases of complex waste streams (e.g., synthetic dyes, petroleum hydrocarbons, heavy metals, and plastics) whose treatment can be costly, energy-intensive, and often incomplete using conventional physicochemical methods. 'Mycoremediation' defined as fungi mediated remediation, or their secreted materials/enzymes offers compelling advantages. These advantages stem across the extensive mycelial networks for matrix penetration, non-specific oxidative enzyme systems that transform lignin-like xenobiotics, and cell-wall chemistries that sorb metal ions. This review synthesizes mechanistic foundations on fungal enzymes (laccases; class II peroxidases such as manganese peroxidase and lignin peroxidase; biosorption and biomineralization), bioengineering strategies (CRISPR/Cas editing, artificial consortia), process intensification (immobilized-laccase reactors; whole-cell formats), and applications across textile dye effluents, petroleum-impacted soils/sediments, heavy-metal bearing wastewaters/soils, and polymer-rich wastes. Emerging evidence shows robust lab and mesocosm performance like rapid dye decolorization in fungal cartridge systems, significant alteration of petroleum (saturate, aromatic, resin and asphaltene-SARA) fractions under estuarine salinities, and high-capacity metal biosorption, while systematic verification for plastics remains a priority. Fungi sustainability assessments identify life-cycle hot spots in enzyme production and immobilization supports; techno-economic analyses suggest feasibility pathways when biocatalyst durability and reuse are optimized. This review also delves into regulatory frameworks for contained use and deliberate environmental release of engineered fungi, shaping the near-term deployments toward contained bioreactors. It concludes by projecting the combination of bioengineering (strain/secretome control), reactorization (immobilized catalysts, modular beds), and standardized metrics (toxicity, mass balance, life-cycle assessment-LCA/techno-economic analysis-TEA) for accelerating the transition of mycoremediation from promising prototypes to field-validated, scalable technologies for industrial waste treatment.},
}

@article {pmid41634489,
year = {2026},
author = {Araújo, MRB and Dos Santos, LS and Viana, MVC and Sousa, EG and Prates, FD and Perini, HF and da Silva, MV and da Silva Sousa, J and Brenig, B and Andrade, BS and Mattos-Guaraldi, AL and de Oliveira Sant'Anna, L and Ramos, JN and de Oliveira Vinhal, AL and de Castro Soares, S and Azevedo, V},
title = {Comparative genomics and molecular characterization of a multidrug-resistant Corynebacterium glucuronolyticum isolated for the first time from the human genitourinary tract in Latin America.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {57},
number = {1},
pages = {49},
pmid = {41634489},
issn = {1678-4405},
abstract = {UNLABELLED: Although Corynebacterium glucuronolyticum has been associated with human infections, its pathogenic potential remains poorly understood. Here, we describe the first case in Latin America of C. glucuronolyticum isolated from the human urogenital tract. The strain, designated IHP2022, was identified by MALDI-TOF MS (99% probability) and exhibited resistance to benzylpenicillin, clindamycin, and tetracycline, characterizing a multidrug-resistant (MDR) phenotype. Genomic analysis revealed a 2.88-Mb genome with 59.04% G + C content and no plasmids. Comparative genomic analysis, including 11 other publicly available genomes, demonstrated high genetic diversity and positioned IHP2022 close to strain p3-SID752 from the USA, suggesting a broad geographical distribution. The genome harbored multiple virulence and resistance genes, as well as a Type I-E CRISPR-Cas system. Functional annotation and pangenome analysis identified 4,027 gene families categorized into core, shell, and cloud components. By integrating phenotypic and genomic data, this study provides the first in-depth characterization of an MDR C. glucuronolyticum strain minimizing current knowledge gaps and contributing to a better understanding of its pathogenic potential.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-025-01822-7.},
}

@article {pmid41633991,
year = {2026},
author = {Kumari, B and Damodaran, AP and Guiblet, WM and Xiao, MS and Behera, AK and On, TA and McIntosh, CE and Teszler, M and Holloway, C and Le, S and Parab, N and Zhao, Y and Aregger, M and Gonatopoulos-Pournatzis, T},
title = {Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1218},
pmid = {41633991},
issn = {2041-1723},
support = {ZIA BC012033/ImNIH/Intramural NIH HHS/United States ; ZIA BC012101/ImNIH/Intramural NIH HHS/United States ; HHSN261200800001C/RC/CCR NIH HHS/United States ; HHSN261200800001E/CA/NCI NIH HHS/United States ; 1ZIABC012101//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 1ZIABC012033//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 75N91019D00024/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Exons/genetics ; *Alternative Splicing/genetics ; *Single-Cell Analysis/methods ; CRISPR-Cas Systems/genetics ; Gene Expression Profiling/methods ; Cell Cycle/genetics ; Sequence Deletion ; Transcriptome ; Gene Expression Regulation ; },
abstract = {Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we develop scCHyMErA-Seq, a scalable CRISPR-based exon deletion screening platform integrated with 10x Genomics single-cell transcriptomic readouts. This tool enables efficient exon deletion while simultaneously capturing Cas9/Cas12a guides and polyadenylated transcripts at single-cell resolution. Applying scCHyMErA-Seq to high-throughput profiling of alternative cassette exons, we identify numerous exons with pronounced regulatory effects on gene expression and cell cycle progression. Analysis of the alternative NRF1 exon-7 demonstrates that its inclusion modulates NRF1's regulatory function by influencing its recruitment to the promoters of target genes. Importantly, gene expression profiles generated using scCHyMErA-Seq accurately recapitulate findings from traditional, labor-intensive orthogonal methods, while offering enhanced scalability and efficiency. Overall, scCHyMErA-Seq represents a versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.},
}

Humans
*Exons/genetics
*Alternative Splicing/genetics
*Single-Cell Analysis/methods
CRISPR-Cas Systems/genetics
Gene Expression Profiling/methods
Cell Cycle/genetics
Sequence Deletion
Transcriptome
Gene Expression Regulation

@article {pmid41611149,
year = {2026},
author = {Setia, A and Patil, D and Randhave, NV and Vaishali, and Verma, N and Rani, K and Kale, A and Wagh, B and Kumar, V and Malik, AK and Sharon Mano Pappu, J and Deshmukh, K and Muthu, MS},
title = {CRISPR/Cas9 genome editing-based preclinical theranostics, biomarkers and drug delivery systems for cancer applications.},
journal = {International journal of biological macromolecules},
volume = {345},
number = {},
pages = {150582},
doi = {10.1016/j.ijbiomac.2026.150582},
pmid = {41611149},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *Neoplasms/genetics/therapy/diagnosis/drug therapy ; *Drug Delivery Systems/methods ; *Biomarkers, Tumor/genetics ; *Theranostic Nanomedicine/methods ; Animals ; Genetic Therapy/methods ; },
abstract = {Cancer development is driven by diverse genetic aberrations, underscoring the importance of innovative approaches like gene therapy for effective treatment. The CRISPR/Cas9 gene-editing system has all the makings of a game-changing technique for future disease treatment, owing to its pinpoint accuracy and efficiency in deleting disease-causing genes or correcting damaging base mutations. A number of efficient Cas9 variants and derivatives were recently designed to tackle the intricate genomic modifications that accompany illnesses. In addition, CRISPR/Cas9 based systems are increasingly explored for biomarker sensing and cancer diagnostics. Early identification, real-time monitoring, and therapy stratification are made possible by CRISPR-driven biosensors, which can detect circulating tumor DNA, microRNAs, or exosomal RNA with high specificity and sensitivity. Furthermore, a variety of stimuli-responsive delivery strategies, including chemical and peptide-assisted systems, light-activated mechanisms, glutathione-sensitive carriers, and pH-responsive platforms, have been explored to improve intracellular release efficiency thereby enhancing the precision of CRISPR/Cas9-mediated gene editing in cancer therapy. The CRISPR/Cas9-enabled theranostic platforms employ engineered nanocarriers to simultaneously deliver gene-editing tools and imaging agents, thereby facilitating synchronized treatment monitoring and improved therapeutic precision. This review emphasizes the transformative potential of CRISPR/Cas9-integrated theranostics, which combine targeted gene editing with advanced imaging for enhanced therapeutic monitoring and efficacy in cancer treatment.},
}

*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
*Neoplasms/genetics/therapy/diagnosis/drug therapy
*Drug Delivery Systems/methods
*Biomarkers, Tumor/genetics
*Theranostic Nanomedicine/methods
Animals
Genetic Therapy/methods

@article {pmid41508115,
year = {2026},
author = {Xiao, Y and Li, X and Jiang, L and Zhao, Y and Wang, L and Feng, Y},
title = {Construction of Escherichia coli L-isoleucine cell factories based on propionate pathway.},
journal = {Journal of biological engineering},
volume = {20},
number = {1},
pages = {28},
pmid = {41508115},
issn = {1754-1611},
abstract = {UNLABELLED: Isoleucine, an essential branched-chain amino acid with broad applications in food, pharmaceuticals, and feed, is predominantly produced via the microbial threonine pathway, which suffers from catalytic complexity and regulatory inefficiency. The propionate pathway offers a streamlined alternative but remains unexplored for L-isoleucine biosynthesis. Here, we engineered Escherichia coli to establish the first propionate pathway-based L-isoleucine cell factory. Critical enzymes—propionyl-CoA synthase (PCS), propionyl-CoA transferase (PCT), and α-ketobutyrate synthase (OBS)—were identified for converting propionate to α-ketobutyrate. Key genes (prpE from Salmonella, pctcP from Gibberella, pctcN from Clostridium propionicum, and nifJ from Moorella thermoacetica) were integrated with the propionate transporter (prpP) and carbonic anhydrase (can) to enhance substrate utilization. ILE-5a, derived from E. coli BW25113 with deletions in brnQ, livJ, and livK, and containing specific insertions, was further modified to create ILE-5b with an additional deletion in yjiP. Plasmid-based expression in these strains, ILE-5a and ILE-5b, yielded top producers ILE-5a-P10 and ILE-5b-P10, which achieved L-isoleucine titers of 304 mg/L and 235 mg/L, respectively, in shake-flask fermentation using glucose and propionate as carbon sources. To stabilize production, the optimal gene set (pctN, nifJ, prpP, can) was genomically integrated via a transposon-encoded CRISPR-Cas system, generating mutants ILE-5a-P11 and ILE-5b-P11. Response surface methodology-optimized medium and 3-L fed-batch fermentation further elevated titers to 1.13 g/L (ILE-5a-P11) and 11.33 g/L (ILE-5b-P11). This study pioneers the propionate pathway for efficient L-isoleucine production, demonstrating its industrial potential through systematic metabolic engineering and process optimization.To our knowledge, this is the first study to design and demonstrate an E. coli platform for isoleucine production that simultaneously leverages the glutamate and propionate precursors.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13036-025-00609-6.},
}

@article {pmid41345278,
year = {2026},
author = {de la Rosa, C and Kendirli, A and Baygün, S and Bauernschmitt, F and Thomann, AS and Kisioglu, I and Beckmann, D and Carpentier Solorio, Y and Pfaffenstaller, V and Tai, YH and Mehraein, N and Sanchez, P and Spieth, L and Gerdes, LA and Beltran, E and Dornmair, K and Simons, M and Peters, A and Schmidt-Supprian, M and Kerschensteiner, M},
title = {In vivo CRISPR screen reveals regulation of macrophage states in neuroinflammation.},
journal = {Nature neuroscience},
volume = {29},
number = {2},
pages = {493-509},
pmid = {41345278},
issn = {1546-1726},
support = {259373024//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 408885537//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 239283807//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *Macrophages/metabolism ; Mice ; Cytokines/metabolism/genetics ; Mice, Inbred C57BL ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Multiple Sclerosis/immunology/genetics ; Humans ; *Neuroinflammatory Diseases/genetics ; CRISPR-Cas Systems ; *Encephalomyelitis, Autoimmune, Experimental/immunology ; },
abstract = {Here we established an in vivo CRISPR screening pipeline using genetically editable progenitor cells to dissect macrophage regulation in mouse models of multiple sclerosis (MS). Screening over 100 cytokine receptors and signaling molecules identified interferon-γ, tumor necrosis factor, granulocyte-macrophage colony-stimulating factor and transforming growth factor-β as essential regulators of macrophage polarization in vivo. Single-cell transcriptomics confirmed that transferred progenitor cells generate all blood-derived CNS myeloid cell populations, enabling Perturb-seq analysis of cytokine actions in neuroinflammation. Combined with biosensor expression, our approach allows monitoring cytokine effects on myeloid cell migration, debris phagocytosis and oxidative activity in vivo. Comparative transcriptomic analyses revealed conserved neuroinflammatory cytokine signatures across myeloid populations, CNS compartments and species, elucidating cytokine cues shaping myeloid function in the cerebrospinal fluid and parenchyma of individuals with MS. This versatile pipeline thus provides a scalable framework for high-resolution analysis of macrophage states and uncovers the cytokine signals that underlie their regulation in MS and MS models.},
}

Animals
*Macrophages/metabolism
Mice
Cytokines/metabolism/genetics
Mice, Inbred C57BL
*Clustered Regularly Interspaced Short Palindromic Repeats
*Multiple Sclerosis/immunology/genetics
Humans
*Neuroinflammatory Diseases/genetics
CRISPR-Cas Systems
*Encephalomyelitis, Autoimmune, Experimental/immunology

@article {pmid41633939,
year = {2026},
author = {Lin, L and Zhang, JJ and Liu, BH and Du, S and Zhang, YQ and Yang, Y and Li, C and Dong, CC and He, YB and Wang, Q and Wang, HY and Shao, CW},
title = {Epigenetic editing of marine medaka (Oryzias melastigma) fgf2 using CRISPR/dCas9-Tet1CD.},
journal = {Zoological research},
volume = {47},
number = {1},
pages = {263-278},
doi = {10.24272/j.issn.2095-8137.2025.089},
pmid = {41633939},
issn = {2095-8137},
mesh = {Animals ; *Oryzias/genetics ; *Gene Editing/veterinary ; *Epigenesis, Genetic ; *Fibroblast Growth Factor 2/genetics/metabolism ; *CRISPR-Cas Systems ; Epigenome Editing ; },
abstract = {CRISPR/dCas9-mediated epigenetic editing offers a versatile approach for transcriptional regulation without introducing DNA strand breaks. Although this strategy has been explored in a limited number of species, its application in aquatic vertebrates remains largely uncharacterized. In this study, ten-eleven translocation methylcytosine dioxygenase 1 (tet1) was cloned and molecularly characterized in marine medaka (Oryzias melastigma). Decitabine treatment identified fibroblast growth factor 2 (fgf2) as a methylation-sensitive gene, with a regulatory CpG island located within its promoter region. Subsequently, a CRISPR/dCas9-Tet1CD activation system was constructed by fusing the catalytic domain of Tet1 (Tet1CD, Ala1352-Thr2034) to dCas9, enabling locus-specific DNA demethylation. Targeting fgf2, this CRISPR/dCas9-Tet1CD system induced efficient and selective demethylation of the CpG island, resulting in a maximal 2.41-fold increase in fgf2 transcript levels. Whole-genome bisulfite sequencing and transcriptomic analysis confirmed high on-target precision with minimal off-target effects. Epigenetic activation of fgf2 further modulated downstream gene networks associated with growth, promoting durable transcriptional enhancement and increased cellular proliferation. Collectively, these results establish a robust and highly specific epigenetic editing platform in marine medaka, providing a powerful tool for functional genomics studies and regulatory element analysis in aquatic models.},
}

Animals
*Oryzias/genetics
*Gene Editing/veterinary
*Epigenesis, Genetic
*Fibroblast Growth Factor 2/genetics/metabolism
*CRISPR-Cas Systems
Epigenome Editing

@article {pmid41632443,
year = {2026},
author = {Mašlaňová, I and Nováková, D and Švec, P and Kovařovic, V and Sedláček, I and Botka, T and Šedo, O and Neumann-Schaal, M and Vives, J and Doškař, J and Pantůček, R},
title = {Genomic and taxonomic characterization of strain CCM 2573: Uncovering unique genetic features and description of Macrococcus caseolyticus subsp. lactis subsp. nov.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {41632443},
issn = {1874-9356},
abstract = {Strain CCM 2573 is a Gram-positive bacterium that has been intensively studied in the past due to its distinct chemotaxonomic properties, but its reliable taxonomic classification has not been satisfactorily clarified. Whole-genome sequencing and comparative genomic analyses performed in this study revealed that the strain belongs to the Macrococcus caseolyticus phylogenetic clade. Genome-to-genome comparisons confirmed the closest relationship to the type strains of M. caseolyticus subsp. hominis CCM 7927[T] and M. caseolyticus subsp. caseolyticus DSM 20597[T]. However, the strain harbored unique genomic elements distinguishing it from its nearest phylogenetic neighbors. Its accessory genome contains dozens of insertion sequences, a 92-kbp composite transposon with unique palindromic repeat loci associated with a CRISPR-Cas adaptive immune system, a pseudo-staphylococcal chromosome cassette, and several additional genomic islets. Unlike other macrococci, strain CCM 2573 exhibits a specific peptidoglycan composition (L-Lys-Gly2-Ser2-Gly) and shows a higher phylogenetic divergence of aminoacyltransferases (FemABX) involved in interpeptide bridge synthesis. In addition, it reveals distinct biochemical characteristics from both subspecies of M. caseolyticus, particularly in its ability to produce acid from galactose, cellobiose, melezitose, and turanose, as well as in its susceptibility to novobiocin. The MALDI-TOF mass spectra enable differentiation of the strain from other type strains of the genus Macrococcus. The results of polyphasic taxonomy obtained in this study showed that strain CCM 2573 belongs to the species M. caseolyticus, but it is distinct from both validly named M. caseolyticus subspecies. We propose to assign the analyzed strain as a new subspecies, Macrococcus caseolyticus subsp. lactis subsp. nov. The type strain is CCM 2573[T] (= DSM 20227[T]).},
}

@article {pmid41632281,
year = {2026},
author = {Li, J and Zhu, M and Hu, G and Chen, X and Xue, H and Zhang, Y and Wang, Y and Li, Z and Xu, D and Zhai, M and Zhou, G and Cui, C and Zhao, C and Qin, R and Wu, Y and Cui, F and Sun, H},
title = {Gene editing and association analysis of circadian clock gene TaPRR59 highlights its importance in yield-related traits in wheat.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {2},
pages = {58},
pmid = {41632281},
issn = {1432-2242},
support = {32272119//National Natural Science Foundation of China/ ; 32072051//National Natural Science Foundation of China/ ; ZR2022YQ19//Shandong Provincial Fund for Excellent Young Scholars/ ; 32101726//National Natural Science Foundation for Young Scholars of China/ ; ZR2022MC119//Natural Science Foundation of Shandong Province, China/ ; 20230119//Taishan scholar young expert/ ; ZR2019ZD16//Major Basic Research Project of Natural Science Foundation of Shandong Province, China/ ; 2022LZG002-2//Key R & D Program of Shandong Province, China (Major Innovation Project)/ ; 2023ZDCX023//Yantai science and technology plan project/ ; IPGS2025-083//The Innovation Project for graduate students of Ludong University/ ; },
mesh = {*Triticum/genetics/growth & development ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; *Circadian Clocks/genetics ; Gene Expression Regulation, Plant ; Haplotypes ; Phenotype ; Plant Breeding ; *Genes, Plant ; CRISPR-Cas Systems ; },
abstract = {Mutations in TaPRR59 impact transcript levels of some key flowering genes and show earlier heading time and reduced plant height. Favorable haplotype TaPRR59-A1-Hapla was positively selected in wheat breeding programs. The circadian clock system is a crucial endogenous rhythmic regulatory mechanism with a significant role in plant growth and development. The pseudo-response regulator (PRR) family is a pivotal component of circadian networks. In the present study, we cloned the wheat PRR family member TaPRR59 and investigated its function using gene editing, transcriptome sequencing, haplotype analysis, and association analysis. The expression profile of TaPRR59 over a 24-h period exhibited a diurnal rhythmic expression pattern. Luciferase transient transcriptional assay demonstrated that TaPRR59 acts as a transcriptional repressor in the nucleus. The taprr59-ABD-KO gene-edited lines produced using the CRISPR/Cas9 genome-editing system had earlier heading time and reduced plant height. Overexpression of TaPRR59-D1 in rice significantly delayed the heading date, reduced plant height and thousand-grain weight, and increased the number of grains per panicle. Transcriptome analysis revealed the transcript levels of several key flowering genes and chlorophyll a-b binding protein-related genes were up- or down-regulated in the taprr59 mutant plants. Association analysis showed that natural variations at TaPRR59-A1, TaPRR59-B1, and TaPRR59-D1 were significantly associated with yield traits such as plant height, thousand-grain weight, and heading date. Geographical analysis showed distinctive distribution characteristics of TaPRR59 haplotypes in different agroecological production zones. Additionally, the significant difference in frequency of the favorable haplotype TaPRR59-A1-Hapla between landraces and modern cultivars indicates that it has been subject to directional selection during wheat breeding. This research provided novel insights into the influence of the circadian clock system on agronomic traits and provided useful molecular markers and genetic resources for wheat breeding.},
}

*Triticum/genetics/growth & development
*Gene Editing
*Plant Proteins/genetics/metabolism
*Circadian Clocks/genetics
Gene Expression Regulation, Plant
Haplotypes
Phenotype
Plant Breeding
*Genes, Plant
CRISPR-Cas Systems

@article {pmid41630767,
year = {2025},
author = {Molaghi, MAA and Atiyah, WR and Saeed, AAR},
title = {Novel CRISPR/Cas12-based assay for the rapid and accurate detection of donkey meat.},
journal = {Open veterinary journal},
volume = {15},
number = {11},
pages = {5682-5688},
pmid = {41630767},
issn = {2218-6050},
mesh = {Animals ; *Equidae/genetics ; *Meat/analysis ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Polymerase Chain Reaction/veterinary/methods ; },
abstract = {BACKGROUND: Precise identification of items originating from animals is required for monitoring the authenticity of livestock commodities. Nucleic acid-based detection methods, including polymerase chain reaction, are highly accurate tests for detecting meat fraud. However, these tests require costly devices and highly skilled personnel.

AIM: This study aims to develop a precise and rapid test based on deoxyribonucleic acid (DNA) for detecting meat from donkeys.

METHODS: The assay was developed by combining Clustered regularly interspaced short palindromic repeat /Cas12 with RPA and a lateral flow device. A conserved sequence of the mitochondrial D-loop gene was used as the target gene. The assessment of the assay focused on its sensitivity, specificity, and completion time.

RESULTS: The results show that the assay can detect donkey meat in the tested samples with 100% specificity with no cross-reactivity with other species, and no false-positive results were recorded. Furthermore, the assay has shown high sensitivity and to detect as little as 5 ng of input DNA. Moreover, the results, including DNA extraction, were obtained in less than 40 minutes and did not require any specialized equipment.

CONCLUSION: The assay is a good option for the rapid and precise detection of donkey meat. Further optimization and studies are needed to enable the direct detection of donkey meat without DNA extraction.},
}

Animals
*Equidae/genetics
*Meat/analysis
Sensitivity and Specificity
*CRISPR-Cas Systems
Polymerase Chain Reaction/veterinary/methods

@article {pmid41630633,
year = {2026},
author = {Lee, SY and Park, HH},
title = {Structural Basis of Recognition of Anti-CRISPR Operon by Aca3.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {3},
pages = {e71512},
doi = {10.1096/fj.202502389RR},
pmid = {41630633},
issn = {1530-6860},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; RS-2025-16065724//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Operon ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Viral Proteins/genetics/metabolism/chemistry ; Bacteriophages/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Promoter Regions, Genetic ; },
abstract = {The CRISPR-Cas system equips bacteria with adaptive immunity by storing fragments of invading nucleic acids in CRISPR loci and deploying Cas proteins to recognize and degrade matching sequences. In turn, bacteriophages have evolved small anti-CRISPR (Acr) that neutralize diverse CRISPR-Cas types. Acr genes are often co-encoded with transcriptional regulators called anti-CRISPR-associated (Aca) proteins, which suppress acr expression. Although 13 Aca families have been identified through bioinformatic analysis, detailed information on their target DNA-binding mechanisms and the inhibition of acr expression remains limited. Here, we report the high-resolution structure of Aca3 and delineate its DNA-binding interface. We demonstrate that Aca3 selectively recognizes inverted repeats upstream of its cognate acr gene, AcrIIC1. Mutational analyses of key helix-turn-helix residues confirm their essential roles in promoter engagement. Together, these results reveal the molecular basis for Aca3-mediated control of anti-CRISPR expression and expand our understanding of regulatory strategies that phages employ to modulate host CRISPR-Cas immunity.},
}

*CRISPR-Cas Systems/genetics
*Operon
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Viral Proteins/genetics/metabolism/chemistry
Bacteriophages/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism/chemistry
Promoter Regions, Genetic

@article {pmid41629994,
year = {2026},
author = {Papaioannou, NY and Patsali, P and Klermund, J and Papasavva, PL and Andrieux, G and Koniali, L and Naiisseh, B and Christou, S and Sitarou, M and Kleanthous, M and Cathomen, T and Lederer, CW},
title = {Functional correction and genome integrity with duplex base editing of β-thalassemic hematopoietic stem cells.},
journal = {Genome biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13059-026-03974-7},
pmid = {41629994},
issn = {1474-760X},
support = {EXCELLENCE/1216/0092//Research and Innovation Foundation/ ; CA21113 GenE-HumDi//COST Actions/ ; New Infrastructure for the Diagnosis and Therapy of Patients//Norway Grants 2014-2021/ ; },
abstract = {BACKGROUND: Beta-thalassemia is among the most common monogenic disorders, posing a major global health challenge. Editing of genetic modifiers, such as BCL11A erythroid enhancer and HBG promoters, enhances fetal hemoglobin expression and confers major therapeutic potential. Double-strand-break (DSB)-independent genome editing tools, such as base editors (BE), are potentially safer and better suited for multiplexed application than DSB-dependent CRISPR/Cas technology. However, harmful on- and off-target events remain a concern and must be excluded before clinical application, including chromosomal rearrangements invisible to standard detection technologies.

RESULTS: Using primary patient-derived CD34[+] cells from three donors, we investigate simplex and duplex BE-based disruption of the BCL11A erythroid enhancer and the BCL11A binding site (-115 bp) on the HBG promoter for DNA-level and functional studies at the RNA, protein, and morphological level. Analyses include direct comparison to DSB-based editing, the current clinically applied standard, and CAST-seq to assess recombination events, allowing wider inferences on relative safety. RNA-seq analyses for clones of primary CD34[+] cells across all treatments confirm peak HBG induction for duplex BE and comparable effects on apoptotic and immune response signatures. Overall, duplex BE produces robust γ-globin and fetal hemoglobin induction, improves functional correction over simplex editing and results in low incidence of genomic alterations in both target loci.

CONCLUSIONS: Duplex BE targeting both BCL11A erythroid enhancer and HBG promoter enables functional correction and genome integrity. Our study highlights the efficacy, safety, and therapeutic potential of the present duplex BE approach.},
}

@article {pmid41629464,
year = {2026},
author = {},
title = {Reprogramming CRISPR-Cas enzymes for customized genome editing.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {41629464},
issn = {1546-1696},
}

@article {pmid41629462,
year = {2026},
author = {Nayfach, S and Bhatnagar, A and Novichkov, A and Kim, N and Hoffnagle, AM and Hussain, R and Estevam, GO and Hill, E and Ruffolo, JA and Silverstein, RA and Gallagher, J and Kleinstiver, BP and Meeske, AJ and Cameron, P and Madani, A},
title = {Customizing CRISPR-Cas PAM specificity with protein language models.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {41629462},
issn = {1546-1696},
abstract = {CRISPR-Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, greatly limiting the range of targetable sequences in a genome. Although engineering strategies to alter PAM specificity exist, they typically require labor-intensive, iterative experimentation. We introduce an evolution-informed deep learning model, Protein2PAM, to efficiently guide the design of Cas protein variants tailored to recognize specific PAMs. Trained on a dataset of over 45,000 CRISPR-Cas PAMs, Protein2PAM rapidly and accurately predicts PAM specificity directly from Cas proteins across type I, II and V CRISPR-Cas systems. Using in silico mutagenesis, the model identifies residues critical for PAM recognition in Cas9 without using structural information. We use Protein2PAM to computationally evolve Nme1Cas9, generating variants with broadened PAM recognition and up to a 50-fold increase in PAM cleavage rates compared to the wild type in vitro. Our machine learning approach allows Cas enzymes to target sequences that were previously inaccessible because of PAM constraints, potentially increasing target flexibility in personalized genome editing.},
}

@article {pmid41629044,
year = {2026},
author = {Sharma, S and Karna, SKL and Khanal, S and Pokharel, YR},
title = {Rapid detection of measles virus RNA from clinical specimens by using RT-LAMP coupled with CRISPR/Cas12b via fluorescence and lateral flow biosensor readouts: A proof-of-concept study.},
journal = {Analytica chimica acta},
volume = {1389},
number = {},
pages = {345081},
doi = {10.1016/j.aca.2026.345081},
pmid = {41629044},
issn = {1873-4324},
mesh = {*Measles virus/genetics/isolation & purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Fluorescence ; *Measles/diagnosis/virology ; Proof of Concept Study ; Animals ; Chlorocebus aethiops ; Vero Cells ; },
abstract = {BACKGROUND: Rapid laboratory confirmation of suspected cases is essential for measles control, but current methods require a complex laboratory infrastructure. We developed loop-mediated isothermal amplification coupled with CRISPR-Cas-mediated diagnostic (LAmCaD), a novel two-pot diagnostic platform that integrates rapid nucleic acid extraction, RT-LAMP amplification, and in-house purified AapCas12b as a rapid test to detect measles RNA that could be used in settings lacking laboratory infrastructure.

METHODS: LAmCaD is based on dual detection modalities, fluorescence and lateral flow biosensor readouts. The LAmCaD assay was evaluated for analytical sensitivity and specificity using RNA from Vero/hSLAM-grown measles virus, and diagnostic evaluation was performed using patient samples, compared with standard RT-PCR. The cross-genotype detection capability was assessed across epidemiologically relevant measles genotypes D8, D4, and B3.

RESULTS: The in-house purified protein AapCas12b from Alicyclobacillus acidiphilus exhibited strong cis and trans cleavage activities, eliminating dependence on commercial enzyme preparations. The platform enables the use of clinical samples from patients through rapid nucleic acid extraction that eliminates the need for RNA purification steps, allowing direct use of extracted material in RT-LAMP reactions. RT-LAMP alone achieved analytical sensitivity of ∼10[3] copies, while the complete protocol detected measles virus at ∼10[5] copies by fluorescence and ∼10[4] copies by lateral flow detection. Diagnostic evaluation demonstrated sensitivity of 64.00 %, specificity of 92.59 %, and negative predictive value of 99.95 % with an overall accuracy of 92.56 %. ROC curve analysis revealed an AUC of 0.717, indicating fair discriminatory performance. The assay demonstrated moderate agreement with RT-PCR (κ = 0.6) and successfully identified genotypes D8, D4, and B3. The entire testing process took 90 min to complete.

CONCLUSIONS: This proof-of-concept LAmCaD platform establishes the foundation for a cost-effective, field-deployable diagnostic test without relying on commercial enzymes or complex sample processing. The platform could facilitate the rapid confirmation of measles cases in resource-limited and/or remote settings, thereby contributing to global measles elimination goals. Conducted within the WHO South-East Asia Region, this study is particularly relevant to the region's 2023 elimination target, addressing current surveillance gaps and specimen transport challenges that hinder efforts to eliminate the disease.},
}

*Measles virus/genetics/isolation & purification
Humans
*Nucleic Acid Amplification Techniques/methods
*RNA, Viral/analysis/genetics
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*Molecular Diagnostic Techniques/methods
Fluorescence
*Measles/diagnosis/virology
Proof of Concept Study
Animals
Chlorocebus aethiops
Vero Cells

@article {pmid41628256,
year = {2026},
author = {Luo, Y and Dong, Q and Yi, S and Zhang, W and Du, Y and Fang, Q and Zhang, W and Ouyang, K and Chen, Y and Yin, Y and Wei, Z and Qin, Y and Huang, W},
title = {Genome-wide CRISPR screening identifies Annexin A1 as a facilitator of porcine astrovirus entry.},
journal = {PLoS pathogens},
volume = {22},
number = {2},
pages = {e1013943},
doi = {10.1371/journal.ppat.1013943},
pmid = {41628256},
issn = {1553-7374},
mesh = {Animals ; Swine ; *Virus Internalization ; CRISPR-Cas Systems ; *Annexin A1/metabolism/genetics ; *Swine Diseases/virology/genetics/metabolism ; *Astroviridae Infections/virology/metabolism/genetics ; *Mamastrovirus/physiology/genetics ; },
abstract = {Porcine astrovirus (PAstV) is an important and widespread pathogen in swine, linked to diarrheal outbreaks and extraintestinal disease. How PAstV enters host cells has remained unclear, and no cellular factor has been defined for PAstV entry. Here, a genome-wide CRISPR-Cas9 loss-of-function screen in porcine epithelial cells identifies Annexin A1 (ANXA1) as a host factor that facilitates PAstV entry. Genetic ablation or pharmacological/antibody blockade of ANXA1 reduces binding, lowers early viral RNA and capsid signals, and delays the rise of progeny, whereas re-expression restores susceptibility. Biochemical assays and surface plasmon resonance indicate a direct interaction between ANXA1 and the acidic C-terminal domain of the PAstV ORF2 capsid protein, and imaging shows ANXA1 co-localizes with incoming particles at the cell surface and supports attachment and uptake. Loss of ANXA1 does not alter infection by the non-astrovirus panel tested, indicating selectivity for PAstV under our conditions. Notably, infection is reduced but not abolished in ANXA1-deficient cells, consistent with additional entry factors acting alongside ANXA1. These findings position ANXA1 as an entry cofactor for PAstV and provide a mechanistic basis to refine models of astrovirus host-cell recognition.},
}

Animals
Swine
*Virus Internalization
CRISPR-Cas Systems
*Annexin A1/metabolism/genetics
*Swine Diseases/virology/genetics/metabolism
*Astroviridae Infections/virology/metabolism/genetics
*Mamastrovirus/physiology/genetics

@article {pmid41605211,
year = {2026},
author = {Lin, J and Lin, Y and Liu, N and Cao, W and Zhang, J and Wen, S and Zhang, Y and Liao, W and Hong, Z and Lin, Y and Liu, Q and Liu, H and Li, Q and Chen, B and Li, M and Luo, Z and Yang, L and Yang, Y and Zheng, SH and Wang, Y and Chu, H and Hu, Y and Qin, Y and Luo, BX and Tian, S and Chen, Y and Yan, T and Yang, L and Wang, H and Liu, T and Jiang, Y and Lu, Z},
title = {AAVLINK: A potent DNA-recombination method for large cargo delivery in gene therapy.},
journal = {Cell},
volume = {189},
number = {3},
pages = {969-986.e17},
doi = {10.1016/j.cell.2025.12.039},
pmid = {41605211},
issn = {1097-4172},
mesh = {*Dependovirus/genetics ; Animals ; *Genetic Therapy/methods ; Mice ; Genetic Vectors/genetics ; Humans ; *Recombination, Genetic ; *Gene Transfer Techniques ; Nerve Tissue Proteins/genetics/metabolism ; HEK293 Cells ; CRISPR-Cas Systems ; Transduction, Genetic/methods ; },
abstract = {Delivery of therapeutic genes is essential for successful gene therapy. Adeno-associated viruses (AAVs) are a prime vector for carrying gene cargoes. However, the limited packaging capacity of AAVs poses a major challenge for large gene transduction. Here, we devised a strategy termed AAV with translocation linkage (AAVLINK), leveraging Cre/lox-mediated intermolecular DNA recombination to overcome cargo size constraints. This AAVLINK strategy enabled superior gene segmentation flexibility, robust gene reconstitution efficiency, and a marked reduction in truncated protein products. AAVLINK drove expression of intact Shank3 or SCN1A and rescued behavior and seizure phenotypes of mutant mice, respectively. Moreover, we generated AAVLINK2.0 with destabilized Cre to address biosafety concerns. Importantly, we used AAVLINK to build a vector bank for 193 large genetic-disorder-associated genes and 5 CRISPR-based tools with verified gene reconstitution. Altogether, our study establishes a robust method to facilitate delivery of large gene cargoes using AAVs.},
}

*Dependovirus/genetics
Animals
*Genetic Therapy/methods
Mice
Genetic Vectors/genetics
Humans
*Recombination, Genetic
*Gene Transfer Techniques
Nerve Tissue Proteins/genetics/metabolism
HEK293 Cells
CRISPR-Cas Systems
Transduction, Genetic/methods

@article {pmid41519897,
year = {2026},
author = {Boutin, J and Fayet, S and Marin, V and Bergès, C and Riandière, M and Toutain, J and Lamrissi-Garcia, I and Thibault, C and Cappellen, D and Dabernat, S and Poulet, A and Francillette, M and Droin, N and Debeissat, C and Brunet de la Grange, P and Moreau-Gaudry, F and Bedel, A},
title = {Single-cell multiplex approaches deeply map ON-target CRISPR-genotoxicity and reveal its mitigation by palbociclib and long-term engraftment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1429},
pmid = {41519897},
issn = {2041-1723},
mesh = {*Piperazines/pharmacology ; Animals ; *Pyridines/pharmacology ; Humans ; *Single-Cell Analysis/methods ; Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; Polymorphism, Single Nucleotide ; Hematopoietic Stem Cells/drug effects/metabolism ; Loss of Heterozygosity ; Female ; },
abstract = {Genome editing by CRISPR-Cas9-nuclease is promising for gene therapy. However, safety concerns remain. Monitoring ON-target genotoxicity is essential, especially to assay megabasic rearrangements at the targeted locus. Here, we developed a combined single-cell resolution approach with DNA sequencing focused on single nucleotide polymorphism (scSNP-DNAseq), micronuclei and LOH cytometry-reporter assays. This sensitive multiplexed strategy enables the sensitive monitoring of CRISPR-mediated genotoxicity in primary cells. Using this approach, we detect, map and characterize various types of induced-losses of heterozygosity and assess editing-associated chromosomal instability. Importantly, palbociclib prevents the appearance of such genomic rearrangements in hematopoietic stem cells without impairing cell fate or graft capability. Conversely, short-term risk is significantly increased with DNA-PKcs inhibitor AZD7648. Fortunately, targeting HBG1/2p, scSNP-DNA-seq reveals that ON-target genotoxic events are no longer detectable after long-term xenografts. This work demonstrates that scSNP-DNA-seq should be routinely implemented to monitor chromosomal rearrangements before and after CRISPR-edited cell infusions.},
}

*Piperazines/pharmacology
Animals
*Pyridines/pharmacology
Humans
*Single-Cell Analysis/methods
Gene Editing/methods
*CRISPR-Cas Systems/genetics
Mice
Polymorphism, Single Nucleotide
Hematopoietic Stem Cells/drug effects/metabolism
Loss of Heterozygosity
Female

@article {pmid41519779,
year = {2026},
author = {Lim, MYT and Tan, C and Subhramanyam, CS and Teo, SJ and DeFalco, L and Pasaribu, SK and Koh, CH and Rayamajhi, D and Chi, J and Li, S and Wee, KB and Roy, S and Huber, RG and Aw, SS},
title = {A programmable ribozyme for RNA signal transduction.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1428},
pmid = {41519779},
issn = {2041-1723},
support = {H20C6a0034//Agency for Science, Technology and Research (A*STAR)/ ; UIBR//Agency for Science, Technology and Research (A*STAR)/ ; Cell and Gene Therapy Flagship Grant C253623010//Agency for Science, Technology and Research (A*STAR)/ ; IAF-PP H20C6a0034//Agency for Science, Technology and Research (A*STAR)/ ; OFYIRG16may045//MOH | National Medical Research Council (NMRC)/ ; Core funding//A*STAR | Institute of Molecular and Cell Biology (Institute of Molecular and Cell Biology - A STAR)/ ; },
mesh = {*RNA, Catalytic/metabolism/genetics/chemistry ; Humans ; Animals ; Zebrafish/genetics/embryology ; Aptamers, Nucleotide/metabolism/genetics ; CRISPR-Cas Systems/genetics ; *Signal Transduction/genetics ; Gene Editing/methods ; HEK293 Cells ; MicroRNAs/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA/metabolism/genetics ; },
abstract = {RNA detection applications can be augmented if a sensed RNA can be directly functionally transduced. However, there is no generalisable approach that allows an RNA trigger itself to directly activate diverse non-coding RNA effectors. Here, we report engineering of a programmable, RNA trigger-activated, dual-site self-cleaving ribozyme with modular sensing domain and cleavage product. This platform, UNlocked by Activating RNA (UNBAR), is entirely encoded within one RNA strand. The ribozyme can be designed to be almost completely inactive in absence of trigger, and to exhibit single-nucleotide trigger specificity. UNBAR ribozymes carry out cell-free sensing and protein-free amplification of microRNA and viral RNA sequences, and trigger-dependent release of ncRNA effectors sgRNA, shRNA and aptamer. We demonstrate RNA detection and functional transduction by a cleaved aptamer, whose fluorescence can be directly read out as a function of trigger RNA. We further engineer the ribozyme for function in cells, and demonstrate trigger-dependent regulation of CRISPR-Cas9 editing by sgRNA-embedded ribozymes in zebrafish embryos and human cells. UNBAR is a first-in-class modality with potential to be developed into a versatile platform for synthetic biology, diagnostics and gene regulation.},
}

*RNA, Catalytic/metabolism/genetics/chemistry
Humans
Animals
Zebrafish/genetics/embryology
Aptamers, Nucleotide/metabolism/genetics
CRISPR-Cas Systems/genetics
*Signal Transduction/genetics
Gene Editing/methods
HEK293 Cells
MicroRNAs/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*RNA/metabolism/genetics

@article {pmid41478283,
year = {2026},
author = {Zhang, X and Zhang, Y and Liu, X and Liu, C and Liu, Y and He, Y and Qiu, Y and Sun, L and Hu, J and Gao, Y and Wei, W and Liu, J},
title = {FOCAS: Transcriptome-wide screening of individual m[6]A sites functionally dissects epitranscriptomic control of gene expression in cancer.},
journal = {Cell},
volume = {189},
number = {3},
pages = {922-938.e23},
doi = {10.1016/j.cell.2025.11.037},
pmid = {41478283},
issn = {1097-4172},
mesh = {Humans ; *Adenosine/analogs & derivatives/metabolism/genetics ; *Neoplasms/genetics/metabolism ; *Transcriptome/genetics ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems/genetics ; RNA, Messenger/metabolism/genetics ; RNA, Untranslated/metabolism/genetics ; Epigenesis, Genetic ; },
abstract = {Although N[6]-methyladenosine (m[6]A) is a pervasive RNA modification essential for gene regulation, dissecting the functions of individual m[6]A sites remains technically challenging. To overcome this, we developed functional m[6]A sites detection by CRISPR-dCas13b-FTO screening (FOCAS), a CRISPR-dCas13b-based platform enabling high-throughput, site-specific functional screening of m[6]A. Applying FOCAS to four human cancer cell lines identified 4,475 m[6]A-regulated genes influencing cell fitness via both mRNAs and non-coding RNAs (ncRNAs), many of which are newly linked to cancer and exhibit dynamic developmental expression. FOCAS uncovered context-dependent and reader-specific effects of m[6]A within the same gene, revealing its intricate regulatory logic. We further uncovered universal and cell-type-specific m[6]A patterns, with unique sites enriched in ncRNAs and universal ones in transcription-related genes. In SMMC-7721 cells, we identified m[6]A-regulated transcriptional networks that demonstrated extensive epitranscriptome-transcriptome crosstalk. Overall, this study established a powerful, unbiased approach for the functional dissection of m[6]A, advancing the understanding of its complexity and therapeutic relevance in cancers.},
}

Humans
*Adenosine/analogs & derivatives/metabolism/genetics
*Neoplasms/genetics/metabolism
*Transcriptome/genetics
Cell Line, Tumor
*Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems/genetics
RNA, Messenger/metabolism/genetics
RNA, Untranslated/metabolism/genetics
Epigenesis, Genetic

@article {pmid41418537,
year = {2026},
author = {Tan, D and Ye, Y and Miao, D and Zhao, C and Wu, S and Shi, J and Yang, J and Fang, K and Lu, F and Lv, Q and Gong, J and Yang, H and Xiao, W and Xiong, Z and Zhang, X and Ruan, H},
title = {UBL3 governs VEGFR inhibitor resistance by activating NOTCH signaling in renal cell carcinoma.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {85},
number = {},
pages = {101332},
doi = {10.1016/j.drup.2025.101332},
pmid = {41418537},
issn = {1532-2084},
mesh = {Humans ; *Carcinoma, Renal Cell/drug therapy/pathology/genetics ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Kidney Neoplasms/drug therapy/pathology/genetics ; Signal Transduction/drug effects ; Animals ; Cell Line, Tumor ; Mice ; Xenograft Model Antitumor Assays ; *Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors ; *Protein Kinase Inhibitors/pharmacology ; Sunitinib/pharmacology ; Pyridines/pharmacology ; Receptors, Notch/metabolism ; Apoptosis/drug effects ; Mice, Nude ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Anilides ; },
abstract = {BACKGROUND: Targeted therapy is the first-line treatment for patients with metastatic renal cell carcinoma (RCC), with vascular endothelial growth factor receptor inhibitors (VEGFRis) constituting the bulk of regimens used. Although the repertoire of VEGFRis for RCC now spans from sunitinib to cabozantinib, resistance to treatments has emerged as a common and prominent challenge. Thus, identifying novel therapeutic targets has become essential for enhancing the antitumor efficacy of current treatments and inhibiting RCC progression.

METHOD: To investigate the potential mechanisms underlying VEGFRi resistance in RCC, we performed a genome-wide CRISPR/Cas9 library screen under sunitinib and cabozantinib treatment and identified UBL3 as a key driver of VEGFRi resistance in RCC cells. The critical role of UBL3 in promoting VEGFRi resistance was validated using CCK8 assays, flow cytometry, TUNEL assays, and bioinformatics analyses. To elucidate the molecular mechanisms underlying UBL3, we utilized western blotting, RNA sequencing, chromatin immunoprecipitation, small extracellular vesicles (sEVs) isolation, and Astral-DIA proteomics. The contribution of UBL3 to VEGFRi resistance was further confirmed through comprehensive in vitro and in vivo experiments.

RESULTS: UBL3 was confirmed to suppress apoptosis and promote VEGFRi resistance through NOTCH signaling activation. Further investigations highlighted the importance of NOTCH signaling in VEGFRi resistance in RCC via the NOTCH-PTEN-AKT and NOTCH-FOS pathways and revealed the mechanisms by which UBL3 activated NOTCH signaling. On the one hand, UBL3 formed complex with NOTCH2 and ADAM17 simultaneously, accelerating ADAM17-mediated cleavage of NOTCH2. On the other hand, UBL3-modified NOTCH2 was sorted into sEVs, which were taken up by recipient cells, activating NOTCH signaling and thereby transmitting VEGFRi resistance. Finally, lipid nanoparticle-mediated delivery of the CRISPR/Cas9 knockout system targeting UBL3 effectively restored the sensitivity of RCC tumors to VEGFRis.

CONCLUSION: This study emphasized the importance of UBL3 in VEGFRi resistance in RCC and proposed that UBL3 activated NOTCH signaling through two distinct pathways, thereby suppressing cancer apoptosis and promoting resistance to VEGFRis. These findings provided a solid scientific foundation and paved the way for the development of novel therapeutic strategies for patients with advanced RCC.},
}

Humans
*Carcinoma, Renal Cell/drug therapy/pathology/genetics
*Drug Resistance, Neoplasm/genetics/drug effects
*Kidney Neoplasms/drug therapy/pathology/genetics
Signal Transduction/drug effects
Animals
Cell Line, Tumor
Mice
Xenograft Model Antitumor Assays
*Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors
*Protein Kinase Inhibitors/pharmacology
Sunitinib/pharmacology
Pyridines/pharmacology
Receptors, Notch/metabolism
Apoptosis/drug effects
Mice, Nude
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic
Anilides

@article {pmid41416398,
year = {2026},
author = {Hoang, TS and Faraji, F and Mendez-Molina, AN and Adame-Garcia, SR and Sato, K and Ishikawa, T and Vo, PTT and Ramirez, SI and Anguiano Quiroz, PY and Guo, T and Fan, K and Wu, X and Molinolo, AA and Cohen, EEW and Mali, P and Lippman, SM and Gutkind, JS},
title = {Genome-wide CRISPR Screening Reveals a PKA-Driven Resistance Mechanism to Metformin for Oral Cancer Prevention That Can Be Exploited by Combination with NSAIDs.},
journal = {Cancer prevention research (Philadelphia, Pa.)},
volume = {19},
number = {2},
pages = {79-92},
pmid = {41416398},
issn = {1940-6215},
support = {T32 CA121938/CA/NCI NIH HHS/United States ; R01 DE035393/DE/NIDCR NIH HHS/United States ; R25 CA221779/CA/NCI NIH HHS/United States ; U54 CA274502/CA/NCI NIH HHS/United States ; U01 CA290479/CA/NCI NIH HHS/United States ; R01 DE026644/DE/NIDCR NIH HHS/United States ; T32 DC000028/DC/NIDCD NIH HHS/United States ; R01DE026644//National Institute of Dental and Craniofacial Research (NIDR)/ ; U01CA290479//National Cancer Institute (NCI)/ ; SU2C-FARF-FFF//Stand Up To Cancer (SU2C)/ ; 308268//Stand Up To Cancer (SU2C)/ ; T32DT4965//Tobacco-Related Disease Research Program (TRDRP)/ ; T32CA121938//National Cancer Institute (NCI)/ ; T32DC000028//National Institute on Deafness and Other Communication Disorders (NIDCD)/ ; 1061310//American Head and Neck Society (AHNS)/ ; //Takeda Science Foundation (TSF)/ ; //Japan Society for the Promotion of Science (JSPS)/ ; //Rotary Foundation (Rotary)/ ; R25CA221779//National Cancer Institute (NCI)/ ; T34DT8340//Tobacco-Related Disease Research Program (TRDRP)/ ; },
mesh = {*Metformin/pharmacology/therapeutic use ; Humans ; *Anti-Inflammatory Agents, Non-Steroidal/pharmacology/therapeutic use ; *Cyclic AMP-Dependent Protein Kinases/metabolism ; *Mouth Neoplasms/prevention & control/genetics/pathology ; *Drug Resistance, Neoplasm/genetics/drug effects ; CRISPR-Cas Systems ; *Squamous Cell Carcinoma of Head and Neck/genetics/prevention & control/pathology/drug therapy ; Cell Line, Tumor ; Mice ; Drug Synergism ; Animals ; Cell Proliferation/drug effects ; *Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use ; Signal Transduction/drug effects ; Cyclooxygenase 2 Inhibitors/pharmacology ; },
abstract = {UNLABELLED: Head and neck squamous cell carcinoma (HNSCC) is among the 10 most common cancers worldwide and is associated with high morbidity and poor survival. Diminished HNSCC outcomes are often related to delayed diagnosis and treatment of occult progression of premalignant lesions, underscoring the need for effective and low-risk chemoprevention strategies. In this regard, metformin has shown promising clinical activity for HNSCC prevention. In this study, we performed a genome-wide CRISPR/Cas9 screen of metformin-treated HNSCC cells and identified the activation of PKA signaling as the top resistance pathway. We show that metformin mediates PKA activation in HNSCC cells and that PKA inhibition, when combined with metformin treatment, synergistically inhibits HNSCC growth. We found that metformin-induced PKA activation is mediated by a prostaglandin E2 autocrine loop, which can be blocked using cyclooxygenase-2 (COX2) inhibitors. Importantly, COX2 inhibition using nonsteroidal anti-inflammatory drugs (NSAID) combined with metformin treatment synergistically inhibits HNSCC cell growth and prevents the progression of oral premalignant lesions into invasive HNSCC in a model of tobacco-driven oral carcinogenesis. Together, these findings demonstrate that metformin and NSAID combination therapy may represent a promising therapeutic strategy for HNSCC chemoprevention.

PREVENTION RELEVANCE: Our findings reveal that using metformin for head and neck cancer chemoprevention leads to compensatory activation of a PKA-driven resistance mechanism that can be blocked by cotreatment with NSAIDs. These findings provide a rationale for combining metformin with NSAIDs as a precision head and neck cancer chemoprevention strategy.},
}

*Metformin/pharmacology/therapeutic use
Humans
*Anti-Inflammatory Agents, Non-Steroidal/pharmacology/therapeutic use
*Cyclic AMP-Dependent Protein Kinases/metabolism
*Mouth Neoplasms/prevention & control/genetics/pathology
*Drug Resistance, Neoplasm/genetics/drug effects
CRISPR-Cas Systems
*Squamous Cell Carcinoma of Head and Neck/genetics/prevention & control/pathology/drug therapy
Cell Line, Tumor
Mice
Drug Synergism
Animals
Cell Proliferation/drug effects
*Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use
Signal Transduction/drug effects
Cyclooxygenase 2 Inhibitors/pharmacology

@article {pmid41317988,
year = {2026},
author = {Arya, SK and Goodman, CL and Palli, SR},
title = {The expanding toolkit of insect cell culture: a new era in biotechnology.},
journal = {Current opinion in insect science},
volume = {74},
number = {},
pages = {101465},
doi = {10.1016/j.cois.2025.101465},
pmid = {41317988},
issn = {2214-5753},
mesh = {Animals ; *Insecta/genetics/cytology ; *Biotechnology/methods ; *Cell Culture Techniques/methods ; Cell Line ; Baculoviridae/genetics ; CRISPR-Cas Systems ; },
abstract = {Insect cell culture has become an essential platform in modern biotechnology, valued for its safety, scalability, and ability to perform complex post-translational modifications. This review highlights the latest and most important advances in the field. We focus on efforts at developing and engineering new insect cell lines, innovations in expression systems, especially the baculovirus expression vector system and the transformative impact of CRISPR/Cas9-based genome editing. Additionally, we explore breakthroughs that improve the efficiency of recombinant protein production and discuss key challenges such as viral contamination and expression instability. Collectively, these developments mark an important step forward in insect cell biotechnology and are expected to enhance the efficiency and scalability of producing vaccines and biopharmaceuticals. Together, these innovations illustrate a transition from cataloging cell line development to understanding the mechanisms and engineering principles driving these advances. This review not only summarizes recent progress but also provides perspective on how foundational lepidopteran models have guided innovations now extending into dipteran, hemipteran, and hymenopteran systems, shaping the future of insect biotechnology.},
}

Animals
*Insecta/genetics/cytology
*Biotechnology/methods
*Cell Culture Techniques/methods
Cell Line
Baculoviridae/genetics
CRISPR-Cas Systems

@article {pmid41629010,
year = {2026},
author = {Saini, A and Sharma, N and Sharma, N and Kumari, N and Sharma, M and Singh, B and Thakur, AK},
title = {Precision pest management: Genome editing tools, specifically CRISPR/Cas9 and future prospects.},
journal = {Pesticide biochemistry and physiology},
volume = {218},
number = {},
pages = {106941},
doi = {10.1016/j.pestbp.2026.106941},
pmid = {41629010},
issn = {1095-9939},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Insecta/genetics ; *Pest Control, Biological/methods ; *Pest Control/methods ; Insect Control/methods ; },
abstract = {The growing resistance to synthetic insecticides and Bt toxins, alongside persistent crop losses despite heavy pesticide application, highlights the urgent need for safer, sustainable and efficient pest management strategies. This review presents genome editing as a precise and versatile approach to reduce pest impact by altering fertility, feeding patterns or vulnerability, while protecting beneficial organisms. Among the genome editing tools, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) is one of the most promising genome editing techniques in insects. It facilitates targeted functional studies, integration with RNAi and dual-expression systems and gene drive applications. Deployment is envisioned in two phases, initial laboratory modification followed by regulated field release, with a strong emphasis on biosafety through terminator genes, marked individuals for gene flow monitoring, optimized dosages, stringent screening and long-term ecological surveillance, along with transparency and adherence to international safety protocols. Significant challenges encompass delivery efficiency, identification of edits, off-target mutations, dose-related efficacy and sterility, unstable transmission and resistance development. Innovations such as base and prime editing minimize unintended mutations by circumventing double-stranded breaks (DSBs), while paratransgenic strategies targeting gut symbionts offer supplementary avenues; plant-mediated insect gene editing emerges as a promising frontier. Overall, carefully regulated trials aligned with policy frameworks and stakeholder involvement are vital to assess effectiveness in natural environments and achieve targeted, dependable and ecologically responsible pest control.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Animals
Insecta/genetics
*Pest Control, Biological/methods
*Pest Control/methods
Insect Control/methods

@article {pmid41628563,
year = {2026},
author = {Liang, L and Xu, B and Xiao, S and Mu, X and Zhao, S and Tian, J},
title = {A new split DNA-based activation of CRISPR/Cas12a for amplification-free and dual-stimulus responsive detection and precise imaging of miRNA-221.},
journal = {Talanta},
volume = {303},
number = {},
pages = {129443},
doi = {10.1016/j.talanta.2026.129443},
pmid = {41628563},
issn = {1873-3573},
abstract = {The CRISPR/Cas12a system is a genome editing technology that has been widely applied in biosensing and molecular diagnostics. However, the detection and regulation of its core components remain challenging. Therefore, we constructed a new split DNA-based activation method for the regulation of CRISPR/Cas12a, and based on that, an APE1-assisted activation CRISPR/Cas12a system for miRNA detection and a precise imaging method was also developed without amplification and complex design. Two split DNA were used as activators and embedded in two hairpins. When APE1 and miRNA-221 were simultaneously input, the DNA logic gate was started, thus releasing the determinant activation chain to activate the trans-shearing activity of the CRISPR/Cas12a system, so that the fluorescent probe signal can be significantly recovered. Different cleavage-activated chain hairpins were designed, and the influence on the trans-shear activity of CRISPR/Cas12a and the activation effect were discussed. And the method was successfully applied to detect the expression levels of miRNA-221 in cell lysates. The detection limit for miRNA-221 is 9.71 pmol/L (S/N = 3). At the same time, the method was applied for precise imaging of miRNA-221 within different cells and can effectively distinguish tumor cells. This study combines the regulation of the CRISPR/Cas system by split activators with the advantages of dual-responsive DNA logic circuits. The dual-response activation design effectively reduces false positive signals, thereby enhancing the detection and imaging accuracy. This method provides a novel design concept for utilizing split-DNA activation of the CRISPR/Cas system for nucleic acid detection and cell imaging.},
}

@article {pmid41627753,
year = {2026},
author = {Arnould, K and El Kadri, M and Hervé, P and Asencio, C and Plazolles, N and Monic, S and Morel, CA and Rivière, L and Bringaud, F and Tetaud, E},
title = {Precision Without Selection: A Marker-Free CRISPR/Cas9-Based Protocol for Multiplexed Genome Editing in Trypanosomatids.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3014},
number = {},
pages = {65-78},
pmid = {41627753},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Protozoan ; Electroporation ; *Trypanosomatina/genetics ; Ribonucleoproteins/genetics ; },
abstract = {The CRISPR-Cas9 system has become a valuable tool for genome editing in trypanosomatid parasites such as Trypanosoma and Leishmania species. Although these organisms have been genetically engineered for a long time using homologous recombination, CRISPR/Cas9 offers improved efficiency for genome editing. However, conventional strategies employing stable Cas9 expression require the persistent use of a specific genetic background (i.e., strains expressing Cas9), depend on selectable resistance markers, compromise genomic stability, and are not readily applicable to diverse strain backgrounds. Herein, we report an optimized marker-free CRISPR/Cas9 method based on transient ribonucleoprotein (RNP) delivery that overcomes these drawbacks. Our method eliminates the need for plasmid integration or antibiotic selection while maintaining high editing efficiency. The protocol comprises the following steps: (1) design of the guide RNA (gRNA), (2) design of the repair template (cassette), (3) assembly of the ribonucleoprotein (RNP) complex, (4) delivery by electroporation, and (5) clonal screening through PCR and sequencing. The procedure permits rapid (≤3 weeks) production of homozygous mutant lines in wild-type strains, including low-density culture strains. The reproducibility and ease of the technique render it particularly suited for multiplexed editing of polyploid genomes, multi-gene families, and several different genes at once, as well as validation of the essential nature of genes. Although designed for trypanosomatids, the workflow can be adapted to other kinetoplastids, offering a flexible platform for functional genomics.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
*Genome, Protozoan
Electroporation
*Trypanosomatina/genetics
Ribonucleoproteins/genetics

@article {pmid41627331,
year = {2026},
author = {Roy, N and Debnath, P and Srivastava, S and Gaur, HS},
title = {Recent developments in CRISPR/Cas9 genome editing research for edible fungiculture.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {36},
pmid = {41627331},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Genome, Fungal ; *Agaricales/genetics/growth & development ; },
abstract = {Fungiculture refers to the deliberate cultivation or agricultural practice involving the growth and management of fungi. The practice encompasses the intentional culture of diverse species of macrofungi, including mushrooms and truffles, within controlled habitats or under specified conditions, in order to fulfill human requirements especially for food purpose. As the global market for edible mushrooms grows quickly, it is becoming increasingly necessary to grow novel and improved strains of edible fungi. Growing and breeding edible fungi using traditional methods is both time-consuming and difficult. So, there is a need for evolving advanced techniques at a molecular level which can help breeding of edible fungi with much better efficiency. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system is one of the most effective techniques for accurately cutting and modifying the genomes of edible fungi. In this review, we discuss how genome editing using CRISPR/Cas9 has been utilized in many edible fungal species such as Pleurotus ostreatus, Agaricus bisporus, Cordyceps militaris, Ganoderma lucidum, Flammulina filiformis, Lentinula edodes, and others for their target specific breeding. We also discuss the working mechanism of the above-mentioned system in these mushroom species, and also the advantages and limitations of using this system in mushrooms.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Genome, Fungal
*Agaricales/genetics/growth & development

@article {pmid41627751,
year = {2026},
author = {Nagamine, S and Oishi, R and Nakazawa, M},
title = {Engineering Wax Ester Composition in Euglena gracilis Using Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3014},
number = {},
pages = {37-49},
pmid = {41627751},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Euglena gracilis/genetics/metabolism ; CRISPR-Cas Systems ; *Metabolic Engineering/methods ; *Esters/metabolism/chemistry ; *Waxes/metabolism/chemistry ; },
abstract = {Genome editing technologies have significantly expanded the potential for metabolic engineering in non-model organisms. In Euglena gracilis, genome editing methods using Cas9 and Cas12a were reported in 2019 and 2024, respectively, and are increasingly being applied to modify metabolic functions. This chapter provides a detailed protocol for CRISPR/Cas9-based genome editing that enables stable modification of wax ester composition under anaerobic conditions. By targeting key enzymes in the reversed β-oxidation pathway, the method allows the generation of knockout mutants with altered wax ester chain lengths. Beyond this application, the protocol supports reproducible and stable genetic modification of E. gracilis metabolism. It can be extended to the engineering of other biosynthetic pathways and is compatible with future integration of knock-in strategies. The approach offers a practical basis for the broader use of E. gracilis as a green chassis organism in synthetic biology and biomanufacturing.},
}

*Gene Editing/methods
*Euglena gracilis/genetics/metabolism
CRISPR-Cas Systems
*Metabolic Engineering/methods
*Esters/metabolism/chemistry
*Waxes/metabolism/chemistry

@article {pmid41581816,
year = {2026},
author = {Xiao, Y and Yang, J and Yang, W and Yuan, M and Zhang, Y and Liu, J and Zhang, Y and Zhu, H and Luo, G},
title = {A rapid on-site diagnostic method for goose parvovirus disease based on recombinase polymerase amplification and CRISPR/AsCas12a.},
journal = {International journal of biological macromolecules},
volume = {343},
number = {Pt 2},
pages = {150496},
doi = {10.1016/j.ijbiomac.2026.150496},
pmid = {41581816},
issn = {1879-0003},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Geese/virology ; *Parvoviridae Infections/diagnosis/veterinary/virology ; *Parvovirus/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Poultry Diseases/diagnosis/virology ; Ducks/virology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Goose parvovirus (GPV) is a highly pathogenic and lethal virus responsible for Derzsy's disease in goslings and ducklings, significantly influencing the economic viability of waterfowl farming. This necessitates the development of rapid diagnostic techniques for effective disease management. In this study, an optimized clustered regularly interspaced short palindromic repeats (CRISPR)/Acidaminococcus sp. CRISPR associated nuclease 12a (AsCas12a) system was developed for the diagnosis of GPV. The study determined that the optimal conditions for the CRISPR/Cas12a-based fluorescence assay were 20 nM AsCas12a, 5 nM crRNA, and 5 nM single-stranded DNA (ssDNA), whereas the lateral flow assay (LFA) required 20 nM AsCas12a and 4 nM crRNA. Moreover, the fluorescence-based assay and LFA achieved minimum detection limits of 7.8 copies/μL and 78 copies/μL, respectively, representing 1000-fold and 100-fold improvements over conventional PCR methods. Both detection methods exhibited high specificity and demonstrated no cross-reactivity with other prevalent waterfowl pathogens, such as duck plague virus, duck hepatitis viruses, H5 avian influenza virus, waterfowl astrovirus, reovirus, Muscovy duck parvovirus, and novel GPV. The results of the LFA were in complete concordance with laboratory qPCR analyses, thereby affirming their reliability for clinical diagnostics. In conclusion, we have successfully developed a dual-readout GPV detection system utilizing CRISPR/Cas12a technology, which holds significant promise for the early surveillance and containment of GPV outbreaks.},
}

Animals
*CRISPR-Cas Systems/genetics
*Geese/virology
*Parvoviridae Infections/diagnosis/veterinary/virology
*Parvovirus/genetics
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism
*Poultry Diseases/diagnosis/virology
Ducks/virology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41577322,
year = {2026},
author = {Wang, L and Miao, M and Bao, L and Chu, J and Zhou, J and Song, W and Cai, P and Cheng, C and Xu, H and Wang, T and Zhao, R and Wang, H and Liu, F and Xu, M and Tian, G},
title = {CRISPR/Cas9-based genome-wide screen reveals a synergistic effect of Irinotecan and USP1 inhibitor in colorectal cancer.},
journal = {European journal of pharmacology},
volume = {1015},
number = {},
pages = {178558},
doi = {10.1016/j.ejphar.2026.178558},
pmid = {41577322},
issn = {1879-0712},
mesh = {Humans ; *Irinotecan/pharmacology/therapeutic use ; *Colorectal Neoplasms/drug therapy/genetics/pathology ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Ubiquitin-Specific Proteases/antagonists & inhibitors/metabolism ; Drug Synergism ; Cell Proliferation/drug effects ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Drug Resistance, Neoplasm/drug effects ; HCT116 Cells ; Gene Expression Regulation, Neoplastic/drug effects ; Antineoplastic Combined Chemotherapy Protocols/pharmacology ; },
abstract = {Irinotecan resistance remains a significant challenge in metastatic colorectal cancer (mCRC) therapy. To address this, we identified USP1 as a synthetic lethal partner of Irinotecan through genome-wide CRISPR/Cas9 screening in HCT-116 cells. Combining the USP1 inhibitor I-138 with Irinotecan in HCT-116, HT-29, and SW620 cell lines significantly reduced IC50, suppressed proliferation, and diminished colony formation compared to monotherapy, demonstrating a synergistic effect (combination index CI < 1). The synergistic therapeutic efficacy was further validated in the xenograft mouse model. Mechanistic studies revealed that I-138 significantly upregulated pCREB (Ser133), concurrently dynamically regulating the activity of USP1, FANCD2/FANCI, and PCNA upon DNA damage response and repair. RNA sequencing further highlighted the enrichment of cAMP, PI3K-AKT, and Wnt pathways, which are all linked to CREB activity in the combination group. These findings establish USP1 inhibition as a promising strategy to overcome Irinotecan resistance through the combination strategy, providing a novel therapeutic avenue for CRC.},
}

Humans
*Irinotecan/pharmacology/therapeutic use
*Colorectal Neoplasms/drug therapy/genetics/pathology
Animals
Mice
*CRISPR-Cas Systems/genetics
*Ubiquitin-Specific Proteases/antagonists & inhibitors/metabolism
Drug Synergism
Cell Proliferation/drug effects
Xenograft Model Antitumor Assays
Cell Line, Tumor
Drug Resistance, Neoplasm/drug effects
HCT116 Cells
Gene Expression Regulation, Neoplastic/drug effects
Antineoplastic Combined Chemotherapy Protocols/pharmacology