@article {pmid40914913,
year = {2026},
author = {McFarlane, GR and Whitaker, K and Plett, KL and O'Rourke, B and Bogema, DR},
title = {Optimising Guide RNA Production for Multiplexed Cas9-Targeted Nanopore Sequencing to Detect Pathogens.},
journal = {Molecular biotechnology},
volume = {68},
number = {5},
pages = {2399-2409},
pmid = {40914913},
issn = {1559-0305},
support = {A-023//Centre for Invasive Species Solutions/ ; },
mesh = {*Nanopore Sequencing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Triticum/microbiology ; *Fungi/genetics/pathogenicity/isolation & purification ; },
abstract = {In agriculture, biosecurity, and human health, the rapid and accurate detection of pathogens and pests is crucial. Our study investigates the sensitivity and practicality of six guide RNA (gRNA) production methods for use in Nanopore Cas9-targeted sequencing (nCATS), focusing on their implications for multiplexed pathogen and pest detection. Each production method generated a library of eight gRNAs capable of excising ~ 1.6 kb fragments of the 5.8S_rRNA-ITS2-28S_rRNA regions of five economically significant wheat fungal pathogens. Through comparative analysis, we evaluated the efficacy of commercially synthesised and in-house in vitro-transcribed gRNAs, assessing their impact on sequencing enrichment outcomes. Our findings highlight differences amongst the methods in terms of gRNA yield, integrity, performance, and costs. Our best performing gRNA production method was able to successfully identify all target sequences across the 0.96 to 8.4 pg ranges we tested with coverage ranging from 66 to 2037X. This study highlights the challenges and opportunities in implementing nCATS for multiplexed pathogen and pest detection, offering insights into the development of cost-effective and reliable gRNA production strategies for nCATS.},
}

*Nanopore Sequencing/methods
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Triticum/microbiology
*Fungi/genetics/pathogenicity/isolation & purification

@article {pmid41137959,
year = {2026},
author = {Chen, Y and Wang, Z and Lei, X and Liu, Y and Cai, Y and Wang, C and Jin, J and Ding, X},
title = {Efficient Construction of Recombinant CHO Cells for Stable Expression of Therapeutic Proteins Based on New Hotspot 'LOC'.},
journal = {Molecular biotechnology},
volume = {68},
number = {5},
pages = {2527-2537},
pmid = {41137959},
issn = {1559-0305},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Recombinant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cricetinae ; *Gene Editing/methods ; Humans ; Green Fluorescent Proteins/genetics/metabolism ; Interferon-alpha/genetics/metabolism ; Interferon alpha-2/genetics ; },
abstract = {Efficient generation of stably express therapeutic recombinant proteins in Chinese hamster ovary (CHO) cell lines remains as a major challenge in biologics development. Among available strategies, CRISPR/Cas9-mediated homology-directed repair (HDR) has emerged as a highly efficient method for inserting genes of interest (GOIs) into predefined genomic 'hotspot.' In this study, we report such a 'hotspot' (LOC site) which has discovered previously by our research team, which is a non-coding RNA region within the CHO-K1 genome at position 1689 of NW_003626341.1, located in the LOC103162981 gene. Utilizing this 'hotspot,' the study implemented an integrated platform combining CRISPR/Cas9 genome editing with Bxb1 recombinase-mediated cassette exchange (RMCE) to enable efficient GOIs integration and followed by sustained expression. First, to create a fluorescent landing pad (LP) cell by inserting a Bxb1 attP site, an EGFP cassette and a promoter trap into LOC site via CRISPR/Cas9. Then, LP cell undergoes subculture for 60 consecutive generations, and the fluorescence was consistently observed, illustrating the capacity of LOC site for stable EGFP expression. Donor cassettes harboring GOIs flanked by Bxb1 attB sites were then easily swapped in through RMCE. This platform enabled one‑step insertion and stable expression of therapeutic target proteins ranging from 12 to 190 kDa including human serum albumin (HSA), interferon alpha-2b (INFα2b), recombinant factor VII (rFVII), monoclonal antibodies against the epidermal growth factor receptor (anti-EGFR, AE01), and a disintegrin and metalloproteinase with thrombospondin type 1 motif, 13 (ADAMST13). Here, we efficiently generated HSA CHO cell clones within three weeks; HSA titers reached 220 mg/L in shake‑flask batch culture conditions. Collectively, this study establishes LOC site as a 'hotspot' and shows that the CRISPR/Cas9‑Bxb1 pipeline markedly accelerates CHO cell‑line development for biologics manufacturing.},
}

Animals
CHO Cells
Cricetulus
*Recombinant Proteins/genetics/metabolism
CRISPR-Cas Systems
Cricetinae
*Gene Editing/methods
Humans
Green Fluorescent Proteins/genetics/metabolism
Interferon-alpha/genetics/metabolism
Interferon alpha-2/genetics

@article {pmid41162805,
year = {2026},
author = {Jatwani, S and Aggarwal, F and Jadhav, RR and Yadav, R and Sarma, SJ and Khare, D},
title = {Engineered CRISPR-Cas9 Vector for Efficient Agrobacterium-Mediated Transformation of Phanerochaete chrysosporium.},
journal = {Molecular biotechnology},
volume = {68},
number = {5},
pages = {2549-2559},
pmid = {41162805},
issn = {1559-0305},
mesh = {*Phanerochaete/genetics ; *Transformation, Genetic ; *Genetic Vectors/genetics ; *CRISPR-Cas Systems ; *Agrobacterium/genetics ; Gene Editing/methods ; DNA, Bacterial/genetics ; Luminescent Proteins/genetics/metabolism ; Hygromycin B/pharmacology/analogs & derivatives ; Red Fluorescent Protein ; Promoter Regions, Genetic ; Acetophenones ; },
abstract = {Phanerochaete chrysosporium is a model white-rot-fungus with exceptional lignolytic activity, but its genetic manipulation has remained a major challenge due to low transformation efficiencies. Here, we report the development of an optimized Agrobacterium-mediated transformation (AMT) protocol optimized for this species. A CRISPR-Cas9 compatible binary vector was modified by replacing the mCherry cassette with a strong sGFP reporter under the constitutive 35S promoter, enabling reliable identification of transformants. Using mycelial disks as starting material and applying a sandwich selection method, we achieved stable integration of T-DNA carrying the hygromycin resistance and sGFP genes. Transformation efficiency was influenced by co-cultivation parameters, with low-temperature incubation (23°C) and the addition of acetosyringone significantly improving recovery of resistant colonies. Stable expression of sGFP was observed in transformed mycelia, confirming the reproducibility of the system. Although CRISPR-Cas9-mediated gene editing was not demonstrated in this study, our results provide proof-of-concept that a Cas9-based vector can be stably maintained in P. chrysosporium and drive heterologous gene expression. This work represents the first step toward establishing a functional genome editing platform in this ligninolytic fungus. By expanding the molecular toolbox for P. chrysosporium, the optimized transformation strategy lays the foundation for metabolic engineering aimed at lignin valorization, bioplastics, biofuels, and high-value biochemical production within a sustainable circular bioeconomy framework.},
}

*Phanerochaete/genetics
*Transformation, Genetic
*Genetic Vectors/genetics
*CRISPR-Cas Systems
*Agrobacterium/genetics
Gene Editing/methods
DNA, Bacterial/genetics
Luminescent Proteins/genetics/metabolism
Hygromycin B/pharmacology/analogs & derivatives
Red Fluorescent Protein
Promoter Regions, Genetic
Acetophenones

@article {pmid41264211,
year = {2026},
author = {Kim, S and Park, YG and Choi, JH and Moon, SH},
title = {CRISPR Technology for Livestock Improvement: Advances and Future Directions.},
journal = {Molecular biotechnology},
volume = {68},
number = {5},
pages = {2185-2200},
pmid = {41264211},
issn = {1559-0305},
support = {RS-2023-00220207//Ministry of Science and ICT, South Korea/ ; 25A0203L1//Ministry of Science and ICT, South Korea/ ; Graduate Research Scholarship in 2023//Chung-Ang University/ ; },
mesh = {Animals ; *Livestock/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Breeding ; Animals, Genetically Modified/genetics ; },
abstract = {CRISPR-Cas9 technology is revolutionizing genetic engineering by enabling precise genome modifications across a wide range of organisms, particularly in livestock. This review focuses on livestock improvement and the most recent transformative developments in the application of CRISPR-based genome editing techniques. We analyzed productivity improvements achieved by editing growth factor genes, immunogenic resistance enhancement through the editing of immune loci, and ecological footprint reduction for sustainability. This review also addresses the intricate ethical and regulatory issues posed by the application of CRISPR technology in animal breeding, including concerns about animal welfare violations, unintended off-target effects, and impacts on public perception. Furthermore, we discuss key untapped CRISPR targets in livestock genetics enabled by precision gene editing, the emerging integration of CRISPR with artificial intelligence, and the need for collaboration to address complex ethical and regulatory challenges related to applying CRISPR in animal breeding.},
}

Animals
*Livestock/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
Breeding
Animals, Genetically Modified/genetics

@article {pmid41329461,
year = {2026},
author = {Nguyen, AH and Quang, MT},
title = {CRISPR/Cas9 Genome Editing in Oncology: Mechanisms, Therapeutic Platforms and Translational Challenges.},
journal = {Molecular biotechnology},
volume = {68},
number = {5},
pages = {2201-2229},
pmid = {41329461},
issn = {1559-0305},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy ; *Gene Editing/methods ; Animals ; Genetic Therapy/methods ; Translational Research, Biomedical ; },
abstract = {The CRISPR/Cas9 genome editing technology has had a significant impact on cancer research and therapeutic development, providing unprecedented precision in manipulating cancer-associated genes. Although this review focuses on Cas9, we situate it within the broader CRISPR landscape that includes DNA-targeting effectors (Cas9/Cas12), RNA-targeting systems such as Cas13, and type III systems with dual DNA and RNA activity, modalities that expand both experimental and therapeutic possibilities. This comprehensive review examines the current applications of CRISPR/Cas9 in oncology, including its mechanisms and the challenges associated with its clinical translation. Knockout, interference, and activation CRISPR screening platforms have transformed functional genomics by systematically interrogating gene function, identifying therapeutic vulnerabilities, and clarifying resistance mechanisms across diverse cancer phenotypes. This technology has also reshaped cancer modeling, enabling precise recapitulation of disease-relevant mutations from engineered cell lines to patient-derived xenografts that capture tumor heterogeneity and microenvironmental interactions. Notably, the integration of CRISPR/Cas9 with CAR-T therapy has enabled multiplex editing to eliminate alloreactivity, overcome checkpoint-mediated exhaustion, and engineer universal CAR-T cells. Emerging in vivo strategies that directly generate or reprogram CAR-T cells in patients via targeted viral and nonviral delivery underscore accelerating translational momentum. However, significant challenges, including off-target mutagenesis, delivery barriers, p53-mediated selective pressure favoring potentially oncogenic populations, and Cas9 immunogenicity, continue to hinder clinical translation. These limitations necessitate high-fidelity nucleases, optimized guide designs, and improved delivery systems. The future of CRISPR/Cas9 in cancer therapy will depend on technological innovation, comprehensive safety frameworks, and rigorous clinical evaluation as next-generation editing modalities advance toward transformative precision oncology.},
}

Humans
*CRISPR-Cas Systems/genetics
*Neoplasms/genetics/therapy
*Gene Editing/methods
Animals
Genetic Therapy/methods
Translational Research, Biomedical

@article {pmid42060320,
year = {2026},
author = {Zhong, N and Wang, M and Jiang, W and Li, G and Miao, J and Yin, H and Vanhnaseng, P and Gong, J and Yu, Z and Han, X},
title = {One-pot CRISPR/Cas12b-LAMP platform for dual-mode detection of Pasteurella multocida.},
journal = {Letters in applied microbiology},
volume = {79},
number = {5},
pages = {},
doi = {10.1093/lambio/ovag044},
pmid = {42060320},
issn = {1472-765X},
support = {2024-02-08-00-12-F00051//Shanghai Agriculture Applied Technology Development Program/ ; 2023YFD1800700//National Key Research and Development Program of China/ ; 2025I0030//External Cooperation Program of Fujian Science and Technology Plan Project/ ; },
mesh = {*Pasteurella multocida/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Animals ; *Molecular Diagnostic Techniques/methods ; Milk/microbiology ; *Pasteurella Infections/diagnosis/microbiology ; Sensitivity and Specificity ; Clustered Regularly Interspaced Short Palindromic Repeats ; Limit of Detection ; },
abstract = {Pasteurella multocida is a significant bacterial pathogen that poses a significant threat to public health and causes substantial economic losses. Existing detection methods for P. multocida have limitations, including time-consuming and technically complex methods. Here, we describe a simple and accurate detection platform that combines loop-mediated isothermal amplification (LAMP) with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system. By using heparin sodium to modulate the cis-cleavage activity of Cas12b, we developed a one-pot LAMP-CRISPR/Cas12b assay in a single closed tube. The assay achieved a detection limit of 5.0 × 101 CFU ml-1 and showed no cross-reactivity with other bacterial species, indicating high sensitivity and specificity. Furthermore, we validated the clinical utility of the platform using milk samples artificially contaminated with P. multocida, which successfully detected P. multocida in the LAMP-CRISPR/Cas12b results. In summary, this study establishes a novel and robust detection system for P. multocida and highlights its potential for nucleic acid-based diagnostics in practical applications.},
}

*Pasteurella multocida/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Animals
*Molecular Diagnostic Techniques/methods
Milk/microbiology
*Pasteurella Infections/diagnosis/microbiology
Sensitivity and Specificity
Clustered Regularly Interspaced Short Palindromic Repeats
Limit of Detection

@article {pmid42126187,
year = {2026},
author = {Huang, J and Ma, K and Ding, S and Wang, Y and Xiong, J and Yi, J and Zhang, J and He, Z and Huang, L and Ren, X and Zhou, J and Chen, X and Liu, L and Qi, W and Wang, S and Liao, M},
title = {A CRISPR activation screen identifies CH25H as a restriction factor against influenza viruses by targeting accessible cholesterol.},
journal = {Emerging microbes & infections},
volume = {15},
number = {1},
pages = {2651464},
doi = {10.1080/22221751.2026.2651464},
pmid = {42126187},
issn = {2222-1751},
mesh = {*Cholesterol/metabolism ; Animals ; Humans ; *Steroid Hydroxylases/genetics/metabolism ; *Influenza A Virus, H7N9 Subtype/physiology ; Virus Internalization/drug effects ; *Influenza, Human/virology ; Mice ; CRISPR-Cas Systems ; Endoplasmic Reticulum/metabolism ; Cell Membrane/metabolism ; Dogs ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Influenza A viruses (IAVs) cause severe outbreaks with high mortality in birds and humans. A deeper understanding of cell-intrinsic defense mechanisms against influenza viruses is therefore crucial for developing novel antiviral strategies. Herein, we perform a genome-wide CRISPR activation screen to systematically elucidate host restriction factors against influenza A (H7N9) virus. Among multiple candidates, cholesterol 25-hydroxylase (CH25H) is shown to be induced by influenza virus infection and inhibit viral membrane fusion. Notably, our previous work demonstrated that CH25H blocks the entry of plasma membrane-fusing viruses such as coronaviruses. This inhibition occurs by relocating accessible cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER). Here, we extend this finding and show that the same mechanism works against endocytosis-dependent viruses such as influenza viruses. The exogenous supplementation of cholesterol can restore depleted accessible cholesterol and reverse the CH25H-mediated restriction. Additionally, we prove that acyl-CoA:cholesterol acyltransferase (ACAT) is required to recruit the accessible cholesterol in this process. However, how hydrophobic accessible cholesterol is transported remains unclear. Here, we demonstrate that GRAMD1/Aster-mediated non-vesicular cholesterol transport is utilized to mobilize accessible cholesterol upon stimulation of CH25H. 25-hydroxycholesterol (25HC), the catalytic product of CH25H, is a natural metabolite that potently inhibits influenza virus infection both in vitro and in vivo. These findings underscore the promising therapeutic potential of 25HC against influenza viruses.},
}

*Cholesterol/metabolism
Animals
Humans
*Steroid Hydroxylases/genetics/metabolism
*Influenza A Virus, H7N9 Subtype/physiology
Virus Internalization/drug effects
*Influenza, Human/virology
Mice
CRISPR-Cas Systems
Endoplasmic Reticulum/metabolism
Cell Membrane/metabolism
Dogs
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42126246,
year = {2026},
author = {Song, R and Yin, C and Chen, B and Qu, B and Qiao, W and Li, R and Gao, Y and Song, X},
title = {Machine Learning-Assisted Portable Ai BOX Based on RPA-CRISPR/Cas12a for Rapid On-Site Detection of Foodborne Pathogens.},
journal = {Analytical chemistry},
volume = {98},
number = {20},
pages = {14913-14927},
doi = {10.1021/acs.analchem.6c00303},
pmid = {42126246},
issn = {1520-6882},
mesh = {*Machine Learning ; *CRISPR-Cas Systems ; *Food Microbiology ; *Listeria monocytogenes/isolation & purification/genetics ; },
abstract = {Foodborne pathogens present a major threat to global public health. However, conventional detection methods and equipment are often unsuitable for the on-site and timely monitoring of these pathogens. To overcome this critical limitation and establish a rapid detection workflow, we developed the portable smart Ai BOX (artificial intelligence BOX). This device is a compact, palm-sized, internet of things (IoT)-enabled instrument that utilizes isothermal fluorescence diagnostics and weighs only 180 g. The Ai BOX features an optimized minimalist industrial design, ultralow power consumption, and a high-sensitivity optical sensing system. The device performs real-time fluorescence detection, with results automatically interpreted and transmitted to a dedicated mobile application (APP) via an integrated smart camera, enabling comprehensive food monitoring. Furthermore, the incorporation of artificial intelligence and machine learning (ML) algorithms significantly enhances the processing capability of the RPA-CRISPR/Cas12a fluorescence signal, thereby ensuring superior detection accuracy. The Ai BOX is ideally suited for on-site point-of-care testing (POCT) of foodborne pathogens. By integrating the one-pot-RPA-CRISPR/Cas12a method, the device achieves an exceptionally low limit of detection (LOD) of 1 × 10[1] CFU/mL for Listeria monocytogenes. In tests using simulated samples, it demonstrated 100% sensitivity and specificity. Consequently, the Ai BOX exhibits promising application potential for diverse public and personal health scenarios, including the detection of meat adulteration, food contamination, and wastewater monitoring.},
}

*Machine Learning
*CRISPR-Cas Systems
*Food Microbiology
*Listeria monocytogenes/isolation & purification/genetics

@article {pmid42133534,
year = {2026},
author = {Liu, C and Feng, M and Yu, H and Zhang, X and Li, Y and Sui, G and Jing, W and Cheng, X},
title = {CRISPR-Cas12a2-Based Multiplexed Diagnostic for Rapid and Highly Sensitive Detection of Respiratory Viruses.},
journal = {Analytical chemistry},
volume = {98},
number = {20},
pages = {14775-14787},
doi = {10.1021/acs.analchem.5c07791},
pmid = {42133534},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Lab-On-A-Chip Devices ; *Respiratory Syncytial Viruses/isolation & purification/genetics ; Molecular Diagnostic Techniques ; *Influenza A virus/isolation & purification/genetics ; },
abstract = {Infectious diseases severely threaten global public health security, necessitating rapid and highly sensitive diagnosis. This study presents a novel multiplex diagnostic platform combining transcription-mediated amplification (TMA) with the CRISPR-Cas12a2 system for rapid and highly sensitive detection of respiratory viruses. The assay uses an integrated microfluidic chip, which can simultaneously identify influenza A/B and respiratory syncytial viruses (RSV-A/B) with optimized CRISPR RNAs and isothermal amplification, achieving detection limits as low as 10[2] copies/μL within 60 min. The detection system showed excellent specificity; nonspecific reactions were not observed in the presence of nucleic acids from other respiratory pathogens. Clinical validation using nasopharyngeal swabs demonstrated high concordance with real-time quantitative reverse transcription polymerase chain reaction, with most positive samples detected within 40 min. The system eliminates DNA amplification steps, reduces contamination risk, and simplifies the workflow. Using two-step reactions on a centrifugal microfluidic chip, the TMA-CRISPR-Cas12a2 platform offers a promising integrated platform for multiplex respiratory pathogen screening, thereby supporting timely diagnosis and outbreak management.},
}

*CRISPR-Cas Systems/genetics
Humans
Nucleic Acid Amplification Techniques/methods
Limit of Detection
Lab-On-A-Chip Devices
*Respiratory Syncytial Viruses/isolation & purification/genetics
Molecular Diagnostic Techniques
*Influenza A virus/isolation & purification/genetics

@article {pmid42184721,
year = {2026},
author = {da Silva, GE and Obst, S and Carvalho, P and Forner, J and Ruf, S and Saibo, NJM and Bock, R},
title = {Generation of a recipient line for Rubisco engineering by multiplex genome editing in tobacco.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {4},
pages = {e70930},
doi = {10.1111/tpj.70930},
pmid = {42184721},
issn = {1365-313X},
support = {//Max-Planck-Gesellschaft/ ; UIDB/04551/2020//Fundação para a Ciência e a Tecnologia/ ; PD/BD/138096/2018//Fundação para a Ciência e a Tecnologia/ ; },
mesh = {*Ribulose-Bisphosphate Carboxylase/genetics/metabolism ; *Nicotiana/genetics/enzymology ; *Gene Editing/methods ; CRISPR-Cas Systems ; Photosynthesis/genetics ; Plants, Genetically Modified/genetics ; Plant Proteins/genetics/metabolism ; Genetic Engineering/methods ; },
abstract = {Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) is the primary CO2-fixing enzyme on our planet. Its slow kinetics and poor discrimination between carbon dioxide and oxygen as substrates severely limit the efficiency of photosynthetic carbon fixation by plants. Attempts to improve Rubisco by genetic engineering have been hampered by the complexity of the Rubisco system, with a gene family for the enzyme's small subunit being encoded in the nuclear genome and the gene for the large subunit residing in the plastid (chloroplast) genome. Another nuclear gene family encodes the enzyme Rubisco activase, which is required to facilitate catalysis in the active site of the enzyme. The Rubisco subunits and the activase have co-evolved, making single-gene replacements largely fruitless and typically resulting in dysfunctional Rubiscos. Here, we have generated a dedicated plant line for Rubisco engineering that lacks nuclear genes for components of the Rubisco system. Using multiplex genome editing by CRISPR-Cas9 in the diploid tobacco species Nicotiana sylvestris, we have knocked out six loci encoding the small subunit of Rubisco and three loci encoding Rubisco activase. The generated mutants are incapable of autotrophic growth, and grafting experiments are underway to obtain transgene-free T1 progeny. The recipient line produced here provides a clean genetic background, in which heterologous Rubisco systems from other organisms can be readily implemented and systematically tested in comparative functional studies. It greatly simplifies Rubisco engineering and creates new opportunities for future efforts to enhance photosynthetic carbon assimilation and increase crop yields.},
}

*Ribulose-Bisphosphate Carboxylase/genetics/metabolism
*Nicotiana/genetics/enzymology
*Gene Editing/methods
CRISPR-Cas Systems
Photosynthesis/genetics
Plants, Genetically Modified/genetics
Plant Proteins/genetics/metabolism
Genetic Engineering/methods

@article {pmid42185623,
year = {2026},
author = {Tsai, FY and Sternberg, SH},
title = {Flying under the radar: immune-evasive DNA for genome engineering.},
journal = {Cell research},
volume = {},
number = {},
pages = {},
pmid = {42185623},
issn = {1748-7838},
support = {RM1HG009490//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01EB027793//U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; CAREER 2239685//NSF | BIO | Division of Biological Infrastructure (DBI)/ ; },
}

@article {pmid42187217,
year = {2026},
author = {Awotundun, TA and Samson, OJ and Olanbiwoninu, AA},
title = {Viruses that heal: harnessing bacteriophages in the era of antibiotic resistance.},
journal = {Voprosy virusologii},
volume = {71},
number = {2},
pages = {91-108},
doi = {10.36233/0507-4088-366},
pmid = {42187217},
issn = {2411-2097},
mesh = {*Bacteriophages/genetics/pathogenicity ; Humans ; *Phage Therapy/methods ; *Bacteria/virology/genetics ; *Bacterial Infections/therapy/genetics/microbiology/virology ; Anti-Bacterial Agents/therapeutic use ; Genetic Engineering ; CRISPR-Cas Systems/genetics ; *Drug Resistance, Microbial/genetics ; },
abstract = {The global rise in antimicrobial resistance (AMR) poses an urgent threat to public health, and novel alternatives to traditional antibiotics are needed. One of the most promising options is bacteriophages, viruses that infect and destroy bacteria. Once overshadowed by the discovery of antibiotics, phage therapy is now regaining attention, driven by advances in genomics, synthetic biology, and targeted medicine. This review examines the biology, diversity, and therapeutic use of bacteriophages in treating bacterial infections, especially those caused by multidrug-resistant pathogens. It also discusses how phages act through natural mechanisms, such as lytic enzymes (holins, endolysins, and muralysins), and highlights new genetic engineering techniques, such as CRISPR-Cas systems, phage recombineering, and synthetic genome reboots. In addition to clinical applications, we evaluate phages as biocontrol agents for food safety, environmental sanitation, and biofilm management. Additionally, the article explores key issues in phage therapy, including regulatory frameworks, formulation stability, dynamics of phage-host resistance, and the importance of rapid diagnosis. When properly integrated into modern health and biotechnology practices, bacteriophages offer significant potential and a sustainable solution to the global challenge of antimicrobial resistance.},
}

*Bacteriophages/genetics/pathogenicity
Humans
*Phage Therapy/methods
*Bacteria/virology/genetics
*Bacterial Infections/therapy/genetics/microbiology/virology
Anti-Bacterial Agents/therapeutic use
Genetic Engineering
CRISPR-Cas Systems/genetics
*Drug Resistance, Microbial/genetics

@article {pmid42187489,
year = {2026},
author = {Liu, T and Guo, H and Yu, M and Peng, J and Guan, L and Xie, S and Hao, X and Yang, Y},
title = {A Magnetic-Assisted CRISPR-Cas12a Biosensor Incorporating a Y-DNA Probe for Sensitive Detection of Schistosoma japonicum Eggs.},
journal = {Biosensors},
volume = {16},
number = {5},
pages = {},
doi = {10.3390/bios16050293},
pmid = {42187489},
issn = {2079-6374},
support = {22277047//National Natural Science Foundation of China/ ; 82160631//National Natural Science Foundation of China/ ; 20252BAC250153//Jiangxi Provincial Natural Science Foundation/ ; },
mesh = {Animals ; *Biosensing Techniques ; *Schistosoma japonicum/isolation & purification ; *CRISPR-Cas Systems ; DNA Probes ; Rabbits ; Ovum ; Magnetics ; Aptamers, Nucleotide ; },
abstract = {Schistosomiasis, caused by Schistosoma species, is notoriously difficult to accurately diagnose with conventional methods. In this study, we present an innovative biosensor that integrates CRISPR-Cas12a technology with nucleic acid aptamers for the highly sensitive detection of Schistosoma japonicum eggs. The biosensor leverages a Y-shaped DNA structure (Y-DNA) that incorporates an aptamer specific to S. japonicum eggs, along with an activator DNA and a segment for immobilization on magnetic nanomaterials. Upon target recognition, the Y-DNA releases the activator, which triggers the collateral cleavage activity of Cas12a, enabling the direct detection of eggs. This system demonstrates remarkable sensitivity, being capable of detecting individual eggs in infected rabbit serum and feces. Moreover, it effectively distinguishes the eggs of S. japonicum from those of other parasitic species. The simplicity, high sensitivity, and rapid detection of our biosensor offer significant potential for improving the diagnosis of schistosomiasis, providing a novel, reliable tool for early detection in clinical settings.},
}

Animals
*Biosensing Techniques
*Schistosoma japonicum/isolation & purification
*CRISPR-Cas Systems
DNA Probes
Rabbits
Ovum
Magnetics
Aptamers, Nucleotide

@article {pmid42187493,
year = {2026},
author = {Bao, C and Zhang, H and Jiang, L and Liu, T and Liu, W and Qi, Q and Ren, X and Fu, H and Sun, M},
title = {Synthetic Biology-Enabled Biosensing Platforms for Point-of-Care In Vitro Diagnostics: Programmable Modules, Clinical Applications, and Translational Challenges.},
journal = {Biosensors},
volume = {16},
number = {5},
pages = {},
doi = {10.3390/bios16050297},
pmid = {42187493},
issn = {2079-6374},
support = {JYBS2025011LK//Jilin Medical University Institutional Research Project/ ; No. S202413706005, No. 202113706067, and No. 202513706018).//National Innovation and Entrepreneurship training Program for College Students/ ; 20250204071YY//the Science and Technology Department of Jilin Province/ ; },
mesh = {*Biosensing Techniques ; *Synthetic Biology ; Humans ; *Point-of-Care Systems ; CRISPR-Cas Systems ; Rapid Diagnostic Tests ; },
abstract = {Synthetic biology is reshaping in vitro diagnostics (IVD) by enabling programmable and modular biosensing elements that can be integrated into point-of-care testing (POCT) platforms. Compared with conventional assays that depend on fixed chemistries and centralized instrumentation, synthetic biology-based systems offer adaptable molecular recognition, tunable signal processing, and flexible readout formats for decentralized diagnostics. In this review, we present synthetic biology-enabled IVD as programmable biosensing platforms organized into four functional layers: molecular recognition, signal transduction and amplification, output generation, and system integration. We discuss four major enabling modules, including cell-free protein synthesis (CFPS) systems, aptamer and riboswitch sensors, CRISPR-Cas diagnostic platforms, and microfluidic integration technologies. We summarize representative clinical applications from 2021 to 2025 in infectious disease detection, cancer biomarker analysis, and drug metabolism/toxicity screening. In addition, we examine practical considerations beyond analytical sensitivity, including matrix tolerance, workflow complexity, manufacturability, quantitative capability, and regulatory readiness. Finally, we highlight future directions for programmable diagnostics, including AI-assisted biosensor design, multimodal readouts, interoperable platform architectures, and real-world clinical validation.},
}

*Biosensing Techniques
*Synthetic Biology
Humans
*Point-of-Care Systems
CRISPR-Cas Systems
Rapid Diagnostic Tests

@article {pmid42187704,
year = {2026},
author = {Jiang, C and Yang, D and Sun, C and Ren, X and Li, T and Wu, J and Tian, J and Feng, M and Yao, Y and Song, J and Weng, X and Mu, Y},
title = {An Episomal Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System for Transgene-Free Multiplex Gene Editing in Pig Cells.},
journal = {Biology},
volume = {15},
number = {10},
pages = {},
doi = {10.3390/biology15100742},
pmid = {42187704},
issn = {2079-7737},
support = {2022YFA1105402//Key Project of Natural Science Foundation of Heilongjiang Province of China/ ; 32272885//National Natural Science Foundation of China/ ; },
abstract = {Despite significant advancements in CRISPR/Cas-based genome editing technology over the past decade, achieving simultaneous homozygous gene editing at multiple targets in primary cells remains a major challenge. In this study, we developed and constructed a CRISPR multi-gene targeting system that integrates episomal vectors with tRNA-sgRNA array technology. This approach leverages scaffold/matrix attachment region (S/MAR) sequences to enable sustained episomal expression of both Cas9 and single-guide RNAs (sgRNAs) without genomic integration, thereby enhancing gene editing efficiency. For simultaneous editing of multiple loci, we used the tRNA-sgRNA architecture to process multiple sgRNAs from a single vector. Using this system in porcine fetal fibroblasts, we achieved concurrent editing of six genes, namely ANXA7, GSK3A, ENTPD6, SIRT3, CYP20A1, and SOCS2, in individual cells. These edited cells supported normal development following somatic cell nuclear transfer, yielding blastocysts with unaltered developmental competence. Collectively, our findings establish a framework for the application of CRISPR/Cas9 in gene-edited pigs, facilitating the generation of multi-gene-edited animals for biomedical and agricultural applications.},
}

@article {pmid42187710,
year = {2026},
author = {Singh, S and Tiwari, H and Singh, M and Gautam, V and Gautam, A and Gautam, HK},
title = {Expanding the Microbial Genomic Landscape and Biotechnological Applications of CRISPR-Cas Systems.},
journal = {Biology},
volume = {15},
number = {10},
pages = {},
doi = {10.3390/biology15100748},
pmid = {42187710},
issn = {2079-7737},
support = {(File No.: ANRF/IRG/2025/000135/LS)//Anusandhan National Research Foundation (ANRF)/ ; CST/D-1187//Council of Science and Technology, Uttar Pradesh, India (CST-UP)/ ; },
abstract = {The CRISPR-Cas systems, identified initially as adaptive immune mechanisms in bacteria and archaea against viral threats, have rapidly evolved into transformative tools in genetic engineering and biotechnology. These RNA-guided systems are broadly classified into Class 1, comprising multi-subunit complexes, and Class 2, characterized by compact single-effector protein, such as Cas9, Cas12, and Cas13. Their remarkable structural and functional diversity enables microorganisms to adapt to diverse ecological niches, offering a vast repertoire of genome-editing strategies. Beyond their natural role in maintaining genome integrity and defense, CRISPR-Cas systems have been extensively repurposed for precise genome modification, transcriptional regulation, epigenetic editing, and nucleic acid detection. Recent advances in computational mining of microbial genomes and metagenomes have uncovered a broad range of novel CRISPR effectors with unique properties, distinct protospacer adjacent motif (PAM) requirements, RNA-targeting capabilities, miniature architectures, and promiscuous cleavage activities that significantly expand the molecular biology toolkit. The development of CRISPR-based technologies such as base editing, prime editing, gene knock-in/out, and live-cell DNA/RNA imaging exemplifies the versatility of these systems. Despite the challenges associated with delivering complex Class 1 systems, both classes are now being actively harnessed across diverse microbial platforms. Concurrently, the CRISPR-Cas research, particularly for guide RNA (gRNA) design and activity prediction, has revolutionized target specificity and editing efficiency. This review presents a comprehensive overview of CRISPR-Cas system diversity, their genomic landscape in microorganisms, and their cutting-edge biotechnological applications. It also emphasizes the transformative potential of CRISPR in synthetic biology, therapeutics, diagnostics, environmental remediation, and agriculture, while also addressing the ethical and biosafety considerations surrounding its deployment. As CRISPR-Cas systems continue to evolve, they stand at the forefront of innovations that bridge natural microbial immunity with engineered precision tools for next-generation biotechnology.},
}

@article {pmid42189993,
year = {2026},
author = {Sabol, AL and Mengiste, AA and Singh, P and Sreekanth, V and Hendel, SJ and Tran, MTN and Barybin, AM and Chaudhary, S and Harris, RM and Liivak, KE and Severance, ZC and Locicero, CM and Kailass, K and Lee, C and Xu, LQ and Butty, VL and Choudhary, A and Shoulders, MD},
title = {Anti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {22},
pages = {e2536003123},
doi = {10.1073/pnas.2536003123},
pmid = {42189993},
issn = {1091-6490},
support = {N66001-17-2-4055//DOD | ARPA | Defense Sciences Office, DARPA (DSO)/ ; R35GM136354//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01GM132825//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01DK132900//HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/ ; R01GM137606//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 2330699//National Science Foundation (NSF)/ ; 587836-2024//Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; DRG-2539-24//Damon Runyon Cancer Research Foundation (DRCRF)/ ; P30-CA14051//HHS | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Directed Molecular Evolution/methods ; *CRISPR-Cas Systems/genetics ; Streptococcus pyogenes/genetics ; Mutation ; Adenoviridae/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Engineering CRISPR-Cas systems for improved or altered function is critical to both research and therapeutic applications. Unfortunately, most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate Streptococcus pyogenes Cas9 variants with both increased and decreased DNA binding capacity and nearly 1,000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns, the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells.},
}

Humans
*Directed Molecular Evolution/methods
*CRISPR-Cas Systems/genetics
Streptococcus pyogenes/genetics
Mutation
Adenoviridae/genetics
HEK293 Cells
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42190501,
year = {2026},
author = {Adams, BG and Wu, J},
title = {Imaging genome dynamics in real time with CRISPR-based technologies.},
journal = {Current opinion in chemical biology},
volume = {93},
number = {},
pages = {102700},
doi = {10.1016/j.cbpa.2026.102700},
pmid = {42190501},
issn = {1879-0402},
abstract = {Gene expression is a fundamental aspect of cellular function, driving diverse biological processes and disease. Dynamic interactions between genomic loci play an essential role in gene regulation. Therefore, visualizing the spatiotemporal dynamics of these interactions is vital to elucidating their function. CRISPR-Cas technology has enabled many powerful techniques for dynamic genome imaging. Recently, new methods for imaging single and multiple loci in live cells have been developed. This review describes the most recent advancements in CRISPR-based genome imaging, covering background reduction, signal amplification, and guide RNA tiling approaches. Fluorescence microscopy techniques complementing CRISPR-based imaging methods are also discussed.},
}

@article {pmid42173700,
year = {2026},
author = {Ramachandran, H and Dobner, J and Nguyen, T and Binder, S and Tolle, I and Vykhlyantseva, I and Krutmann, J and Miccio, A and Staerk, C and Brusson, M and Kontarakis, Z and Prigione, A and Rossi, A},
title = {CleanFinder: a scalable framework for comprehensive genome editing analysis.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.04.024},
pmid = {42173700},
issn = {1879-3096},
abstract = {Genome editing often generates complex mixtures of alleles rather than single, predefined outcomes. Resolving these heterogeneous edits across diverse editing modalities, sequencing platforms, and multiplexed designs remains a persistent analytical challenge. To address this, we developed CleanFinder, a browser-native framework for genotyping genome editing outcomes using a constrained semi-global alignment strategy. Context-aware alignment modes support a broad spectrum of editing scenarios, including indels, base substitutions, and complex prime editing modifications across nuclear and mitochondrial targets. Additional modules include an optional turbo mode for high-throughput heuristic alignment in exploratory workflows and an allele-aware module that leverages heterozygous single-nucleotide polymorphisms to detect allelic dropout. To evaluate scalability and practical performance, we applied CleanFinder to a primary small-molecule screen of 1849 compounds in HEK293T cells. The software efficiently processed the dataset, enabling high-throughput comparison of editing outcomes and nomination of candidate compounds for follow-up analysis. Together, CleanFinder provides a flexible and scalable platform for genome editing analysis, enabling detailed genotyping and systematic comparison of editing outcomes across diverse edit types and genomic contexts.},
}

@article {pmid42176206,
year = {2026},
author = {Dias, RG and Freitas, FPM and Barbosa, SL and Assis, JVMG and Entringer, TL and Fonseca, JSC and Romanizio, ME and Pimentel, BBZ and Campos-Galvão, MEM and Vieira, NM and Fietto, LG and Zsögön, A and da Silveira, WB},
title = {10 years of CRISPR/CAS genomic engineering in Yarrowia lipolytica.},
journal = {Bioprocess and biosystems engineering},
volume = {},
number = {},
pages = {},
pmid = {42176206},
issn = {1615-7605},
abstract = {Yarrowia lipolytica is a versatile cell factory widely used in bioprocesses for producing lipids, organic acids, and other high-value compounds. Historically, its genetic engineering was constrained by low homologous recombination (HR) efficiency and the predominance of non-homologous end joining (NHEJ), limiting strain development and metabolic pathway optimization. The advent of CRISPR-Cas technologies has revolutionized genome editing in Y. lipolytica, enabling precise, efficient, and multiplexed modifications. Innovations such as pCAS1yl and pCRISPRyl plasmids, along with genomic Cas9 integration, have enhanced targeted editing efficiency. CRISPR applications now extend to metabolic engineering for lipids, itaconic acid, erythritol, and other compounds. Beyond canonical Cas9, alternative systems, including CRISPRa, Cas12a, base editors, and sgRNA libraries, provide increased flexibility and functional diversity. Central to these approaches is the rational design of guide RNAs (gRNAs), supported by bioinformatics platforms such as CHOPCHOP v3, CRISPOR, CCTop, and Cas-OFFinder, which assist in target site selection, off-target prediction, and editing optimization. This review summarizes the main CRISPR/Cas9 applications in Y. lipolytica, highlighting key engineered strains and emphasizing the critical role of bioinformatics in improving editing strategies. We also propose a pipeline for systematic gRNA design based on published evidence and discuss future perspectives, including the integration of machine learning, artificial intelligence, and emerging CRISPR variants to further advance yeast metabolic engineering.},
}

@article {pmid42178819,
year = {2026},
author = {Yadav, A and Saini, A and Dilbaghi, N and Yadav, N},
title = {Advances in Nano-Enabled Biosensing Technology for Gastric Cancer Diagnosis: Mechanistic Insights and Translational Perspectives.},
journal = {Critical reviews in analytical chemistry},
volume = {},
number = {},
pages = {1-28},
doi = {10.1080/10408347.2026.2672560},
pmid = {42178819},
issn = {1547-6510},
abstract = {Gastric cancer (GC) remains a critical burden on healthcare, affecting millions of people annually. Helicobacter pylori infection is a critical contributor to GC. The diagnostic confirmation of GC is generally obtained at late stages owing to the delayed onset of symptoms. Early detection can significantly improve the disease outcomes. Several approaches, like endoscopy, MRI, and computed tomography, are conventionally employed. But they often have certain drawbacks, such as less accessibility, invasiveness, and potential false results. Delayed diagnosis and poor prognosis by conventional strategies have underlined the need for an efficient and precise solution. These obstacles can be mitigated by implementing advanced biosensing platforms. The amalgamation of nanotechnology, machine learning, and advanced computational intelligence has extensively evolved sensor technology. This review offers a holistic overview of GC pathogenicity and conventional diagnostics with special emphasis on recently fabricated biosensors. Advanced biosensing platforms, like CRISPR-Cas, smartphone-integrated, breath-based, and ingestible biosensors, are also explored. This review further highlights the translational perspectives along with the increasing role of AI and advanced algorithms. With a critical discussion on key challenges, this article provides a future roadmap for the detection of GC biomarkers. Significant innovations are needed to translate biosensors into a state-of-the-art technique in GC diagnostics.},
}

@article {pmid42178964,
year = {2026},
author = {Sha, T and Zhao, D and Zhao, X and Lu, Y and Wang, R and Liu, J and Li, Y and Li, S and Chen, M and Bi, C and Zhang, X},
title = {Mutant-Initiated Structure-Guided Refinement Enables Second-Generation Compact IscB Genome Editors.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {10},
pages = {e70392},
doi = {10.1002/cbic.70392},
pmid = {42178964},
issn = {1439-7633},
support = {XDC0110200//the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 32225031//National Science Fund for Distinguished Young Scholar/ ; 32488301//the Basic Science Center Project of the National Natural Science Foundation of China/ ; 32271483//National Natural Science Foundation of China/ ; 2022177//Youth Innovation Promotion Association CAS/ ; },
mesh = {*Gene Editing/methods ; Humans ; Mutation ; DNA/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Compact RNA-guided nucleases such as IscB represent an attractive foundation for next-generation genome editors, yet their application in mammalian cells has been constrained by suboptimal activity. Here, instead of re-engineering enzymes, we establish a mutant-initiated, structure-guided optimization strategy to generate second-generation high-activity IscB editors. Using AlphaFold3 to model the engineered IscB*-ωRNA-DNA complex, we reveal remodeling of the nucleic-acid-binding interface induced by activity-enhancing substitutions. Guided by this predicted structure, we perform a focused mutational scan and identify V367 as an activity hotspot. Saturation mutagenesis at this position yields a single substitution, V367Y (IscB*-Act), which increases mean editing efficiency by 34% and achieves up to 2.1-fold improvement across endogenous targets in mammalian cells. Importantly, the V367Y substitution is transferable to an IscB-based adenine base editor, elevating A-to-G conversion by 68% on average and up to 4.46-fold at individual loci without altering the intrinsic editing window. Targeted off-target profiling at loci suggests that V367Y does not substantially increase off-target indels or A-to-G conversion. Together, our work demonstrates a practical framework for second-generation refinement of compact genome editors, bridging deep-learning-enabled structural prediction with interpretable protein engineering, and expands the functional potential of miniature IscB systems for both nuclease and base editing applications.},
}

*Gene Editing/methods
Humans
Mutation
DNA/chemistry/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid42179772,
year = {2026},
author = {Vats, P and Baweja, B and Saini, C and Kushwah, AS and Kumar, A and Srivastava, SK and Nema, R},
title = {An overview of CRISPR-artificial intelligence theranostics: Current and emerging applications.},
journal = {Biomaterials translational},
volume = {7},
number = {1},
pages = {79-120},
pmid = {42179772},
issn = {2096-112X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics are revolutionizing precision medicine by enabling highly sensitive detection of nucleic acid and protein biomarkers. Building on these capabilities, CRISPR-based theranostics now aim to unify real-time disease detection with targeted therapeutic interventions. However, traditional CRISPR diagnostics face several limitations, including restricted multiplexing, off-target effects, and challenges in delivery efficiency. To overcome these issues, artificial intelligence (AI) has significantly enhanced CRISPR platforms by enabling intelligent guide RNA (gRNA) design, interpretation of complex biosensor outputs, and facilitation of rapid clinical decision-making. Machine learning tools such as DeepCRISPR, Azimuth 2.0, DeepHF, and CRISPRpred support the development of highly specific gRNAs, reduce off-target events, and personalize genome-editing strategies based on individual genomic profiles. Recently, by combining CRISPR systems with nanomaterials, fluorescence-based detection, and electrochemical sensing, researchers have developed advanced biosensors capable of detecting a broad spectrum of disease biomarkers, from cancer-associated nucleic acids to viral and genetic disorders. These advances support both diagnostics and gene therapy, enabling accurate, low-cost testing at home, in point-of-care settings, and in resource-limited environments. Together, the integration of AI and CRISPR is accelerating biomarker discovery and the development of intelligent, adaptive therapeutic platforms. New point-of-care diagnostic tests (POCTs) based on CRISPR-AI are essential for early screening of high-mortality diseases, and CRISPR-based diagnostic assays have emerged as powerful, versatile alternatives to traditional nucleic acid tests, offering rapid, programmable, and portable diagnostic solutions. This review explores the evolution of CRISPR-AI theranostic systems, current and emerging POCT applications. It highlights the technological, clinical, and ethical challenges shaping their translation into next-generation precision diagnostics.},
}

@article {pmid42180316,
year = {2026},
author = {Chen, X and Zhang, M and Yang, L and Chen, Y and Chi, Y and Zhao, Y and Ma, Z and Li, Y and Wang, X},
title = {CRISPR spacer profiling and prophage mining reveal diverse bacteriophages associated with Streptococcus Mutans.},
journal = {Journal of oral microbiology},
volume = {18},
number = {1},
pages = {2674332},
pmid = {42180316},
issn = {2000-2297},
abstract = {BACKGROUND: Streptococcus mutans is a key cariogenic bacterium. Current antimicrobials lack species specificity, while phage-based approaches remain experimental and require more S. mutans phage isolates.

OBJECTIVE: To profile the diversity of S. mutans-associated phages and strain-level heterogeneity in phage exposure using genome-informed CRISPR spacer and prophage analyses.

MATERIALS AND METHODS: We compiled 944 publicly available S. mutans genomes and dereplicated them into 735 non-redundant strains. CRISPR-Cas systems, spacers, spacer targets, and putative prophages were identified, quality-assessed, and functionally annotated. Phylogenetic relationships of (pro)phages were evaluated using terminase large subunit proteins, and comparative genomics compared spacer-positive and spacer-negative strains.

RESULTS: CRISPR systems were detected in 548/735 strains, yielding 14,263 spacers, 1,864 phage-targeting spacers mapped to 110 viral genomes, including 41 cultured isolates, 51 metagenome-assembled phages, and 18 uncultured viral genomes. The most frequently targeted cultured phage was phiKSM96, whereas metagenome-assembled Caudoviricetes ctNo011 showed broader targeting. Prophage mining identified 186 regions in 130 strains, including 37 of ≥ medium quality and elements related to ctNo011 and phiKSM96. TerL phylogeny showed that most high-quality endogenous prophages clustered with phiKSM96 and ctNo011.

CONCLUSION: These findings reveal a vast, uncultivated phage repertoire targeting S. mutans, providing a critical genomic roadmap to guide the future isolation of novel phages for caries prevention.},
}

@article {pmid42180538,
year = {2026},
author = {Yang, J and Chen, Y and Chen, X and Ping, Y},
title = {Non-viral delivery of genome-editing tools for treatment of genetic disorders.},
journal = {Acta pharmaceutica Sinica. B},
volume = {16},
number = {5},
pages = {2903-2928},
pmid = {42180538},
issn = {2211-3835},
abstract = {Pathogenic mutations within protein-coding regions of genomic DNA can disrupt protein structure and lead to hereditary disorders. Genome-editing technologies, particularly those based on clustered, regularly interspaced, short palindromic repeats-associated protein (CRISPR-Cas), are promising therapeutic tools for correcting genetic abnormalities. To date, viral delivery vectors for genome-editing biomacromolecules have shown numerous promises in treating genetic disorders. However, safe viral delivery for genome-editing components remains challenging, largely due to the immunogenicity of viruses. As an alternative, non-viral delivery systems are emerging as a safer choice and may offer solutions to address the safety challenges. In this review, we first introduce CRISPR-Cas9-based genome editing tools and their delivery formats. Then, we outline the pathology of major genetic disorders and both preclinical and clinical approaches for these diseases by therapeutic genome editing, and provide an overview of current non-viral delivery strategies and their potential to overcome existing limitations. Finally, we discuss the current challenges and future outlooks of non-viral delivery of gene-editing components in treating genetic diseases.},
}

@article {pmid42182021,
year = {2026},
author = {Li, X and Huang, Y and Zhang, X and Du, L and Qiu, Y and Jiang, L},
title = {Recent advances in rapid multiplex detection of nucleic acid markers using RPA and CRISPR-Cas.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1810544},
pmid = {42182021},
issn = {1664-302X},
abstract = {The integration of recombinase polymerase amplification (RPA) with CRISPR-Cas systems has emerged as a powerful platform for rapid multiplex nucleic acid detection. Compared with quantitative polymerase chain reaction (qPCR) and Next-generation sequencing (NGS), RPA-CRISPR operates isothermally (37 °C-42 °C), requires minimal equipment, and achieves attomolar sensitivity in 20-90 min via collateral cleavage. Recent multiplex strategies, namely two-tube, spatial separation one-tube, and homogeneous one-pot, they have overcome crosstalk and enabled highly multiplexed detection in complex food matrices such as poultry, milk, and lettuce. These approaches are particularly suited for foodborne pathogen screening (e.g., Salmonella, Listeria), antimicrobial resistance profiling, and on-site surveillance, aligning with the scope of research at the frontier of food microbiology diagnostics. Despite advances, challenges persist in standardization, matrix inhibition, and regulatory approval. This mini-review summarizes recent advances (2020-2025) in RPA-CRISPR multiplex detection, outlines future directions for clinical implementation and food safety deployment, and provides guidance for subsequent research on its practical applications in these fields.},
}

@article {pmid42182524,
year = {2026},
author = {Huang, X and Sun, X and Dong, X and Tang, Y and Xu, S and Wu, Y and Hu, S and Ren, Y and Tu, Q and Zhang, Y},
title = {Prospects and challenges in using engineered lactic acid bacteria in aquaculture applications.},
journal = {Engineering microbiology},
volume = {6},
number = {2},
pages = {100275},
pmid = {42182524},
issn = {2667-3703},
abstract = {Despite the considerable potential of lactic acid bacteria (LAB) as probiotics, there is a fundamental gap between the functional limitations of wild-type strains and the complex demands of aquaculture. Modular and intelligent engineering strategies are the primary avenues for bridging this gap. This article systematically reviews the strategies and advances in the application of genetically engineered LAB. Technologies, including clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems, Red/ET recombination, and functional modifications have significantly enhanced the targeted delivery, environmental tolerance, and multiple probiotic functions of LAB, successfully yielding engineered strains such as oral vaccine strains expressing pathogen antigens, antimicrobial peptide-high-yielding antibacterial strains, and nitrite-degrading water-improving strains. These engineered strains have demonstrated superior performance in disease prevention, growth promotion, and environmental remediation compared to wild-type strains in the farming of tilapia, shrimp, and shellfish. However, challenges, such as plasmid instability, biosafety risks, and regulatory barriers, remain unresolved. Future research should focus on multi-omics-guided precision design, development of environmentally responsive genetic circuits, and full-cycle risk assessment, promoting engineered LAB as a core solution for sustainable aquaculture through collaboration across industries, academia, and research.},
}

@article {pmid42184324,
year = {2026},
author = {Aliev, TI and Imatdinov, AR and Prudnikova, EY and Imatdinov, IR},
title = {[Experimental Selection of Effective sgRNAs for MmCas12m Targeting the Region of the Start Codon of the HIV-1 gag Gene].},
journal = {Molekuliarnaia biologiia},
volume = {60},
number = {1},
pages = {120-131},
doi = {10.7868/S3034555326010073},
pmid = {42184324},
issn = {0026-8984},
mesh = {*HIV-1/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Codon, Initiator/genetics ; *Gene Editing ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *gag Gene Products, Human Immunodeficiency Virus/genetics/metabolism ; *HIV Infections/genetics/therapy ; },
abstract = {HIV-1 remains a threat to global health. There is no effective vaccine or drug for a complete cure of HIV infection. Work continues on the development of gene therapy drugs against HIV-1. The use of DNA base editors delivered to the editing site by CRISPR-Cas systems has shown some success in the field of HIV-1 gene therapy. The MmCas12m isoform obtained from Mycolicibacterium mucogenicum can become a promising platform for this task. MmCas12m has a compact size, the ability to bind strongly to the target DNA sequence, and an absence of nuclease activity. Thus, MmCas12m can act not only as a platform for the delivery of DNA base editors, but also as an inhibitor of transcription from HIV-1 proviral DNA. We experimentally selected in vitro the most effective sgRNAs for MmCas12m to target the start codon of the gag HIV-1, the product of which is important in virion assembly. Of the nine sgRNAs we selected, four showed statistically significant effectiveness in targeting the desired region of the HIV-1 genome. To test the effectiveness of each sgRNA, we have developed a system suitable for evaluating the binding of any Cas protein to the target site of HIV-1 genome editing.},
}

*HIV-1/genetics/metabolism
Humans
*CRISPR-Cas Systems
*Codon, Initiator/genetics
*Gene Editing
*RNA, Guide, CRISPR-Cas Systems/genetics
*gag Gene Products, Human Immunodeficiency Virus/genetics/metabolism
*HIV Infections/genetics/therapy

@article {pmid42184325,
year = {2026},
author = {Kolesnikova, OA and Svetlakova, AV and Furtak, ED and Zvereva, SD and Kukushkin, ID and Komedchikova, EN and Shipunova, VO},
title = {[Establishment and Validation of Cancer Cell Lines with HER2 Receptor Gene Deletion].},
journal = {Molekuliarnaia biologiia},
volume = {60},
number = {1},
pages = {132-143},
doi = {10.7868/S3034555326010086},
pmid = {42184325},
issn = {0026-8984},
mesh = {Humans ; *Erb-b2 Receptor Tyrosine Kinases/genetics/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Gene Deletion ; Gene Editing ; Cell Proliferation ; },
abstract = {One of the clinically significant molecular targets in oncotheranostics is the receptor tyrosine kinase HER2 (HER2/neu, ERBB2), which is involved in the activation of various cellular programs. The overexpression of this receptor leads to uncontrolled cell proliferation, the initiation of oncogenesis, and is considered one of the most important oncogenic biomarkers. In vitro studies utilizing various HER2-positive cancer cell lines play a crucial role in the development of anti-HER2 targeted drug formulations. These cell lines differ in their structural and metabolic features, as well as in their sensitivity to hormones and other factors; therefore, the selection of an optimal cellular control is essential for the successful testing of HER2-specific agents. In the present study, a deletion in the ERBB2 gene was generated using CRISPR/Cas9 technology, with the success of the editing confirmed by sequencing of the target locus. A reduction in ERBB2 mRNA levels was demonstrated in three cancer cell lines with varying baseline HER2 receptor levels, alongside an alteration in the receptor's functional activity on the cell surface. Assessment of the binding efficiency of a fluorescently labeled HER2-specific antibody to the generated cell clones revealed a decrease in fluorescence intensity by 80.6-fold, 33.7-fold, and 2-fold in the SK-BR-3, SK-OV-3, and A549 cell lines, respectively. The generated cell lines with ERBB2 deletion represent a key tool for testing targeted therapeutics and can be utilized in the development of treatment modalities aimed at HER2-overexpressing malignant neoplasms.},
}

Humans
*Erb-b2 Receptor Tyrosine Kinases/genetics/metabolism
Cell Line, Tumor
CRISPR-Cas Systems
*Gene Deletion
Gene Editing
Cell Proliferation

@article {pmid42184676,
year = {2026},
author = {Li, P and Hu, Q and Li, Y and Li, S and Ruan, Z and Yin, K and Sun, G},
title = {Amplification-free light-activated CRISPR/Cas12a system with nano-amplifier for quantitative detection of non-nucleic acid targets.},
journal = {Biosensors & bioelectronics},
volume = {309},
number = {},
pages = {118844},
doi = {10.1016/j.bios.2026.118844},
pmid = {42184676},
issn = {1873-4235},
abstract = {Quantitative amplification-free CRISPR/Cas diagnosis to non-nucleic acid clinical biomarkers is limited by inefficient signal conversion and suboptimal sensitivity. Here, we present a light-activated CRISPR/Cas12a biosensing platform (LANA) to response this challenge. The LANA arms with a cascade signal enhancing strategy that integrates immunomagnetic enrichment, aptamer-functionalized gold nanoparticles carrying photocleavable activators, and UV-triggered release of activators to initiate Cas12a trans-cleavage. This light-controlled mechanism overcomes steric hindrance caused by surface confinement, provides precise temporal control of signal initiation, and effectively suppresses background activation. Using cardiac troponin I (cTnI) as a model analyte, LANA achieved a detection limit of 50 pg/mL, a wide dynamic range of 0.05-500 ng/mL, demonstrating high sensitivity and reliable quantitative performance with a simple fluorescence readout. Owing to its modular and amplification-free design, the platform can be readily adapted to other protein or small-molecule biomarkers, offering a generalizable and amplification-free framework for sensitive detection of non-nucleic acid targets.},
}

@article {pmid42173587,
year = {2026},
author = {Lu, X and Wang, Y and Che, Y and Li, Y and Nong, B and Ge, Y and Wang, X and Guo, Y and Li, R and Liu, J and Guo, J and Yao, Y and Geng, M},
title = {CRISPR/Cas9 mediated knockout of MeSSI enhances resistant starch content without compromising yield in cassava.},
journal = {Carbohydrate polymers},
volume = {385},
number = {},
pages = {125382},
doi = {10.1016/j.carbpol.2026.125382},
pmid = {42173587},
issn = {1879-1344},
mesh = {*Manihot/genetics/metabolism/chemistry ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Starch/chemistry/metabolism ; *Plant Proteins/genetics/metabolism ; Amylopectin/metabolism/chemistry ; Gene Editing ; Amylose/metabolism ; Gene Expression Regulation, Plant ; *Resistant Starch/metabolism ; },
abstract = {Enhancing resistant starch (RS) content in cassava is vital for developing nutritionally improved, functional food crops. In this study, targeted mutagenesis of the MeSSI gene via CRISPR/Cas9 was conducted to investigate its role in starch biosynthesis and RS accumulation. MeSSI knockout lines exhibited a 6.74-fold increase in RS content and a 16.42% elevation in amylose levels compared to the wild-type, without compromising total starch content or root yield. Starch structural analysis revealed an increased number of smaller granules per amyloplast and a shift in amylopectin chain-length distribution, characterized by reduced short chains (DP 6-12) and enrichment of intermediate and long chains, resulting in a lower branching degree. These modifications were associated with enhanced thermal stability and altered pasting behavior. Transcriptomic profiling indicated compensatory upregulation of AGPase subunits, and glycolytic genes, suggesting a reprogramming of carbon metabolism to sustain starch accumulation. This work identifies MeSSI as a key determinant of amylopectin fine structure and RS formation, providing a precise genome-editing strategy to improve the nutritional profile of cassava.},
}

*Manihot/genetics/metabolism/chemistry
*CRISPR-Cas Systems/genetics
Gene Knockout Techniques
*Starch/chemistry/metabolism
*Plant Proteins/genetics/metabolism
Amylopectin/metabolism/chemistry
Gene Editing
Amylose/metabolism
Gene Expression Regulation, Plant
*Resistant Starch/metabolism

@article {pmid42019459,
year = {2026},
author = {Yun, Q and Gu, M and Li, L and Xia, WH},
title = {Rapid and visual detection of Mycoplasma pneumoniae using a novel ERA-CRISPR/Cas12a-based lateral flow assay.},
journal = {Diagnostic microbiology and infectious disease},
volume = {116},
number = {1},
pages = {117416},
doi = {10.1016/j.diagmicrobio.2026.117416},
pmid = {42019459},
issn = {1879-0070},
mesh = {Humans ; *Mycoplasma pneumoniae/isolation & purification/genetics ; Sensitivity and Specificity ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Child ; Adhesins, Bacterial/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {OBJECTIVE: Mycoplasma pneumoniae (MP) is a leading cause of community-acquired pneumonia in children. Conventional detection methods often lack the speed or accessibility required for point-of-care testing (POCT). This study aims to develop a rapid, highly sensitive, and instrument-free diagnostic platform integrating Enzymatic Rapid Amplification (ERA), CRISPR/Cas12a cleavage, and Lateral Flow Assay (LFA) for visual MP detection.

METHODS: Specific primers targeting the conserved P1 adhesin gene of MP were screened for ERA efficiency. A CRISPR/Cas12a system was designed to specifically recognize the ERA amplicons, triggering the trans-cleavage of a reporter probe. The results were visually interpreted using LFA strips. The assay's limit of detection (LoD), specificity against six common respiratory pathogens, and clinical performance on 80 throat swab samples were evaluated.

RESULTS: The optimized ERA-CRISPR/Cas12a-LFA assay can be completed within 40 minutes. The LoD was established at 200 copies/mL. Specificity testing showed no cross-reactivity with S. pneumoniae, H. influenzae, or other tested pathogens. In clinical validation (n = 80), the assay demonstrated a sensitivity of 96.23% and specificity of 100% compared to qPCR, with a Kappa value of 0.945.

CONCLUSION: The established ERA-CRISPR/Cas12a-LFA method offers a rapid, sensitive, and specific alternative for MP screening. Its minimal equipment requirements make it highly suitable for resource-limited settings and primary care clinics.},
}

Humans
*Mycoplasma pneumoniae/isolation & purification/genetics
Sensitivity and Specificity
*Pneumonia, Mycoplasma/diagnosis/microbiology
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Point-of-Care Testing
Limit of Detection
*Nucleic Acid Amplification Techniques/methods
Child
Adhesins, Bacterial/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42172144,
year = {2026},
author = {Aguilar, G and Sickmann, ME and Bieli, D and Born, G and Affolter, M and Müller, M},
title = {In situ mutational screening and CRISPR interference define apterous cis-regulatory inputs during compartment boundary formation.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {42172144},
issn = {2050-084X},
support = {310030_192659/SNSF_/Swiss National Science Foundation/Switzerland ; 310030B_176400/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Animals ; *Drosophila Proteins/genetics/metabolism ; *Wings, Animal/embryology/growth & development ; *Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; *Drosophila melanogaster/genetics/embryology ; CRISPR-Cas Systems ; Homeodomain Proteins/genetics/metabolism ; Body Patterning/genetics ; Mutation ; },
abstract = {The establishment of tissue axes is fundamental during embryonic development. In the Drosophila wing, the anterior/posterior (AP) and the dorsal/ventral (DV) compartment boundaries provide the basic coordinates around which the tissue develops. These boundaries arise as a result of two lineage decisions, the acquisition of posterior fate by the selector gene engrailed (en) and dorsal fate by the selector gene apterous (ap). While the en expression domain is set up during embryogenesis, ap expression begins only during early wing development. Thus, the correct establishment of the ap expression pattern relative to en must be tightly controlled. Here, we functionally investigate the transcriptional inputs integrated by the early ap enhancer (apE) and their requirement for correct boundary formation. Detailed mutational analyses using CRISPR/Cas revealed a role for apE in positioning the DV boundary relative to the AP boundary, with apE mutants often displaying mirror-image anterior wing duplications. We then designed and applied methods to accomplish tissue-specific enhancer disruption via dCas9 expression. This approach allowed us to dissect the spatiotemporal requirement for apE function, clarifying the mechanism by which apE misregulation leads to AP defects. Base-pair-resolution analyses of apE uncovered a single HOX-binding site essential for wing development that, when mutated, led to wingless flies. We demonstrated that the transcription factors Pointed (Pnt), Homothorax (Hth), and Grain (Grn) are required for apE function, and the HOX gene Antennapedia (Antp) contributes to early wing development. Together, our results provide a comprehensive molecular basis of early ap activation and the developmental consequences of its misregulation, shedding light on how compartmental boundaries are set up during development.},
}

Animals
*Drosophila Proteins/genetics/metabolism
*Wings, Animal/embryology/growth & development
*Gene Expression Regulation, Developmental
*Transcription Factors/genetics/metabolism
*Drosophila melanogaster/genetics/embryology
CRISPR-Cas Systems
Homeodomain Proteins/genetics/metabolism
Body Patterning/genetics
Mutation

@article {pmid42172311,
year = {2026},
author = {Guo, C and Zhang, S and Yerramsetti, R and Zhang, J and Guan, X and Yang, R and Hou, C and Pei, M and Schalper, KT and Liu, X and Li, Z and Perpetua, L and Gan, W and Ibrahim, O and Clark, RA and Liu, C},
title = {Single-nucleotide variant profiling in liquid biopsy with RECO-Cas.},
journal = {Science advances},
volume = {12},
number = {21},
pages = {eaed1757},
pmid = {42172311},
issn = {2375-2548},
mesh = {Humans ; Liquid Biopsy/methods ; *Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems/genetics ; Proto-Oncogene Proteins p21(ras)/genetics ; *Cell-Free Nucleic Acids/genetics/blood ; ErbB Receptors/genetics ; Class I Phosphatidylinositol 3-Kinases/genetics ; *Neoplasms/genetics/diagnosis ; Mutation ; },
abstract = {Mutation detection of cell-free DNA (cfDNA) through liquid biopsy is essential for precision oncology, resistance profiling, and informed clinical decision-making. However, its clinical application has remained limited by the lack of simple, rapid, accurate, and cost-effective detection approaches. Here, we report a recombined DNA construct-activated Cas12a (RECO-Cas) assay for profiling cfDNA mutations. The RECO-Cas assay uses a recombined DNA construct generated from Argonaute-nicked mutant DNA and an artificial DNA activator to selectively trigger CRISPR-Cas12a, enabling 0.01% variant allele frequency sensitivity and single-nucleotide resolution. Using the assay, we detect KRAS, EGFR, and PIK3CA point mutations in cfDNA from clinical plasma samples, demonstrating high sensitivity (90.48%) and excellent specificity (100%). We also apply it to successfully classify and identify KRAS missense variants. RECO-Cas provides a simple, rapid, and affordable solution that is compatible with a compact, wirelessly powered point-of-care diagnostic platform incorporating smartphone-based fluorescence detection. This assay enables highly sensitive and specific detection of low-frequency mutations, facilitating early cancer diagnosis and supporting the development of personalized treatment strategies.},
}

Humans
Liquid Biopsy/methods
*Polymorphism, Single Nucleotide
*CRISPR-Cas Systems/genetics
Proto-Oncogene Proteins p21(ras)/genetics
*Cell-Free Nucleic Acids/genetics/blood
ErbB Receptors/genetics
Class I Phosphatidylinositol 3-Kinases/genetics
*Neoplasms/genetics/diagnosis
Mutation

@article {pmid42173102,
year = {2026},
author = {Peng, S and Xie, W and Zhu, J and Bao, Z},
title = {Multiplex genome engineering: Methodologies and applications.},
journal = {Cell systems},
volume = {},
number = {},
pages = {101614},
doi = {10.1016/j.cels.2026.101614},
pmid = {42173102},
issn = {2405-4720},
abstract = {To facilitate the study of applied genetics and enable a rapid translation of genetic insights, it is highly desirable to concurrently modify many genomic loci in an organism of interest. While single-locus editing is well-established and technically straightforward, multiplex genome engineering (MGE) poses significant technological barriers. With the convergence of low-cost DNA synthesis, advanced genome editing techniques, and laboratory automation, a plethora of MGE methodologies were recently developed and applied in fields ranging from basic research to applied sectors. This review analyzes one-step and iterative MGE methodologies, with an emphasis on recombineering and CRISPR-Cas systems, and showcases emerging paradigm-shifting applications in biomanufacturing, agriculture, and therapeutics. We conclude by analyzing the limitations of existing technologies and discussing future directions for further optimizing MGE to solve system-level problems.},
}

@article {pmid42173386,
year = {2026},
author = {Mishra, S and Rehan, S and Barekzai, AM and Sharma, A and Raghav, A},
title = {Emerging frontiers in genome editing: From CRISPR to next-generation technologies.},
journal = {Methods (San Diego, Calif.)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymeth.2026.05.011},
pmid = {42173386},
issn = {1095-9130},
abstract = {Genome editing has revolutionized molecular biology. It offers precise modification of genetic material across diverse organisms. This review outlines the evolution of genome editing technologies from homologous recombination to advanced Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-9) based systems that now dominate the field. Early methods, such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), established the foundation for site-specific DNA cleavage. However, they were limited by complexity and cost. The advent of the CRISPR-Cas systems, particularly CRISPR-Cas9, transformed the landscape due to their simplicity, high efficiency, and adaptability. Variants such as CRISPR-Cas12a, base editors, and prime editors enhanced editing precision. They enable single-nucleotide modifications and targeted insertions without double-strand breaks. Emerging tools such as CRISPR-associated transposases, recombinase fusions, and RNA-targeting Cas13 enzymes expand the scope of manipulation beyond DNA to RNA. At the same time, epigenome editing, and gene drives present new therapeutic and ecological applications. Efficient delivery systems, both viral (Adeno-Associated Virus (AAV), lentivirus, adenovirus) and non-viral (lipid nanoparticles, gold nanoparticles, DNA nano clews), remain critical for clinical translation. Future directions emphasize artificial intelligence-guided design, retroelement-based integration, and novel biomimetic delivery vehicles to overcome current efficiency and safety barriers. These innovations can help overcome current efficiency and safety barriers. Together, they are propelling genome editing toward precise, programmable, and ethically responsible therapeutic applications. Despite unresolved challenges involving off-target effects, immunogenicity, and germline ethics, genome editing redefines biomedical research, drug development, and disease correction. CRISPR-derived technologies now stand at the forefront of next-generation genetic medicine.},
}

@article {pmid41652852,
year = {2026},
author = {Li, Y and Xu, B and Gao, X and Wang, Y and Liu, X and Xu, R and Li, J and Wei, P and Qin, R},
title = {Tuning Rice Gene Expression via In Situ Promoter Truncations Using a Prime Editing Library.},
journal = {Plant biotechnology journal},
volume = {24},
number = {6},
pages = {3528-3530},
doi = {10.1111/pbi.70587},
pmid = {41652852},
issn = {1467-7652},
support = {2023ZD04074//Biological Breeding - Major Projects/ ; 32400335//National Natural Science Foundation of China/ ; 32572441//National Natural Science Foundation of China/ ; 32570484//National Natural Science Foundation of China/ ; 2024CSJZN01100//Yangtze River Delta Science and Technology Innovation Community Joint Research (Basic Research) Project/ ; 202423110050063//Science and Technology Major Project of Anhui Province/ ; 202423m10050002//Science and Technology Major Project of Anhui Province/ ; 2023n06020020//Science and Technology Major Project of Anhui Province/ ; },
mesh = {*Oryza/genetics ; *Promoter Regions, Genetic/genetics ; *Gene Editing/methods ; *Gene Expression Regulation, Plant/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified ; Gene Library ; },
abstract = {Promoter engineering holds immense potential for fine-tuning gene expression and optimising agronomic traits, yet conventional genome-editing tools face limitations in precision, scalability, and risk mitigation. Here, we develop Prime Editing-mediated Promoter Engineering (PEPE), a DSB-free platform integrating bidirectional Protospacer adjacent motif (PAM) recognition (NGG/CCN) with combinatorial duo-pegRNA strategies to achieve tiled, overlapping deletions across entire plant promoters. Applying PEPE to the 1.8-kb rice D53 promoter, we generated a mutant library with stepwise deletions. Edited alleles showed stable inheritance, and dual-method validation confirmed the precision at junctions. Quantitative profiling revealed functional modularity: core-region deletions suppressed D53 expression by 70%-85%, while a distal deletion (D53-G9C10) paradoxically upregulated transcription 2.2-fold, uncovering a cryptic repressor element. Phenotypic variation corresponded with transcriptional changes, establishing a direct link between cis-regulatory diversity and agronomic traits. By circumventing DSBs and enabling kilobase-scale CRE manipulation, PEPE establishes a robust framework for decoding promoter logic and accelerating trait pyramiding in crops. This study advances plant genome editing by merging precision with scalability, offering transformative potential for breeding climate-resilient, high-yield cultivars.},
}

*Oryza/genetics
*Promoter Regions, Genetic/genetics
*Gene Editing/methods
*Gene Expression Regulation, Plant/genetics
CRISPR-Cas Systems
Plants, Genetically Modified
Gene Library

@article {pmid41734974,
year = {2026},
author = {Ding, Y and Chen, X and Wu, K and Hou, H and Wang, Y and Yan, B and Khursheed, MHUR and Shang, C and Zhang, X and Pan, Y and Wu, L and Li, J},
title = {A Truncated WRKY Protein Enhances Drought Resistance in Wild Tomatoes Through the SlWRKY16-CIP2b-SlSYP121 Module.},
journal = {Plant biotechnology journal},
volume = {24},
number = {6},
pages = {3840-3860},
doi = {10.1111/pbi.70596},
pmid = {41734974},
issn = {1467-7652},
support = {Nos.CSTB2023TIAD-KPX0026//Special Key Project of Technological Innovation and Application Development of Chongqing/ ; CSTB2023TIAD-KPX0025//Special Key Project of Technological Innovation and Application Development of Chongqing/ ; 31872123//National Natural Science Foundation of China/ ; 32172597//National Natural Science Foundation of China/ ; SWU-KQ22041//Fundamental Research Funds for the Central Universities/ ; CARS-23-B08//China Agriculture Research System/ ; },
mesh = {*Solanum lycopersicum/genetics/physiology/metabolism ; *Droughts ; *Plant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Plant ; Polymorphism, Single Nucleotide/genetics ; Stress, Physiological/genetics ; CRISPR-Cas Systems ; Drought Resistance ; },
abstract = {Drought stress is a major abiotic factor that severely affects plant growth and food production. Identifying drought-resistant genes and their regulatory mechanisms is essential for mitigating the negative impacts of drought on plants. In this study, we identified a natural single nucleotide polymorphism (SNP) mutation in SlWRKY16 that is closely linked to drought tolerance in tomato. This SNP leads to the expression of a truncated SlWRKY16 protein. The CRISPR/Cas9 knockout of SlWRKY16, which produces this truncated SlWRKY16 protein, exhibits enhanced drought tolerance, whereas the overexpression lines demonstrate the opposite effect. Yeast two-hybrid screening demonstrated that SlWRKY16 physically interacted with CONSTANS Interacting Protein 2b (CIP2b). The CIP2b knockout mutants displayed increased sensitivity to drought stress. Importantly, this drought-sensitive phenotype was rescued in double mutants (cip2b/slwrky16). RNA-seq analysis revealed that a syntaxin gene (SlSYP121) co-expressed with both SlWRKY16 and CIP2b. Electrophoretic mobility shift assays confirmed that SlWRKY16 directly binds to the promoter of SlSYP121 and represses its expression, while the truncated SlWRKY16 protein failed to bind. Moreover, SlSYP121 acts as a positive regulator of drought tolerance. Our findings further demonstrate that the interaction between CIP2b and SlWRKY16 reduces the binding affinity of SlWRKY16 to the SlSYP121 promoter. This study identified a key SNP associated with differences in drought tolerance between wild and cultivated tomato, elucidated the regulatory function of the SlWRKY16-CIP2b-SlSYP121 module in the tomato drought response, and enhanced our understanding of the molecular mechanisms underlying plant drought resistance.},
}

*Solanum lycopersicum/genetics/physiology/metabolism
*Droughts
*Plant Proteins/genetics/metabolism
*Transcription Factors/genetics/metabolism
Gene Expression Regulation, Plant
Polymorphism, Single Nucleotide/genetics
Stress, Physiological/genetics
CRISPR-Cas Systems
Drought Resistance

@article {pmid41818620,
year = {2026},
author = {Huang, ZJ and Li, FM and Tu, YF and Feng, KK and Li, CL and Tian, SC and Hu, YS and Shao, JW and Liu, ZH},
title = {CRISPR/Cas9-Based Vanadium MXene-Free Radical Spatiotemporally Controlled Nanoreactor for Photothermal-Induced Multi-Effect Synergistic Antitumor Therapy.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {29},
pages = {e22535},
doi = {10.1002/advs.202522535},
pmid = {41818620},
issn = {2198-3844},
mesh = {*Photothermal Therapy/methods ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; Free Radicals ; Cell Line, Tumor ; *Neoplasms/therapy ; Tumor Microenvironment ; Apoptosis ; Nitrites ; Transition Elements ; },
abstract = {Photothermal therapy (PTT), a non-invasive tumor treatment, shows promise but is limited in solid tumors by restricted tissue penetration, thermotolerance, anti-apoptotic and immunosuppressive effects. In this study, tumor microenvironment-responsive nanoplatform VARH was constructed based on MXene. Under NIR-II laser irradiation, VARH achieves a high photothermal conversion efficiency of 44.21%. Loaded 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride decomposes at high temperatures to generate alkyl radicals, synergizing with hydroxyl radicals from V[4+]-catalyzed endogenous H2O2 decomposition, enabling chemodynamic therapy (CDT) and thermal dynamic therapy to enhance tumor cell oxidative damage. Triggered by high glutathione, VARH releases ribonucleoprotein (RNP) complexes to knockout heat shock protein 90 (HSP90), attenuating cellular heat resistance and promoting apoptosis. It also enhances T cell-mediated anti-tumor immunity and, with free radicals, promotes tumor cell immunogenic cell death (ICD), achieving immunotherapeutic multi-effect synergy. Integrating nanotechnology with precise gene editing, this study develops a novel multimodal synergistic therapy system, providing new insights for multi-modal treatment R&D and advancing PTT and free radical-based cancer therapies.},
}

*Photothermal Therapy/methods
Animals
Mice
*CRISPR-Cas Systems/genetics
Humans
Free Radicals
Cell Line, Tumor
*Neoplasms/therapy
Tumor Microenvironment
Apoptosis
Nitrites
Transition Elements

@article {pmid41834768,
year = {2026},
author = {Hou, Y and Wong, DCJ and Wang, L and Kang, Y and Zhou, H and Kafle, S and Liu, Y and Xu, M and Meng, L and Liang, Z and Yu, G and Wang, Q and Xin, H},
title = {A Hierarchical VvbHLH30-VvERF70-VvACS2 Module Orchestrates Ethylene Biosynthesis and Cold Adaptation in Grapevine.},
journal = {Plant biotechnology journal},
volume = {24},
number = {6},
pages = {4317-4341},
doi = {10.1111/pbi.70640},
pmid = {41834768},
issn = {1467-7652},
support = {32272691//National Natural Science Foundation of China/ ; 202403AP140029//Yunnan Provincial Key Research and Development Program/ ; },
mesh = {*Vitis/genetics/metabolism/physiology ; *Ethylenes/biosynthesis ; Gene Expression Regulation, Plant ; *Plant Proteins/metabolism/genetics ; Plant Roots/metabolism/genetics ; Cold Temperature ; Transcription Factors/metabolism/genetics ; Plant Growth Regulators/biosynthesis/metabolism ; Cold-Shock Response/genetics ; CRISPR-Cas Systems ; },
abstract = {Ethylene is a key gaseous phytohormone that plays crucial roles in regulating plant growth, development and stress responses. However, ethylene-associated biosynthetic and transcriptional regulatory mechanisms governing cold-adaptation responses in plants remain poorly understood. In this work, genome-wide analysis from grapevines (Vitis vinifera) identified nine ACS family members, of which VvACS2, VvACS4 and VvACS6 exhibited the most dynamic transcriptional responses to cold stress and were chosen for functional validation. CRISPR-Cas9-mediated knockout and overexpression experiments revealed that VvACS2 is the major contributor to ethylene biosynthesis during cold stress in grapevine roots. Screening time-course cold treatment data from Vitis vinifera and Vitis amurensis roots identified VvERF70 and VvbHLH30 as the only two TFs, among six candidates, that directly regulate VvACS2 expression. Overexpression and CRISPR-Cas9-mediated knockout of VvERF70 or VvbHLH30 in roots further confirmed their contribution to enhanced ethylene production and cold tolerance under low-temperature treatment. Furthermore, the induction of VvACS2 was greatly enhanced when VvERF70 dimerized with VvbHLH30. Notably, VvbHLH30 further positively regulates ethylene biosynthesis under cold stress by interacting with VvERF70 and binding to its promoter. Taken together, we define a hierarchical transcriptional regulatory network where the VvbHLH30-VvERF70-VvACS2 module is pivotal for ethylene biosynthesis and underpins grapevine cold tolerance. This work provides new mechanistic insights into cold adaptation mechanisms and offers novel strategies to mitigate frost damage in agricultural crops.},
}

*Vitis/genetics/metabolism/physiology
*Ethylenes/biosynthesis
Gene Expression Regulation, Plant
*Plant Proteins/metabolism/genetics
Plant Roots/metabolism/genetics
Cold Temperature
Transcription Factors/metabolism/genetics
Plant Growth Regulators/biosynthesis/metabolism
Cold-Shock Response/genetics
CRISPR-Cas Systems

@article {pmid41928521,
year = {2026},
author = {Diao, K and Duff, SMG and Li, H},
title = {An Idea to Explore: Enhancing the Teaching of Genome Editing Through 3D Printed Models of CRISPR-Cas9 Technology.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {54},
number = {3},
pages = {292-296},
doi = {10.1002/bmb.70047},
pmid = {41928521},
issn = {1539-3429},
mesh = {*Gene Editing/methods ; *Printing, Three-Dimensional ; *CRISPR-Cas Systems/genetics ; Humans ; *Teaching ; *Molecular Biology/education ; },
abstract = {Innovative biological discoveries are crucial for addressing global challenges, yet teaching these complex concepts poses significant difficulties due to the complexity of the subject matter as well as limited educational resources and methodologies. Genome editing, specifically with CRISPR-Cas9, represents a convergence of technology, molecular biology, and engineering, enabling precise manipulation of DNA sequences in various organisms. It leverages advanced tools and a deep understanding of molecular biology to target specific genes while applying engineering principles to improve editing mechanisms. However, the complexity of this field poses educational challenges due to a scarcity of accessible resources. To enhance the accessibility of genome editing to scientists and students, we propose utilizing 3D modeling and printing to create tangible models of key components in the genome editing process. By visualizing these molecular structures, our goal is to simplify and enrich the educational experience, making the intricate principles of genome editing more comprehensible and engaging for students.},
}

*Gene Editing/methods
*Printing, Three-Dimensional
*CRISPR-Cas Systems/genetics
Humans
*Teaching
*Molecular Biology/education

@article {pmid41942889,
year = {2026},
author = {Qiu, X and Yuan, L and Liu, X and Li, Y and Ma, X and Du, B and Yuan, M and Li, Z},
title = {A one-pot CRISPR/Cas12b assay for extraction-free and visual detection of Haemophilus influenzae.},
journal = {BMC microbiology},
volume = {26},
number = {1},
pages = {},
pmid = {41942889},
issn = {1471-2180},
support = {2024A102//Chinese Center for Disease Control and Prevention/ ; 2021YFC2301105//National Key Research and Development Program of China/ ; },
mesh = {*Haemophilus influenzae/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Haemophilus Infections/diagnosis/microbiology ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Sputum/microbiology ; DNA, Bacterial/genetics ; },
abstract = {Haemophilus influenzae is a major respiratory pathogen, particularly in pediatric populations, and its rapid and accurate detection is critical for early diagnosis and targeted treatment. Traditional diagnostic methods, such as bacterial culture and PCR, are often time-consuming and require specialized equipment. In this study, we developed the Hi-ExCad assay, a one-pot, extraction-free CRISPR/Cas12b-based system for the rapid, simple, and accurate detection of H. influenzae. The assay integrates loop-mediated isothermal amplification and CRISPR/Cas12b detection in a single reaction, enabling direct detection from clinical sputum samples without the need for nucleic acid extraction. The Hi-ExCad assay demonstrated high specificity, correctly identifying H. influenzae in clinical samples, with a limit of detection of 1 pg of genomic DNA. The assay also exhibited excellent performance in real-time detection and visual result interpretation under blue light, making it highly suitable for point-of-care testing and resource-limited settings. These findings suggest that the Hi-ExCad assay provides a rapid, reliable, and user-friendly method for the detection of H. influenzae, with significant potential for clinical application.},
}

*Haemophilus influenzae/genetics/isolation & purification
Humans
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Haemophilus Infections/diagnosis/microbiology
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Sputum/microbiology
DNA, Bacterial/genetics

@article {pmid41987334,
year = {2026},
author = {Kuo, CY and Harrington, S and Campo-Fernandez, B and Wyman, SK and Wu, X and Zhang, R and Espinoza, A and de Andrade Silva, BJ and Sanchez, JM and Fitz-Gibbon, S and Tseng, CH and Pellegrini, M and Bonner, M and Romero, Z},
title = {Comparative analysis of NSG and NBSGW mice for preclinical evaluation of gene-modified human hematopoietic stem and progenitor cells.},
journal = {Stem cell research & therapy},
volume = {17},
number = {1},
pages = {},
pmid = {41987334},
issn = {1757-6512},
mesh = {Animals ; Humans ; *Hematopoietic Stem Cells/metabolism/cytology ; Mice ; *Hematopoietic Stem Cell Transplantation ; CRISPR-Cas Systems/genetics ; Gene Editing ; Lentivirus/genetics ; Mice, Inbred NOD ; },
abstract = {Humanized mouse models are essential for evaluating the engraftment capacity and genetic integrity of gene-modified hematopoietic stem and progenitor cells (HSPCs). Here, we compared two widely used xenotransplantation platforms, NSG and NBSGW mice, in the context of lentiviral vector (LVV) transduction and CRISPR/Cas9-mediated gene correction. HSPCs harboring high LVV copy numbers exhibited engraftment deficits in NSG mice that were not observed in NBSGW mice. This discrepancy highlights the potential for the NBSGW model to mask safety liabilities of LVV-modified products due to its higher overall levels of human chimerism. In contrast, CRISPR/Cas9 editing with a single-stranded oligodeoxynucleotide donor yielded comparable correction rates in both models, even across decreasing input cell doses, demonstrating that long-term repopulating hematopoietic stem cells (HSCs) retain equivalent engraftment capacity in each strain. Single-cell RNA-sequencing revealed distinct progenitor populations that were markedly under-represented in the NSG model but preserved in NBSGW recipients, emphasizing the greater capacity of NBSGW mice to better support multilineage human hematopoiesis. Together, these findings establish that both NSG and NBSGW mice are suitable for assessing long-term engraftment and gene modification outcomes in human HSPCs. However, the significantly higher percentage of human cell chimerism in the NBSGW model may obscure cell populations with engraftment deficits. Careful selection of in vivo models is therefore critical for rigorous preclinical evaluation of gene therapy products prior to clinical translation.},
}

Animals
Humans
*Hematopoietic Stem Cells/metabolism/cytology
Mice
*Hematopoietic Stem Cell Transplantation
CRISPR-Cas Systems/genetics
Gene Editing
Lentivirus/genetics
Mice, Inbred NOD

@article {pmid42167149,
year = {2026},
author = {Buchholz, F},
title = {INSTALLing recombinase-driven genome writing: Engineered single-stranded donors bypass innate immunity to enable large DNA integration.},
journal = {Molecular cell},
volume = {86},
number = {10},
pages = {1836-1838},
doi = {10.1016/j.molcel.2026.04.018},
pmid = {42167149},
issn = {1097-4164},
mesh = {*Immunity, Innate/genetics ; *DNA, Single-Stranded/genetics/immunology ; Humans ; *Gene Editing/methods ; *Recombinases/genetics/metabolism ; CRISPR-Cas Systems ; *DNA, Circular/genetics/immunology ; },
abstract = {In a recent Nature article, Tou et al.[1] introduce INSTALL, a strategy that couples immune-evasive circular single-stranded DNA (cssDNA) technology with recombinase-based genome editing to overcome innate immune barriers and enable the integration of large DNA cargos.},
}

*Immunity, Innate/genetics
*DNA, Single-Stranded/genetics/immunology
Humans
*Gene Editing/methods
*Recombinases/genetics/metabolism
CRISPR-Cas Systems
*DNA, Circular/genetics/immunology

@article {pmid42167165,
year = {2026},
author = {Weerasinghe, PR and Tsugama, D},
title = {Diverse novel RNA polymerase III promoters and dual-activity promoters identified through motif and transcriptomic analyses across multiple plant species.},
journal = {Biochemical and biophysical research communications},
volume = {825},
number = {},
pages = {153936},
doi = {10.1016/j.bbrc.2026.153936},
pmid = {42167165},
issn = {1090-2104},
abstract = {RNA polymerase III (Pol III) promoters are essential tools for driving small RNAs such as guide RNAs and CRISPR RNAs (crRNAs) in CRISPR-Cas systems, yet their diversity and regulatory potential in major crops remain largely uncharacterized. In this study, we conducted a systematic, promoter motif-based scan for potential Pol III-transcribed genes across nine plant species including eight major crops. We identified 824 non-coding RNA (ncRNA) genes across 14 categories (consisting of 13 ncRNA families and 'Others') as Pol III-transcribed candidates; these included traditional Pol III-transcribed small nuclear RNA (snRNA) families and the families conventionally regarded as Pol II-transcribed. Small RNA sequencing revealed expression variability within families and across species, highlighting the importance of species-specific promoter selection. We also identified several rice ncRNA promoters with potential dual Pol II/Pol III activity and tested them for simultaneously expressing both Cas nucleases (AsCas12f-HKRA and Cas12j-8) and their crRNAs. While gene editing efficiencies in this single-promoter-driven architecture were low, the appearance of GFP signals in reporter assays confirms the functional feasibility of dual-polymerase-mediated transcription in plants. Our findings provide a roadmap for Pol III transcriptional diversity in crops and offer a simplified integrated promoter architecture for the development of compact genome-editing toolsets.},
}

@article {pmid42169298,
year = {2026},
author = {Zhao, G and Wei, J and Li, Y and Zhu, C and Tang, J and Gong, J and Huang, Y},
title = {Quantitative monitoring of Bacillus licheniformis during fermentation using PCR-CRISPR/Cas12a.},
journal = {Food research international (Ottawa, Ont.)},
volume = {237},
number = {},
pages = {119334},
doi = {10.1016/j.foodres.2026.119334},
pmid = {42169298},
issn = {1873-7145},
mesh = {*Fermentation ; *Bacillus licheniformis/genetics/isolation & purification/metabolism ; *CRISPR-Cas Systems ; *Real-Time Polymerase Chain Reaction/methods ; *Food Microbiology/methods ; Limit of Detection ; DNA, Bacterial/genetics ; *Polymerase Chain Reaction/methods ; },
abstract = {Bacillus licheniformis is a key functional bacterium in Baijiu fermentation and plays an essential role in the formation of flavor compounds, making its quantitative detection crucial. This study developed a CRISPR/Cas12a-based microbial nucleic acid quantification (CRMNQ) method to quantify B. licheniformis in Baijiu fermentation. This method combines efficient PCR amplification with highly specific CRISPR/Cas12a recognition, enabling quantitative analysis through end-point fluorescence measurement without the need for a quantitative real-time PCR instrument. Under optimized conditions, a limit of detection (LOD) of 72.3 copies/μL was achieved with high specificity. The method was successfully applied to monitor the dynamics of B. licheniformis in both simulated and in situ samples of Baijiu fermentation, with higher robustness and anti-interference ability compared with the qPCR method. In summary, the method provides an effective and practical tool for highly sensitive and specific detection of B. licheniformis in Baijiu fermentation, facilitating process monitoring and quality control during fermentation.},
}

*Fermentation
*Bacillus licheniformis/genetics/isolation & purification/metabolism
*CRISPR-Cas Systems
*Real-Time Polymerase Chain Reaction/methods
*Food Microbiology/methods
Limit of Detection
DNA, Bacterial/genetics
*Polymerase Chain Reaction/methods

@article {pmid42169395,
year = {2026},
author = {van Roosmalen, RN and Murphy, CD and Sweeney, JB},
title = {A Stereospecific Lactic Acid Exclusion Biosensor for Grass Silage Fed Green Biorefinery.},
journal = {Microbial biotechnology},
volume = {19},
number = {5},
pages = {e70374},
doi = {10.1111/1751-7915.70374},
pmid = {42169395},
issn = {1751-7915},
support = {//European Climate, Enviornment and Infrastructure Executive Agency (CINEA) LIFE Programme/ ; 860477//European Union's Horizon 2020 Research and Innovation Programme AgRefine/ ; LIFE18CCM/IE/001195//Government of Ireland Department of Climate, Energy and the Environment (DCEE)/ ; },
mesh = {*Silage/analysis/microbiology ; *Lactic Acid/analysis/metabolism ; *Biosensing Techniques/methods ; *Escherichia coli/genetics/metabolism ; *Poaceae/metabolism/chemistry ; Gene Knockout Techniques ; Gene Deletion ; Lactate Dehydrogenases ; },
abstract = {Grass silage fed green biorefineries require high quality grass silage leachates to produce high quality products. Real-time, cost-effective methods to monitor key leachate analytes are needed for small-scale decentralised systems. In this study, Escherichia coli mutants, designated JSP0090 and JSP0094, were created to quantify d- and l-lactic acid concentrations in grass leachate samples using an oxygen probe. The genes encoding either d- or l-lactate dehydrogenase were expressed in an exclusion biosensor strain (JSK0115). This strain required successive gene deletions to prevent interference from sugars, amino acids and organic acids present in grass silage leachates. This strain was incapable of catabolising d-lactic acid, l-lactic acid, acetic acid, propionic acid, formic acid, ethanol, glucose, fructose, l-alanine, VFAs, glycerol, mannitol and succinate. Gene knockouts were achieved using P1 phage lysates and CRISPR Cas 9 methods to target key steps in the catabolism of these compounds, with the exception of succinate. For this metabolite, the di-carboxylic acid transporters YaaH, DctA, YchM, DcuA and DcuB had to be deleted. The effectiveness of the biosensors for selectively measuring d- and l-lactic acid was assessed in Austrian and Irish grass silage leachate. The concentrations measured were comparable to those obtained using a commercial enzyme kit.},
}

*Silage/analysis/microbiology
*Lactic Acid/analysis/metabolism
*Biosensing Techniques/methods
*Escherichia coli/genetics/metabolism
*Poaceae/metabolism/chemistry
Gene Knockout Techniques
Gene Deletion
Lactate Dehydrogenases

@article {pmid42170179,
year = {2026},
author = {Karagyaur, M and Averina, O and Bozov, K and Dzhauari, S and Priymak, A and Khaybullina, R and Permyakov, O and Popov, V and Grigorieva, O and Illarionova, M and Shkarina, L and Gulyaev, M and Lebedev, D and Primak, A and Sergiev, P and Semina, E and Klimovich, P and Samokhodskaya, L and Malkov, P and Pirogov, Y and Tsygankov, B and Chaika, Y and Tkachuk, V and Neyfeld, E},
title = {Novel mouse line with D277N mutation in the Plau gene displays autism spectrum disorder-like traits.},
journal = {Frontiers in cell and developmental biology},
volume = {14},
number = {},
pages = {1762737},
pmid = {42170179},
issn = {2296-634X},
abstract = {INTRODUCTION: Genetic technologies provide an opportunity to study the molecular basis of a wide range of hereditary pathologies, including mental disorders. Reproducing of potentially pathogenic genomic variants in cellular and animal models allows establishing their functional significance and possible mechanisms of involvement in the pathogenesis of certain disorders.

METHODS: In this study, a genetic variant of urokinase type plasminogen activator (uPA, gene Plau) was modeled in mice using CRISPR/Cas genome editing tool, enabling a better understanding of the role of this molecule and its associated pathways in brain development. The protease uPA plays an important role in the directed migration of neural progenitors, glial, endothelial and immune cells, it participates in axon guidance and maturation of synaptic connections, activation of growth factors and degradation of the extracellular matrix. To study the contribution of the catalytic function of uPA to brain development, we have created for the first time a mouse line carrying the D277N (rs1243306395) mutation. We assessed social activity, anxiety, memory, problem-solving ability and stress resistance of these mice, as well as histological features of their brains.

RESULTS: Timely and correct functioning of the Plau gene ensures adequate positioning of crucial cellular components in the developing nervous system. According to bioinformatic calculations, the D277N (corresponds to the human single nucleotide variant rs1243306395) substitution that happens due to C-to-T mutation in the murine Plau gene may impair the catalytic activity of the uPA protein. While retaining their ability to find solutions in the escape test, this mouse line is characterized by high levels of anxiety, impaired social behavior, slowed learning dynamics (spatial memory), and impaired adaptation to stressors. This behavioral pattern can potentially be interpreted as autism spectrum disorder Histological analysis of the brain and cerebral cortex in Plau-D277N mice revealed brain volume enlargement and cortical thickening of approximately 10-15% compared to wild-type mice.

DISCUSSION: In this study, we draw attention for the first time to the genomic variant rs1243306395 in the Plau gene as a potential cause of autism spectrum disorder and propose the genetically modified Plau-D277N mouse line as a model object for studying the pathogenesis of this disorder. These models can also be used for the development and testing of promising therapeutic approaches and pharmacological agents.},
}

@article {pmid42171807,
year = {2026},
author = {Li, Z and Guo, H and Yang, Y and Cheng, Q and Dao, L and Liu, J and Ding, P and Wu, P},
title = {An Fe single-atom nanozyme-sensitized RPA-CRISPR/Cas12a biosensor for the early detection of potential microcystins-producing cyanobacteria.},
journal = {Mikrochimica acta},
volume = {193},
number = {6},
pages = {},
pmid = {42171807},
issn = {1436-5073},
support = {2025JJ60624//Natural Science Foundation of Hunan Province/ ; 82504465//National Natural Science Foundation of China/ ; 502044013//Start-up Funds from Central South University/ ; },
mesh = {*Biosensing Techniques/methods ; *Microcystins/analysis/biosynthesis ; *Cyanobacteria/metabolism/genetics/isolation & purification ; *CRISPR-Cas Systems ; Limit of Detection ; *Iron/chemistry ; Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Lakes/microbiology ; },
abstract = {Harmful cyanobacteria are capable of producing hepatotoxic microcystins (MCs), which pose a significant risk to both aquatic ecosystems and public health. Since cyanobacterial strains that produce MCs harbor the mcy gene cluster, monitoring and analyzing the levels of the mcy gene is critical for identifying potential MCs-producing strains and assessing periods of elevated risk. In this study, Fe single-atom nanozymes (Fe SANs) with enhanced peroxidase-like (POD) activity were synthesized, which were then used to form a Fe SANs-DNA-magnetic bead complex (Fe SANs-DNA-MB). By integrating pre-amplification via recombinase polymerase amplification (RPA), trans-cleavage by CRISPR/Cas12a, and catalytic activity by Fe SANs, a novel triple signal amplification biosensor was developed for the detection of the microcystin synthase gene E (mcyE), a critical biomarker for MCs-producing cyanobacteria. The biosensor demonstrated significantly improved analytical performance, achieving a broad dynamic rangefor mcyE detection from 0.1 pM to 20 nM (R2 = 0.99), with a low limit of detection (LOD) of 0.05 pM. Furthermore, the biosensor provides high accuracy, as evidenced by spiked recoveries of mcyE in natural lake water samples ranging from 97.93% to 103.3%. It also enabled the selective identification of the mcyE gene in toxin-producing cyanobacterial strains (FACHB-905 and FACHB-979), thereby effectively distinguishing between toxin-producing and non-toxin-producing cyanobacteria. This innovative approach advances the field of molecular biosensing by providing a highly sensitive platform for the quantitative detection of the mcyE gene, facilitating accurate monitoring of MCs-producing cyanobacteria in environmental contexts. The method holds considerable promise for enhancing water quality management and protecting public health.},
}

*Biosensing Techniques/methods
*Microcystins/analysis/biosynthesis
*Cyanobacteria/metabolism/genetics/isolation & purification
*CRISPR-Cas Systems
Limit of Detection
*Iron/chemistry
Nucleic Acid Amplification Techniques/methods
Bacterial Proteins/genetics
Lakes/microbiology

@article {pmid40962493,
year = {2026},
author = {Ferreira, A},
title = {The future of reproduction in Eve Smith's Off Target and Kira Peikoff's Baby X.},
journal = {Medical humanities},
volume = {52},
number = {2},
pages = {190-198},
doi = {10.1136/medhum-2025-013321},
pmid = {40962493},
issn = {1473-4265},
mesh = {Humans ; *Gene Editing/ethics ; Germ Cells ; *Gametogenesis ; *Reproduction ; *Reproductive Techniques, Assisted/ethics ; CRISPR-Cas Systems ; *Genetic Engineering/ethics ; Genome, Human ; },
abstract = {This essay analyses two novels that explore reproductive scenarios which may be implemented in the near future: human germline genome editing and in vitro gametogenesis (IVG), dramatised in Eve Smith's Off-Target (2022) and Kira Peikoff's Baby X (2024), respectively.Smith's Off Target considers the plethora of medical, ethical, social and familial implications of germline editing, which is used to modify an embryo's genome and enhance it with traits deemed optimal that will be passed on to future generations, in this way effectively creating designer babies and radically interfering with the laws of evolution and genetic inheritance. The novel deals with the potential unintended consequences of genetic engineering, in particular CRISPR/Cas9. Genetic edits, if insufficiently tested, can have off-target effects, alluded to in the name of the novel. Peikoff's Baby X takes the premise of Off Target a step further by introducing IVG, exploring the multiple ramifications of this as yet undeveloped reproductive technology which would radically change the reproductive landscape as we know it.Meanwhile, the science that will enable germline editing and IVG is advancing rapidly, making it imperative to consider the far-reaching repercussions of these technologies, not only in medical terms but specifically with regard to ethical implications for families and society in general. These novels are powerful reminders that the debate about the future applications of genome editing and IVG needs to happen now. The issues raised by the novels will be analysed mainly through a gender studies and bioethics lens.},
}

Humans
*Gene Editing/ethics
Germ Cells
*Gametogenesis
*Reproduction
*Reproductive Techniques, Assisted/ethics
CRISPR-Cas Systems
*Genetic Engineering/ethics
Genome, Human

@article {pmid40973816,
year = {2026},
author = {Janjuha, S and Haenggi, T and Chamberlain, TC and Rothgangl, T and Kissling, L and Wilhelm, M and Mathis, N and Boeck, D and Marquart, K and Ioannidi, E and Moon, WJ and Muramatsu, H and Vadovics, M and Pardi, N and Semple, SC and Tam, YK and Schwank, G},
title = {Spatial profiling of gene editing by in situ sequencing in mice and macaques.},
journal = {Nature biomedical engineering},
volume = {10},
number = {5},
pages = {968-979},
pmid = {40973816},
issn = {2157-846X},
support = {185293/SNSF_/Swiss National Science Foundation/Switzerland ; P01 AI158571/AI/NIAID NIH HHS/United States ; P01 AI172531/AI/NIAID NIH HHS/United States ; R01 AI153064/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Liver/metabolism ; Nanoparticles/chemistry ; Brain/metabolism ; Macaca ; RNA, Messenger/genetics ; Dependovirus/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; Male ; CRISPR-Cas Systems ; Lipids/chemistry ; },
abstract = {Base and prime editing technologies precisely install defined nucleotide edits in both dividing and non-dividing cells, offering potential for correcting pathogenic mutations directly in organisms. However, to fully leverage their therapeutic potential, accurately measuring editing rates with high spatial resolution is crucial. Here we use imaging-based in situ sequencing (ISS) to map base and prime editing events within native tissues. We establish and validate this technology in mouse brains treated with intein-split adenine base editors or prime editors delivered via adeno-associated viral vectors. We next apply ISS in the liver of mice and macaques treated with adenine base editors encoded on lipid nanoparticle-encapsulated mRNA and guide RNA (RNA-LNP). Effective editing was observed across all metabolic zones of liver lobules. Moreover, in experiments where repeated doses of RNA-LNP are administered, the initial dose does not affect the editing efficiency and distribution of the subsequent dose. Our results demonstrate how ISS can visualize gene editing events in vivo and suggest that base editor delivery using RNA-LNP could be used to address a wide spectrum of metabolic liver diseases.},
}

Animals
*Gene Editing/methods
Mice
Liver/metabolism
Nanoparticles/chemistry
Brain/metabolism
Macaca
RNA, Messenger/genetics
Dependovirus/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Mice, Inbred C57BL
Male
CRISPR-Cas Systems
Lipids/chemistry

@article {pmid40983646,
year = {2026},
author = {Siddiqui, SM and Welch, NL and Nguyen, TG and Razmi, A and Chang, T and Senft, R and Arizti-Sanz, J and Mirhashemi, ME and Stirling, DR and Ackerman, CM and Cimini, BA and Blainey, PC and Sabeti, PC and Myhrvold, C},
title = {Bead-based approaches for increased sensitivity and multiplexing of CRISPR diagnostics.},
journal = {Nature biomedical engineering},
volume = {10},
number = {5},
pages = {939-951},
pmid = {40983646},
issn = {2157-846X},
support = {D18AC00006//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; 75D30122C15113//U.S. Department of Health & Human Services | Centers for Disease Control and Prevention (CDC)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Sensitivity and Specificity ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genes, Reporter ; },
abstract = {CRISPR-based diagnostics have emerged as a promising tool for fast, accurate and portable pathogen detection. There has been rapid progress in pre-amplification processes and CRISPR-related enzymes used in these approaches, but the development of reporter systems and reaction platforms has lagged behind. In this paper, we develop bead-based techniques to address these gaps. First, we develop a novel bead-based split-luciferase reporter system with up to 20× sensitivity compared with standard fluorescence-based reporter design in CRISPR diagnostics. Second, we develop a highly deployable, bead-based platform capable of detecting nine distinct viral targets in parallelized, droplet-based reactions, with sensitivity reaching as low as 2.5 copies per µl of input RNA. We demonstrate the enhanced performance of both approaches on synthetic and clinical sample sensitivity, speed, multiplexing and deployability.},
}

*CRISPR-Cas Systems/genetics
Humans
Sensitivity and Specificity
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genes, Reporter

@article {pmid41904145,
year = {2026},
author = {Zhao, DS and Wang, S and Xu, YC and Chen, YT and Farooq, MA and Lin, S and Cao, P and Li, J and Hu, ZW and Li, N and Scarsini, M and Si, C and Li, SM and Yan, X and Pang, Q and Bowler, C and Zou, HX},
title = {Tryptophanol enhances nitrogen assimilation in marine diatoms.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41904145},
issn = {2041-1723},
support = {LY23D060001//Natural Science Foundation of Zhejiang Province (Zhejiang Provincial Natural Science Foundation)/ ; 31670402//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Diatoms/metabolism/genetics/growth & development ; *Nitrogen/metabolism ; *Tryptophan/metabolism/analogs & derivatives ; Peptide Synthases/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Diatoms exhibit high competitive capacity in nitrogen assimilation, but the underlying mechanisms remain unclear. Here, we identify a non-ribosomal peptide synthase-like gene (PtNRPS1) with an atypical domain structure (A-T-R1-R2) in the marine diatom Phaeodactylum tricornutum, crucial for short-term nitrogen assimilation. In vitro enzyme assays show PtNRPS1 catalyzes conversion of L-tryptophan to tryptophanol, a tryptophan-derived indole compound that promotes diatom growth at concentrations far lower than indole-3-acetic acid. Transcriptomic, metabolomic analyses, and stable-isotope analyses indicate tryptophanol enhances short-term nitrogen assimilation. CRISPR-Cas9 knockout of PtNRPS1 abolishes tryptophanol biosynthesis and reduces nitrogen-assimilation enzymes activities, which are restored by exogenous tryptophanol. PtNRPS1 overexpression results in delayed but sustained enzyme elevation. Global distribution of PtNRPS1 homologues in stramenopiles positively correlates with nitrogen-assimilation gene abundance. Our findings suggest tryptophanol, synthesized by a diatom NRPS, accelerates nitrogen assimilation, providing a competitive edge in oceanic nitrogen acquisition.},
}

*Diatoms/metabolism/genetics/growth & development
*Nitrogen/metabolism
*Tryptophan/metabolism/analogs & derivatives
Peptide Synthases/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41920952,
year = {2026},
author = {Nicolia, A and Cuccurullo, A and Tamada, K and Yoneyama, K and Rambla, JL and Granell, A and Camerlengo, F and Festa, G and Francese, G and Contaldi, F and D'Alessandro, A and Rigano, MM and Principio, L and D'Agostino, N and Cardi, T},
title = {Editing strigolactone biosynthesis genes in tomato reveals novel phenotypic effects and highlights D27 as a breeding target for parasitic weed resistance.},
journal = {Plant & cell physiology},
volume = {67},
number = {4},
pages = {667-684},
doi = {10.1093/pcp/pcag042},
pmid = {41920952},
issn = {1471-9053},
support = {DM 15924//Italian Ministry of Agriculture, Food Sovereignty and Forests/ ; 18-05-2018//Italian Ministry of Agriculture, Food Sovereignty and Forests/ ; IJC2020-045612-I//Spanish Ministry of Science and Innovation/ ; PID2022-141438OB-I00//Spanish Ministry/ ; 101000716//EU for Harnesstom/ ; JPMJFR220F//Japan Science and Technology Agency/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/parasitology ; *Lactones/metabolism ; *Plant Weeds/physiology ; *Plant Proteins/genetics/metabolism ; *Gene Editing ; Phenotype ; Germination ; Plant Breeding ; CRISPR-Cas Systems ; Seeds ; Plant Growth Regulators/metabolism/biosynthesis ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Genes, Plant ; *Heterocyclic Compounds, 3-Ring/metabolism ; },
abstract = {Parasitic weed infestations represent an increasing threat to agriculture worldwide, especially in the Mediterranean region. Phelipanche ramosa (L.) and Phelipanche aegyptiaca (Pers.) (broomrapes) cause severe yield losses in field-grown tomato (Solanum lycopersicum L.). Strigolactones (SLs) are apocarotenoid phytohormones that not only play a critical role in plant physiology and development but also act as the primary germination signals for parasitic weed seeds. In this study, we generated CRISPR/Cas9 tomato knock-out (KO) lines targeting the SlD27 gene and three other key genes involved in SL biosynthesis (SlCCD7, SlCCD8, and SlMAX1), all in the same genetic background. All the edited lines exhibited undetectable SL levels in root exudates, leading to a strong reduction in the in vitro germination of Phelipanche spp. seeds. Consistently, reduced parasitism was also observed in vivo when Sld27 lines were tested. A comprehensive evaluation of morphological, reproductive, and fruit-related traits revealed gene-specific phenotypic effects, including changes in vegetative growth, fruit set, fruit development, and volatilome. Specifically, KO of two carotenoid cleavage dioxygenases and SlMAX1 affected shoot architecture, fruit development, and the production of volatile organic compounds during fruit ripening. In contrast, the newly developed Sld27 lines in this study displayed a mild phenotype generally comparable to nonedited control plants and likely due to the expression of SlD27 paralogues. Overall, our results indicate that SlD27 represents a promising breeding target for enhancing resistance to parasitic weeds in tomato while minimizing negative impacts on plant development and fruit quality.},
}

*Solanum lycopersicum/genetics/metabolism/parasitology
*Lactones/metabolism
*Plant Weeds/physiology
*Plant Proteins/genetics/metabolism
*Gene Editing
Phenotype
Germination
Plant Breeding
CRISPR-Cas Systems
Seeds
Plant Growth Regulators/metabolism/biosynthesis
Gene Expression Regulation, Plant
Gene Knockout Techniques
Genes, Plant
*Heterocyclic Compounds, 3-Ring/metabolism

@article {pmid41997156,
year = {2026},
author = {Peng, J and Chan, DCT and Chu, HY and Fong, JHC and Lam, YK and Cheung, MSH and Leung, W and Choi, GCG and Wong, ASL},
title = {Rapid customization of base editors via machine learning-powered combinatorial mutagenesis.},
journal = {Molecular cell},
volume = {86},
number = {10},
pages = {1839-1855.e10},
doi = {10.1016/j.molcel.2026.03.030},
pmid = {41997156},
issn = {1097-4164},
mesh = {Humans ; *Machine Learning ; *Gene Editing/methods ; *Mutagenesis ; Mutation ; *DNA/genetics/metabolism ; CRISPR-Cas Systems ; Deep Learning ; Nucleotide Motifs ; Cytosine/metabolism ; },
abstract = {Being able to control the complementarity and hindrance between target DNA and base editor proteins enables precise, bystander-free editing. Here, we combined combinatorial mutagenesis with machine learning to analyze and engineer these interactions at scale. By profiling DNA motif preferences across 160,000 evoAPOBEC1 and 64 million TadA variants in human cells, we used as little as 0.004% of the mutational landscape to make predictions. This identified variants with motif-specific activity and eliminated residual adenine editing in cytosine base editors. In correcting >800 disease-associated mutations, our variants outperformed previous versions in precluding unintended edits at purine motifs, achieving undetectable bystander edits in 50% of cases. Additionally, a pre-trained, structure-based deep learning model predicted functional TadA variants with 63% success across 20[26] variants spanning 26 amino acid sites, without experimental data and in a single prediction round. These approaches streamline the re-engineering of base editors for enhanced precision tailored to specific targets.},
}

Humans
*Machine Learning
*Gene Editing/methods
*Mutagenesis
Mutation
*DNA/genetics/metabolism
CRISPR-Cas Systems
Deep Learning
Nucleotide Motifs
Cytosine/metabolism

@article {pmid42017790,
year = {2026},
author = {Gopinath, A and Shen, L and Ouellette, SP},
title = {CRISPRi and beyond: studying essential gene function in the obligate intracellular bacterium Chlamydia trachomatis.},
journal = {Journal of bacteriology},
volume = {208},
number = {5},
pages = {e0005926},
pmid = {42017790},
issn = {1098-5530},
support = {R35 GM151971/GM/NIGMS NIH HHS/United States ; R21AI175651/NH/NIH HHS/United States ; R35GM151971/NH/NIH HHS/United States ; },
mesh = {*Chlamydia trachomatis/genetics/metabolism ; *Genes, Essential ; Gene Expression Regulation, Bacterial ; *CRISPR-Cas Systems ; Bacterial Proteins/genetics/metabolism ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Chlamydia Infections/microbiology ; },
abstract = {Chlamydia trachomatis is an obligate intracellular bacterium that is the leading cause of bacterial sexually transmitted infections (STIs) and preventable infectious blindness. Its unique biphasic developmental cycle comprises an infectious but non-dividing elementary body and a replicative but non-infectious reticulate body. C. trachomatis possesses a reduced genome where more than half of the open reading frames (ORFs) are predicted to code for essential genes, abrogation of which with traditional chromosomal disruption methods is expected to block bacterial growth and developmental cycle progression. However, understanding the function of such genes is critical to expand our knowledge of chlamydial biology and reveal new therapeutic targets. This review aims to compare and contrast four systems developed in the past 5 years for studying essential genes in Chlamydia. These include systems to conditionally knock down or knockout a target gene product using CRISPR interference (CRISPRi), inducible small RNAs (sRNA), fluorescence-reported allelic exchange mutagenesis (FRAEM) with inducible complementation of the target gene, and dependence on plasmid expression (DOPE).},
}

*Chlamydia trachomatis/genetics/metabolism
*Genes, Essential
Gene Expression Regulation, Bacterial
*CRISPR-Cas Systems
Bacterial Proteins/genetics/metabolism
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats
Chlamydia Infections/microbiology

@article {pmid42070443,
year = {2026},
author = {Awais, M and Chen, Y and Bibi, S and Wu, J and Zhang, W and Tlili, I and Hu, J},
title = {Breaking boundaries for PFAS surveillance in water reservoirs: CRISPR-electrochemical synergies from MXene to microfluidics- A review.},
journal = {Biosensors & bioelectronics},
volume = {307},
number = {},
pages = {118756},
doi = {10.1016/j.bios.2026.118756},
pmid = {42070443},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods/instrumentation ; CRISPR-Cas Systems ; *Water Pollutants, Chemical/analysis/isolation & purification ; *Fluorocarbons/analysis/isolation & purification ; Lab-On-A-Chip Devices ; Environmental Monitoring/methods ; Electrochemical Techniques/methods ; Nitrites ; Transition Elements ; },
abstract = {The hydro-spherical contamination of water resources by per- and polyfluoroalkyl substances (PFAS) demands analytical technologies that transcend the limitations of current methods, which struggle to simultaneously achieve ultra-sensitivity, specificity, portability, and low cost. Therefore, this review advances next-generation PFAS surveillance by proposing a framework built on the synergistic convergence of molecularly engineered sulfonate-MXenes, CRISPR-Cas12a, and microfluidic automation. The manuscript critically analyzes how sulfonate-terminated Ti3C2Tx MXenes achieve picomolar affinity and rapid preconcentration of PFAS through biomimetic binding architectures. It also details the mechanism by which CRISPR-Cas12a, guided by PFAS-specific aptamers, enables single-molecule discrimination with attomolar sensitivity. Finally, this review paper demonstrates how microfluidic networks orchestrate this synergy, miniaturizing the entire assay into a portable, multiplexed platform that reduces analysis time from hours to minutes. Synergistically unifying breakthroughs in nanomaterials, synthetic biology, and lab-on-a-chip design, this work provides both a methodological blueprint for ultrasensitive PFAS sensors and a relevant roadmap for implementing proactive, decentralized water quality monitoring.},
}

*Biosensing Techniques/methods/instrumentation
CRISPR-Cas Systems
*Water Pollutants, Chemical/analysis/isolation & purification
*Fluorocarbons/analysis/isolation & purification
Lab-On-A-Chip Devices
Environmental Monitoring/methods
Electrochemical Techniques/methods
Nitrites
Transition Elements

@article {pmid42090813,
year = {2026},
author = {Hyeon, LS and Yang, JW and Bae, S and Yoo, JS and Kim, E and Lee, SS and Kang, S},
title = {Rapid and sensitive detection of blaKPC and blaNDM in carbapenemase-producing Enterobacterales using a one-pot recombinase polymerase amplification-CRISPR/Cas12a system with modified protospacer adjacent motifs.},
journal = {Biosensors & bioelectronics},
volume = {307},
number = {},
pages = {118725},
doi = {10.1016/j.bios.2026.118725},
pmid = {42090813},
issn = {1873-4235},
mesh = {*beta-Lactamases/genetics/isolation & purification ; *Bacterial Proteins/genetics/isolation & purification ; Humans ; *Biosensing Techniques ; CRISPR-Cas Systems/genetics ; *Enterobacteriaceae/genetics/enzymology/isolation & purification ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Point-of-Care Systems ; *Carbapenem-Resistant Enterobacteriaceae/genetics/isolation & purification ; },
abstract = {Carbapenem-resistant Enterobacterales pose a critical global health threat because of their rapid transmission and resistance to last-line antibiotics. To address this threat, rapid and user-friendly point-of-care (POC) detection is essential. Quantitative polymerase chain reaction (qPCR), while providing high sensitivity, relies on expensive equipment and skilled personnel, hindering its practical use in resource-limited settings. Here, we aimed to develop an optimized one-pot RPA-CRISPR/Cas12a (RCCS) system with enhanced diagnostic performance to provide a reliable and user-friendly platform for rapid carbapenemase gene screening in both clinical and resource-limited settings. The developed assay was used for the detection of blaKPC and blaNDM genes and was integrated into a portable diagnostic device. By using suboptimal protospacer adjacent motif sequences, the assay provided a streamlined workflow and substantially enhanced detection sensitivity. This platform achieved a limit of detection of 10[-17] M for blaKPC and 10[-16] M for blaNDM within 30 min. Validation with 44 clinical samples demonstrated that the assay had 100% sensitivity and specificity, matching the effectiveness of qPCR. The one-pot RCCS platform offers a robust and highly sensitive POC solution for on-site testing.},
}

*beta-Lactamases/genetics/isolation & purification
*Bacterial Proteins/genetics/isolation & purification
Humans
*Biosensing Techniques
CRISPR-Cas Systems/genetics
*Enterobacteriaceae/genetics/enzymology/isolation & purification
Nucleic Acid Amplification Techniques
Limit of Detection
Point-of-Care Systems
*Carbapenem-Resistant Enterobacteriaceae/genetics/isolation & purification

@article {pmid42106044,
year = {2026},
author = {Dutta, TK and Ray, S and Akhil, VS and Rupinikrishna, K and Chauhan, U and Dutta, A and Vijayan, J and Iquebal, MA and Chinnusamy, V},
title = {A synthetic guide RNA scaffold enhanced CRISPR/Cas9 editing efficiency in plants across multiple gene targets.},
journal = {International journal of biological macromolecules},
volume = {365},
number = {},
pages = {152456},
doi = {10.1016/j.ijbiomac.2026.152456},
pmid = {42106044},
issn = {1879-0003},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Arabidopsis/genetics ; Oryza/genetics ; *Plants/genetics ; },
abstract = {CRISPR/Cas9 mediated genome editing is a highly powerful and versatile tool for accelerating crop improvement. The editing efficiency of CRISPR/Cas9 system in planta has been highly variable owing to the variable binding affinity between native CRISPR RNA and Cas9 protein in vivo. In plant systems, systematic, large-scale engineering and benchmarking of guide RNA (gRNA) scaffold variants is still relatively limited compared with work in mammalian systems, despite several important studies demonstrating that scaffold and expression-cassette engineering can substantially improve CRISPR/Cas9 efficacy. Current study addresses the limitations of commonly used gRNA scaffold architecture by incorporating a stabilized stem-loop RAR (tetra loop) extension and a transcription-termination site mutation, resulting in improved RNA folding, increased Cas9 binding affinity, and enhanced in vivo editing outcomes. The synthesized scaffold boosted CRISPR/Cas9 efficiency in monocot or dicot plants across the 19 diverse target sites in Arabidopsis, rice and tomato. Furthermore, the synthetic scaffold is compatible with multiplex genome editing architectures, including polycistronic tRNA-gRNA (PTG) expression systems, enabling efficient simultaneous targeting of multiple genomic loci. The findings of this study have broad applications in precision plant breeding, functional genomics, and agricultural biotechnology, facilitating reliable gene modification across diverse plant species and transformation platforms.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
Arabidopsis/genetics
Oryza/genetics
*Plants/genetics

@article {pmid42120728,
year = {2026},
author = {DiPersio, JF and Koehne, G and Shah, NN and Bernard, L and Suh, HC and Koura, D and Tamari, R and Mushtaq, MU and Maakaron, J and Rimando, J and Kennedy, VE and Patel, SS and Hudson, C and Loken, MR and Slapak, CA and Lloyd, DM and Stanizzi, DA and Lee-Sundlov, MM and Thosar, S and Mundelboim, G and Guo, G and Ge, HG and Li, BE and Xavier-Ferrucio, J and Hyzy, SL and Lin, MI and Raffel, GD and Cooper, BW},
title = {CRISPR-Cas9 CD33-deleted allogeneic hematopoietic cell transplantation with gemtuzumab ozogamicin maintenance in AML: a phase 1/2 trial.},
journal = {Nature medicine},
volume = {32},
number = {5},
pages = {1763-1772},
pmid = {42120728},
issn = {1546-170X},
mesh = {Humans ; *Hematopoietic Stem Cell Transplantation/methods/adverse effects ; *Gemtuzumab/therapeutic use/administration & dosage/adverse effects ; *Leukemia, Myeloid, Acute/therapy/genetics/drug therapy ; *Sialic Acid Binding Ig-like Lectin 3/genetics ; Middle Aged ; Female ; Male ; Adult ; Aged ; *CRISPR-Cas Systems/genetics ; Transplantation, Homologous ; Graft vs Host Disease ; Myelodysplastic Syndromes/therapy/genetics ; },
abstract = {Patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are likely to relapse despite allogenic hematopoietic cell transplantation (HCT). Post-HCT preventative maintenance can be limited by toxicity toward the normal donor cells. Tremtelectogene empogeditemcel (trem-cel) is a CRISPR-Cas9 gene-edited allogeneic HCT product lacking CD33, designed to shield the donor graft from cytotoxicity of subsequent CD33-targeted therapies such as gemtuzumab ozogamicin (GO). In this multicenter, phase 1/2a, open-label study, adult patients with AML/MDS with high relapse risk received trem-cel after myeloablative conditioning followed by GO maintenance (0.5-2.0 mg m[-][2] day 1 per 28-day cycles). Patients receiving trem-cel were assessed for the primary safety endpoint of neutrophil engraftment by day 28 and secondary endpoints including time to neutrophil engraftment, incidence of graft-versus-host disease and graft failure, transplant-related mortality, percentage of CD33-negative myeloid cells and survival. Patients receiving trem-cel and GO were assessed for the additional secondary endpoints of safety of maintenance GO with trem-cel and pharmacokinetics of GO after trem-cel transplant. All 30 patients receiving trem-cel achieved the primary safety endpoint of neutrophil engraftment by day 28 with a median engraftment time of 10 days (95% confidence interval: 9-10). Nineteen patients received GO maintenance in phase 1 dose escalation (n = 15) and in phase 2 dose expansion (n = 4). The trial was stopped early, and this is the final report on the trial including the completed phase 1 portion. GO treatment was safely tolerated up to the recommended phase 2 dose of 2 mg m[-][2], and no prolonged high-grade cytopenias were observed. The most common adverse events were cytopenias and infections. Three cases of transplant-related mortality were observed due to renal failure, sepsis and sinusoidal obstruction syndrome, respectively. In summary, trem-cel demonstrated safe, rapid, robust engraftment, and GO maintenance was administered without prolonged hematologic toxicity. ClinicalTrials.gov identifier: NCT04849910 .},
}

Humans
*Hematopoietic Stem Cell Transplantation/methods/adverse effects
*Gemtuzumab/therapeutic use/administration & dosage/adverse effects
*Leukemia, Myeloid, Acute/therapy/genetics/drug therapy
*Sialic Acid Binding Ig-like Lectin 3/genetics
Middle Aged
Female
Male
Adult
Aged
*CRISPR-Cas Systems/genetics
Transplantation, Homologous
Graft vs Host Disease
Myelodysplastic Syndromes/therapy/genetics

@article {pmid42159719,
year = {2026},
author = {Wu, B and Luo, H and Xie, H and Zhao, C and Xie, P and Yang, X and Shi, J and Huang, W and Fang, Z},
title = {Cis-regulatory editing of SD1 promoter enhances TCP19-mediated repression to optimize plant height in Kam sweet rice.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {6},
pages = {},
pmid = {42159719},
issn = {1432-2242},
mesh = {*Oryza/genetics/growth & development ; *Gene Editing ; *Promoter Regions, Genetic ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Transcription Factors/genetics/metabolism ; Gibberellins/metabolism ; Phenotype ; Plants, Genetically Modified ; Plant Breeding ; },
abstract = {This study demonstrates that CRISPR-mediated cis-regulatory element editing (CRE editing) of the SD1 promoter effectively reduces plant height in Kam sweet rice, without compromising yield or grain quality, offering a precise strategy for crop improvement. Kam sweet rice, a unique aromatic variety, faces challenges with excessive plant height and suboptimal yield. This study explores a precision breeding approach by strengthening an endogenous TCP19-SD1 repression module through CRISPR-Cas9-mediated CRE editing to modulate the expression of the SD1 gene, a key regulator of gibberellin biosynthesis and stem elongation. By introducing an adenine insertion in the GGCCCCCC cis-regulatory element in the SD1 promoter, we enhanced the binding affinity of the transcription factor TCP19, resulting in down-regulated SD1 expression. This led to a reduction in gibberellin levels, shortening internodes, and reducing plant height. Phenotypic evaluations revealed that the edited lines exhibited significantly shorter plant height while maintaining grain yield and nitrogen utilization efficiency compared to wild-type plants. Microscopic analysis of the internodes confirmed that the reduced plant height correlated with decreased cell length. Transcriptomic studies indicated that CRE editing modulated a network of genes involved in both gibberellin and auxin signaling pathways, critical for plant growth. Importantly, the genetic modification did not adversely affect grain quality. This study demonstrates the potential of strengthening endogenous transcriptional repression via CRE editing as a precise alternative to conventional gene knockout techniques, offering a powerful strategy for optimizing complex agronomic traits in rice, with applications in modern crop breeding strategies.},
}

*Oryza/genetics/growth & development
*Gene Editing
*Promoter Regions, Genetic
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
*Transcription Factors/genetics/metabolism
Gibberellins/metabolism
Phenotype
Plants, Genetically Modified
Plant Breeding

@article {pmid42162502,
year = {2026},
author = {Makhijani, S and Alasiri, G and Quadri, MSA and Ingle, RG},
title = {From mechanism to medicine: CRISPR‒Cas9 delivery strategies, therapeutic applications and translation challenges.},
journal = {Discover nano},
volume = {21},
number = {1},
pages = {},
pmid = {42162502},
issn = {2731-9229},
abstract = {The advent of CRISPR‒Cas technology has revolutionized genome editing by providing unprecedented precision, efficiency, and versatility in genetic manipulation. This revolutionary system originated from bacterial adaptive immunity. It allows the programmed modification of DNA sequences in a highly specific way, as long as the desired modification site contains an enzyme-compatible protospacer adjacent motif (PAM) sequence. Recent innovations with CRISPR‒Cas systems have focused on optimizing delivery methods, addressing challenges and expanding their therapeutic applications. Delivery approaches, including viral vectors, lipid nanoparticles, and other nonviral vectors, have been improved to increase target specificity while minimizing off-target effects, ensuring safe and efficient In Vivo genome editing. The applications of CRISPR‒Cas technology include the correction of genetic disorders, engineering of immune cells for cancer therapy, and combating viral infections. However, significant challenges in the form of unintended edits, immune responses, and delivery to specific tissues or organs remain formidable barriers to clinical translation. New CRISPR variants, such as base editing, prime editing, and epigenome editing, provide solutions to improve accuracy and broaden intervention. This review covers recent progress in various CRISPR delivery methodologies, discusses broadening therapeutic applications, highlights ongoing challenges, and describes the future trajectory of this disruptive technology.},
}

@article {pmid42164567,
year = {2025},
author = {Li, X and Zhang, Z and Zhu, J and Zhang, Y and Guo, Y},
title = {Innovative Applications and Challenges of Isothermal Amplification Technology in miRNA Detection.},
journal = {Current genomics},
volume = {26},
number = {6},
pages = {581-598},
pmid = {42164567},
issn = {1389-2029},
abstract = {A class of endogenous non-coding RNAs with a length of roughly 18-25 nucleotides is known as microRNAs (miRNAs). They have been established as the best biomarkers for early cancer diagnosis, molecular subtyping, and prognostic evaluation. They also play important roles in important biological processes like cell proliferation, differentiation, apoptosis, and tumorigenesis through post-transcriptional regulatory networks. Traditional detection technologies (such as quantitative Polymerase Chain Reaction (qPCR), Northern blot, and microarrays) exhibit high sensitivity, yet they rely on expensive thermocycling equipment, involve cumbersome procedures, and are time-consuming, making it difficult to meet the demands of clinical point-of-care testing. In recent years, isothermal amplification technologies, represented by loop-mediated isothermal amplification (LAMP), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), and Exponential Amplification Reaction (EXPAR), have emerged as a research frontier in the field of miRNA detection, by virtue of their advantages including elimination of thermal cycling requirements, fast reaction kinetics, high sensitivity, and simple operation. This study aims to systematically organize the core principles, latest advancements, and integrated innovation patterns of isothermal amplification-based miRNA detection technologies, and provide support for the development of miRNA detection platforms suitable for clinical point-of-care testing.},
}

@article {pmid42164670,
year = {2026},
author = {Islam, MS and Fan, J and Vabna, MA and Haque, N and Monir, SB and Li, Y and Nime, I and Acharjee, M and Pan, F},
title = {Phage enabled precision drug delivery: dual function platforms for therapeutics and genetic cargo transport.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1812871},
pmid = {42164670},
issn = {1664-302X},
abstract = {The rising concern of antimicrobial resistance, coupled with the continually challenging management of complicated diseases such as cancer, has provided momentum toward precision molecular medicine. This review provides an overview of bacteriophage enabled strategies encompassing both conventional antibacterial applications and advanced bioengineered delivery systems. Recent advances in phage therapy include the use of tailored phage formulations, phage immobilization approaches and phage antibiotic combinations to achieve targeted bacterial lysis particularly against multidrug-resistant pathogens and biofilm-associated infections. Beyond their intrinsic antibacterial activity, phages can be genetically and chemically engineered as nanoscale scaffolds. Phage display technologies enable the incorporation of targeting ligands for selective binding to specific tissues including tumor cells. Furthermore, phage capsids can be modified to encapsulate and deliver diverse therapeutic payloads such as small-molecule drugs, nucleic acids and gene-editing systems such as CRISPR-Cas, thereby expanding their utility beyond infectious diseases. The integration of phage biology with nanobiotechnology positions these viral platforms at the forefront of next generation therapeutics. Engineered phages have demonstrated potential as precision delivery vectors for cytotoxic agents, immunomodulators and genetic material with improved specificity and reduced off-target effects. Emerging strategies including phage antibiotic conjugates and enzyme functionalized phages further enhance therapeutic efficacy and facilitate penetration of physiological barriers. Collectively, phage-based platforms represent a versatile and transformative approach with significant implications for the treatment of infectious, oncologic and genetic disorders, supporting the advancement of targeted and personalized medicine.},
}

@article {pmid42164832,
year = {2026},
author = {Kumar, A and Krishan, B and Dhiman, S and Sharma, A and Thadiyan, V and Azmi, W},
title = {Beyond antibiotics: innovative and translational strategies to overcome antimicrobial resistance.},
journal = {3 Biotech},
volume = {16},
number = {6},
pages = {201},
pmid = {42164832},
issn = {2190-572X},
abstract = {The rapid rise of antimicrobial resistance demands therapeutic strategies that extend beyond conventional antibiotics. However, most existing reviews describe emerging alternatives without systematically linking their mechanistic advances to translational readiness and clinical implementation barriers. This review addresses this gap by integrating evidence across multiple beyond-antibiotic approaches, including antimicrobial peptides, bacteriophage therapy, CRISPR-based antimicrobials, nanotechnology-enabled delivery systems, anti-virulence agents, host-directed immunotherapies, microbiome modulation (engineered probiotics and fecal microbiota transplantation), and drug-repurposing or combination therapies. The principal contribution of this synthesis is a comparative framework that maps mechanisms of action, engineering innovations, and translational evidence across these diverse strategies. Advances such as peptidomimetics, engineered phages, and nanoparticle carriers that enhance stability, targeting, and therapeutic efficacy are highlighted, along with synergistic strategies including phage-antibiotic and CRISPR-nanocarrier combinations. The review further identifies major barriers limiting clinical translation, including delivery efficiency, toxicity and ecological concerns, large-scale production challenges, cost, inconsistent clinical outcomes, and regulatory fragmentation for biologics and live therapeutics. To facilitate clinical implementation, the study proposes a translational roadmap emphasizing standardized evaluation assays, physiologically relevant infection models, integrated rapid diagnostics, and regulatory frameworks tailored for emerging antimicrobial platforms, thereby supporting the development of sustainable therapies for the post-antibiotic era.},
}

@article {pmid42164989,
year = {2025},
author = {Taumar, D and Singh, AP and Sharma, H and Chaudhary, V},
title = {RNA Modifications as Drug Targets: Unlocking the Therapeutic Potential of the Epitranscriptome.},
journal = {Current genomics},
volume = {26},
number = {7},
pages = {802-818},
pmid = {42164989},
issn = {1389-2029},
abstract = {INTRODUCTION: The epitranscriptome covers reversible changes to RNA. These changes help control gene expression by making RNA more stable, easier to use, or more prone to degradation. They are increasingly implicated in disease development and offer a promising target for treatment. This review covers RNA alterations and their potential clinical applications.

METHODS: The systematic analysis of the peer-reviewed literature was performed, including experimental, clinical, and computational research. The biochemical properties and biological functions were used to classify RNA modifications. In addition, we evaluated current therapeutic options, including small molecules, CRISPR/Cas-based technologies, and RNA-targeted approaches.

RESULTS: RNA undergoes immense changes, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine. By these modifications, the genes are regulated by coordinated writers, erasers, and readers. Targeted therapeutics aim to regulate changes in cancer, neurodegenerative disease, and viral diseases. Several limitations remain, including overly general framing, suboptimal clarity in conveying the message, and inadequate response mechanisms.

DISCUSSION: The epitranscriptome provides an additional regulatory layer with significant therapeutic applications. New techniques appear to be successful. Researchers, in turn, should be more specific. These therapies require improved delivery vehicles and reduced side effects not associated with the treatment to be safe and effective for the patient.

CONCLUSION: Therapy targeting RNA-based modifications is a breakthrough in disease treatment. These chemical modifications affect gene function and expression patterns. The next step in the research requires overcoming current limitations to fully realize the therapeutic potential of epitranscriptomics-based interventions.},
}

@article {pmid42165133,
year = {2026},
author = {Liu, B and Klatt, D and Harris, C and McGuinness, M and Brendel, C and Williams, DA},
title = {Optimized lentivirus-derived virus-like particles for efficient delivery of Cas9-based genome editors.},
journal = {Nucleic acids research},
volume = {54},
number = {10},
pages = {},
doi = {10.1093/nar/gkag500},
pmid = {42165133},
issn = {1362-4962},
support = {INV-021791/GATES/Gates Foundation/United States ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; Proprotein Convertase 9/genetics ; Animals ; *Lentivirus/genetics ; HEK293 Cells ; Mice ; Genetic Vectors/genetics ; Ribonucleoproteins/genetics ; Virion/genetics ; },
abstract = {Implementation of therapeutic genome editing requires a potent, versatile, and transient delivery system to enable safe and effective in vivo applications. Here, we report on an optimized virus-like particle (VLP) platform for protein-based delivery of Cas9 ribonucleoproteins and Cas9-derived base editors and prime editors, termed LV-VLP-MA, that enables flexible editor deployment. By systematically engineering a panel of truncated Gag-Cas9 fusion variants, we identify a minimal MA-Cas9 configuration that maximizes editor packaging while effectively preserving efficient particle production and functional delivery. Systematic refinement of VLP production parameters enhances particle yield, supporting robust editing activity across diverse genomic targets. Importantly, systemic administration of LV-VLP-MA mediates efficient in vivo editing of the Pcsk9 locus with functional target suppression, establishing proof-of-concept for therapeutic application. Together, these results define a programmable, modular VLP-based platform that combines potency, flexibility, and transient delivery to expand the scope of in vivo genome engineering for therapeutic development.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism
Proprotein Convertase 9/genetics
Animals
*Lentivirus/genetics
HEK293 Cells
Mice
Genetic Vectors/genetics
Ribonucleoproteins/genetics
Virion/genetics

@article {pmid42165845,
year = {2026},
author = {Oxendine, J and Ibarra-Reyes, E and Ma, J and Li, C and Baron, S and Hwang, AE and Wang, R and Rodriguez-Leal, D},
title = {Optimization of Agrobacterium-mediated transformation of commercial heirloom tomato cultivars to develop novel traits via CRISPR/Cas9 genome editing.},
journal = {Planta},
volume = {264},
number = {1},
pages = {},
pmid = {42165845},
issn = {1432-2048},
support = {89365//California Tomato Research Institute/ ; MD-PSLA-243084//University of Maryland/ ; Startup funds//University of Maryland/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Solanum lycopersicum/genetics/growth & development ; Plants, Genetically Modified/genetics ; *Agrobacterium/genetics ; Transformation, Genetic ; Plant Breeding/methods ; Genome, Plant ; },
abstract = {Trait development for commercial heirloom tomatoes can be advanced by optimization of tissue culture and transformation via Agrobacterium and CRISPR/Cas9 mutagenesis. Genetic improvement using new genome editing approaches relies on the efficient delivery of the CRISPR/Cas system in the vegetable crop tomato. Previous protocols for tomato transformation have primarily focused on a handful of cultivars (M82, Ailsa Craig, Microtom, Sweet-100) with very little commercial relevance, and it is not clear if these protocols can be implemented directly in other commercially relevant varieties. Heirloom tomatoes are sought for their deep and diverse flavor but have not been subjected to systematic crop improvement via conventional breeding or biotechnology approaches such as transgenesis or genome editing. Therefore, we tested the transformation and regeneration capacity of six different heirloom cultivars known for their superior taste and market relevance in the US. Subsequently, we optimized rooting conditions and used the GRF4-GIF1 chimeric developmental regulator to successfully recover transgenic plants. Finally, we evaluated the efficiency of targeted genetic modification using the CRISPR/Cas9 genome editing system in several of these cultivars. We demonstrate that our optimizations led to successful transformation of several heirloom varieties, including the generation of edited plants for target genes modifying plant architecture and flowering time. Our results set the foundation for a biotechnology platform to deliver improved traits to local and regional heirloom varieties using genome editing.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Solanum lycopersicum/genetics/growth & development
Plants, Genetically Modified/genetics
*Agrobacterium/genetics
Transformation, Genetic
Plant Breeding/methods
Genome, Plant

@article {pmid42165978,
year = {2026},
author = {Zhu, B},
title = {Nanomaterial-nucleic acid probe synergy: accelerating rapid pathogen detection and antimicrobial susceptibility testing in bloodstream infections.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {42165978},
issn = {1874-9356},
abstract = {Bloodstream infections (BSIs) remain among the most lethal clinical syndromes, driven in large part by diagnostic delays that compel empiric, broad-spectrum antimicrobial therapy and expose patients to avoidable toxicity and resistance selection. Conventional blood culture-based workflows, although diagnostically definitive, are intrinsically slow, often requiring 24-72 h, and are therefore poorly matched to the time-critical demands of sepsis management, where each hour of delayed appropriate therapy measurably increases mortality. In this context, nano-enabled nucleic acid diagnostics represent a promising but largely preclinical strategy for improving analytical sensitivity and turnaround time. This critical translational review examines how engineered nanomaterials spanning plasmonic and magnetic nanoparticles, fluorescent quantum dots, upconversion nanoparticles, and two-dimensional materials synergize with programmable nucleic acid recognizers, including aptamers, CRISPR/Cas effectors, DNAzymes, and conformational probes, to enable rapid, ultrasensitive detection of pathogens and resistance determinants directly from whole blood. Rather than reviewing nanomaterials and nucleic acid probes as separate toolkits, this article focuses on how their co-design at the nano-bio interface enables clinically actionable whole-blood diagnostics. We elucidate how convergence engineering at the nano-bio interface governs signal amplification, background suppression, and assay robustness in complex biological matrices. Particular emphasis is placed on front-end enrichment strategies, optical and magnetic transduction mechanisms, and multiplexed readout architectures that together enable species-level identification and early antimicrobial susceptibility profiling within clinically relevant timeframes, typically ~ 1-6 h in research settings. Beyond analytical performance, we critically assess interconnected translational barriers including batch-to-batch reproducibility, standardization of bioconjugation protocols, antifouling strategies, and evolving regulatory frameworks, which collectively govern the trajectory from laboratory innovation to clinical adoption. At present, direct-from-blood phenotypic antimicrobial susceptibility testing remains technically challenging, and clinical adoption is limited by reproducibility, matrix tolerance, and workflow integration. By integrating mechanistic insight with clinical positioning, this review frames nano-probe diagnostics as promising candidates for next-generation BSI management that may support more timely and precise therapy once analytical robustness, standardization, and clinical validation are achieved.},
}

@article {pmid42166245,
year = {2026},
author = {Lim, J and Van, AB and Wester, M and Koprowski, K and Valera, E and Bashir, R},
title = {Amplification-free dual-blocking autocatalytic CRISPR-Cascade for attomolar DNA detection with low nonspecific signal.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {21},
pages = {e2537414123},
doi = {10.1073/pnas.2537414123},
pmid = {42166245},
issn = {1091-6490},
support = {R01EB032725 A//HHS | NIH (NIH)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *DNA/analysis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *DNA, Bacterial/genetics ; Ribonucleoproteins/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Autocatalytic CRISPR architecture offers amplification-free nucleic acid detection by directly linking target recognition to self-reinforcing ribonucleoprotein (RNP) generation. However, spontaneous background activation remains a key barrier, because strand invasion or unwinding events can initiate unintended amplification and diminish assay specificity. Here, we introduce a dual-blocking CRISPR-Cascade design that independently cages both the guide RNA and trigger DNA, establishing an intrinsic AND gate to raise the effective kinetic barrier for unintended RNP formation. This strategy suppresses leakage by approximately 3- to 18-fold relative to single blocking configurations in full Cascade reactions, while preserving rapid detection (10 min), achieving single-copy sensitivity, and enabling quantitative detection. When paired with a competitive guide RNA decoy, the system further reduces background signals without affecting true target detection. Finally, we demonstrate robust Methicillin-resistant Staphylococcus aureus detection from whole blood in under 40 min including the sample purification and extraction. These results establish dual-blocking as a generalizable molecular gating framework for constructing leakage-resistant, amplification-free CRISPR systems suitable for rapid and decentralized diagnostics.},
}

*CRISPR-Cas Systems/genetics
Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
*DNA/analysis/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*DNA, Bacterial/genetics
Ribonucleoproteins/genetics
Nucleic Acid Amplification Techniques/methods

@article {pmid42166840,
year = {2026},
author = {Simonneau, B and Baghdoyan, S and Cailleret, M and Simon, S and Ruckebusch, O and Vrablikova, B and Giraud-Triboult, K and Kassar, LE and Fanen, P and Duriez, B},
title = {Generation of two iPSC lines carrying two cystic fibrosis rare intronic mutations c.1585-1G>A and c.1680-886A>G in the CFTR gene of the parental line PCIi033-A using CRISPR/Cas tools.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {104016},
doi = {10.1016/j.scr.2026.104016},
pmid = {42166840},
issn = {1876-7753},
abstract = {CFTR gene mutations are responsible for Cystic Fibrosis. For half a decade, a triple therapy has been available for patients carrying the most frequent mutation: p.F508del. Among classified mutations, intronic mutations are rare, and no therapeutic strategies have yet been developed for such patients. We genome edited the parental iPSC line PCIi033-A to introduce two intronic mutations: A.s.Cas12a for c.1585-1G>A (PCIi033-A-8) and SpCas9 for c.1680-886A>G (PCIi033-A-9). Both cell lines have normal morphology and karyotype, conserved pluripotency, and differentiate into the three germ layers. Obtaining iPSC-derived mutant lung epithelia would be an efficient tool for testing new therapeutic strategies.},
}

@article {pmid41937145,
year = {2026},
author = {Wu, X and Jin, S and Pan, Y and Zhen, W and Yu, S and Zhang, Y and Xu, F and Wang, R and Wu, M and Sun, W and Xu, J and Zang, X and Zhang, H},
title = {Immune-evasive stem cells: engineering tolerance and reprogramming microenvironments for regenerative therapy.},
journal = {Stem cell research & therapy},
volume = {17},
number = {1},
pages = {},
pmid = {41937145},
issn = {1757-6512},
support = {2023xkfyts01//the 2023 Disciplinary Construction Project in the School of Dentistry, Anhui Medical University/ ; YQYB2024013//Anhui Province Outstanding Young Teachers Development Program/ ; },
mesh = {Humans ; *Regenerative Medicine/methods ; Gene Editing ; *Immune Tolerance ; Animals ; *Cellular Reprogramming ; *Induced Pluripotent Stem Cells/immunology ; CRISPR-Cas Systems ; *Stem Cell Transplantation ; },
abstract = {Stem cell transplantation (SCT) holds significant promise for regenerative medicine, yet immune rejection remains a major obstacle. To address this, recent advances leverage CRISPR/Cas9 to engineer hypoimmunogenic induced pluripotent stem cells. These modified cells lack classical immune recognition markers (HLA class I/II) yet retain immune-tolerant molecules such as HLA-E, HLA-G, and CD47, enabling their universal use across different individuals. Additionally, mesenchymal stem cell-derived exosomes and immune checkpoint modulators (e.g., PD-L1) have shown clinical effectiveness by reducing graft-versus-host disease and autoimmune reactions. They achieve this through mechanisms such as suppressing inflammatory T-cell activation, promoting regulatory T-cell expansion, and modulating macrophage polarization. Despite these advances, several challenges remain. One key concern is the potential tumorigenic risk caused by genomic instability during genome editing and long-term cell expansion. Emerging precision editing platforms, including base editing and prime editing, provide strategies to reduce double-strand DNA break-induced chromosomal rearrangements and improve genomic safety. Future research priorities include integrating AI-based immune profiling, precision genome editing, and advanced 3D-bioprinting technologies. Together, these innovations represent a paradigm shift toward developing safer, more effective, universally compatible stem cell therapies for diseases previously deemed untreatable.},
}

Humans
*Regenerative Medicine/methods
Gene Editing
*Immune Tolerance
Animals
*Cellular Reprogramming
*Induced Pluripotent Stem Cells/immunology
CRISPR-Cas Systems
*Stem Cell Transplantation

@article {pmid41975095,
year = {2026},
author = {Guan, K and Ocampo, RF and Matheus Carnevali, PB and Castelle, CJ and Gonzalez-Osorio, L and Castanzo, DT and Thomas, NC and Brothers, M and Dangerfield, TL and Hooper, MM and West, MS and Appleby, NM and Krudop, I and Lamothe, RC and Aliaga Goltsman, DS and Alexander, LM and Butterfield, CN and Johnson, KA and Brown, CT and Taylor, DW},
title = {Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency.},
journal = {Nature structural & molecular biology},
volume = {33},
number = {5},
pages = {756-767},
pmid = {41975095},
issn = {1545-9985},
support = {R35 GM138348/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/chemistry/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *Eubacteriales/enzymology/genetics ; Models, Molecular ; HEK293 Cells ; },
abstract = {Miniature CRISPR-Cas12f nucleases are attractive candidates for therapeutic genome editing because of their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here we identify and characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Alistipes sp. Cas12f (Al3Cas12f), which supports robust genome editing in human cells. Through structural, biochemical and kinetic analyses, we compare Al3Cas12f to two recently described orthologs, Oscillibacter sp. Cas12f and Ruminiclostridium herbifermentans Cas12f. These orthologs present divergent architectures and regulatory features governing protospacer-adjacent motif recognition, guide RNA (gRNA) binding, dimerization and DNA cleavage. Notably, Al3Cas12f achieves efficient R-loop formation through a stable dimer interface and a naturally optimized gRNA. Leveraging these structural insights, we generate an engineered Al3Cas12f variant (RKK) that increases editing and improves activity across several tested genomic loci. By overcoming locus-dependent variability and an apparent potency threshold, this engineered compact editor seems to expand the feasibility of low-dose, AAV-compatible therapeutic genome editing. Our results elucidate mechanistic determinants of Cas12f activity and offer a framework for engineering compact genome editors that may bear therapeutic potential.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/chemistry/metabolism
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
*Eubacteriales/enzymology/genetics
Models, Molecular
HEK293 Cells

@article {pmid42156407,
year = {2026},
author = {Sa, Y and Liu, C and Yang, L and Yue, L and Zhu, L and Guo, Y and Wang, R and Wang, Y and Feng, Y and Wang, Y and Zhang, Y and Wang, W and Xie, Y},
title = {Structural basis for dual mechanism of Cas2/3 nuclease inhibition by anti-CRISPR protein AcrIF19.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-73156-3},
pmid = {42156407},
issn = {2041-1723},
support = {32301058//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32371329, 32301055，32400565，32560225//National Natural Science Foundation of China (National Science Foundation of China)/ ; ZR202211190090//Natural Science Foundation of Shandong Province (Shandong Provincial Natural Science Foundation)/ ; tsqn202306146//Taishan Scholar Project of Shandong Province/ ; },
abstract = {CRISPR-Cas systems are prokaryotic immune mechanisms often targeted by phage-encoded anti-CRISPR (Acr) proteins. This study characterizes AcrIF19, a potent inhibitor of the type I-F system in Pectobacterium atrosepticum. The cryo-EM structure of the apo Cas2/3 and Cas2/3-AcrIF19 complex reveals a dual inhibitory mechanism. AcrIF19 employs a negatively charged β2-β3 loop to sterically occlude the non-target DNA strand entry channel, acting as a competitive inhibitor to disrupt Cas2/3 recruitment. Concurrently, this steric occlusion impedes ssDNA-mediated allosteric activation, which locks the critical helix-like loop motif in an inhibitory conformation and thereby abrogates DNA cleavage activity. AcrIF19 represents an anti-CRISPR protein inhibiting Cas2/3 via two different mechanisms, integrating a competitive ssDNA inhibitor with an allosteric blockade to suppress both target recruitment and DNA cleavage.},
}

@article {pmid42157911,
year = {2025},
author = {Waqar, Z and Sethi, P and Jain, D and Singh, K and Alsaidan, OA and Alzarea, SI and Gupta, JK and Saxena, S and Sharma, MC},
title = {Precision Medicine in Neurodegenerative Diseases: Genomic Approaches to Target Amyloid-β, Tau, and Alpha-Synuclein Pathways.},
journal = {Current genomics},
volume = {26},
number = {6},
pages = {469-494},
pmid = {42157911},
issn = {1389-2029},
abstract = {Neurodegenerative diseases, including Alzheimer's and Parkinson's disease, are characterized by the pathological aggregation of proteins such as amyloid-β, tau, and alpha-synuclein. These hallmark proteins play central roles in disease progression and represent promising targets for therapeutic intervention. Advances in precision medicine, driven by genomic technologies such as CRISPR-Cas systems, RNA-based therapies, and high-throughput sequencing, have enabled the development of tailored strategies to modulate these pathological pathways. This review examines the integration of genomic approaches in targeting amyloid-β, tau, and alpha-synuclein, emphasizing their potential to mitigate disease progression and improve patient outcomes. We highlight current progress in preclinical and clinical studies, discuss challenges associated with translating these therapies into clinical practice, and explore future directions for achieving therapeutic precision in neurodegenerative disorders. By examining the interplay of genetic, molecular, and therapeutic innovations, this review underscores the transformative potential of genomic medicine in addressing the unmet needs of neurodegenerative disease treatment.},
}

@article {pmid42158919,
year = {2026},
author = {Zhou, C and Arimura, SI},
title = {TALENs and Related Technologies for Editing Nuclear and Organellar Genomes in a Model Plant, Arabidopsis thaliana.},
journal = {Bio-protocol},
volume = {16},
number = {9},
pages = {e5668},
pmid = {42158919},
issn = {2331-8325},
abstract = {Plant genome editing is a powerful approach for modifying plant DNA to investigate gene function and to engineer desirable traits. Several genome-editing technologies have been developed, among which CRISPR/Cas systems and transcription activator-like effector nucleases (TALENs) are widely used to introduce targeted double-stranded DNA breaks. While CRISPR/Cas systems are highly efficient for nuclear genome editing, their application to plant organellar genomes remains limited, largely due to difficulties in guide RNA delivery into mitochondria and chloroplasts. Here, we present a detailed and reproducible protocol for constructing TALEN-based binary vectors for targeted genome editing in Arabidopsis thaliana. This protocol describes the assembly of TALE repeat arrays, the generation of nuclear-, mitochondrial-, and plastid-targeted TALEN expression vectors using MultiSite Gateway cloning, and subsequent Agrobacterium-mediated plant transformation and genotyping. The workflow enables the production of nTALENs, mitoTALENs, and ptpTALENs using a unified vector design strategy. In addition, the protocol briefly outlines the construction principles of TALE-based cytidine deaminases (TALECDs) for targeted C-to-T base editing in plant organellar genomes. The protocol provides a flexible and robust framework for plant nuclear and organellar genome editing and can be readily adapted to different target genes and experimental purposes. Its modular design and compatibility with standard molecular cloning techniques make it accessible to laboratories aiming to perform precise genome manipulation in plants. Key features • Requires experience in basic molecular cloning and Arabidopsis transformation; suitable for laboratories performing plant nuclear and organellar genome editing. • Enables construction of nuclear-, mitochondrial-, and plastid-targeted TALENs using a unified MultiSite Gateway-based vector system. • Provides a modular workflow for assembling large TALEN binary vectors compatible with Agrobacterium-mediated transformation in Arabidopsis thaliana. • Includes optional extension to TALE-based cytidine deaminases for targeted C-to-T base editing in plant mitochondrial and plastid genomes.},
}

@article {pmid42159153,
year = {2026},
author = {Gonçalves, LDS and Domingues, WB and Nunes, LS and Blödorn, EB and Dellagostin, EN and Martins, AWDS and Acosta, IB and Corcini, CD and Varela, AS and Fróes, CN and Campos, VF},
title = {First Report of CRISPR-Cas9 Ribonucleoprotein Delivery Into Teleost Spermatozoa With Preserved Membrane and Genomic Integrity.},
journal = {Molecular reproduction and development},
volume = {93},
number = {5},
pages = {e70114},
doi = {10.1002/mrd.70114},
pmid = {42159153},
issn = {1098-2795},
support = {23/2551-0000181-0//Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul/ ; 22/2551-0001645-6//Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul/ ; 24/2551-0002158-2//Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul/ ; 440636/2022-1//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 407610/2024-3//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; AUXPE #2537/2018//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; },
mesh = {Animals ; Male ; *Spermatozoa/metabolism/cytology ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Cichlids/genetics ; *Cell Membrane/metabolism ; Electroporation/methods ; Sperm Motility ; DNA Fragmentation ; },
abstract = {Delivery of large molecular complexes into spermatozoa remains a challenge in reproductive biotechnology. Teleost sperm possess highly compacted chromatin and minimal cytoplasmic volume, limiting intracellular access of ribonucleoprotein assemblies. Here, we evaluated whether CRISPR-Cas9 ribonucleoprotein (RNP) complexes can be introduced into Nile tilapia spermatozoa using capillary electroporation while preserving cellular integrity. RNP uptake was quantified via EGFP fluorescence, and sperm performance was assessed through motility, membrane integrity, mitochondrial activity, and DNA fragmentation analyses. Approximately 10%-13% of spermatozoa exhibited detectable EGFP signal following electroporation. Increased pulse numbers reduced motility and mitochondrial activity, whereas membrane integrity was preserved and no increase in DNA fragmentation was detected. A single pulse (1250 V, 40 ms) provided the best balance between RNP uptake and functional preservation. These findings support capillary electroporation as a minimally disruptive strategy for CRISPR-RNP delivery in teleost reproductive biology.},
}

Animals
Male
*Spermatozoa/metabolism/cytology
*Ribonucleoproteins/genetics/metabolism
*CRISPR-Cas Systems
*Cichlids/genetics
*Cell Membrane/metabolism
Electroporation/methods
Sperm Motility
DNA Fragmentation

@article {pmid40601102,
year = {2026},
author = {Jong, HL and Yuen, KS and Jin, DY and Hoe, SLL and Ideris, A and Tan, CH and Lam, SK and Lim, YM and Cheong, SK},
title = {Generation of Leucine-Rich Repeat Kinase 2 (LRRK2) Knockout Neuroblastoma Cells SH-SY5Y by CRISPR/Cas9-Mediated Genome Editing.},
journal = {Biochemical genetics},
volume = {64},
number = {3},
pages = {4423-4455},
pmid = {40601102},
issn = {1573-4927},
mesh = {Humans ; *Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Neuroblastoma/genetics/pathology ; Cell Line, Tumor ; *Gene Knockout Techniques/methods ; },
abstract = {Leucine-rich repeat kinase 2 (LRRK2) is associated with Parkinson's disease, despite its low expression in the brain. Pathogenic mutations in LRRK2 enhance kinase activity and contribute to the disease's pathogenesis. Neuroblastoma SH-SY5Y cells, which also exhibit low LRRK2 expression, are extensively used as a model for Parkinson's disease. While less prominent, low-expression genes can play crucial roles in cellular processes, development, and disease. Knocking out such genes poses specific challenges, including difficulties in detection, incomplete knockout, and compensatory mechanisms that can obscure phenotypic changes. This study develops a strategy to knockout low-expression LRRK2 in SH-SY5Y cells effectively. Our approach employs a double-cut and multiple guide RNAs strategy, optimized electroporation parameters to enhance CRISPR/Cas9 plasmid delivery, refined clonal expansion technique, and a sensitive protein detection protocol. We successfully generate LRRK2 knockout SH-SY5Y cells using CRISPR/Cas9, with the knockout efficiency validated by PCR analysis, sequencing, and Western blot analysis.},
}

Humans
*Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
*Neuroblastoma/genetics/pathology
Cell Line, Tumor
*Gene Knockout Techniques/methods

@article {pmid40853436,
year = {2026},
author = {Yıldız, Bİ and Karabağ, K},
title = {Targeted Gene Editing in Honey Bees Using Liposome-Based CRISPR-Cas9.},
journal = {Biochemical genetics},
volume = {64},
number = {3},
pages = {4477-4492},
pmid = {40853436},
issn = {1573-4927},
support = {FDK-2022-6006//Akdeniz Üniversitesi/ ; },
mesh = {Animals ; Bees/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Liposomes/chemistry ; Male ; },
abstract = {Genome editing technologies have revolutionized molecular biology, enabling precise manipulation of gene functions across diverse organisms. In this study, we introduce a novel liposome-mediated delivery system for CRISPR-Cas9 components targeting the Juvenile Hormone Acid Methyltransferase (JHAMT) gene in honey bees (Apis mellifera anatoliaca). This approach leverages drone sperm cells as vectors for CRISPR-Cas9 transfection, overcoming the technical challenges of embryo microinjection in honey bees, such as low survival rates and labor-intensive procedures. The study involved artificial insemination of queen bees with transfected sperm and subsequent evaluation of gene-editing efficiency across generations.Our findings demonstrate the successful generation of both heterozygous and homozygous mutants, with gene-editing efficiencies reaching approximately 43%. This innovative method highlights the potential of liposome-mediated delivery systems for non-invasive, efficient, and scalable genome editing in eusocial insects. The results pave the way for broader applications in honey bee genetic research, offering a viable alternative to traditional methods. Furthermore, this study underscores the importance of genetic tools in advancing apiculture and addressing ecological challenges linked to pollinator health.},
}

Animals
Bees/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Liposomes/chemistry
Male

@article {pmid41793179,
year = {2026},
author = {Dong, K and Hu, H and Wang, H and Zheng, Z and Cheng, S and Shu, W and Liu, R and Xin, X and Huang, S and Qian, D and Xiao, X and Fu, Q and Wang, H},
title = {A Single-Enzyme Activated CRISPR-Cas12a Nano System via Subtly Balanced dsDNA for Kinetic-Gated UDG Detection and Spatiotemporal Cellular Imaging.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {28},
pages = {e23400},
doi = {10.1002/advs.202523400},
pmid = {41793179},
issn = {2198-3844},
support = {2023YFC2705400//The National Key Research and Development Plan/ ; 82472965//National Natural Science Foundation project/ ; W2521097//National Natural Science Foundation project/ ; 2024EIA002//Hubei Provincial Central Government-Guided Local Science and Technology Fund Development Project/ ; 2025AFD286//Hubei Provincial National Natural Science Foundation/ ; 2025XHYN047//Free Innovation Pre-research Fund of Union Hospital/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/metabolism/genetics ; *Uracil-DNA Glycosidase/metabolism/genetics ; Kinetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; DNA Repair ; Bacterial Proteins ; },
abstract = {Uracil-DNA glycosylase (UDG) is a key enzyme in base excision repair and an important biomarker for genomic stability and disease. In many reported sensing systems, uracil excision is coupled to signal generation through additional downstream BER processing steps, resulting in an indirect readout of UDG activity. Here, we report a single-enzyme activated CRISPR-Cas12a nanosystem driven by a subtly balanced double-stranded DNA (dsDNA) substrate. This dsDNA serves as a kinetic gatekeeper that maintains Cas12a in an inert state until UDG-mediated uracil excision disrupts the balance, lowering the energy barrier for crRNA invasion and initiating Cas12a trans-cleavage. This conformationally gated mechanism directly converts a uracil excision event into an amplified CRISPR response without requiring sequential enzymatic processing. The system achieves a 1840-fold discrimination ratio and an ultralow detection limit of 5 × 10[-7] U/mL. Furthermore, a genetically encoded variant enables nuclear localization of Cas12a and dsDNA sensors for in situ imaging of endogenous UDG. The platform visualizes UDG dynamics across distinct cell cycle phases, realizing spatiotemporal mapping of repair activity in living cells. This work introduces a new activation paradigm for CRISPR-Cas12a via subtly balanced dsDNA and establishes a generalizable strategy for precise molecular sensing in complex biological environments.},
}

*CRISPR-Cas Systems/genetics
*DNA/metabolism/genetics
*Uracil-DNA Glycosidase/metabolism/genetics
Kinetics
Humans
*Endodeoxyribonucleases/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/genetics
DNA Repair
Bacterial Proteins

@article {pmid41806318,
year = {2026},
author = {Sun, J and Yang, X and Jiang, W and Ji, C and Wu, Y and Sun, H and Liu, X and Yamamoto, M and Tsukamoto, T and Nomura, S and Zhao, J and Ruan, Y and Li, H and Wang, X},
title = {In Vivo CRISPR Screening Identifies the Glutamate Receptor GRIA2 as Promoting Peritoneal Metastasis of Gastric Cancer via Calcium-Dependent β-Catenin Activation.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {28},
pages = {e21746},
doi = {10.1002/advs.202521746},
pmid = {41806318},
issn = {2198-3844},
support = {ZY2024-003//Scientific Research Foundation of Zhongshan Hospital/ ; },
mesh = {*Stomach Neoplasms/genetics/pathology/metabolism ; Animals ; Humans ; *Receptors, AMPA/genetics/metabolism ; Mice ; *Peritoneal Neoplasms/secondary/genetics/metabolism ; *beta Catenin/metabolism/genetics ; Cell Line, Tumor ; *Calcium/metabolism ; Cell Movement/genetics ; Wnt Signaling Pathway/genetics ; CRISPR-Cas Systems/genetics ; Glycogen Synthase Kinase 3 beta/metabolism ; },
abstract = {Peritoneal metastasis is the most lethal manifestation of gastric cancer, with a median survival of less than one year, highlighting the need for new therapeutic targets. Through an in vivo genome-wide CRISPR/Cas9 screen, we identified GRIA2, an AMPA-type glutamate receptor subunit, as a key driver of peritoneal metastasis. GRIA2 promotes gastric cancer cell migration, invasion, stemness, and adhesion to mesothelial cells in a glutamate-dependent manner. Mechanistically, glutamate activates GRIA2, enhancing its interaction with GSK-3β and inducing calcium influx, inhibiting GSK-3β kinase activity and stabilizing β-catenin, thereby activating the Wnt/β-catenin signaling pathway. Single-cell RNA sequencing revealed that cancer-associated fibroblasts are the primary source of glutamate in the peritoneal microenvironment, which establishes a paracrine axis that enhances GRIA2-driven metastasis. Pharmacological inhibition of AMPA receptors with NBQX and Selurampanel suppressed peritoneal metastasis in both cell line-derived and patient-derived organoid xenograft (PDOX) mouse models. In clinical analysis, GRIA2 expression in peritoneal metastases correlated with the levels of β-catenin and phosphorylated GSK-3β (serine 9), with high GRIA2 expression predicting poor prognosis. These findings suggest that GRIA2 is a novel therapeutic target, and AMPA receptor antagonists are promising agents for treating gastric cancer peritoneal metastasis.},
}

*Stomach Neoplasms/genetics/pathology/metabolism
Animals
Humans
*Receptors, AMPA/genetics/metabolism
Mice
*Peritoneal Neoplasms/secondary/genetics/metabolism
*beta Catenin/metabolism/genetics
Cell Line, Tumor
*Calcium/metabolism
Cell Movement/genetics
Wnt Signaling Pathway/genetics
CRISPR-Cas Systems/genetics
Glycogen Synthase Kinase 3 beta/metabolism

@article {pmid41957628,
year = {2026},
author = {Yang, W and Wu, J and Zhou, Y and Yu, Z and Shang, X and Huang, J},
title = {PfVPS4, an ESCRT AAA-ATPase, is essential for asexual proliferation and gametocyte sexual conversion in Plasmodium falciparum.},
journal = {Parasites & vectors},
volume = {19},
number = {1},
pages = {},
pmid = {41957628},
issn = {1756-3305},
support = {32170071//National Natural Science Foundation of China/ ; 32200450//National Natural Science Foundation of China/ ; 32300051//National Natural Science Foundation of China/ ; 2023JJ40798//Natural Science Foundation of Hunan Province/ ; 2023JJ30651//Natural Science Foundation of Hunan Province/ ; },
mesh = {*Plasmodium falciparum/genetics/growth & development/enzymology/physiology ; *Endosomal Sorting Complexes Required for Transport/genetics/metabolism ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Adenosine Triphosphatases/genetics/metabolism ; Gene Knockdown Techniques ; Erythrocytes/parasitology ; Reproduction, Asexual ; Humans ; *ATPases Associated with Diverse Cellular Activities/genetics/metabolism ; Life Cycle Stages ; },
abstract = {BACKGROUND: Malaria, caused by Plasmodium spp., remains a major global health threat. Among them, Plasmodium falciparum is the most pathogenic, and its asexual intraerythrocytic proliferation is the pathological basis. This process has enormous biosynthetic demands and highly relies on the coordinated function of the endomembrane and vesicular transport systems. The transition from asexual proliferation to sexual differentiation similarly involves remodeling of internal membrane complexes, membrane reshaping, and precise protein sorting. In eukaryotic cells, the Endosomal Sorting Complexes Required for Transport (ESCRT) complex is a core machinery for membrane remodeling and endosomal development. However, how the ESCRT system regulates the complex life cycle of Plasmodium, particularly during intraerythrocytic proliferation and sexual conversion, remains an important unresolved question.

METHODS: In this study, using Plasmodium falciparum as a model system, we applied CRISPR-Cas9-mediated homologous recombination to achieve conditional knockdown of PfVPS4, the core ATPase of the ESCRT complex - vacuolar protein sorting-associated protein 4 (PfVPS4). Western blotting and immunofluorescence assays were used to assess PfVPS4 abundance and subcellular localization. Tightly synchronized cultures were used to evaluate its effects on parasite growth, merozoite numbers, and gametocyte conversion rate. In vitro protein purification, enzyme kinetics, and site-directed mutagenesis were performed to identify the impact of key residues on PfVPS4 ATPase activity and to validate the synergistic activation by its cofactor PfVta1. In addition, multiple sequence alignment and AlphaFold3 modeling were used to predict and display structural features before and after mutation of key sites.

RESULTS: We successfully generated conditional knockdown lines in both Pf3D7 and PfNF54 parasite strains, enabling effective knockdown at different stages of the intraerythrocytic cycle and during gametocytogenesis. Knockdown of PfVPS4 led to an 84% reduction in asexual progeny parasite numbers, decreased merozoite numbers, and a 46% reduction in gametocyte conversion rate, without affecting subsequent gametocyte maturation. Biochemical assays showed that PfVPS4 ATPase activity is optimal at pH 7.5 and 37°C, and is dependent on Mg²⁺, with a Vmax of 2.23 ± 0.053 U/mg and a Km of 0.086 mM. Site-directed mutagenesis validated the essential role of the canonical catalytic residues (D213, E214) and the species-specific key residues (T161, I288) in maintaining enzymatic activity, and confirmed that the cofactor PfVta1 significantly enhances PfVPS4 activity.

CONCLUSION: PfVPS4 is essential for normal asexual blood-stage replication and efficient sexual conversion in Plasmodium falciparum. Its knockdown severely disrupts intraerythrocytic proliferative homeostasis and reduces gametocyte conversion, indicating that this protein has a broader role in coordinating parasite proliferation and transmission. Given its essentiality, species‑specific residues, and regulation by PfVta1, PfVPS4 and its complex are attractive antimalarial drug targets.},
}

*Plasmodium falciparum/genetics/growth & development/enzymology/physiology
*Endosomal Sorting Complexes Required for Transport/genetics/metabolism
*Protozoan Proteins/genetics/metabolism
CRISPR-Cas Systems
*Adenosine Triphosphatases/genetics/metabolism
Gene Knockdown Techniques
Erythrocytes/parasitology
Reproduction, Asexual
Humans
*ATPases Associated with Diverse Cellular Activities/genetics/metabolism
Life Cycle Stages

@article {pmid41975467,
year = {2026},
author = {Song, HY and Cao, H and Huang, SB and Elsheikha, HM and Zheng, Z and Lu, XS and Tian, X and Zheng, XN and Zhu, XQ},
title = {Functional characterization of 11 novel rhoptry proteins in the type I RH strain of Toxoplasma gondii using the CRISPR-Cas9 system.},
journal = {Parasites & vectors},
volume = {19},
number = {1},
pages = {},
pmid = {41975467},
issn = {1756-3305},
support = {2022Y309//the Graduate Innovation Project of Shanxi Province/ ; U2202201//the NSFC-Yunnan Joint Fund/ ; 2021YFC2300800 and 2021YFC2300802//National Key Research and Development Program of China/ ; 2021XG001//the Special Research Fund of Shanxi Agricultural University for High-level Talents/ ; },
mesh = {*Toxoplasma/genetics/pathogenicity/metabolism ; *Protozoan Proteins/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; Mice ; Virulence ; Gene Editing ; Female ; Virulence Factors/genetics ; Mice, Inbred BALB C ; Gene Knockout Techniques ; Toxoplasmosis/parasitology ; },
abstract = {BACKGROUND: Rhoptry proteins (ROPs) are secreted effectors that play important roles in the virulence of Toxoplasma gondii by facilitating host cell invasion and immune modulation. Although many ROPs have been predicted, their specific functions remain largely unexplored. This study investigates the roles of 11 previously uncharacterized ROPs in T. gondii biology, with a focus on their contributions to virulence.

METHODS: Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing was employed to generate epitope-tagged and knockout mutants for each candidate ROP in the T. gondii RHΔku80 strain. Subcellular localization was determined via immunofluorescence microscopy in both tachyzoite and bradyzoite stages. In vitro assays assessed parasite invasion, replication, egress, and plaque formation. In vivo virulence was evaluated in mouse infection models. To explore molecular mechanisms underlying virulence attenuation, we performed transcriptomic profiling of RHΔrop64 and RHΔrop65 knockout strains.

RESULTS: All 11 candidate ROPs exhibited rhoptry localization in both tachyzoite and bradyzoite stages. Despite no apparent in vitro growth defects, deletion of ROP64 and ROP65 led to significant attenuation of virulence in mice, with ROP64 showing the most pronounced effect. Transcriptome analysis revealed downregulation of key immune-modulatory genes, including ROP5, ROP39, TgIST, and PLP1. In addition, RHΔrop64 exhibited broader suppression of ROPs than RHΔrop65, suggesting it has a more pronounced role in immune modulation.

CONCLUSIONS: ROP64 and ROP65 are critical to T. gondii virulence, likely through modulation of the parasite's immune-evasive machinery. Their regulatory influence on effector expression underscores their importance in host adaptation. Importantly, the RHΔrop64 mutant displays characteristics of an attenuated strain with potential for vaccine development against toxoplasmosis.},
}

*Toxoplasma/genetics/pathogenicity/metabolism
*Protozoan Proteins/genetics/metabolism
Animals
*CRISPR-Cas Systems
Mice
Virulence
Gene Editing
Female
Virulence Factors/genetics
Mice, Inbred BALB C
Gene Knockout Techniques
Toxoplasmosis/parasitology

@article {pmid41999750,
year = {2026},
author = {Thege, FI and Hoskins, A and Kramer, A and Salim, I and Seetharaman, A and Fowlkes, N and Rajapakshe, KI and Maitra, A and Wörmann, SM},
title = {An autochthonous CRISPR activation screening platform for characterizing tissue-specific oncogene selection.},
journal = {Cell reports. Medicine},
volume = {7},
number = {5},
pages = {102759},
doi = {10.1016/j.xcrm.2026.102759},
pmid = {41999750},
issn = {2666-3791},
mesh = {Animals ; *Oncogenes/genetics ; Humans ; Mice ; *Lung Neoplasms/genetics/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; *Pancreatic Neoplasms/genetics/pathology ; Organ Specificity/genetics ; },
abstract = {Human adenocarcinomas exhibit tissue-specific mutation and copy-number patterns that suggest diverse selective pressures and distinct oncogene dependencies. Here, we use our FiCASCan platform to test whether in vivo CRISPR activation screening can recapitulate oncogene selection during tumor initiation. Using CRISPRa-competent PPKS mice and intranasal or intraductal delivery of pooled lentivirus, we screen frequently amplified and mutated genes in autochthonous lung and pancreas cancer models. We observe strong selection for Egfr, Myc, Sox2, and Pik3cb activation in lung tumors and near-complete dominance of Myc in pancreatic tumors, revealing striking tissue-specific differences. In our model, Sox2 activation suppresses Nkx2-1 signaling and drives aggressive mucinous lung adenocarcinoma. MYC activation in the pancreas mirrors MYC amplification in human PDAC, including the emergence of an immune-cold microenvironment. Overall, our findings show that in vivo CRISPR activation screening faithfully captures oncogene selection and provides a powerful approach for studying tumor initiation and progression.},
}

Animals
*Oncogenes/genetics
Humans
Mice
*Lung Neoplasms/genetics/pathology
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics
*Pancreatic Neoplasms/genetics/pathology
Organ Specificity/genetics

@article {pmid42083769,
year = {2026},
author = {Chen, Z and Mao, K and Meng, H and Gao, C and Lv, H and Li, X and Zheng, Q and Yang, Z and Hamza, IA and Tu, C and Zhang, H},
title = {String-Powered Microfluidic Chip Integrating Heparin-Mediated One-Pot RT-RPA/CRISPR-Cas12a for Multiplex Detection of HFMD Viruses.},
journal = {Analytical chemistry},
volume = {98},
number = {19},
pages = {14315-14329},
doi = {10.1021/acs.analchem.6c00663},
pmid = {42083769},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *Hand, Foot and Mouth Disease/virology/diagnosis ; *Heparin/chemistry ; Humans ; *Lab-On-A-Chip Devices ; Wastewater/virology ; Nucleic Acid Amplification Techniques ; },
abstract = {Hand, foot, and mouth disease (HFMD) is a common childhood infection caused by enteroviruses, which exhibit distinct regional and seasonal epidemiological patterns. Wastewater-based epidemiology is a crucial tool for monitoring population infection dynamics and viral subtype distribution. However, the lack of effective on-site viral detection methods limits timely early warning and effective surveillance of infectious disease outbreaks. This study developed a one-pot RT-RPA/CRISPR-Cas12a assay-based, string-powered flywheel microfluidic chip for the multiplex detection of HFMD viruses in wastewater. First, by leveraging the regulatory effect of heparin sodium on CRISPR/Cas12a activity, a one-pot RT-RPA/CRISPR-Cas12a system was constructed to detect four major subtypes of HFMD virus (EV-A71, CV-A16, CV-A6, and CV-A10). Subsequently, this method was integrated into a pull-wire, flywheel-type, dual-axis centrifugal microfluidic chip, named the Heparin-Inhibited CRISPR-Associated System Chip (HICAS-Chip), enabling integrated enrichment, purification, elution, and multiplexed detection. The HICAS-Chip allowed visual detection of nucleic acids at 10 aM sensitivity within 1 h, corresponding to the sensitivity of the one-pot RT-RPA/CRISPR-Cas12a assay. During a year-long wastewater monitoring program in Guiyang City, China, the HICAS-Chip identified EV-A71 and CV-A10 as the predominant circulating subtypes, with incidence peaks observed in June, November, and December. The wastewater detection results obtained using HICAS-Chip showed high concordance (95.83%) with RT-qPCR assays. This platform provides an efficient portable device for the early detection and continuous monitoring of HFMD epidemic trends by wastewater-based epidemiology.},
}

*CRISPR-Cas Systems
*Hand, Foot and Mouth Disease/virology/diagnosis
*Heparin/chemistry
Humans
*Lab-On-A-Chip Devices
Wastewater/virology
Nucleic Acid Amplification Techniques

@article {pmid42087437,
year = {2026},
author = {Yang, Y and Liu, Y and Xu, H and Zhou, F and Ji, X and He, Z},
title = {CRISPR/Cas13a-Driven Catalytic Hairpin Assembly of a Quantum Dot Nanobeacon for Viral RNA Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {19},
pages = {14163-14172},
doi = {10.1021/acs.analchem.5c08146},
pmid = {42087437},
issn = {1520-6882},
mesh = {*Quantum Dots/chemistry ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Influenza A Virus, H1N1 Subtype/genetics/isolation & purification ; SARS-CoV-2/genetics/isolation & purification ; Limit of Detection ; Humans ; Nucleic Acid Amplification Techniques ; COVID-19/diagnosis ; },
abstract = {Influenza A (H1N1), which is a highly contagious respiratory pathogen, poses a serious public health threat. Its effective control necessitates rapid and accurate detection methods. Here, we developed a novel quantum dot (QDs) nanobeacon-based biosensor that synergistically integrates the precise target recognition and trans-cleavage activity of CRISPR/Cas13a with the isothermal, enzyme-free signal amplification power of catalytic hairpin assembly (CHA) for sensitive detection of H1N1 RNA. This system achieves dual signal amplification, enabling detection with a remarkably low limit of detection (LOD) of 95.2 aM within 40 min. Excellent specificity distinguishes H1N1 from other common influenza viruses. Crucially, the programmability of crRNA endows the method with broad versatility. Beyond influenza virus detection, it was successfully applied to SARS-CoV-2 RNA detection, achieving an LOD of 87.0 aM, demonstrating its potential for diverse pathogen diagnostics. These results collectively demonstrate the high performance and adaptability of this diagnostic platform, highlighting its significant promise for future applications in the rapid and sensitive detection of diverse viral pathogens.},
}

*Quantum Dots/chemistry
*RNA, Viral/analysis/genetics
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*Influenza A Virus, H1N1 Subtype/genetics/isolation & purification
SARS-CoV-2/genetics/isolation & purification
Limit of Detection
Humans
Nucleic Acid Amplification Techniques
COVID-19/diagnosis

@article {pmid42153625,
year = {2026},
author = {Djouad, ME and Bendaha, MEA and Belaouni, HA and Bensalah, F},
title = {Comparative genomics of Lacticaseibacillus paracasei strain 69Bis reveals encoded probiotic traits and genomic specificities.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnag060},
pmid = {42153625},
issn = {1574-6968},
abstract = {This study provides a comprehensive characterization of Lacticaseibacillus paracasei strain 69Bis, isolated from traditional fermented goat milk collected in Biskra (southeast Algeria). Phenotypic investigations revealed robust probiotic traits across all isolates, including NaCl tolerance at 10%, acid resistance at pH 2.0 for 3 h, bile tolerance at 0.3%, and survival in simulated gastrointestinal digestion. Adhesion properties included high auto-aggregation and hydrophobicity. The strain exhibited potent antimicrobial activity, particularly against Pseudomonas aeruginosa. Whole-genome sequencing yielded a genome of 2.9 Mbp, with 46.38% GC and 2,969 protein-coding sequences, enriched in carbohydrate and amino acid metabolism. Functional annotation highlighted genomic features associated with probiotic functions, including adhesion factors (acm, fimA, lapA), stress response pathways, and exopolysaccharide synthesis (eps gene family). Three biosynthetic gene clusters were detected, encoding bacteriocins and terpene synthesis. The absence of known acquired antimicrobial resistance genes and the susceptibility to all tested antibiotics indicate a favourable safety profile. The presence of CRISPR-Cas systems was confirmed, which may contribute to genomic stability and protection against phages. Comparative genomics, through genome-based taxonomy, confirmed the assignment to Lacticaseibacillus paracasei. Pan-genome analysis of 122 genomes from the same species (including 69Bis) revealed an open pan-genome (6,878 genes, 25.76% core), along with several unique and rare determinants linked to probiotic traits in 69Bis. Overall, these results position strain 69Bis as a promising candidate for use in functional foods, particularly in the context of nutrition in arid regions.},
}

@article {pmid42153634,
year = {2026},
author = {Shang, W and Lyu, Z and Chen, G},
title = {Harnessing Nature's Algorithm: From Test Tubes to Autonomous In Vivo Evolution.},
journal = {Biotechnology journal},
volume = {21},
number = {5},
pages = {e70237},
doi = {10.1002/biot.70237},
pmid = {42153634},
issn = {1860-7314},
support = {2024JJ4053//Hunan Provincial Natural Science Foundation/ ; 2025PT5021//Scientific Research Program of FuRong Laboratory/ ; },
mesh = {*Directed Molecular Evolution/methods ; *Algorithms ; Machine Learning ; Mutagenesis ; CRISPR-Cas Systems ; },
abstract = {Directed evolution (DE) enables the engineering of biomolecules without prior structural knowledge. However, traditional step-wise DE is constrained by limited screening throughput. To more efficiently navigate epistatic fitness landscapes, the field is increasingly adopting autonomous, continuous in vivo evolution systems. This review critically examines the molecular architectures and engineering principles driving this transition. We evaluate strategies for continuous genetic diversification-ranging from orthogonal replication systems (e.g., OrthoRep, T7-ORACLE) to CRISPR-guided mutagenesis (e.g., EvolvR)-with a focus on the fundamental trade-off between mutational load and host viability. Furthermore, we analyze the biophysical constraints of screening and the kinetic demands of coupling real-time selection with ultra-fast mutagenesis, as exemplified by phage-assisted continuous evolution (PACE). Crucially, we explore the functional integration of machine learning (ML), highlighting how active learning models and zero-shot predictions via protein language models (PLMs) can resolve epistatic complexities and mitigate the latency of next-generation sequencing. Finally, we discuss the multidimensional hardware and algorithmic bottlenecks currently impeding the realization of fully closed-loop biofoundries, and assess the strategic implications of these technologies for accelerating the engineering of complex therapeutics.},
}

*Directed Molecular Evolution/methods
*Algorithms
Machine Learning
Mutagenesis
CRISPR-Cas Systems

@article {pmid42153784,
year = {2026},
author = {Estay-Ahumada, CE and Roux, M and Ciocca, D and El-Kholti, N and Birling, MC and Rossolillo, P and Felder-Schmittbuhl, MP and Hicks, D},
title = {Abca4 Knockdown in the Cone-Rich Rodent Psammomys Obesus Leads to Stargardt's Disease-Like Progressive Retinal Degeneration.},
journal = {Investigative ophthalmology & visual science},
volume = {67},
number = {5},
pages = {47},
doi = {10.1167/iovs.67.5.47},
pmid = {42153784},
issn = {1552-5783},
mesh = {Animals ; Gerbillinae ; *ATP-Binding Cassette Transporters/genetics ; Electroretinography ; Stargardt Disease/genetics ; Disease Models, Animal ; *Retinal Cone Photoreceptor Cells/metabolism/pathology ; Tomography, Optical Coherence ; *Macular Degeneration/genetics/congenital/metabolism ; *Retinal Degeneration/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; Genetic Vectors ; Dependovirus/genetics ; },
abstract = {PURPOSE: Mutations in the gene ABCA4 coding for photoreceptor-specific ATP-binding cassette subfamily A member 4, are responsible for Stargardts disease type 1 (STGD1), the most common form of inherited macular degeneration. We recently showed that injection of viral vectors expressing CRISPR/Cas9 tools directed against Abca4 into young Sand Rat (Psammomys obesus) eyes led to extensive structural and functional retinal degeneration resembling STGD1. Here we provide further evidence that this is highly likely due to specific knockdown of Abca4 and not off-target errors.

METHODS: We performed subretinal injections of Adeno-Associated Virus-CRISPR/Cas9-Abca4 constructs into postnatal (∼P15) Psammomys obesus. Eyes were examined by noninvasive exploration (ocular coherence tomography, fundus and electroretinography) at 15-60 days after injection. Additionally, subgroups were euthanized over the same time period, and ocular tissue was used for immunochemical analyses.

RESULTS: RNAscope analysis of injected eyes showed knockdown of Abca4, rhodopsin and cone transducin mRNA in transduced regions; neighbouring tissue that was not transduced showed robust expression of all three. Injection of control AAV, expressing CAS9 alone, induced only mild glial activation. Statistically significant decreases in visual responses to light flashes were only seen in eyes injected with the fully active CRISPR/Cas9-Abca4 probes.

CONCLUSIONS: Taken together, these data rule out off-target effects as responsible for the observed degeneration, and indicate that Psammomys obesus faithfully recapitulates many of the features seen in human STGD1, thus positioning it as an important research opportunity to further explore genotype-phenotype relationships and test putative therapeutic approaches.},
}

Animals
Gerbillinae
*ATP-Binding Cassette Transporters/genetics
Electroretinography
Stargardt Disease/genetics
Disease Models, Animal
*Retinal Cone Photoreceptor Cells/metabolism/pathology
Tomography, Optical Coherence
*Macular Degeneration/genetics/congenital/metabolism
*Retinal Degeneration/genetics/metabolism
CRISPR-Cas Systems
Gene Knockdown Techniques
Genetic Vectors
Dependovirus/genetics

@article {pmid42154426,
year = {2026},
author = {Mayuri, K and Saravanan, KM and Somala, CS and Selvaraj, C and Anand, T and Vickram, S},
title = {CRISPR-Cas and Infectious Diseases: A Decade of Translational Advances in Molecular Biotechnology.},
journal = {Biochemical genetics},
volume = {},
number = {},
pages = {},
pmid = {42154426},
issn = {1573-4927},
abstract = {CRISPR-Cas systems have emerged as a versatile tool for diagnosing, treating, and preventing infectious diseases. This review highlights translational advancements in CRISPR-Cas-based applications, concentrating on the past decades in diagnostics, therapeutic genome editing, and vaccine development. The article highlights key platforms like DETECTR and SHERLOCK, which enable rapid, sensitive pathogen detection, and explores CRISPR-Cas9 systems in therapeutic strategies for directly targeting viral genomes and combating antimicrobial resistance. It also examines the role of CRISPR-Cas9 in engineering live-attenuated and personalized neoantigen vaccines. Principal findings demonstrate a clear progression from experimental proof-of-concept to preclinical applications primarily in CRISPR-based diagnostics and the engineering of live-attenuated vaccine candidates, whereas translation in CRISPR-based therapeutics and personalized neoantigen vaccines for infectious diseases remains at earlier, more exploratory stages. CRISPR-based diagnostics have progressed further toward clinical evaluation than therapeutics due to delivery and safety constraints, while personalized neoantigen vaccines are included mainly as an emerging, comparative concept for infectious diseases rather than a mature application. This review uniquely integrates CRISPR-based diagnostics, therapeutics, and vaccine development within a single infectious disease framework, critically assesses their current maturity, and systematically highlights technical, regulatory, and ethical barriers alongside realistic future priorities. The review concludes that while CRISPR-Cas holds transformative potential for infectious disease management, significant challenges in delivery efficiency, off-target effects, and ethical regulation must be addressed to ensure safe and equitable clinical translation.},
}

@article {pmid41884948,
year = {2026},
author = {Ekstrand, F and Ruhrmann, S and Bacos, K and Bartel, S and Jellema, P and Rots, MG and Ling, C and Prinz, CN},
title = {Nanopore Electroporation: A New Delivery Method Within the Field of Epigenetic Editing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {28},
pages = {e13858},
doi = {10.1002/smll.202513858},
pmid = {41884948},
issn = {1613-6829},
support = {682206//ERC CoG NanoPokers/ ; //Swedish Research Council (VR)/ ; //Novo Nordisk foundation/ ; //Region Skåne/ ; Dnr 2009-1039//Strategic Research Area Exodiab/ ; //The Crafoord Foundation/ ; //NanoLund/ ; //The Swedish Diabetes Foundation/ ; ITM-17//The Swedish foundation for Strategic Research/ ; IRC15-0067//The Swedish foundation for Strategic Research/ ; },
mesh = {*Electroporation/methods ; *Nanopores ; *Gene Editing/methods ; *Epigenesis, Genetic/genetics ; CRISPR-Cas Systems/genetics ; Animals ; Humans ; Insulin-Secreting Cells/metabolism ; Insulin/genetics/metabolism ; Epigenome Editing ; },
abstract = {Epigenetic modifications influence gene expression and contribute to type 2 diabetes (T2D), but establishing causality requires targeted modulation of specific genes. CRISPR-dCas9-based tools offer this potential, yet β-cells are notoriously difficult to transfect, and efficient, non-viral delivery methods are lacking. Here, we developed nanopore-mediated electroporation to deliver a CRISPR interference (CRISPRi) system to clonal INS1 β-cells, achieving targeted downregulation of insulin expression. Cells were seeded atop a nanopore substrate with CRISPRi plasmids in solution below. Mild electric pulses generated transient nanoscale pores in the membrane, enabling electrophoretic delivery of plasmids into the cytosol while preserving high cell viability. The CRISPRi system comprised the transcriptional repressor Krueppel-associated Box Domain (KRAB) fused to an inactive Cas9 (dCas9), guided to the transcription start site of the insulin-1 gene (Ins1) by a single guide RNA (sgRNA). After transfection, Ins1 expression was significantly reduced, demonstrating effective modulation of gene expression in this difficult-to-transfect cell type. This nanopore electroporation approach provides a robust, safe, and efficient platform for delivering CRISPR-dCas9-based epigenetic editors in pancreatic β-cells. By enabling precise gene regulation, it opens avenues for mechanistic studies of epigenetic contributions to T2D and potentially other challenging cell systems.},
}

*Electroporation/methods
*Nanopores
*Gene Editing/methods
*Epigenesis, Genetic/genetics
CRISPR-Cas Systems/genetics
Animals
Humans
Insulin-Secreting Cells/metabolism
Insulin/genetics/metabolism
Epigenome Editing

@article {pmid41950922,
year = {2026},
author = {Kang, T and Bleris, L},
title = {Cellular-state control using ribozyme-scaffolded miRNA-sensing and CRISPR-mediated actuation.},
journal = {Cell reports methods},
volume = {6},
number = {5},
pages = {101379},
doi = {10.1016/j.crmeth.2026.101379},
pmid = {41950922},
issn = {2667-2375},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *RNA, Catalytic/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Epithelial-Mesenchymal Transition/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Line, Tumor ; },
abstract = {Cellular transitions between states are fundamental to development, adaptation, and pathological processes, but monitoring and guiding these transitions using endogenous signals remain challenging. MicroRNAs (miRNAs) represent a powerful modality, as distinct cell states are characterized by unique miRNA expression signatures. Here, we introduce a state-specific miRNA-directed CRISPR system for detecting and responding to epithelial-to-mesenchymal transition (EMT), a critical process in development, wound healing, and cancer metastasis. This system leverages EMT-specific miRNAs to regulate activation of type II polymerase-driven ribozyme-single-guide RNA (sgRNA) constructs, which direct CRISPR-based effectors to modulate gene expression. Using this approach, we demonstrate selective elimination of cells that have undergone mesenchymal transition and dynamic filtering of cell populations. This system provides a versatile platform for precise activation of CRISPR-Cas9 effectors using endogenous, state-specific cues. Integrating miRNA signatures with CRISPR technology to monitor, modulate, and reprogram cell-state transitions paves the way for applications in regenerative medicine, cancer therapy, and beyond.},
}

*MicroRNAs/genetics/metabolism
Humans
*RNA, Catalytic/metabolism/genetics
*CRISPR-Cas Systems/genetics
Epithelial-Mesenchymal Transition/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Line, Tumor

@article {pmid42000058,
year = {2026},
author = {Carvalho, C and Hérivaux, A and Mello, T and Bastide, F and Thomas, O and Guillemette, T and Saulnier, P and Souza Dos Santos, AL and Papon, N and Bouchara, JP},
title = {TRXR2, a thioredoxin reductase-encoding gene, contributes to protection against the oxidative stress and virulence in Scedosporium apiospermum.},
journal = {Microbial pathogenesis},
volume = {216},
number = {},
pages = {108498},
doi = {10.1016/j.micpath.2026.108498},
pmid = {42000058},
issn = {1096-1208},
mesh = {*Oxidative Stress ; *Scedosporium/pathogenicity/genetics/enzymology/drug effects ; Virulence/genetics ; Animals ; Hyphae ; *Thioredoxin-Disulfide Reductase/genetics/metabolism ; *Thioredoxin Reductase 2/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Fungal Proteins/genetics/metabolism ; Spores, Fungal ; Cell Wall/metabolism/ultrastructure ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Antifungal Agents/pharmacology ; Larva/microbiology ; Gene Expression Regulation, Fungal ; Benzene Derivatives ; },
abstract = {Scedosporium apiospermum has received an increased attention over the past decades, especially because of its low susceptibility to current antifungals and its clinical relevance in cystic fibrosis. Nevertheless, little is still known about its pathogenic mechanisms. During the inflammatory reaction, macrophages and neutrophils release antimicrobial compounds, especially reactive oxygen species (ROS). To cope with ROS, pathogens have developed various strategies, including synthesis of some immunoprotective secondary metabolites, and enzymatic mechanisms relying on antioxidant enzymes. A recent transcriptomic study showed that the TRXR2 gene, encoding one of its two thioredoxin reductases (TrxRs), is overexpressed upon exposure to oxidative stress. To investigate the role of TrxR2, the encoding gene was disrupted using the CRISPR-Cas9 technology. An ultrastructural study revealed that TRXR2 gene knock-out induced the loss of the outer cell wall layer of hyphae, thereby increasing their surface hydrophobicity. The effects on the tolerance of the fungus to chemically-induced oxidative stresses were also investigated, revealing a higher susceptibility to cumene hydroperoxide. Additionally, an overexpression of BoyT gene (encoding the other TrxR) was seen as a compensatory mechanism, but bioinformatic analysis also suggested distinct roles for the two TrxRs in relation with distinct subcellular localizations. Finally, conidia exhibited a reduced capacity to adhere to epithelial cells and a diminished virulence in the Galleria mellonella model. In conclusion, these results suggest that TrxR2 plays a contributory role in the pathogenicity of S. apiospermum, but is not a major determinant of its virulence, which justifies future studies on the other components of the thioredoxin system.},
}

*Oxidative Stress
*Scedosporium/pathogenicity/genetics/enzymology/drug effects
Virulence/genetics
Animals
Hyphae
*Thioredoxin-Disulfide Reductase/genetics/metabolism
*Thioredoxin Reductase 2/genetics/metabolism
Reactive Oxygen Species/metabolism
Fungal Proteins/genetics/metabolism
Spores, Fungal
Cell Wall/metabolism/ultrastructure
CRISPR-Cas Systems
Gene Knockout Techniques
Antifungal Agents/pharmacology
Larva/microbiology
Gene Expression Regulation, Fungal
Benzene Derivatives

@article {pmid42019502,
year = {2026},
author = {Vereecke, N and Behrmann, M and Khare, A and Dekker, JP},
title = {Improved Cas9-targeted nanopore sequencing facilitates ultra-deep analysis of genomic variation.},
journal = {Cell reports methods},
volume = {6},
number = {5},
pages = {101410},
doi = {10.1016/j.crmeth.2026.101410},
pmid = {42019502},
issn = {2667-2375},
mesh = {*High-Throughput Nucleotide Sequencing/methods ; *Nanopore Sequencing/methods ; Staphylococcus aureus/genetics ; *CRISPR-Cas Systems/genetics ; Bacteroides fragilis/genetics ; *Nanopores ; *Genomics/methods ; *CRISPR-Associated Protein 9/metabolism ; Genome, Bacterial ; *Genetic Variation ; },
abstract = {We present nanopore adapter-enriched Cas9-targeted sequencing (nAECATS), a method permitting inexpensive, ultra-deep, selective long-read sequencing of targeted regions in native, unamplified DNA. This method modifies previous Cas9-targeted sequencing approaches through the inclusion of a bead-based capture step that exploits the poly(T)8 stretch within the R10.4.1 ligation adapter for additional purification. Testing on a 10 kb Bacteroides fragilis genomic region achieved 90% on-target yield with 51,000× coverage from a single Flongle flow cell (353-fold increase versus whole-genome sequencing). Applied to a variable-length (>20 kb) Staphylococcus aureus genomic target containing dynamic gene amplifications conferring antibiotic resistance, nAECATS achieved 46,000× coverage and 74% on-target yield, revealing 2-4 tandem amplifications at single-cell resolution. While efficiency decreased with longer fragments (up to 41 kb), substantial enrichment improvements were demonstrated. We anticipate that nAECATS ultra-deep sequencing will find broad application for a wide range of biological questions in pro- and eukaryotic (epi)genomics and microbiology.},
}

*High-Throughput Nucleotide Sequencing/methods
*Nanopore Sequencing/methods
Staphylococcus aureus/genetics
*CRISPR-Cas Systems/genetics
Bacteroides fragilis/genetics
*Nanopores
*Genomics/methods
*CRISPR-Associated Protein 9/metabolism
Genome, Bacterial
*Genetic Variation

@article {pmid42053710,
year = {2026},
author = {Panchuk, IO and Grigorieva, OV and Kurshakova, EV and Nagieva, SE and Shchagina, OA and Levchenko, OA and Pozhitnova, VO and Voronina, ES and Tabakov, VY and Smirnikhina, SA and Lavrov, AV},
title = {Generation of an Induced Pluripotent Stem Cell Line from a Duchenne Muscular Dystrophy Patient Carrying the DMD p.Ser429Ter (c.1286C>G) Nonsense Mutation.},
journal = {Bulletin of experimental biology and medicine},
volume = {180},
number = {4},
pages = {548-554},
pmid = {42053710},
issn = {1573-8221},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Muscular Dystrophy, Duchenne/genetics/pathology/metabolism ; Kruppel-Like Factor 4 ; *Codon, Nonsense/genetics ; Cell Differentiation ; *Dystrophin/genetics/metabolism ; Cell Line ; Male ; Fibroblasts/metabolism/cytology ; Octamer Transcription Factor-3/genetics/metabolism ; CRISPR-Cas Systems ; Cellular Reprogramming ; Sendai virus/genetics ; },
abstract = {An induced pluripotent stem cell (iPSC) line was successfully generated from dermal fibroblasts of a patient with Duchenne muscular dystrophy (DMD) harboring the pathogenic nonsense variant c.1286C>G (p.Ser429Ter) in the DMD gene using non-integrating Sendai virus reprogramming. The iPSC clone exhibited typical pluripotent stem cell morphology, expressed key pluripotency markers (OCT4, SSEA4, NANOG, and TRA-1-60), and retained trilineage differentiation potential. The cell line had a normal karyotype, and elimination of reprogramming vectors (OCT3/4, SOX2, KLF4, and c-MYC) was confirmed. This isogenic cell model provides a valuable platform for investigating DMD pathogenesis associated with this specific mutation and for developing targeted therapeutic approaches, including CRISPR/Cas9-mediated gene correction.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology/pathology
*Muscular Dystrophy, Duchenne/genetics/pathology/metabolism
Kruppel-Like Factor 4
*Codon, Nonsense/genetics
Cell Differentiation
*Dystrophin/genetics/metabolism
Cell Line
Male
Fibroblasts/metabolism/cytology
Octamer Transcription Factor-3/genetics/metabolism
CRISPR-Cas Systems
Cellular Reprogramming
Sendai virus/genetics

@article {pmid42080223,
year = {2026},
author = {Zhao, S and Huang, S and Li, N and Wang, N and Yu, Z and Liang, X and Yang, Z and Guo, H},
title = {Piperazine-Derived Diamine Lipid Nanoparticles Targeting to the Liver for Delivering Clustered Regularly Interspaced Short Palindromic Repeat Editing of PCSK9 to Durably Maintain Plasmatic Low-Density Lipoprotein Cholesterol in Low Levels.},
journal = {ACS applied bio materials},
volume = {9},
number = {10},
pages = {4465-4476},
doi = {10.1021/acsabm.6c00032},
pmid = {42080223},
issn = {2576-6422},
mesh = {*Proprotein Convertase 9/genetics/metabolism ; *Nanoparticles/chemistry ; Humans ; Animals ; Mice ; *Gene Editing ; *Liver/metabolism/drug effects ; *Cholesterol, LDL/blood ; *Piperazine/chemistry ; *Lipids/chemistry ; *Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Particle Size ; Materials Testing ; Mice, Inbred C57BL ; Hep G2 Cells ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cardiovascular diseases remain a leading cause of morbidity and mortality worldwide, and durable suppression of low-density lipoprotein cholesterol (LDL-C) via genome editing represents a promising therapeutic strategy. Here, we report the rational design of a piperazine-derived bis-tertiary amine ionizable lipid (M10) and its optimized lipid nanoparticle formulation (M10-F4) for efficient and liver-targeted CRISPR/Cas9 delivery. Compared with benchmark lipids such as SM-102, M10 enables a reduced molar ratio of ionizable lipid while maintaining high nucleic acid encapsulation efficiency (>80%) and forming stable, spherical nanoparticles. The piperazine-based multi-cationic core confers an optimized apparent pKa of 6.56, facilitating endosomal escape through enhanced protonation under acidic conditions. Confocal microscopy in HepG2 and Huh-7 cells reveals efficient cellular uptake and enhanced cytosolic release of RNA cargo with minimal lysosomal entrapment. In vivo, M10-F4 exhibits strong liver tropism following systemic administration. A single intravenous dose mediates robust PCSK9 gene editing in C57BL/6 mice, resulting in sustained reductions of circulating PCSK9 and LDL-C levels under both normal and high-fat diet conditions for up to 48 days, accompanied by decreased hepatic PCSK9 expression. Importantly, acute safety evaluation in BALB/c mice showed no obvious signs of short-term systemic toxicity, including stable body weight, minimal induction of inflammatory cytokines (IL-6, TNF-α, and CXCL-10), no significant elevation of liver enzymes, and normal gross organ morphology. Collectively, this work establishes M10-F4 as a molecularly engineered, liver-targeted LNP platform in which ionizable lipid architecture and formulation composition enable effective in vivo genome editing with favorable tolerability, highlighting the importance of rational materials design at the materials-bio interface for cardiometabolic gene-editing applications.},
}

*Proprotein Convertase 9/genetics/metabolism
*Nanoparticles/chemistry
Humans
Animals
Mice
*Gene Editing
*Liver/metabolism/drug effects
*Cholesterol, LDL/blood
*Piperazine/chemistry
*Lipids/chemistry
*Biocompatible Materials/chemistry/pharmacology/chemical synthesis
Particle Size
Materials Testing
Mice, Inbred C57BL
Hep G2 Cells
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42119627,
year = {2026},
author = {Guo, K and Yu, S and Yang, N and Wang, X and Liu, Q and Fu, Y and Liu, J},
title = {Serial passage is associated with virulence attenuation in Neospora caninum and transcriptomic remodeling of invasion- and chronic-stage-associated pathways.},
journal = {Microbial pathogenesis},
volume = {216},
number = {},
pages = {108549},
doi = {10.1016/j.micpath.2026.108549},
pmid = {42119627},
issn = {1096-1208},
mesh = {*Neospora/pathogenicity/genetics/growth & development ; Virulence/genetics ; Animals ; Gene Expression Profiling ; *Transcriptome ; Serial Passage ; Cattle ; Protozoan Proteins/genetics ; Host-Parasite Interactions/genetics ; Coccidiosis/parasitology/veterinary ; CRISPR-Cas Systems ; },
abstract = {N. caninum is an obligate intracellular apicomplexan parasite and a major cause of abortion in cattle worldwide. However, the molecular basis underlying virulence attenuation during long-term in vitro passage remains poorly understood. Here, we performed comparative transcriptomic profiling between the virulent Nc1-09 strain and its attenuated derivative Nc1 to investigate transcriptional changes associated with virulence reduction. A total of 487 differentially expressed genes (DEGs) were identified. Functional enrichment analyses revealed prominent changes in pathways related to protein modification, host-parasite interaction, motility, and metabolism. Notably, multiple members of the surface antigen glycoprotein (SRS) family displayed marked transcriptional shifts, consistent with altered host cell interaction and immune-related functions. CRISPR/Cas9-mediated disruption of three representative DEGs (NCLIV_016320, NCLIV_024950, and NCLIV_022830) did not reproduce the attenuated phenotype, suggesting that disruption of these individual genes is insufficient to account for the full attenuated phenotype under the conditions tested. Gene set enrichment analysis further demonstrated systematic downregulation of protein modification-associated pathways in the attenuated strain. Overall, the data indicate that virulence attenuation in Neospora caninum （N. caninum） is associated with coordinated transcriptional remodeling across multiple functional pathways rather than discrete gene loss.},
}

*Neospora/pathogenicity/genetics/growth & development
Virulence/genetics
Animals
Gene Expression Profiling
*Transcriptome
Serial Passage
Cattle
Protozoan Proteins/genetics
Host-Parasite Interactions/genetics
Coccidiosis/parasitology/veterinary
CRISPR-Cas Systems

@article {pmid42144912,
year = {2026},
author = {Guo, X and Tian, T and Zhou, X},
title = {Split crRNAs Enhance Cas12a Diagnostic Performance.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {10},
pages = {e70383},
doi = {10.1002/cbic.70383},
pmid = {42144912},
issn = {1439-7633},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {CRISPR RNA (crRNA) is the key guide molecule in the CRISPR-Cas12a system, directing the Cas protein to recognize target sequences. It consists of a repeat-derived stem loop that binds Cas12a and helps stabilize the ribonucleoprotein complex, and a spacer region that base-pairs with the target and determines recognition specificity. Recently, multiple studies have shown that crRNAs can be split and reassembled in vitro in diverse ways. These split-and-reconfigured strategies have enabled detection schemes that are more flexible than full-length crRNAs, cover a broader range of targets, and achieve higher signal-to-background ratios. Here, we focus on split crRNA strategies for CRISPR-Cas12a and systematically summarize existing split crRNA-based detection platforms. We outline their design principles, reaction mechanisms, and performance features, and we synthesize how these approaches improve key metrics-including target scope, sensitivity, specificity, and controllability. Finally, we discuss the major advantages and current limitations of split crRNA strategies and highlight directions for further design optimization and translational applications. Schematic overview of split crRNA strategies for enhancing CRISPR-Dx performance. These advances are mainly reflected in four aspects: broadening the target range, thereby enabling Cas12a to be applied to the detection of short RNAs, structured RNAs, and certain non-nucleic acid targets; improving sensitivity by enhancing detection signals through reassembly-dependent activation, cascade amplification, or auxiliary activation strategies; increasing specificity by strengthening the discrimination of single-nucleotide differences through stepwise recognition and conditional assembly; and enhancing controllability by achieving on-demand activation of Cas12a activity via light, enzymes, small molecules, or proximity effects.},
}

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

@article {pmid42145154,
year = {2026},
author = {Dong, R and Zhang, Y and Yuan, G and Yue, X and Ding, Y and Zeng, X and Xiao, H},
title = {A Novel Strategy to Produce CAR-γδ T Cells via Site-Directed Gene Integration by a Combination of CRISPR/Cas9 and AAV.},
journal = {Cancer medicine},
volume = {15},
number = {5},
pages = {e71918},
doi = {10.1002/cam4.71918},
pmid = {42145154},
issn = {2045-7634},
support = {82170141//National Natural Science Foundation of China/ ; 82470209//National Natural Science Foundation of China/ ; },
mesh = {*Dependovirus/genetics ; Animals ; *CRISPR-Cas Systems ; Humans ; Mice ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism ; *T-Lymphocytes/immunology/metabolism ; Genetic Vectors/genetics ; Cell Line, Tumor ; },
abstract = {Chimeric antigen receptor (CAR)-αβ T cells are commonly employed in tumor therapy but hindered by some limitations. γδ T cells are promising substrates for CAR therapy for their major histocompatibility complex (MHC)-unrestricted recognition manner and innate immune function. Here, we established a novel method for generating CAR-γδ T cells. We utilized the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) method to interrupt the TCR delta chain constant region (TRDC) sequence, followed by the site-directed insertion of the CAR sequence into the TRDC locus via homologous complementation mediated by adeno-associated virus (AAV) gene delivery. We optimized electroporation parameters for Cas9/ribonucleoproteins (RNP) delivery and infection conditions for CAR-gene carrying AAV in γδ T cells. These optimizations facilitated efficient TCR knockout and site-directed CAR insertion, ultimately yielding functional CAR-γδ T cells. In vitro experiments demonstrated that these newly prepared CAR-γδ T cells could stimulate cytokine production, kill tumor cells as well as exhibit robust proliferative potential and memory-like phenotype. These state-of-the-art CAR-γδ T cells could reduce tumor burden and extend the survival period of tumor-bearing mice.},
}

*Dependovirus/genetics
Animals
*CRISPR-Cas Systems
Humans
Mice
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Receptors, Antigen, T-Cell, gamma-delta/genetics/immunology/metabolism
*T-Lymphocytes/immunology/metabolism
Genetic Vectors/genetics
Cell Line, Tumor

@article {pmid42145193,
year = {2026},
author = {Yin, XY and Wang, SZ},
title = {High-throughput identification methods of genomic functional variation in post-GWAS era and their application in agricultural animals.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {5},
pages = {451-470},
doi = {10.16288/j.yczz.25-319},
pmid = {42145193},
issn = {0253-9772},
mesh = {Animals ; *Genome-Wide Association Study/methods ; *Genetic Variation ; Gene Editing ; *Genomics/methods ; CRISPR-Cas Systems ; },
abstract = {Genome-wide association study (GWAS) has identified a large number of genetic variations that are significantly associated with human diseases and animal and plant economic traits. However, the majority of these variants are located in non-coding regions of the genome, which makes it challenging to accurately pinpoint functional variants of biological significance from a vast number of candidate loci. In the post-GWAS era, high-throughput analytical approaches, such as high-throughput reporter gene analysis, CRISPR/Cas9-based gene editing technologies, and epigenetic analyses, have become powerful tools for systematically uncovering functional variants in the genome. These methods not only enable efficient identification of functional variants but also help elucidate the mechanisms through which they regulate gene expression, thereby clarifying the molecular basis underlying trait formation or disease pathogenesis. In this review, we systematically summarize current high-throughput strategies for identifying functional genomic variants, highlight their applications and recent advances in major agricultural animal species, and outline future research directions, with the aim of providing a reference for subsequent studies in related fields.},
}

Animals
*Genome-Wide Association Study/methods
*Genetic Variation
Gene Editing
*Genomics/methods
CRISPR-Cas Systems