@article {pmid40121084,
year = {2025},
author = {Zhao, A and Chan, MM},
title = {Cloning and validating systems for high throughput molecular recording.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {453-473},
doi = {10.1016/bs.mie.2025.01.015},
pmid = {40121084},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems ; *Cloning, Molecular/methods ; Humans ; *Gene Editing/methods ; Animals ; Single-Cell Analysis/methods ; Cell Lineage/genetics ; Mice ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Molecular recording technologies record and store information about cellular history. Lineage tracing is one form of molecular recording and produces information describing cellular trajectories during mammalian development, differentiation and maintenance of adult stem cell niches, and tumor evolution. Our molecular recorder technology utilizes CRISPR-Cas9 barcode editing to generate mutations in genomically integrated, engineered DNA cassettes, which are read out by single-cell RNA sequencing and used to produce high-resolution lineage trees. Here, we describe optimized cloning and validation procedures to construct the molecular recorder lineage tracing system. We include information on considerations of technology design, cloning procedures, the generation of lineage tracing cell lines, and time course experiments to assess their performance.},
}

*CRISPR-Cas Systems
*Cloning, Molecular/methods
Humans
*Gene Editing/methods
Animals
Single-Cell Analysis/methods
Cell Lineage/genetics
Mice
High-Throughput Nucleotide Sequencing/methods

@article {pmid40121083,
year = {2025},
author = {Gould, SI and Sánchez-Rivera, FJ},
title = {Using Prime Editing Guide Generator (PEGG) for high-throughput generation of prime editing sensor libraries.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {437-451},
doi = {10.1016/bs.mie.2025.01.006},
pmid = {40121083},
issn = {1557-7988},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Software ; Gene Library ; Humans ; High-Throughput Screening Assays/methods/instrumentation ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Prime editing enables the generation of nearly any small genetic variant. However, the process of prime editing guide RNA (pegRNA) design is challenging and requires automated computational design tools. We developed Prime Editing Guide Generator (PEGG), a fast, flexible, and user-friendly Python package that enables the rapid generation of pegRNA and pegRNA-sensor libraries. Here, we describe the installation and use of PEGG (https://pegg.readthedocs.io) to rapidly generate custom pegRNA-sensor libraries for use in high-throughput prime editing screens.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
CRISPR-Cas Systems
Software
Gene Library
Humans
High-Throughput Screening Assays/methods/instrumentation
High-Throughput Nucleotide Sequencing/methods

@article {pmid40121082,
year = {2025},
author = {Liu, C and Cheng, S and Zhu, J and Zhou, L and Chen, J},
title = {A quick guide to evaluating prime editing efficiency in mammalian cells.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {419-436},
doi = {10.1016/bs.mie.2025.01.016},
pmid = {40121082},
issn = {1557-7988},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; Animals ; Transfection/methods ; Core Binding Factor Alpha 2 Subunit/genetics ; Plasmids/genetics ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {According to the Clinvar database, modeling the diseases associated with pathogenic mutations requires the installation of base substitutions, small insertions or deletions. Prime editor (PE) was recently developed to precisely install any base substitutions and/or small insertions/deletions (indels) in mammalian cells and animals without requiring DSBs or donor DNA templates. PE also offers greater editing and targeting flexibility compared to other precision CRISPR editing methods because the versatile editing information is encoded in the reverse-transcription template of its prime editing guide RNA. However, optimal PE system selection and experimental design can be complex, and there are various factors that can affect PE efficiency. This chapter serves as a rapid entry-level guideline for the application of PE, providing an experimental framework for using PE at a specific genomic locus. RUNX1 was selected as a representative target site to illustrate the detailed methodology for constructing PE plasmids and the process of transfecting these plasmids into 293FT cells. We further examined the efficiency of PE-mediated genome editing in mammalian cells by using next-generation sequencing.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells
Animals
Transfection/methods
Core Binding Factor Alpha 2 Subunit/genetics
Plasmids/genetics
High-Throughput Nucleotide Sequencing/methods

@article {pmid40121081,
year = {2025},
author = {Hibshman, GN and Taylor, DW},
title = {Visualizing the conformational landscape of CRISPR-Cas9 through kinetics-informed structural studies.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {41-53},
doi = {10.1016/bs.mie.2025.01.004},
pmid = {40121081},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems ; *Cryoelectron Microscopy/methods ; Kinetics ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *Gene Editing/methods ; Protein Conformation ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; DNA Cleavage ; Humans ; DNA/chemistry/genetics/metabolism ; },
abstract = {CRISPR-Cas9 has transformed genome editing through its programmability and versatility. Its DNA cleavage activity involves dynamic conformational changes during gRNA binding, DNA recognition, R-loop formation, and endonuclease activation. Understanding these molecular transitions is critical for improving the specificity and efficiency of Cas9, but this remains challenging precisely due to these rapid structural rearrangements. Early structural studies provided foundational insights but were limited to static states under catalytically inactive conditions. Cryo-EM has since enabled visualization of the dynamic nature of active Cas9, by enriching for specific conformations. This chapter introduces a kinetics-informed cryo-EM approach to capture the stepwise activation of Cas9 in real time. With thorough kinetic analyses, such as stopped-flow measurements of R-loop formation, we describe how to identify optimal timepoints to visualize key conformational states with cryo-EM. Integration of kinetic and structural data enables precise mapping of the conformational landscape of Cas9 and other dynamic enzymes, advancing our understanding of their molecular mechanisms and providing a framework for engineering enhanced variants.},
}

*CRISPR-Cas Systems
*Cryoelectron Microscopy/methods
Kinetics
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
*Gene Editing/methods
Protein Conformation
RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
DNA Cleavage
Humans
DNA/chemistry/genetics/metabolism

@article {pmid40121080,
year = {2025},
author = {Zhang, H and Ji, Q},
title = {Prime editing in bacteria with BacPE.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {405-418},
doi = {10.1016/bs.mie.2025.01.026},
pmid = {40121080},
issn = {1557-7988},
mesh = {*Escherichia coli/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Bacterial ; },
abstract = {Programmable genome editing technologies have revolutionized the ability of researchers to alter the genomes of microorganisms in a straightforward and efficient manner, significantly advancing the field of microbiology. To date, several CRISPR-Cas-based genome-editing systems have been developed for use in E. coli, including CRISPR/Cas9, base editing, and prime editing technologies. In this chapter, we describe the design and experimental application of BacPE, a variant of prime editing technology optimized for E. coli. BacPE facilitates the introduction of point mutations, insertions, and deletions without the need for double-strand DNA breaks. We demonstrate that BacPE is a powerful tool for genome editing in E. coli and highlight its potential applicability to other bacterial species.},
}

*Escherichia coli/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Genome, Bacterial

@article {pmid40121079,
year = {2025},
author = {Osgood, NRB and Zawalick, NM and Sawyer, CB and Cowan, QT and Gu, S and Mawson, SJ and Ranzau, BL and Li, L and Gymrek, M and Goren, A and Komor, AC},
title = {Genome editing with programmable base editors in human cells.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {351-404},
doi = {10.1016/bs.mie.2025.01.001},
pmid = {40121079},
issn = {1557-7988},
mesh = {Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; Transfection/methods ; Plasmids/genetics ; HEK293 Cells ; Genome, Human ; },
abstract = {Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.},
}

Humans
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
Transfection/methods
Plasmids/genetics
HEK293 Cells
Genome, Human

@article {pmid40121078,
year = {2025},
author = {Mach, RQ and Miller, SM},
title = {Bacterial directed evolution of CRISPR base editors.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {317-350},
doi = {10.1016/bs.mie.2025.01.003},
pmid = {40121078},
issn = {1557-7988},
mesh = {*Gene Editing/methods ; *Directed Molecular Evolution/methods ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Base editing and other precision editing agents have transformed the utility and therapeutic potential of CRISPR-based genome editing. While some native enzymes edit efficiently with their nature-derived function, many enzymes require rational engineering or directed evolution to enhance the compatibility with mammalian cell genome editing. While many methods of engineering and directed evolution exist, plate-based discrete evolution offers an ideal balance between ease of use and engineering power. Here, we describe a detailed method for the bacterial directed evolution of CRISPR base editors that compounds technical ease with flexibility of application.},
}

*Gene Editing/methods
*Directed Molecular Evolution/methods
*CRISPR-Cas Systems
Escherichia coli/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40121077,
year = {2025},
author = {McAndrew, MJ and King, MB and Lapinaite, A},
title = {Preparation of high-purity RNPs of CRISPR-based DNA base editors.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {277-315},
doi = {10.1016/bs.mie.2025.01.019},
pmid = {40121077},
issn = {1557-7988},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; DNA/genetics/chemistry/isolation & purification ; Ribonucleoproteins/genetics ; },
abstract = {Since their introduction, CRISPR-based DNA base editors (BEs) have become essential in the field of precision genome editing, revolutionizing the correction of pathogenic SNPs for both basic research and therapeutic applications. As this technology advances, more laboratories are implementing these tools into their workflow. The delivery of BEs as BE-guide RNA complexes (RNPs), rather than as mRNA or plasmids, has been shown to exhibit lower off-target effects, establishing it as the preferred method of delivery. However, there are no protocols describing in detail how to obtain high-purity and highly active BE RNPs. Here, we offer a comprehensive guide for the expression, purification, RNP reconstitution, and in vitro activity assessment of TadA-based BEs. The protocol includes guidance on performing activity assays using commercial denaturing gels, which is convenient and uses standard molecular biology equipment. This allows for rapid quality control testing of reconstituted BE RNPs prior to more expensive and time-consuming in vivo genome editing experiments. Overall, this protocol aims to empower more laboratories to generate tailored BE RNPs for diverse in vitro and in vivo applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
DNA/genetics/chemistry/isolation & purification
Ribonucleoproteins/genetics

@article {pmid40121076,
year = {2025},
author = {Grimm, MS and Myhrvold, C},
title = {Using CRISPR for viral nucleic acid detection.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {245-275},
doi = {10.1016/bs.mie.2025.01.031},
pmid = {40121076},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems ; Humans ; *RNA, Viral/genetics/isolation & purification/analysis ; Viruses/genetics/isolation & purification ; },
abstract = {Pathogenic microorganisms, such as viruses, have threatened human health and will continue to contribute to future epidemics and pandemics, highlighting the importance of developing effective diagnostics. To contain viral outbreaks within populations, fast and early diagnosis of infected individuals is essential. Although current standard methods are highly sensitive and specific, like RT-qPCR, some can have slow turnaround times, which can hinder the prevention of viral transmission. The discovery of CRISPR-Cas systems in bacteria and archaea initially revolutionized the world of genome editing. Intriguingly, CRISPR-Cas enzymes also have the ability to detect nucleic acids with high sensitivity and specificity, which sparked the interest of researchers to also explore their potential in diagnosis of viral pathogens. In particular, the CRISPR-Cas13 system has been used as a tool for detecting viral nucleic acids. Cas13's capability to detect both target RNA and non-specific RNAs has led to the development of detection methods that leverage these characteristics through designing specific detection read-outs. Optimization of viral sample collection, amplification steps and the detection process within the Cas13 detection workflow has resulted in assays with high sensitivity, rapid turnaround times and the capacity for large-scale implementation. This review focuses on the significant innovations of various CRISPR-Cas13-based viral nucleic acid detection methods, comparing their strengths and weaknesses while highlighting Cas13's great potential as a tool for viral diagnostics.},
}

*CRISPR-Cas Systems
Humans
*RNA, Viral/genetics/isolation & purification/analysis
Viruses/genetics/isolation & purification

@article {pmid40121075,
year = {2025},
author = {Omura, SN and Nureki, O},
title = {General and robust sample preparation strategies for cryo-EM studies of CRISPR-Cas9 and Cas12 enzymes.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {23-39},
doi = {10.1016/bs.mie.2025.01.052},
pmid = {40121075},
issn = {1557-7988},
mesh = {*Cryoelectron Microscopy/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/chemistry/metabolism ; Gene Editing/methods ; CRISPR-Associated Protein 9/genetics/chemistry/metabolism ; Bacterial Proteins/genetics/chemistry/metabolism ; Endodeoxyribonucleases ; },
abstract = {Cas9 and Cas12 are RNA-guided DNA endonucleases derived from prokaryotic CRISPR-Cas adaptive immune systems that have been repurposed as versatile genome-engineering tools. Computational mining of genomes and metagenomes has expanded the diversity of Cas9 and Cas12 enzymes that can be used to develop versatile, orthogonal molecular toolboxes. Structural information is pivotal to uncovering the precise molecular mechanisms of newly discovered Cas enzymes and providing a foundation for their application in genome editing. In this chapter, we describe detailed protocols for the preparation of Cas9 and Cas12 enzymes for cryo-electron microscopy. These methods will enable fast and robust structural determination of newly discovered Cas9 and Cas12 enzymes, which will enhance the understanding of diverse CRISPR-Cas effectors and provide a molecular framework for expanding CRISPR-based genome-editing technologies.},
}

*Cryoelectron Microscopy/methods
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/chemistry/metabolism
Gene Editing/methods
CRISPR-Associated Protein 9/genetics/chemistry/metabolism
Bacterial Proteins/genetics/chemistry/metabolism
Endodeoxyribonucleases

@article {pmid40121074,
year = {2025},
author = {Chai, HX and Bamert, RS and Knott, GJ},
title = {Methods for Cas13a expression and purification for use in CRISPR diagnostics.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {225-244},
doi = {10.1016/bs.mie.2025.01.030},
pmid = {40121074},
issn = {1557-7988},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Leptotrichia/genetics/isolation & purification ; Humans ; *COVID-19/diagnosis/virology ; CRISPR-Associated Proteins/genetics/isolation & purification/metabolism ; Bacterial Proteins/genetics/isolation & purification ; RNA, Viral/genetics/isolation & purification ; COVID-19 Nucleic Acid Testing/methods ; },
abstract = {The threat of emerging infectious diseases (e.g., SARS-CoV-2 the RNA virus responsible for the COVID-19 pandemic) has highlighted the importance of accurate and rapid testing for screening, patient diagnosis, and effective treatment of infectious disease. Nucleic acid diagnostic tools such as qPCR are considered the gold standard, providing a sensitive, accurate, and robust method of detection. However, these conventional diagnostic platforms are resource intensive, limited in some applications, and are almost always confined to laboratory settings. With the increasing demand for low-cost, rapid, and accurate point-of-care diagnostics, CRISPR-based systems have emerged as powerful tools to augment detection capabilities. Of note is the potent RNA detection enzyme, Leptotrichia buccalis (Lbu) Cas13a, which is capable of rapid RNA detection in complex mixtures with or without pre-amplification. To support its wide-spread use, we describe a detailed method for the expression, purification, and validation of LbuCas13a for use in molecular diagnostics.},
}

*SARS-CoV-2/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*Leptotrichia/genetics/isolation & purification
Humans
*COVID-19/diagnosis/virology
CRISPR-Associated Proteins/genetics/isolation & purification/metabolism
Bacterial Proteins/genetics/isolation & purification
RNA, Viral/genetics/isolation & purification
COVID-19 Nucleic Acid Testing/methods

@article {pmid40121072,
year = {2025},
author = {Druteika, G and Karvelis, T and Šikšnys, V},
title = {Experimental strategy for characterization of novel TnpB orthologs.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {183-195},
doi = {10.1016/bs.mie.2025.01.056},
pmid = {40121072},
issn = {1557-7988},
mesh = {*Gene Editing/methods ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; DNA Transposable Elements ; Bacterial Proteins/genetics/metabolism/chemistry ; },
abstract = {TnpB proteins encoded in IS200/IS605 and IS607 mobile genetic elements are among the most widespread proteins in the microbial world. They function as RNA-guided DNA nucleases that play a critical role in transposon proliferation and are the predecessors of CRISPR-Cas12 effector proteins of the type V CRISPR-Cas family. Small size of TnpB nucleases makes them an attractive alternative for larger Cas9 and Cas12 proteins in genome editing applications. However, only a small fraction of TnpB nucleases characterized to date are active in human cells, highlighting the need to identify new TnpB variants that can function as genome editors. Here, we present an experimental pipeline for the characterization of TnpB proteins by combining in silico analysis with in vitro assays. To validate it we determined guide RNA and identified TAM for a set of TnpB orthologs. The proposed workflow can be employed for rapid screening and characterization of the huge TnpB protein family to identify novel TnpB variants that might expand the genome editing toolbox.},
}

*Gene Editing/methods
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
DNA Transposable Elements
Bacterial Proteins/genetics/metabolism/chemistry

@article {pmid40121071,
year = {2025},
author = {Schut, FT and Hallmark, T and Dmytrenko, O and Jackson, RN and Beisel, CL},
title = {Purification and in vivo, cell-free, and in vitro characterization of CRISPR-Cas12a2.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {143-181},
doi = {10.1016/bs.mie.2025.01.032},
pmid = {40121071},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism ; *Escherichia coli/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Cell-Free System ; Bacterial Proteins/genetics/metabolism/chemistry ; Endodeoxyribonucleases/genetics/metabolism/chemistry ; },
abstract = {The CRISPR-associated (Cas) nuclease Cas12a2 from Sulfuricurvum sp. PC08-66 (SuCas12a2) binds RNA targets with a complementary guide (g)RNA. Target RNA binding causes a major conformational rearrangement in Cas12a2 that activates a RuvC nuclease domain to collaterally cleave RNA, ssDNA and dsDNA, arresting growth and providing population-level immunity. Here, we report in vivo, cell-free, and in vitro methods to characterize the collateral cleavage activity of SuCas12a2 as well as a procedure for gRNA design. As part of the in vivo methods, we describe how to capture growth arrest through plasmid interference and induction of an SOS DNA damage response in the bacterium Escherichia coli. We further apply cell-free transcription-translation to affirm collateral cleavage activity triggered by an expressed RNA target. Finally, as part of the in vitro methods, we describe how to purify active nuclease and subsequently conduct biochemical cleavage assays. In total, the outlined methods should accelerate the exploration of SuCas12a2 and other related Cas nucleases, revealing new features of CRISPR biology and helping develop new CRISPR technologies for molecular diagnostics and other applications.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/metabolism
*Escherichia coli/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Cell-Free System
Bacterial Proteins/genetics/metabolism/chemistry
Endodeoxyribonucleases/genetics/metabolism/chemistry

@article {pmid40121070,
year = {2025},
author = {Nguyen, GT and Raju, A and Sashital, DG},
title = {Analysis of metal-dependent DNA nicking activities by Cas endonucleases.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {117-142},
doi = {10.1016/bs.mie.2025.01.034},
pmid = {40121070},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *DNA/metabolism/chemistry ; CRISPR-Associated Protein 9/metabolism/genetics ; Metals/metabolism/chemistry ; Endodeoxyribonucleases/metabolism/chemistry/genetics ; DNA Cleavage ; Endonucleases/metabolism ; Gene Editing/methods ; Bacterial Proteins/metabolism/genetics/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; },
abstract = {CRISPR-Cas systems use RNA-guided CRISPR-associated (Cas) effectors to neutralize infections in bacteria and archaea. In class 2 CRISPR-Cas systems, Cas9 and Cas12 are single-protein Cas effectors that target double-stranded DNA based on complementarity to the guide RNA before cleaving the target DNA using metal-dependent endonuclease domains. Cas9 and Cas12 proteins can be readily programmed to target any DNA of interest by changing the guiding RNA sequence and have been co-opted for genome editing and other biotechnology purposes. The effect of metal ion concentration is an essential consideration in the physiological role of Cas immunity effectors as well as the biotechnological applications of Cas endonucleases. In this chapter, we describe methods for studying the effect of variable divalent metal ion conditions on the DNA binding and cleavage activities of well-studied Cas9 and Cas12a proteins.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics
*DNA/metabolism/chemistry
CRISPR-Associated Protein 9/metabolism/genetics
Metals/metabolism/chemistry
Endodeoxyribonucleases/metabolism/chemistry/genetics
DNA Cleavage
Endonucleases/metabolism
Gene Editing/methods
Bacterial Proteins/metabolism/genetics/chemistry
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics

@article {pmid40121069,
year = {2025},
author = {Marin-Gonzalez, A and Rybczynski, AT and Zou, RS and Ha, T},
title = {Measuring double-strand break repair events in mammalian cells with multi-target CRISPR.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {1-22},
doi = {10.1016/bs.mie.2025.01.011},
pmid = {40121069},
issn = {1557-7988},
mesh = {*DNA Breaks, Double-Stranded ; Humans ; *CRISPR-Cas Systems ; *DNA Repair ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; High-Throughput Nucleotide Sequencing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {A mechanistic understanding of the different pathways involved in the repair of DSBs is a timely, yet challenging task. CRISPR-Cas9 is a powerful tool to induce DNA double-strand breaks (DSB) at defined genomic locations to study the ensuing repair response, but Cas9 studies are typically limited by i) low-throughput induction of DSB, by targeting only one or a few genomic sites, or ii) the use of genetically integrated reporter systems, which do not always reflect endogenous phenotypes. To address these limitations, we developed multi-target CRISPR, a Cas9-based tool to controllably induce DSBs in high-throughput at endogenous sites, by leveraging repetitive genomic regions. In this Chapter, we describe how to design and execute a multi-target CRISPR experiment. We also detail how to analyze next-generation sequencing data for characterization of DSB repair events at multiple cut sites. We envision that multi-target CRISPR will become a valuable tool for the study of mammalian DSB repair mechanisms.},
}

*DNA Breaks, Double-Stranded
Humans
*CRISPR-Cas Systems
*DNA Repair
Animals
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
High-Throughput Nucleotide Sequencing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods

@article {pmid40119750,
year = {2025},
author = {Li, L and Mueller, YM and Hioki, K and den Dekker, RJH and Brouwers-Haspels, I and Mezzanotte, L and Maas, A and Erkeland, S and Katsikis, PD},
title = {A novel TOX-nanoluciferase reporter mouse for exploring modulators of T cell exhaustion.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {},
number = {},
pages = {},
doi = {10.1093/jimmun/vkaf009},
pmid = {40119750},
issn = {1550-6606},
support = {128371//department of Immunology/ ; 201906210055//China Scholarship Council/ ; },
abstract = {Cytotoxic T cell (CTL) exhaustion is driven by chronic T cell receptor (TCR) stimulation, leading to a dysfunctional state of cells. Exhausted CTLs exhibit diminished effector function against chronic infections and cancers. Therefore, reducing CTL exhaustion may re-establish effective adaptive immune responses. One feature of exhausted CTLs is the sustained and stable expression of transcription factor thymocyte selection-associated high mobility group box (TOX). Downregulating TOX expression in CD8+ T cells enhances their antitumor activities and improves immune checkpoint blockade (ICB) efficiency. We generated a reporter transgenic mouse to rapidly detect the expression of TOX by measuring luciferase activity. We knocked in a reporter cassette containing NanoLuc bioluminescent luciferase (Nluc) into the Tox gene locus by CRISPR/Cas9 (Tox-NLuc mice). We further generated Tox-NLuc-OT-I mice by crossing Tox-NLuc mice with OT-I mice, which allows the induction of CTL exhaustion in vitro by repeated stimulation of CD8+ T cells with OVA (257-264) peptide. Luciferase assays showed that higher luminescent signals were detected in exhausted CTLs compared to non-exhausted CTLs, which can be visualized by bioluminescence imaging. Bioluminescence changes were confirmed by measuring TOX expression by flow cytometry. The luminescence in exhausted CTLs decreased significantly when cells treated with ibrutinib and bryostatin-1, drugs that were found to directly modulate T cell exhaustion and decrease TOX expression. In summary, we have developed a novel TOX-nanoluciferase-based reporter system that can be used to monitor TOX expression and may facilitate the screening of molecules that modulate CTL exhaustion.},
}

@article {pmid40119729,
year = {2025},
author = {Mao, S and Wu, R and Luo, W and Qin, J and Chen, AK},
title = {Spuriously transcribed RNAs from CRISPR-sgRNA expression plasmids scaffold biomolecular condensate formation and hamper accurate genomic imaging.},
journal = {Nucleic acids research},
volume = {53},
number = {6},
pages = {},
doi = {10.1093/nar/gkaf192},
pmid = {40119729},
issn = {1362-4962},
support = {2021YFA1100201//National Key R&D Program of China/ ; 22177001//National Natural Science Foundation of China/ ; },
mesh = {*Plasmids/genetics ; *CRISPR-Cas Systems ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Transcription, Genetic ; Cell Nucleus/genetics/metabolism ; Genomics/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging tools that utilize fluorescently tagged single-guide RNAs (sgRNAs) have enabled versatile analysis of the dynamics of single genomic loci, but the accuracy may be hindered by nonspecific subnuclear probe accumulation, generating false-positive foci in cell nuclei. By examining the subcellular localizations of sgRNA expression plasmids, their RNA transcripts, and several RNA-binding proteins, we found that spuriously transcribed (cryptic) transcripts, produced by sgRNA expression plasmids, are the major contributors of false-positive signals, independent of sgRNA scaffold design or effector probe (i.e. RNA aptamer- or oligonucleotide-based probes) used. These transcripts interact with the paraspeckle core proteins, but not with the sgRNA expression plasmids or the paraspeckle RNA scaffold NEAT1_2, to form nuclear bodies that display liquid-like properties including sphericality, fusion competence, and sensitivity to 1,6-hexanediol. Transfecting sgRNA transcription units (i.e. sgRNA expression cassettes), lacking the plasmid backbones, reduces false-positive signals and enhances genomic imaging accuracy. Overall, this study unveils previously undescribed activities of cryptic plasmid transcripts and presents an easy-to-adapt strategy that can potentially improve the precision of CRISPR-based imaging systems that implement fluorescently tagged sgRNAs.},
}

*Plasmids/genetics
*CRISPR-Cas Systems
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
Transcription, Genetic
Cell Nucleus/genetics/metabolism
Genomics/methods
Clustered Regularly Interspaced Short Palindromic Repeats
RNA-Binding Proteins/metabolism/genetics

@article {pmid40119107,
year = {2025},
author = {Kim, S and Matsushita, Y and Katagiri, T and Maseda, H},
title = {Efficiency of genome editing using modified single-stranded oligodeoxyribonucleotides in human cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {9764},
pmid = {40119107},
issn = {2045-2322},
support = {JP20ck0106410//Japan Agency for Medical Research and Development/ ; JP20K20640//Japan Society for the Promotion of Science/ ; },
mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; *Oligodeoxyribonucleotides/chemistry/genetics ; *Oligonucleotides/genetics ; DNA, Single-Stranded/genetics ; CRISPR-Cas Systems ; Genome, Human ; },
abstract = {Single-stranded oligodeoxyribonucleotide (ssODN) gene editing has emerged as a promising therapeutic strategy. However, further improvements in efficiency are desired for practical application. The effects of strand length and locked nucleic acid (LNA) modification on ssODN genome editing were investigated by introducing an assay cassette into the genome of HEK293T cells and measuring precise base deletions of eight bases. The introduction of LNAs into ssODNs, five pairs of LNAs at 25-35 nt from the centre and one pair at 20-25 nt, showed approximately 18-fold higher efficiency than unmodified ssODNs of the same length in the study using 70 nt ssODNs. In addition, genome editing efficiency was further improved when LNAs were introduced at the same positions as the 70 nt ssODN, which showed the highest efficiency for the 90 nt ssODN. However, in some cases, the same number of LNA modifications could conversely reduce the efficiency, and the modification positions in the ssODN method were successfully optimised in the present study. Furthermore, the oligo DNA was shown to be effective not only for deletions but also for base substitutions, with an editing efficiency of 0.63% per cell.},
}

Humans
*Gene Editing/methods
HEK293 Cells
*Oligodeoxyribonucleotides/chemistry/genetics
*Oligonucleotides/genetics
DNA, Single-Stranded/genetics
CRISPR-Cas Systems
Genome, Human

@article {pmid40118112,
year = {2025},
author = {Ma, X and Miao, L and Liu, X},
title = {Teach plants to fish based on CRISPR-Cas system self-evolution.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70066},
pmid = {40118112},
issn = {1467-7652},
support = {32171453//National Natural Science Foundation of China/ ; 1610392020001//Central Public-interest Scientific Institution Basal Research Fund/ ; 251804006//High-level Talent Research Start-up Project Funding of Henan Academy of Sciences/ ; },
}

@article {pmid40117303,
year = {2025},
author = {Hren, A and Lollini, N and Carper, DL and Abraham, PE and Cameron, JC and Fox, JM and Eckert, CA},
title = {High-density CRISPRi screens reveal diverse routes to improved acclimation in cyanobacteria.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {12},
pages = {e2412625122},
doi = {10.1073/pnas.2412625122},
pmid = {40117303},
issn = {1091-6490},
support = {DE-SC0018368//U.S. Department of Energy (DOE)/ ; W911NF-18-1-0159//DOD | USA | AFC | CCDC | Army Research Office (ARO)/ ; },
mesh = {*Synechococcus/genetics/metabolism/growth & development/physiology ; *Acclimatization/genetics ; *Photosynthesis/genetics ; Gene Expression Regulation, Bacterial ; CRISPR-Cas Systems ; Cyanobacteria/genetics/metabolism/physiology ; Light ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Cyanobacteria are the oldest form of photosynthetic life on Earth and contribute to primary production in nearly every habitat, from permafrost to hot springs. Despite longstanding interest in the acclimation of these microbes, it remains poorly understood and challenging to rewire. This study uses a high-density, genome-wide CRISPR interference screen to examine the influence of gene-specific transcriptional variation on the growth of Synechococcus sp. PCC 7002 under environmental extremes. Surprisingly, many partial knockdowns enhanced fitness under cold monochromatic conditions. Transcriptional repression of genes for core subunits of the NDH-1 complex, which are important for photosynthesis and carbon uptake, improved growth rates under both red and blue light but at distinct, color-specific optima. Most genes with fitness-improving knockdowns were distinct to each light color, and dual-target transcriptional repression produced nonadditive effects. Findings reveal diverse routes to improved acclimation in cyanobacteria (e.g., attenuation of genes involved in CO2 uptake, light harvesting, translation, and purine metabolism) and provide an approach for using gradients in sgRNA activity to pinpoint biochemically influential transcriptional changes in cells.},
}

*Synechococcus/genetics/metabolism/growth & development/physiology
*Acclimatization/genetics
*Photosynthesis/genetics
Gene Expression Regulation, Bacterial
CRISPR-Cas Systems
Cyanobacteria/genetics/metabolism/physiology
Light
Bacterial Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40115958,
year = {2025},
author = {Farinati, S and Soria Garcia, AF and Draga, S and Vannozzi, A and Palumbo, F and Scariolo, F and Gabelli, G and Barcaccia, G},
title = {Unlocking male sterility in horticultural crops through gene editing technology for precision breeding applications: presentation of a case study in tomato.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1549136},
pmid = {40115958},
issn = {1664-462X},
abstract = {Plant male sterility (MS) refers to the failure of the production of functional anthers, viable pollen grains and/or fertile sperm cells. This feature has great potential in horticultural crops for the exploitation of heterosis through the development of F1 hybrid varieties. MS in plants can occur spontaneously or can be induced artificially by exploiting biotechnological tools, such as the editing of genes involved in spore formation or pollen development. The success of such an approach strongly depends both on preliminary knowledge of the involved genes and on effective procedures for in vitro transfection/regeneration of whole plants. Furthermore, according to previous studies based on CRISPR/Cas9 technology, the efficacy of targeting and the resulting mutation profile are critically influenced by intrinsic factors, such as the CRISPR target primary sequence sites and chromatin signatures, which are often associated with varying levels of chromatin accessibility across different genomic regions. This relationship underscores the complexity of CRISPR-based genome editing and highlights the need to identify a precise suitable target. Our paper reports the results obtained for site-specific in vivo mutagenesis via a CRISPR/Cas9-mediated strategy applied to the MYB80 gene, which is a promising target for implementing male sterility in horticultural crops. We highlight the main steps that play a key role in the whole experimental pipeline, which aims at the generation of CRISPR/Cas-edited DNA-free tomato plants. This goal was achieved via protoplast-based technology and by directly delivering a ribonucleoprotein complex consisting of the Cas9 protein and in vitro synthesized single guide RNAs that can target different positions of the gene under investigation. Overall findings and insights are presented and critically discussed.},
}

@article {pmid40114706,
year = {2025},
author = {Maalouf, KE and Frederick, DM and Sharma, N and Haidar, EA and Xiao, T and Han, JS and Mahamdeh, MS and Soberman, RJ and Rufino-Ramos, D and Kleinstiver, BP and Jinnah, HA and Vaine, CA and Bragg, DC and Breyne, K},
title = {Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {1},
pages = {102466},
pmid = {40114706},
issn = {2162-2531},
abstract = {DYT1 dystonia is a neurological movement disorder characterized by a dominant 3-base pair deletion (ΔGAG) in the TOR1A gene. This study demonstrates a gene-editing approach that selectively targets the ΔGAG mutation in the TOR1A DYT1 allele while safeguarding the wild-type (WT) TOR1A allele. We optimized an adeno-associated virus (AAV) vector-compatible variant of the Staphylococcus aureus Cas9 nuclease ortholog (SaCas9-KKH) in DYT1 patient-derived human neuronal progenitor cells (hNPCs). On-target editing of the TOR1A DYT1 allele was confirmed at the genomic level from brain tissue in a xenograft mouse model. To avoid brain biopsy for demonstrating TOR1A DYT1 editing, we developed a non-invasive monitoring method using extracellular RNA (exRNA). TOR1A exRNA was retrieved from the extracellular vesicle (EV) secretions of hNPCs and plasma samples, indicating whether the donor was a TOR1A DYT1 carrier. This technique enabled us to assess AAV-mediated disruption of the TOR1A DYT1 allele in the brains of mice using blood samples.},
}

@article {pmid40058712,
year = {2025},
author = {Song, Y and Li, J and Luan, X and Li, A and Liu, N and Wu, ZH and Yang, W and Gao, W and Zheng, X and Zhang, XQ},
title = {OsCHR728 encodes a chromatin remodeling factor involved in seed size and grain chalkiness in rice.},
journal = {Gene},
volume = {951},
number = {},
pages = {149396},
doi = {10.1016/j.gene.2025.149396},
pmid = {40058712},
issn = {1879-0038},
mesh = {*Oryza/genetics/growth & development/metabolism ; *Chromatin Assembly and Disassembly ; *Seeds/genetics/growth & development/metabolism ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; *Edible Grain/genetics/metabolism/growth & development ; Gene Knockout Techniques ; Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Endosperm/genetics/metabolism ; Adenosine Triphosphatases ; },
abstract = {The Imitation Switch (ISWI)ATP-dependent chromatin remodeling factor proteins regulate various developmental processes, spanning from flowering to stress response. However, researches on the roles of ISWI subfamily genes in rice have been limited. This study investigated the expression profile of the rice chromatin remodeler OsCHR728, encoding an ISWI protein, across various tissues and developing stages. Our findings reveal that OsCHR728 is highly accumulated during early stage of the panicle development. We generated OsCHR728 knockout (KO) lines in rice using CRISPR/Cas9 technology. These mutant lines displayed smaller grain size compared to the wild type (Zhonghua 11, ZH11). Expression analysis revealed a significant downregulation of the transcript levels of five genes associated with cell cycle regulation in KO grains compared to the wild type, consistent with the reduced cell number in the mutant grains. Additionally, total free amino acid levels were higher in the KO mutant compared to the wild type, consequently enhancing the nutritional quality of the KO mutant grains. The mature endosperm of the KO mutant exhibited a reduced percentage of chalky grains and less chalkiness, suggesting an improvement in the appearance quality of the KO mutant. These results suggest that chromatin remodeling factor OsCHR728 plays a role in grain development, potentially providing a new avenue to enhance both the appearance and nutritional quality of rice cultivars.},
}

*Oryza/genetics/growth & development/metabolism
*Chromatin Assembly and Disassembly
*Seeds/genetics/growth & development/metabolism
*Plant Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
*Edible Grain/genetics/metabolism/growth & development
Gene Knockout Techniques
Transcription Factors/genetics/metabolism
CRISPR-Cas Systems
Endosperm/genetics/metabolism
Adenosine Triphosphatases

@article {pmid40054218,
year = {2025},
author = {Zhong, J and Xu, Z and Peng, J and Guan, L and Li, J and Zhou, Z and Zhang, Y and Zhang, J and Liu, S and Yang, Y and Hao, X},
title = {A CRISPR/Cas13a system based on a dumbbell-shaped hairpin combined with DNA-PAINT to establish the DCP-platform for highly sensitive detection of Hantaan virus RNA.},
journal = {Talanta},
volume = {291},
number = {},
pages = {127852},
doi = {10.1016/j.talanta.2025.127852},
pmid = {40054218},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Viral/analysis/genetics ; *Hantaan virus/genetics ; DNA/chemistry ; Limit of Detection ; Inverted Repeat Sequences ; },
abstract = {Rapid and sensitive detection of specific RNA sequences is crucial for clinical diagnosis, surveillance, and biotechnology applications. Currently, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard for RNA detection; however, it is associated with long processing time, complex procedures, and a high false-positive rate. To address these challenges, we developed a novel sensing platform based on CRISPR/Cas13a that incorporates a dumbbell-shaped hairpin and DNA-PAINT for rapid, highly specific, and sensitive RNA analysis. By leveraging the CRISPR/Cas13a system, this platform enables the cleavage of dumbbell-shaped hairpins, which subsequently allows the cleaved primers to initiate cyclic amplification of fluorescent signals. These signals are further enhanced by the binding and dissociation phenomena inherent to DNA-PAINT technology, ultimately achieving remarkable triple signal amplification. Additionally, the system effectively discriminates Hantaan virus RNA from Seoul virus in real samples. Importantly, the platform can be easily adapted for the detection of other RNAs by simply reconfiguring the hybridization region of crRNA. In conclusion, this platform represents a "five-in-one" RNA detection approach that integrates reliability, versatility, robustness, high specificity, and superior quantitative capabilities. It provides novel insights for direct RNA detection based on CRISPR/Cas13a and demonstrates significant potential for advancement in viral diagnostics.},
}

*CRISPR-Cas Systems/genetics
*RNA, Viral/analysis/genetics
*Hantaan virus/genetics
DNA/chemistry
Limit of Detection
Inverted Repeat Sequences

@article {pmid40054213,
year = {2025},
author = {Du, H and Zhou, YY and Wu, JW and Wu, YY and Yan, J and Tan, XC and Feng, DF and Huang, KJ},
title = {CRISPR/Cas12a trans-cleavage cascading dual-template exponential amplification reaction for electrochemiluminescent detection of 17β-estradiol in milk.},
journal = {Talanta},
volume = {291},
number = {},
pages = {127873},
doi = {10.1016/j.talanta.2025.127873},
pmid = {40054213},
issn = {1873-3573},
mesh = {*Milk/chemistry ; *Estradiol/analysis/chemistry ; Animals ; *Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; *Metal-Organic Frameworks/chemistry ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Biosensing Techniques/methods ; Limit of Detection ; },
abstract = {17β-Estradiol (E2) is a common environmental estrogen that can interfere with the endocrine systems of humans and animals, and poses a carcinogenic risk even at picomolar concentrations. In this study, a functionalized Ru(bpy)3[2+]-embedded metal-organic framework (ZnRuMOF) is synthesized, in which Ru(bpy)3[2+] served as an electrochemiluminescence (ECL) indicator and the porous structure of ZnRuMOF acts as a nanoreactor to enhance the ECL signal. Based on this, we developed an E2 detection method combining a highly specific CRISPR-Cas12a system and dual-template exponential amplification. This method utilizes the trans-cleavage activity of CRISPR-Cas12a to control a light switch, achieving precise and ultra-sensitive detection of E2. The sensing platform demonstrates excellent performance in detecting E2 concentrations ranging from 1 fg mL[-1] to 150 ng mL[-1], with a detection limit of 0.27 fg mL[-1] (S/N = 3). This study provides a reliable approach for diagnosing and treating diseases related to E2, aiming to protect environmental quality and human health.},
}

*Milk/chemistry
*Estradiol/analysis/chemistry
Animals
*Luminescent Measurements/methods
*Electrochemical Techniques/methods
*Metal-Organic Frameworks/chemistry
*CRISPR-Cas Systems
Nucleic Acid Amplification Techniques/methods
Biosensing Techniques/methods
Limit of Detection

@article {pmid40049000,
year = {2025},
author = {Gao, S and Guo, J and Wang, T and Xu, L},
title = {A rapid visual detection method for Sugarcane streak mosaic virus based on one-tube RPA-CRISPR/Cas12a.},
journal = {Talanta},
volume = {291},
number = {},
pages = {127888},
doi = {10.1016/j.talanta.2025.127888},
pmid = {40049000},
issn = {1873-3573},
mesh = {*Saccharum/virology/chemistry ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; Plant Diseases/virology ; Plant Leaves/virology/chemistry ; Potyvirus/genetics/isolation & purification ; Potyviridae/genetics ; },
abstract = {Sugarcane is the most important crop for sugar production. Sugarcane streak mosaic virus (SCSMV) triggered sugarcane mosaic disease can lead to substantial reductions in both yield and sucrose content. In the process of disease prevention and control, target pathogen detection technology is indispensable. However, traditional detection methods are time-consuming and require expensive equipment, making them less efficient for timely disease control and unfavorable to disease resistance breeding. Here, we introduce a novel detection technology that combines recombinase polymerase amplification (RPA) with CRISPR-Cas12a. The method utilizes crude extracts from sugarcane leaves as the reaction template, significantly simplifying and expediting the preparation process. By combining RPA and CRISPR-Cas12a in a single reaction tube, the risk of aerosol contamination has decreased markedly. The entire process, from sample preparation to result interpretation, only takes 50 min, and the reaction equipment only a water bath pot, and results can be blue light spectrometer or UV flashlight assessed visually. Importantly, the method demonstrates high sensitivity, detecting a minimum of 50 copies of the plasmid, which surpasses the sensitivity of reverse transcription polymerase chain reaction (RT-PCR) and is comparable to quantitative RT-PCR (RT-qPCR). The method exhibits excellent specificity, showing no cross-reactivity with other common sugarcane viruses, including Sugarcane mosaic virus, Sugarcane yellow leaf virus, and Sorghum mosaic virus. The practicality of this technique was validated through the detection of leaf crude extracts from 40 field samples. The detection results were consistent with those obtained from RT-PCR and RT-qPCR using leaf RNA as the template, indicating its suitability for laboratory detection and field applications.},
}

*Saccharum/virology/chemistry
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism
Plant Diseases/virology
Plant Leaves/virology/chemistry
Potyvirus/genetics/isolation & purification
Potyviridae/genetics

@article {pmid40024134,
year = {2025},
author = {Xiong, M and Wang, Y and Lu, S and Lubanga, N and Li, T and Li, Z and He, B and Li, Y},
title = {Space-coded microchip for multiplexed respiratory virus detection via CRISPR-Cas12a and RPA.},
journal = {Talanta},
volume = {291},
number = {},
pages = {127815},
doi = {10.1016/j.talanta.2025.127815},
pmid = {40024134},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Lab-On-A-Chip Devices ; Recombinases/metabolism ; Respiratory Tract Infections/virology/diagnosis ; Limit of Detection ; Endodeoxyribonucleases/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; },
abstract = {Multiple infections of respiratory viruses are common in patients with clinical respiratory diseases, but current detection methods still have problems such as complex equipment and long detection time. Rapid, low-cost, and on-site detection of human respiratory viruses is crucial for both clinical diagnosis and population screening. In this research, we created a space-coded microfluidic chip (SC-Chip) for the recognition of nine respiratory viruses: influenza A virus, influenza B virus, severe acute respiratory syndrome coronavirus 2, human coronavirus OC43, human coronavirus NL63, human coronavirus HKU1, human respiratory syncytial virus, human parainfluenza virus, and human metapneumovirus. For the first time, a comprehensive sequence comparison among these viruses was performed to design the recombinase polymerase amplification (RPA) primers and Cas12a-crRNAs. The SC-Chip partitions samples amplified by RPA into spatially coded wells preloaded with CRISPR-Cas12a detection reagents, enabling the identification of all nine viral targets in a single test using a single fluorescence probe. The chip-based assay displays 9 respiratory viruses in less than 40 min with a minimum detection limit at a concentration of 10[-18] M (∼1 copy/reaction). Additionally, the efficacy of the method was assessed through its application to 35 clinical patient samples identified as being at risk for respiratory virus infection, yielding a sensitivity of 90 % and a specificity of 100 %. In summary, this space-coded microfluidic CRISPR system offers several advantages, including ease of operation, cost-effectiveness, and rapid data acquisition, thereby holding great potential for multiplexed detection of nucleic acid targets in a clinical setting.},
}

*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*Lab-On-A-Chip Devices
Recombinases/metabolism
Respiratory Tract Infections/virology/diagnosis
Limit of Detection
Endodeoxyribonucleases/genetics
CRISPR-Associated Proteins/genetics
Bacterial Proteins

@article {pmid39955894,
year = {2025},
author = {Kishimoto, H and Miyazaki, K and Omori, M and Higuchi, K and Shirasaka, Y and Inoue, K},
title = {Establishment of endogenous canine MUC1 knock-out MDCKII cells using CRISPR-Cas9 and evaluation of drug permeation.},
journal = {Drug metabolism and pharmacokinetics},
volume = {61},
number = {},
pages = {101051},
doi = {10.1016/j.dmpk.2025.101051},
pmid = {39955894},
issn = {1880-0920},
mesh = {Dogs ; Animals ; Madin Darby Canine Kidney Cells ; *CRISPR-Cas Systems ; *Mucin-1/metabolism/genetics ; Gene Knockout Techniques/methods ; ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism/genetics ; Cell Membrane Permeability ; Permeability ; },
abstract = {Most orally administered drugs are absorbed by simple diffusion across the intestinal epithelium. Monolayers of MDCKII cells and parallel artificial membrane permeability assay are widely used to evaluate simple diffusion as an in vitro model; however, these models do not account for the contribution of mucus glycoprotein, which may play a significant role in drug permeation. We focused on the role of MUC1, a membrane-bound mucin that is found on the luminal surface of the gastrointestinal epithelium, in the simple diffusion of lipophilic drugs. We generated endogenous canine Mdr1 (cMdr1) and Muc1 (cMuc1) knock-out MDCKII cells by genomic editing and evaluated the effect of cMuc1 on the simple diffusion of various drugs. The absence of cMuc1 significantly increased the membrane permeation of lipophilic drugs, such as griseofulvin as well as paclitaxel and rhodamine 123, substrates of the MDR1 efflux transporter, which suggests that cMuc1 is one of the key factors that modulate the membrane permeation of these drugs. Taken together, we successfully established MDCKII cell lines with a complete knock-out of endogenous cMuc1 and cMdr1 expressions. This provides a novel in vitro model system for studying the mechanisms underlying drug absorption and transport, with potential applications for drug development.},
}

Dogs
Animals
Madin Darby Canine Kidney Cells
*CRISPR-Cas Systems
*Mucin-1/metabolism/genetics
Gene Knockout Techniques/methods
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism/genetics
Cell Membrane Permeability
Permeability

@article {pmid40114372,
year = {2025},
author = {Pulgarin, DV and Pelo, N and Ferrandiz, L and Tršelič, T and Nyberg, WA and Bowlin, G and Espinosa, A},
title = {Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.},
journal = {Nucleic acids research},
volume = {53},
number = {6},
pages = {},
pmid = {40114372},
issn = {1362-4962},
support = {F31AR072502/NH/NIH HHS/United States ; //Whitaker Foundation/ ; 200992//Cancerfonden/ ; //Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse/ ; //Cancerfonden/ ; },
mesh = {*Optogenetics/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *T-Lymphocytes/metabolism ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; Humans ; *Light ; Mice ; HEK293 Cells ; Mice, Inbred C57BL ; },
abstract = {There is currently a lack of tools capable of perturbing genes in both a precise and a spatiotemporal fashion. The flexibility of CRISPR (clustered regularly interspaced short palindromic repeats), coupled with light's unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here, we present a new optogenetic CRISPR tool (Blue Light-inducible Universal VPR-Improved Production of RGRs, BLU-VIPR) that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of guide RNA (gRNA) production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor (VPR-EL222) and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single messenger RNA transcript. This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR, including indels, CRISPRa, and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing, achieving optogenetic gene editing in T lymphocytes in vivo.},
}

*Optogenetics/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*T-Lymphocytes/metabolism
*Gene Editing/methods
Animals
*CRISPR-Cas Systems
Humans
*Light
Mice
HEK293 Cells
Mice, Inbred C57BL

@article {pmid40113632,
year = {2025},
author = {Xavier, KVM and de Oliveira Luz, AC and Silva-Junior, JW and de Melo, BST and de Aragão Batista, MV and de Albuquerque Silva, AM and de Queiroz Balbino, V and Leal-Balbino, TC},
title = {Molecular epidemiological study of Pseudomonas aeruginosa strains isolated from hospitals in Brazil by MLST and CRISPR/Cas system analysis.},
journal = {Molecular genetics and genomics : MGG},
volume = {300},
number = {1},
pages = {33},
pmid = {40113632},
issn = {1617-4623},
mesh = {*Pseudomonas aeruginosa/genetics/isolation & purification ; Brazil/epidemiology ; *Multilocus Sequence Typing ; Humans ; *CRISPR-Cas Systems/genetics ; *Pseudomonas Infections/epidemiology/microbiology ; *Molecular Epidemiology ; Hospitals ; Phylogeny ; },
abstract = {The CRISPR/Cas system defends bacteria and archaea against invasive pathogens, such as phages, establishing an immunological memory from this interaction. Pseudomonas aeruginosa, an opportunistic pathogen, represents a significant public health concern due to its multidrug resistance. This study conducted a molecular epidemiological analysis of clinical isolates of Pseudomonas aeruginosa in Brazil using multilocus sequence typing (MLST) and characterization of CRISPR/Cas system. Most P. aeruginosa isolates harbored the type I-F CRISPR/Cas system (83%), with a subset also exhibiting the type I-E system. Additionally, some isolates presented incomplete CRISPR/Cas systems in their secondary loci. Notably, the isolate Pae93 exhibited a genetic composition rich in phage-related proteins proximal to the orphan CRISPR locus. The identification and characterization of spacer sequences, including previously undocumented ones, revealed a remarkable diversity of predatory mobile genetic elements (MGEs) among the P. aeruginosa isolates studied. The spacer sequences were incorporated into the MGE library. Additionally, the study identified the existence of prophages and anti-CRISPR genes. Two new sequence types (STs 3383 and 3384) were identified and added to the PubMLST database. No discernible correlation was established between the observed STs and the previously delineated CRISPR genotypes. However, the CRISPR system remains valuable for elucidating specific interactions between microorganisms and MGEs. The Brazilian population of clinical P. aeruginosa isolates was shown to be genetically heterogeneous with a non-clonal distribution, as revealed by MLST analysis. The presence of high-risk clones, such as ST 244 and ST 235, underscores the importance of robust epidemiological surveillance and infection control strategies for P. aeruginosa, especially in healthcare settings. This study significantly contributes to the understanding of the molecular epidemiology of these isolates in Brazil.},
}

*Pseudomonas aeruginosa/genetics/isolation & purification
Brazil/epidemiology
*Multilocus Sequence Typing
Humans
*CRISPR-Cas Systems/genetics
*Pseudomonas Infections/epidemiology/microbiology
*Molecular Epidemiology
Hospitals
Phylogeny

@article {pmid40113426,
year = {2025},
author = {Song, WJ and Zhang, F and Wang, ZS and Tian, JF and Niu, RF},
title = {[The application of ANXA2 gene knockout mouse models in lung cancer metastasis].},
journal = {Zhonghua zhong liu za zhi [Chinese journal of oncology]},
volume = {47},
number = {3},
pages = {254-261},
doi = {10.3760/cma.j.cn112152-20240705-00277},
pmid = {40113426},
issn = {0253-3766},
support = {81372844//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Annexin A2/genetics/metabolism ; Mice ; *Lung Neoplasms/genetics/pathology/metabolism ; *Mice, Knockout ; *Carcinoma, Lewis Lung/genetics/metabolism/pathology ; Disease Models, Animal ; Neoplasm Metastasis ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; Survival Rate ; },
abstract = {Objective: ANXA2 plays a crucial role in cancer metastasis, but its mechanism is not yet fully understood. Therefore, it is necessary to establish an ANXA2 gene knockout mouse model to provide an effective tool for subsequent studies on ANXA2-related mechanisms. Methods: A gene knockout mouse model was constructed using CRISPR/Cas9 technology. The model was validated through tissue DNA extraction followed by polymerase chain reaction (PCR), sequencing, and western blot to confirm ANXA2 genotype and protein expression. The successfully constructed models were divided into a model group and a wild-type (WT) group for the creation of a mouse tail vein injection Lewis lung carcinoma (LLC) metastasis model. Metastatic foci formation was monitored using in vivo imaging technology, and the survival rates of the two groups were compared. Results: An sgRNA sequence targeting the first exon of ANXA2 was designed, and 16 founder mice were obtained through microinjection. Through consanguineous hybridization, 30 homozygous offspring were ultimately acquired. After establishing the strains of the mouse model, mice were divided into the ANXA2 knockout group and the WT group, with 8 mice in each group. An LLC lung metastasis model was established in both groups. Compared with the WT group, the number of metastatic foci was significantly increased in the ANXA2 knockout group (7 vs. 1), and the fluorescence intensity was stronger in the WT group than in the knockout group (P=0.002). Using the GEPIA2 database to analyze ANXA2 gene expression in tumor tissues and normal tissues of lung cancer patients, it was found that ANXA2 expression levels were significantly higher in lung cancer tumor tissues compared to normal tissues (P＜0.05). The database included data from 478 lung cancer patients, and patients were stratified into high-expression and low-expression groups based on ANXA2 levels. Compared to the low-expression group, patients in the high-expression group exhibited significantly shorter disease-free survival and overall survival (P＜0.05, respectively). The survival time of mice in the ANXA2 knockout group (median survival time, 43 days) was significantly longer compared to the WT group (median survival time, 26 days; P=0.017). Additionally, ANXA2 expression is significantly associated with the prognosis of lung cancer patients (P=6.4e-14). Conclusions: ANXA2 is closely associated with cancer metastasis and holds potential as a new target for metastasis treatment. Further in-depth research will greatly facilitate the transition of ANXA2 from basic research to clinical application.},
}

Animals
*Annexin A2/genetics/metabolism
Mice
*Lung Neoplasms/genetics/pathology/metabolism
*Mice, Knockout
*Carcinoma, Lewis Lung/genetics/metabolism/pathology
Disease Models, Animal
Neoplasm Metastasis
CRISPR-Cas Systems
Mice, Inbred C57BL
Survival Rate

@article {pmid40113237,
year = {2025},
author = {Robertson, NR and Lee, S and Tafrishi, A and Wheeldon, I},
title = {Advances in CRISPR-enabled genome-wide screens in yeast.},
journal = {FEMS yeast research},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsyr/foaf013},
pmid = {40113237},
issn = {1567-1364},
abstract = {CRISPR-Cas genome-wide screens are powerful tools for unraveling genotype-phenotype relationships, enabling precise manipulation of genes to study and engineer industrially-useful traits. Traditional genetic methods, such as random mutagenesis or RNA interference, often lack the specificity and scalability required for large-scale functional genomic screens. CRISPR systems overcome these limitations by offering precision gene targeting and manipulation, allowing for high-throughput investigations into gene function and interactions. Recent work has shown that CRISPR genome editing is widely adaptable to several yeast species, many of which have natural traits suited for industrial biotechnology. In this review, we discuss recent advances in yeast functional genomics, emphasizing advancements made with CRISPR tools. We discuss how the development and optimization of CRISPR genome-wide screens have enabled a host-first approach to metabolic engineering, which takes advantage of the natural traits of non-conventional yeast - fast growth rates, high stress tolerance, and novel metabolism - to create new production hosts. Lastly, we discuss future directions, including automation and biosensor-driven screens, to enhance high-throughput CRISPR-enabled yeast engineering.},
}

@article {pmid40111512,
year = {2025},
author = {Zhang, L and Li, D and Li, X and Zong, L and Bian, H and Lu, J},
title = {CutIn: a ready-to-use construct for rapid generation of urgently needed transgenic cell lines in emerging infection research.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {67},
pmid = {40111512},
issn = {1438-7948},
support = {202303021222378//Natural Science Foundation of Young Scientists of Shanxi Province/ ; 2022A049//Changzhi People's Hospital Clinical & Basic Innovative Research Project Fund/ ; },
mesh = {Humans ; HeLa Cells ; *CRISPR-Cas Systems ; HEK293 Cells ; Gene Knock-In Techniques/methods ; Dependovirus/genetics ; HCT116 Cells ; Plasmids/genetics/metabolism ; },
abstract = {Site-directed exogenous gene knock-in for stable cell line generation remains a multi-step procedure that heavily relies on expertise. Therefore, there is a need for a competent and easily manageable method, particularly when there is an urgent demand for cell lines, especially for emerging infection research. We present here a universal construct called CutIn that expresses the Cas9 protein and dual sgRNAs targeting a host cell genome locus and the ampicillin resistance (AmpR) gene of a cotransfected donor plasmid commercially available. This construct specifically induces double-strand breaks (DSBs) in cotransfected plasmids and host cell genomes, thereby facilitating whole plasmid integration through nonhomologous end joining (NHEJ) repair mechanisms. As pilot tests, adeno-associated virus integration site 1 (AAVS1) or hypoxanthine phosphoribosyl transferase (HPRT) locus was selected as host genome target, commonly used human cell lines 293T, HeLa and HCT116 were employed. CutIn was subjected for reporter plasmid knock-in in all three cell lines, either AAVS1 and AmpR or HPRT and AmpR loci were efficiently targeted. Fluorescent protein, human angiotensin-converting enzyme 2 (ACE2) and dengue virus (DENV) infection reporter transgenic cells were rapidly obtained via CutIn-mediated whole expression vector integration. This method is designed to be user-friendly and shows potential for supporting the investigation of emerging/re-emerging infectious diseases. Further validation in diverse research contexts will be necessary to fully assess its applicability and effectiveness.},
}

Humans
HeLa Cells
*CRISPR-Cas Systems
HEK293 Cells
Gene Knock-In Techniques/methods
Dependovirus/genetics
HCT116 Cells
Plasmids/genetics/metabolism

@article {pmid40111000,
year = {2025},
author = {Zhou, X and Yang, S and Sun, B and Dong, F and Yin, M and Jiang, Y and Huang, Z and Yang, S},
title = {Implementation of RAGATH RNA-associated DNA Endonucleases as Genome Editing Tool in Escherichia coli.},
journal = {Biotechnology journal},
volume = {20},
number = {3},
pages = {e70005},
doi = {10.1002/biot.70005},
pmid = {40111000},
issn = {1860-7314},
support = {2023YFF1000200//National Key Research and Development Program of China/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *CRISPR-Cas Systems/genetics ; Streptococcus pyogenes/genetics/enzymology ; Endonucleases/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; Genome, Bacterial/genetics ; },
abstract = {The preferred method for Escherichia coli genome editing relies on Cas9 from Streptococcus pyogenes (SpCas9) and λ-Red recombinase. Although SpCas9 is currently the most active RNA-guided DNA endonuclease, a significant number of escapers are often observed, making it inefficient across different sites, particularly when inserting large fragments. In this study, we identified two RAGATH RNA-associated DNA endonucleases (RADs) derived from IS607 transposons. Both of them exhibited high cleavage activity in E. coli. When combined with λ-Red recombinase, they achieved editing efficiencies approaching 100%. Even at target sites where SpCas9 exhibited low editing efficiency, RADs maintained efficiencies ranging from 57% to 94%. Moreover, RADs exhibited higher efficiencies in inserting large fragments in certain cases compared to SpCas9. Taken together, these RAD-based genome editing tools provide viable alternatives to SpCas9, particularly for challenging targets and/or large fragment insertions.},
}

*Gene Editing/methods
*Escherichia coli/genetics
*CRISPR-Cas Systems/genetics
Streptococcus pyogenes/genetics/enzymology
Endonucleases/genetics/metabolism
CRISPR-Associated Protein 9/genetics/metabolism
Genome, Bacterial/genetics

@article {pmid40110345,
year = {2025},
author = {He, C and Li, Y and Liu, J and Li, Z and Li, X and Choi, JW and Li, H and Liu, S and Li, CZ},
title = {Application of CRISPR-Cas System in Human Papillomavirus Detection Using Biosensor Devices and Point-of-Care Technologies.},
journal = {BME frontiers},
volume = {6},
number = {},
pages = {0114},
pmid = {40110345},
issn = {2765-8031},
abstract = {Human papillomavirus (HPV) is the most common virus for genital tract infections. Cervical cancer ranks as the fourth most prevalent cancer globally, with over 99% of cases in women attributed to HPV infection. This infection continues to pose an ongoing threat to public health. Therefore, the development of rapid, high-throughput, and sensitive HPV detection platforms is important, especially in regions with limited access to advanced medical resources. CRISPR-based biosensors, a promising new method for nucleic acid detection, are now rapidly and widely used in basic and applied research and have received much attention in recent years for HPV diagnosis and treatment. In this review, we discuss the mechanisms and functions of the CRISPR-Cas system, focusing on its applications in HPV diagnostics. The review covers CRISPR technologies such as CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas13, along with nucleic acid amplification methods, CRISPR-based signal output systems, and point-of-care testing (POCT) strategies. This comprehensive overview highlights the versatility and potential of CRISPR technologies in HPV detection. We also discuss the numerous CRISPR biosensors developed since the introduction of CRISPR to detect HPV. Finally, we discuss some of the challenges faced in HPV detection by the CRISPR-Cas system.},
}

@article {pmid40108777,
year = {2025},
author = {Ren, C and Bao, Z},
title = {Assessment of Miniature AsCas12f1 Variants for Gene Editing and Activation.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.28978},
pmid = {40108777},
issn = {1097-0290},
support = {//This study was supported by the National Key R&D Program of China (2023YFF1204500), the National Natural Science Foundation of China (22308316), and Fundamental Research Funds for the Central Universities (226-2022-00214 and 226-2023-00085)./ ; },
abstract = {Miniature CRISPR/Cas systems possess delivery advantages for gene therapy. The type V-F Cas12f1 from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been engineered by several studies as compact genome editing tools through protein and single guide RNA (sgRNA) engineering. However, a comparative evaluation of gene editing and activation efficiencies mediated by different AsCas12f1 variants and sgRNA scaffolds is lacking. This study tested combinations of four AsCas12f1 protein variants and six sgRNA scaffolds for their gene editing and transcription activation efficiencies. The protein variant AsCas12f1-HKRA performed the best in gene editing and activation when paired with sgRNA-en_v2.1 scaffold. Furthermore, we validated a super miniature gene activator by fusing a small activation domain to AsCas12f1-HKRA. Our findings recommend using AsCas12f1-HKRA and sgRNA-en_v2.1 for gene editing and activation applications.},
}

@article {pmid40108772,
year = {2025},
author = {Xie, Z and Zhao, S and Deng, R and Tang, X and Feng, L and Xie, S and Pi, Y and Chen, M and Chang, K},
title = {Logic-Measurer: A Multienzyme-Assisted Ultrasensitive Circuit for Logical Detection of Exosomal MicroRNAs.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.5c00258},
pmid = {40108772},
issn = {1936-086X},
abstract = {The logic profiling of exosomal microRNAs (miRNAs) offers broad potential applications in the accurate diagnosis and staging of cancer. However, the logical detection of low-abundance exosomal miRNAs in complex clinical samples remains challenging. This study introduces a logic analysis system termed "Measurer" (a multi-enzyme-assisted ultrasensitive circuit) that offers ultrasensitive and versatile method for detecting multiple exosomal miRNAs. The Logic-Measurer comprises three modules: a stem-loop hairpin-enhanced CRISPR/Cas13a, a polymerase-driven primer exchange reaction, and an exonuclease III-mediated fluorescence output. The efficient Logic-Measurer was switched by the faster rate of trans-cleavage activity of Cas13a due to its improved affinity for hairpin RNA structures. The mechanistic model of hairpin-enhanced CRISPR/Cas13a was confirmed by molecular dynamics simulations. The Logic-Measurer accurately detected exosomal miRNA-21 or miRNA-375 down to 2.1 and 4.4 fM, with superior specificity, and enabled in situ detection of miRNA-21 and miRNA-375 in as low as 1.4 × 10[2] particles/mL exosomes via membrane fusion. In addition, this method demonstrated 87.3 and 82.1% accuracy in the diagnosis and early detection of breast cancer, respectively, among a cohort of 315 individuals. Subsequent subgroup analysis further confirmed the method's ability to accurately differentiate estrogen receptor-positive patients from healthy individuals. Therefore, the Logic-Measurer offers valuable insights into the development of a CRISPR/Cas-based enhanced diagnostic platform and the next generation of diagnostic technology based on enzyme circuits.},
}

@article {pmid40052414,
year = {2025},
author = {Tjallinks, G and Angeleri, N and Nguyen, QT and Mannucci, B and Arentshorst, M and Visser, J and Ram, AFJ and Fraaije, MW and Mattevi, A},
title = {Structural and Mechanistic Characterization of the Flavin-Dependent Monooxygenase and Oxidase Involved in Sorbicillinoid Biosynthesis.},
journal = {ACS chemical biology},
volume = {20},
number = {3},
pages = {646-655},
doi = {10.1021/acschembio.4c00783},
pmid = {40052414},
issn = {1554-8937},
mesh = {*Penicillium chrysogenum/enzymology/metabolism/genetics ; *Mixed Function Oxygenases/metabolism/chemistry/genetics ; Aspergillus niger/enzymology ; Catalytic Domain ; Crystallography, X-Ray ; Oxidoreductases/metabolism/chemistry/genetics ; Dinitrocresols/metabolism/chemistry ; Resorcinols/metabolism/chemistry ; Fungal Proteins/metabolism/chemistry/genetics ; Flavin-Adenine Dinucleotide/metabolism/chemistry ; Protein Conformation ; Flavins/metabolism ; Models, Molecular ; CRISPR-Cas Systems ; },
abstract = {Sorbicillinoids are yellow secondary metabolites synthesized through an elegant combination of enzymatic and spontaneous biochemical processes. The flavin-dependent monooxygenase SorC and oxidase SorD are crucial in this interplay, enabling the generation of a diverse array of functionally complex sorbicillinoids. By solving the crystal structures of SorC and SorD from Penicillium chrysogenum with sorbicillin bound in the active site, we describe the catalytically active binding conformations, crucial for attaining enantioselective and stereoselective control in these enzymatic reactions. The structure of SorC was resolved with the cofactor FAD in its out state, which allowed us to identify key residues that modulate flavin mobility and other conformational changes. Catalytic residues of SorC were also confirmed by detailed characterization of wild-type and several SorC variants. Meanwhile, using a CRISPR/Cas9-based multicopy-genome integration system, we could heterologously express the flavin-dependent oxidase SorD from P. chrysogenum in Aspergillus niger with high yields and purity. This allowed us to obtain the crystal structure of SorD with sorbicillin bound in a viable catalytic conformation. Structural analysis of the obtained complex provided insights into the substrate binding pose and highlighted potentially critical active site residues. Ultimately, having both SorC and SorD at our disposal enabled us to investigate their functions and interplays in the biosynthesis of a vast array of functionally complex sorbicillinoids.},
}

*Penicillium chrysogenum/enzymology/metabolism/genetics
*Mixed Function Oxygenases/metabolism/chemistry/genetics
Aspergillus niger/enzymology
Catalytic Domain
Crystallography, X-Ray
Oxidoreductases/metabolism/chemistry/genetics
Dinitrocresols/metabolism/chemistry
Resorcinols/metabolism/chemistry
Fungal Proteins/metabolism/chemistry/genetics
Flavin-Adenine Dinucleotide/metabolism/chemistry
Protein Conformation
Flavins/metabolism
Models, Molecular
CRISPR-Cas Systems

@article {pmid40014422,
year = {2025},
author = {Bryant, EE and Gong, D and Guo, C and Garces, F and Hubert, R and Chen, I},
title = {An Arrayed CRISPR Screen Identifies Knockout Combinations Improving Antibody Productivity in HEK293 Cells.},
journal = {ACS synthetic biology},
volume = {14},
number = {3},
pages = {855-866},
doi = {10.1021/acssynbio.4c00772},
pmid = {40014422},
issn = {2161-5063},
mesh = {Humans ; HEK293 Cells ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems/genetics ; Antibodies/genetics/metabolism ; },
abstract = {Mammalian cells are used to express complex biologics, such as multispecific antibodies. While multispecifics enable promising new strategies for treating human disease, their production at high expression titer and purity can be challenging. To understand how cells respond to antibody and multispecific expression, five molecules were selected for bulk RNA sequencing (RNA-seq) early after the transfection of a human embryonic kidney 293 (HEK293) host. All five molecules shared a differential expression signature of secretory and protein folding stresses, but this signature was stronger for molecules with low titer. We then designed an arrayed CRISPR knockout screen of 206 differentially expressed target genes and 223 literature-motivated targets to identify knockouts that affect antibody productivity. Eight novel knockout targets were identified that increased expression titers by 20-80%. Notably, seven of these top eight hits were from the differentially expressed set of candidate-gene knockouts. The top knockout target, HIST2H3C, showed evidence for additivity with five other hits, including a knockout combination that increased the titer of a difficult-to-express antibody by up to 100%. Findings for both HIST2H3C and INHBE knockout targets generalized to an alternate HEK293 host expressing an additional antibody and a multispecific host with no meaningful impact on product purity. Thus, we propose HIST2H3C and INHBE disruption as a promising and novel strategy for host-cell engineering to improve antibody and multispecific productivity.},
}

Humans
HEK293 Cells
*Gene Knockout Techniques/methods
*CRISPR-Cas Systems/genetics
Antibodies/genetics/metabolism

@article {pmid39848539,
year = {2025},
author = {Yang, K and Cai, L and Zhao, Y and Cheng, H and Zhou, R},
title = {Optimization of genome editing by CRISPR ribonucleoprotein for high efficiency of germline transmission of Sox9 in zebrafish.},
journal = {New biotechnology},
volume = {86},
number = {},
pages = {47-54},
doi = {10.1016/j.nbt.2025.01.009},
pmid = {39848539},
issn = {1876-4347},
mesh = {Animals ; *Zebrafish/genetics/metabolism/embryology ; *Gene Editing ; *SOX9 Transcription Factor/metabolism/genetics ; *Germ Cells/metabolism/cytology ; *Ribonucleoproteins/metabolism/genetics ; Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Male ; },
abstract = {Primordial germ cells (PGCs) are the first germline stem cells to emerge during early embryonic development and are essential for the propagation and survival of species. Genome editing creates mutagenesis possibilities in vivo, but the generation of precise mutations in PGCs is still challenging. Here, we report an optimized approach for highly efficient genome editing via introducing biallelic variations in early embryos in zebrafish. We adopted an extended, GC-rich, and chemically modified sgRNA along with microinjection of the CRISPR ribonucleoprotein (RNP) complex into the yolk sac at the 1-cell stage. We found that genome editing of Sox9a generated a high proportion of heterozygotes with edited alleles in the F1 generation, indicating biallelic editing. Deep sequencing and mapping the edited cells from early embryos to future tissues revealed that the edited founder has a dominantly edited allele, sox9a M1, accounting for over 99 % of alleles in the testis. Specifically, all offspring of the founder inherited the edited allele, suggesting nearly complete editing of the alleles in early germline cells. Overall, the optimization delineates biallelic editing of sox9a in early embryos and transmission of edited alleles to offspring, thus presenting a method to create a desired genetic mutation line of Sox9a avoiding lengthy traditional crossbreeding.},
}

Animals
*Zebrafish/genetics/metabolism/embryology
*Gene Editing
*SOX9 Transcription Factor/metabolism/genetics
*Germ Cells/metabolism/cytology
*Ribonucleoproteins/metabolism/genetics
Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems
Male

@article {pmid38649638,
year = {2025},
author = {Maroofi, N and Maleki, MSM and Tahmasebi, M and Khorshid, HRK and Modaberi, Y and Najafipour, R and Banan, M},
title = {Detection of CRISPR/Cas9-Mediated Fetal Hemoglobin Reactivation in Erythroblasts Derived from Cord Blood-Hematopoietic Stem Cells.},
journal = {Molecular biotechnology},
volume = {67},
number = {4},
pages = {1695-1706},
pmid = {38649638},
issn = {1559-0305},
support = {1399//Hope Generation Foundation/ ; },
mesh = {*Fetal Hemoglobin/genetics/metabolism ; *Erythroblasts/metabolism/cytology ; *Fetal Blood/cytology/metabolism ; Humans ; *CRISPR-Cas Systems ; *Hematopoietic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; Cell Differentiation ; gamma-Globins/genetics/metabolism ; Repressor Proteins/genetics/metabolism ; beta-Globins/genetics/metabolism ; beta-Thalassemia/genetics/metabolism ; },
abstract = {Reactivation of the fetal hemoglobin (HbF) in adult erythroid cells via genome editing is a strategy for the treatment of β-thalassemia and sickle cell disease. In related reports, the reactivation of HbF is regularly examined in erythroblasts which are generated from the adult CD34[+] hematopoietic stem and progenitor cells (HSPCs). However, the procurement of adult HSPCs, either from the bone-marrow (BM) or from mobilized peripheral-blood (mPB), is difficult. Cord-blood (CB) is a readily available source of HSPCs. CB-HSPCs, however, produce high quantities of HbF following differentiation into the erythroid lineage-a potential drawback in such studies. Here, we have edited the BCL11A enhancer (a well-characterized HbF-quantitative trait loci or QTL) via CRISPR/Cas9 in order to determine whether HbF reactivation could be detected in CB-HSPC-derived erythroblasts. In the edited erythroblasts, insertion/deletion (indel) frequencies of 74.0-80.4% and BCL11A RNA reduction levels of 92.6 ± 5.1% (P < 0.0001) were obtained. In turn, the γ/β-globin transcript ratios were increased from 11.3 ± 1.1-fold to 77.1 ± 2.0-fold, i.e., by 6.8-fold (P < 0.0001)-and the HbF% levels increased from 34.3% in the control population to 43.5% in the BCL11A edited erythroblasts. Our results suggest that γ-globin/HbF reactivation via genome editing can be detected in CB-HSPCs generated erythroblasts-rendering CB-HSPCs a useful model for similar studies.},
}

*Fetal Hemoglobin/genetics/metabolism
*Erythroblasts/metabolism/cytology
*Fetal Blood/cytology/metabolism
Humans
*CRISPR-Cas Systems
*Hematopoietic Stem Cells/metabolism/cytology
*Gene Editing/methods
Cell Differentiation
gamma-Globins/genetics/metabolism
Repressor Proteins/genetics/metabolism
beta-Globins/genetics/metabolism
beta-Thalassemia/genetics/metabolism

@article {pmid40108708,
year = {2025},
author = {Kaeuferle, T and Zwermann, M and Stoll, N and Ferrada-Ernst, P and Jablonowski, L and Zeidler, R and Willier, S and Stenger, D and Yassin, A and Stripecke, R and Feuchtinger, T},
title = {All-in-one CRISPR/Cas-engineered glucocorticoid-receptor knock-out EBV-gp350-CAR knock-in T cells are potent and resistant to dexamethasone.},
journal = {Experimental hematology & oncology},
volume = {14},
number = {1},
pages = {40},
pmid = {40108708},
issn = {2162-3619},
support = {TI07.003//German Centre for Infection Research (DZIF)/ ; AD01.902//German Centre for Infection Research (DZIF)/ ; TTU07.836//German Centre for Infection Research (DZIF)/ ; TTU07.815//German Centre for Infection Research (DZIF)/ ; 70114234//Deutsche Krebshilfe/ ; TRR338//LETSimmun CRC (SFB), Munich-Wuerzburg, Germany/ ; },
abstract = {BACKGROUND: Epstein-Barr virus (EBV) reactivation in immunocompromised patients and post-transplantation is associated with morbidity, mortality and with the onset of a variety of malignant diseases. Adoptive T-cell therapies have emerged as promising therapeutic options, but post-transplant immunosuppression jeopardizes the protective anti-EBV immune surveillance by adoptively transferred T cells.

METHODS: Using an all-in-one CRISPR/Cas-mediated approach, we inserted an anti-EBV (gp350) CAR into the T-cell receptor (TRAC) locus and simultaneously knocked-out the glucocorticoid receptor (GR) on a good manufacturing practice (GMP)-compatible platform.

RESULTS: CAR knock-in (CAR[KI]) was confirmed in primary human T cells on genetic and on protein level with a mean efficiency of 41%. With 83%, additional GR knock-out was highly efficient in CAR[KI] cells. On a functional level CAR[KI]GR[KO] T cells showed target-specific potency in terms of cytokine secretion patterns, proliferative capacity and cytotoxic activity against gp350-expressing target cells. Further, CAR[KI]GR[KO] T cells were insensitive to dexamethasone treatment and maintained T-cell functionality. In contrast, CAR[KI]GR[KO] T cells were sensitive to the GR-independent immunosuppressant cyclosporine A (CsA), thereby providing a rescue treatment for patients in case of safety issues.

CONCLUSIONS: The study lays the proof-of-concept for virus-free all-in-one GMP-manufacturing of glucocorticoid-resistant CAR T-cell products. Further, the glucocorticoid-resistant gp350-CAR T cells can provide a future therapeutic option for high-risk post-transplant patients with EBV-reactivations or patients with EBV-associated pathologies requiring steroid treatment.},
}

@article {pmid40107222,
year = {2025},
author = {Yatera, K and Nishida, C and Mukae, H},
title = {Up-to-date nucleic acid assays for diagnosing respiratory infection.},
journal = {Respiratory investigation},
volume = {63},
number = {3},
pages = {383-393},
doi = {10.1016/j.resinv.2025.03.004},
pmid = {40107222},
issn = {2212-5353},
abstract = {Nucleic acid assays have been widely used as rapid tests for diagnosing respiratory infections during and after the coronavirus disease 2019 (COVID-19) pandemic. An ideal point-of-care diagnostic must be affordable, sensitive, specific, user-friendly, rapid/robust, equipment-free and deliverable (ASSURED), and in addition to improvements to conventional methods based on polymerase chain reaction (PCR), point-of-care testing aiming for "REASSURED" are emerging through integration with microfluidic technology. Compared to conventional immunoassays, nucleic acid assays, especially rapid nucleic acid assays as point-of-care testing, contribute to improvements in various clinical outcomes, such as diagnostic yield, turnaround time, length of hospital stay, disease treatment, and infection control management. Rapid and diverse development of new nucleic acid-based molecular diagnostic technologies, such as those based on the CRISPR/Cas system or biosensor nucleic acid assays, is expected to become increasingly diverse in the future as point-of-care testing. In addition, laboratory-based DNA sequencing technology has been used to perform microbiome analyses over a wide area and is expected to shed light on the pathological mechanisms of various respiratory infectious diseases. One example of the benefits of nucleic acid amplification analysis methods is their ability to reveal the true nature of the bacterial flora in pneumonia lesions. This has been demonstrated based on the results of 16S ribosomal RNA gene sequencing analyses using bronchoalveolar lavage fluid directly obtained from pneumonia lesions in patients with pneumonia.},
}

@article {pmid40106574,
year = {2025},
author = {Wiggers, CRM and Yüzügüldü, B and Tadros, NG and Heavican-Foral, TB and Cho, EY and Eisenbies, ZC and Ozdemir, M and Kulp, SB and Chae, YC and Gutierrez, A and Lohr, JG and Knoechel, B},
title = {Genome-wide CRISPR screen identifies IRF1 and TFAP4 as transcriptional regulators of Galectin-9 in T cell acute lymphoblastic leukemia.},
journal = {Science advances},
volume = {11},
number = {12},
pages = {eads8351},
doi = {10.1126/sciadv.ads8351},
pmid = {40106574},
issn = {2375-2548},
mesh = {Humans ; *Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics/metabolism/pathology ; *Interferon Regulatory Factor-1/metabolism/genetics ; *Galectins/genetics/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Leukemic ; CRISPR-Cas Systems ; Promoter Regions, Genetic ; Enhancer Elements, Genetic ; Protein Binding ; Epigenesis, Genetic ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/metabolism/genetics ; },
abstract = {Galectin-9 is overexpressed in a variety of cancers and associated with worse clinical outcome in some cancers. However, the regulators driving Galectin-9 expression are unknown. Here, we defined the transcriptional regulators and epigenetic circuitry of Galectin-9 in pediatric T cell acute lymphoblastic leukemia (T-ALL), as an example of a disease with strong Galectin-9 expression, in which higher expression was associated with lower overall survival. By performing a genome-wide CRISPR screen, we identified the transcription factors IRF1 and TFAP4 as key regulators for Galectin-9 expression by binding its regulatory elements. Whereas IRF1 was observed exclusively on the promoter, TFAP4 binding was detected at an enhancer solely in T-ALL cells associated with higher Galectin-9 levels. Together, our results show that IRF1 is responsible and indispensable for Galectin-9 expression and TFAP4 further fine-tunes its expression. Our approach, a flow-based genome-wide CRISPR screen complemented by transcription factor binding and enhancer mapping, creates innovative opportunities for understanding and manipulating epigenetic transcriptional regulation in cancer.},
}

Humans
*Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics/metabolism/pathology
*Interferon Regulatory Factor-1/metabolism/genetics
*Galectins/genetics/metabolism
Cell Line, Tumor
Gene Expression Regulation, Leukemic
CRISPR-Cas Systems
Promoter Regions, Genetic
Enhancer Elements, Genetic
Protein Binding
Epigenesis, Genetic
Clustered Regularly Interspaced Short Palindromic Repeats
DNA-Binding Proteins/metabolism/genetics

@article {pmid40105951,
year = {2025},
author = {Yang, P and Feng, J and Chen, J},
title = {Engineered S. cerevisiae construction for high-gravity ethanol production and targeted metabolomics.},
journal = {Applied microbiology and biotechnology},
volume = {109},
number = {1},
pages = {67},
pmid = {40105951},
issn = {1432-0614},
support = {2022047//Hefei Municipal Natural Science Foundation/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Ethanol/metabolism ; *Fermentation ; *Metabolic Engineering/methods ; *Metabolomics ; *CRISPR-Cas Systems ; Hypergravity ; Sucrose/metabolism ; Energy Metabolism ; },
abstract = {Strong sugar tolerance and high bioethanol yield of yeast under high-gravity fermentation have caused great attention in the bioethanol industry. In this study, Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) technology was used to knock out S. cerevisiae GPD2, FPS1, ADH2, DLD3, ERG5, NTH1, and AMS1 to construct engineering strain S. cerevisiae GFADENA. Under high-gravity fermentation with 400 g/L of sucrose, S. cerevisiae GFADENA produced 135 g/L ethanol, which increased 17% compared with the wild-type strain. In addition, S. cerevisiae GFADENA produced 145 g/L of ethanol by simultaneous saccharification and fermentation (SSF) using 400 g/L of corn syrup with a sugar-ethanol conversion rate of 41.1%. Further, the targeted metabolomics involving energy, amino acid, and free fatty acid metabolisms were performed to unravel its molecular mechanisms. The deletion of seven genes in S. cerevisiae GFADENA caused a more significant effect on energy metabolism compared with amino acid and free fatty acid metabolisms based on the significantly different metabolites. Two metabolites α-ketoglutaric acid and fructose-1,6-bisphosphate were the most significantly different upregulation and downregulation metabolites, respectively (p < 0.05). Functions of metabolism, environmental information processing, and genetic information processing were related to sucrose tolerance enhancement and ethanol production increase in S. cerevisiae GFADENA by the regulation of significantly different metabolites. This study provided an effective pathway to increase ethanol yield and enhance sucrose tolerance in S. cerevisiae through bioengineering modification. KEY POINTS: • S. cerevisiae GFADENA with gene deletion was constructed by the CRISPR-Cas9 approach • S. cerevisiae GFADENA could produce ethanol using high-gravity fermentation condition • The ethanol yield of 145 g/L was produced using 400 g/L corn syrup by the SSF method.},
}

*Saccharomyces cerevisiae/genetics/metabolism
*Ethanol/metabolism
*Fermentation
*Metabolic Engineering/methods
*Metabolomics
*CRISPR-Cas Systems
Hypergravity
Sucrose/metabolism
Energy Metabolism

@article {pmid40104595,
year = {2025},
author = {Galanis, A and Papadimitriou, K and Moloney, GM},
title = {Editorial: Omics technologies and bioinformatic tools in probiotic research.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1577852},
pmid = {40104595},
issn = {1664-302X},
}

@article {pmid40103259,
year = {2025},
author = {Fonseca, A and Ishoey, T and Espinoza, C and Marshall, IPG and Nielsen, LP and Gallardo, VA},
title = {Large Filamentous Bacteria Isolated From Sulphidic Sediments Reveal Novel Species and Distinct Energy and Defence Mechanisms for Survival.},
journal = {Environmental microbiology},
volume = {27},
number = {3},
pages = {e70083},
doi = {10.1111/1462-2920.70083},
pmid = {40103259},
issn = {1462-2920},
support = {//the Program "Doctorado en el extranjero Becas Chile" from ANID (former CONICYT)/ ; 1070552//Agencia Nacional de Investigación y Desarrollo/ ; //J. Craig Venter Institute/ ; },
mesh = {*Geologic Sediments/microbiology ; Chile ; *Phylogeny ; Bacteria/genetics/classification/metabolism/isolation & purification ; Genome, Bacterial ; RNA, Ribosomal, 16S/genetics ; Deltaproteobacteria/genetics/classification/isolation & purification/metabolism ; Sulfides/metabolism ; },
abstract = {Various morphotypes of large filamentous bacteria were isolated through micromanipulation from sulphidic sediment mats in the Bay of Concepción, central Chile. This study employed DNA amplification, whole-genome sequencing and bioinformatics analyses to unveil the taxonomic and genomic features of previously unidentified bacteria. The results revealed several novel genera, families and species, including three specimens belonging to Beggiatoales (Beggiatoaceae family), five to Desulfobacterales (Desulfobacteraceae family), two to the Chloroflexi phylum and one to the phylum Firmicutes. Metabolically, Beggiatoaceae bacteria exhibit a flexible and versatile genomic repertoire, enabling them to adapt to variable conditions at the sediment-water interface. All the bacteria demonstrated a mixotrophic mode, gaining energy from both inorganic and organic carbon sources. Except for the Firmicutes bacterium, all others displayed the ability to grow chemolithoautotrophically using H2 and CO2. Remarkably, the reverse tricarboxylic acid (rTCA) and Calvin-Benson-Bassham (CBB) pathways coexisted in one Beggiatoaceae bacterium. Additionally, various defence systems, such as CRISPR-Cas, along with evidence of viral interactions, have been identified. These defence mechanisms suggest that large filamentous bacteria inhabiting sulphidic sediments frequently encounter bacteriophages. Thus, robust defence mechanisms coupled with multicellularity may determine the survival or death of these large bacteria.},
}

*Geologic Sediments/microbiology
Chile
*Phylogeny
Bacteria/genetics/classification/metabolism/isolation & purification
Genome, Bacterial
RNA, Ribosomal, 16S/genetics
Deltaproteobacteria/genetics/classification/isolation & purification/metabolism
Sulfides/metabolism

@article {pmid40103232,
year = {2025},
author = {Lin, SJ and Huang, K and Petree, C and Qin, W and Varshney, P and Varshney, GK},
title = {Optimizing gRNA selection for high-penetrance F0 CRISPR screening for interrogating disease gene function.},
journal = {Nucleic acids research},
volume = {53},
number = {5},
pages = {},
doi = {10.1093/nar/gkaf180},
pmid = {40103232},
issn = {1362-4962},
support = {R21DC020317//US National Institutes of Health/ ; //Oklahoma Medical Research Foundation/ ; },
mesh = {*Zebrafish/genetics ; Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Penetrance ; *CRISPR-Cas Systems ; Humans ; Gene Editing/methods ; Gene Knockout Techniques/methods ; Phenotype ; Neurodevelopmental Disorders/genetics ; },
abstract = {Genes and genetic variants associated with human disease are continually being discovered, but validating their causative roles and mechanisms remains a significant challenge. CRISPR/Cas9 genome editing in model organisms like zebrafish can enable phenotypic characterization of founder generation (F0) knockouts (Crispants), but existing approaches are not amenable to high-throughput genetic screening due to high variability, cost, and low phenotype penetrance. To overcome these challenges, here we provide guide RNA (gRNA) selection rules that enable high phenotypic penetrance of up to three simultaneous knockouts in F0 animals following injection of 1-2 gRNAs per gene. We demonstrate a strong transcriptomic overlap in our F0 knockouts and stable knockout lines that take several months to generate. We systematically evaluated this approach across 324 gRNAs targeting 125 genes and demonstrated its utility in studying epistasis, characterizing paralogous genes, and validating human disease gene phenotypes across multiple tissues. Applying our approach in a high-throughput manner, we screened and identified 10 novel neurodevelopmental disorders and 50 hearing genes not previously studied in zebrafish. Altogether, our approach achieves high phenotypic penetrance using low numbers of gRNAs per gene in F0 zebrafish, offering a robust pipeline for rapidly characterizing candidate human disease genes.},
}

*Zebrafish/genetics
Animals
*RNA, Guide, CRISPR-Cas Systems/genetics
*Penetrance
*CRISPR-Cas Systems
Humans
Gene Editing/methods
Gene Knockout Techniques/methods
Phenotype
Neurodevelopmental Disorders/genetics

@article {pmid40103228,
year = {2025},
author = {Tang, H and Han, S and Jie, Y and Jiang, X and Zhang, Y and Peng, J and Wang, F and Li, X and Zhou, X and Jiang, W and Weng, X},
title = {Enhanced or reversible RNA N6-methyladenosine editing by red/far-red light induction.},
journal = {Nucleic acids research},
volume = {53},
number = {5},
pages = {},
doi = {10.1093/nar/gkaf181},
pmid = {40103228},
issn = {1362-4962},
support = {2023YFC3402200//Ministry of Science and Technology/ ; 92253202//National Natural Science Foundation of China/ ; 2042023kfyq05//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Adenosine/analogs & derivatives/metabolism ; Humans ; *RNA Editing ; *Light ; HEK293 Cells ; CRISPR-Cas Systems ; RNA, Messenger/genetics/metabolism ; Optogenetics/methods ; SOXB1 Transcription Factors/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Cell Differentiation/genetics ; Human Embryonic Stem Cells/metabolism ; Red Light ; },
abstract = {The RNA N6-methyladenosine (m6A) modification is a critical regulator of various biological processes, but precise and dynamic control of m6A remains a challenge. In this work, we present a red/far-red light-inducible m6A editing system that enables efficient and reversible modulation of m6A levels with minimal off-target effects. By engineering the CRISPR dCas13 protein and sgRNA with two pairs of light-inducible heterodimerizing proteins, ΔphyA/FHY1 and Bphp1/PspR2, we achieved targeted recruitment of m6A effectors. This system significantly enhances m6A writing efficiency and allows dynamic regulation of m6A deposition and removal on specific transcripts, such as SOX2 and ACTB. Notably, reversible m6A editing was achieved through cyclic modulation at a single target site, demonstrating the ability to influence mRNA expression and modulate the differentiation state of human embryonic stem cells. This optogenetic platform offers a precise, versatile tool for cyclic and reversible m6A regulation, with broad implications for understanding RNA biology and its potential applications in research and medicine.},
}

*Adenosine/analogs & derivatives/metabolism
Humans
*RNA Editing
*Light
HEK293 Cells
CRISPR-Cas Systems
RNA, Messenger/genetics/metabolism
Optogenetics/methods
SOXB1 Transcription Factors/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Cell Differentiation/genetics
Human Embryonic Stem Cells/metabolism
Red Light

@article {pmid40067805,
year = {2025},
author = {Nonarath, HJT and Simpson, SL and Slobodianuk, TL and Tran, H and Collery, RF and Dinculescu, A and Link, BA},
title = {The USH3A causative gene clarin1 functions in Müller glia to maintain retinal photoreceptors.},
journal = {PLoS genetics},
volume = {21},
number = {3},
pages = {e1011205},
doi = {10.1371/journal.pgen.1011205},
pmid = {40067805},
issn = {1553-7404},
support = {R01 EY026559/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/genetics ; *Ependymoglial Cells/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Usher Syndromes/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Photoreceptor Cells, Vertebrate/metabolism/pathology ; Mutation ; Humans ; Retina/metabolism ; Disease Models, Animal ; Larva/genetics/metabolism ; Neuroglia/metabolism ; },
abstract = {Mutations in CLRN1 cause Usher syndrome type IIIA (USH3A), an autosomal recessive disorder characterized by hearing and vision loss, and often accompanied by vestibular dysfunction. The identity of the cell types responsible for the pathology and mechanisms leading to vision loss in USH3A remains elusive. To address this, we employed CRISPR/Cas9 technology to delete a large region in the coding and untranslated (UTR) region of zebrafish clrn1. The retinas of clrn1 mutant larvae exhibited sensitivity to cell stress, along with age-dependent loss of function and degeneration in the photoreceptor layer. Investigation revealed disorganization in the outer retina in clrn1 mutants, including actin-based structures of the Müller glia and photoreceptor cells. To assess cell-specific contributions to USH3A pathology, we specifically re-expressed clrn1 in either Müller glia or photoreceptor cells. Müller glia re-expression of clrn1 prevented the elevated cell death observed in larval clrn1 mutant zebrafish exposed to high-intensity light. Notably, the degree of phenotypic rescue correlated with the level of Clrn1 re-expression. Surprisingly, high levels of Clrn1 expression enhanced cell death in both wild-type and clrn1 mutant animals. However, rod- or cone-specific Clrn1 re-expression did not reduce the extent of cell death. Taken together, our findings underscore three crucial insights. First, clrn1 mutant zebrafish exhibit key pathological features of USH3A; second, Clrn1 within Müller glia plays a pivotal role in photoreceptor maintenance, with its expression requiring controlled regulation; third, the reliance of photoreceptors on Müller glia suggests a structural support mechanism, possibly through direct interactions between Müller glia and photoreceptors mediated in part by Clrn1 protein.},
}

Animals
*Zebrafish/genetics
*Ependymoglial Cells/metabolism
*Zebrafish Proteins/genetics/metabolism
*Usher Syndromes/genetics/metabolism
*Membrane Proteins/genetics/metabolism
CRISPR-Cas Systems
Photoreceptor Cells, Vertebrate/metabolism/pathology
Mutation
Humans
Retina/metabolism
Disease Models, Animal
Larva/genetics/metabolism
Neuroglia/metabolism

@article {pmid40103227,
year = {2025},
author = {Finocchio, G and Querques, I and Chanez, C and Speichert, KJ and Jinek, M},
title = {Structural basis of TnsC oligomerization and transposase recruitment in type I-B CRISPR-associated transposons.},
journal = {Nucleic acids research},
volume = {53},
number = {5},
pages = {},
doi = {10.1093/nar/gkaf149},
pmid = {40103227},
issn = {1362-4962},
support = {ERC-CoG-820152/ERC_/European Research Council/International ; /HHMI/Howard Hughes Medical Institute/United States ; ALTF 296-2020//EMBO/ ; //University of Zurich/ ; },
mesh = {*Transposases/metabolism/genetics/chemistry ; *DNA Transposable Elements/genetics ; *Cryoelectron Microscopy ; Bacterial Proteins/genetics/metabolism/chemistry ; CRISPR-Cas Systems ; Protein Multimerization ; Models, Molecular ; Protein Binding ; },
abstract = {CRISPR-associated transposon (CAST) systems employ CRISPR-Cas systems as RNA-directed targeting modules for site-specific transposon DNA insertion. Among them, type I CASTs rely on the coordinated action of the guide RNA-bound Cascade complex and the transposon proteins TniQ, TnsC, and TnsAB. The interaction between the transposase TnsAB and the ATPase TnsC is crucial for transposition activity, yet the underlying molecular details have remained elusive. Here, we investigate the type I-B CAST system from Peltigera membranacea cyanobiont. Cryo-electron microscopic structures of TnsC and its complex with the C-terminal region of TnsAB reveal that TnsC forms a heptameric ring that recruits TnsAB by interacting with its C-terminal tail. In vitro binding assays indicate that TnsAB exclusively interacts with the TnsC heptamer without inducing its disassembly, in contrast to type V-K CAST systems. Mutational analysis of key structural features corroborates the significance of TnsC multimerization and TnsB interaction for transposon activity in vivo. Altogether, these findings offer detailed structural and functional insights into the molecular mechanism of type I-B CAST, with the aim of facilitating their development as genome engineering tools.},
}

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

@article {pmid40102860,
year = {2025},
author = {Danti, L and Lundin, K and Nedeczey-Ruzsák, P and Tuuri, T and Tapanainen, JS},
title = {FOXL2 drives the differentiation of supporting gonadal cells in early ovarian development.},
journal = {Reproductive biology and endocrinology : RB&E},
volume = {23},
number = {1},
pages = {44},
pmid = {40102860},
issn = {1477-7827},
mesh = {*Forkhead Box Protein L2/genetics/metabolism ; Female ; Humans ; *Cell Differentiation/genetics ; *Ovary/metabolism/cytology/growth & development ; Gene Expression Regulation, Developmental ; Male ; CRISPR-Cas Systems ; GATA4 Transcription Factor/genetics/metabolism ; Forkhead Transcription Factors/genetics/metabolism ; Wnt4 Protein ; },
abstract = {BACKGROUND: Forkhead box L2 (FOXL2) is a transcription factor from the forkhead box family primarily expressed in the pituitary, ovaries, and eyelids. Human mutations in FOXL2 cause blepharophimosis, ptosis, epicanthus and inversus syndrome (BPES), which can be associated with primary ovarian insufficiency, and is indirectly linked with differences of sex development (DSD). Animal studies have shown the crucial role that FOXL2 plays in the development, function, and maintenance of the ovary as well as in sex determination. However, the specific role of FOXL2 in early human somatic cell ovarian development is largely unknown.

METHODS: In this study, we utilised CRISPR/Cas9 genome activation and a previously published in-house 14-day gonadal differentiation protocol to study the role of FOXL2.

RESULTS: Our results demonstrate that FOXL2 downregulates coelomic epithelial markers GATA4 and LHX9, female gonadal markers RSPO1 and WNT4, and male gonadal markers SOX9, NR0B1 and DHH. The differentially expressed genes were mostly associated with Kyoto encyclopaedia of genes and genomes (KEGG) pathways relating to cell adhesion molecules and gene ontology (GO) pathways relating to extracellular matrix and junction formation. Furthermore, a comparative analysis with existing single cell RNA sequencing data from human in vivo-derived samples elucidated that FOXL2 initiates the downregulation of coelomic epithelial genes GATA4, LHX9 and UPK3B at day 6. By day 8, the genes ARX and GATA2 are transiently upregulated by FOXL2 induction and then downregulated as the genes LGR5, TSPAN8, OSR1 and TAC1 become upregulated.

CONCLUSIONS: These findings suggest that FOXL2 facilitates the exit of differentiating cells from the coelomic epithelium and initially drives them towards a transitional identity before progressing into early supporting gonadal-like cells. The findings of this study significantly advance our understanding of normal gonadal development which can be used as a basis to elucidate pathological gonadal development underlying BPES.},
}

*Forkhead Box Protein L2/genetics/metabolism
Female
Humans
*Cell Differentiation/genetics
*Ovary/metabolism/cytology/growth & development
Gene Expression Regulation, Developmental
Male
CRISPR-Cas Systems
GATA4 Transcription Factor/genetics/metabolism
Forkhead Transcription Factors/genetics/metabolism
Wnt4 Protein

@article {pmid40102626,
year = {2025},
author = {Gürhan, G and Sevinç, K and Aztekin, C and Gayretli, M and Yılmaz, A and Yıldız, AB and Ervatan, EN and Morova, T and Datlı, E and Coleman, OD and Kawamura, A and Lack, NA and Syed, H and Önder, T},
title = {A chromatin-focused CRISPR screen identifies USP22 as a barrier to somatic cell reprogramming.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {454},
pmid = {40102626},
issn = {2399-3642},
mesh = {*Ubiquitin Thiolesterase/genetics/metabolism ; Humans ; *Cellular Reprogramming/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Chromatin/metabolism/genetics ; *CRISPR-Cas Systems ; Fibroblasts/metabolism/cytology ; },
abstract = {Cell-autonomous barriers to reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remain poorly understood. Using a focused CRISPR-Cas9 screen, we identified Ubiquitin-specific peptidase 22 (USP22) as a key chromatin-based barrier to human iPSC derivation. Suppression of USP22 significantly enhances reprogramming efficiency. Surprisingly, this effect is likely to be independent of USP22's deubiquitinase activity or its association with the SAGA complex, as shown through module-specific knockouts, and genetic rescue experiments. USP22 is not required for iPSC derivation or maintenance. Mechanistically, USP22 loss during reprogramming downregulates fibroblast-specific genes while activating pluripotency-associated genes, including DNMT3L, LIN28A, SOX2, and GDF3. Additionally, USP22 loss enhances reprogramming efficiency under naïve stem cell conditions. These findings reveal an unrecognized role for USP22 in maintaining somatic cell identity and repressing pluripotency genes, highlighting its potential as a target to improve reprogramming efficiency.},
}

*Ubiquitin Thiolesterase/genetics/metabolism
Humans
*Cellular Reprogramming/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
*Chromatin/metabolism/genetics
*CRISPR-Cas Systems
Fibroblasts/metabolism/cytology

@article {pmid40102313,
year = {2025},
author = {Fan, R and Luo, S and He, Y and Xiao, Y and Liang, Y and Zhang, L and Li, W and Zhang, Y and Li, L},
title = {Simple and sensitive SERS platform for Staphylococcus aureus one-pot determination by photoactivated CRISPR/Cas12a cascade system and core-shell DNA tetrahedron@AuNP@Fe3O4 reporter.},
journal = {Mikrochimica acta},
volume = {192},
number = {4},
pages = {240},
pmid = {40102313},
issn = {1436-5073},
mesh = {*Staphylococcus aureus/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Gold/chemistry ; *Spectrum Analysis, Raman/methods ; Metal Nanoparticles/chemistry ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; Humans ; CRISPR-Associated Proteins/genetics ; DNA/chemistry ; Endodeoxyribonucleases/chemistry ; Photochemical Processes ; Bacterial Proteins ; },
abstract = {Staphylococcus aureus (S. aureus) is a widely prevalent Gram-positive bacteria that can cause serious infections and diseases in humans and other organisms. Timely detection and treatment in clinical settings is crucial for patient safety and public health. However, current methods for S. aureus detection still face some limitations, such as time-consuming operation, false positives, and labor-intensive available methodology with low sensitivity. Therefore, it is particularly important to develop a rapid, simple, sensitive, and cost-effective method for detecting S. aureus. We developed a SERS platform based on allosteric aptamer-triggered catalytic hairpin assembly (CHA) and photoactivated CRISPR/Cas12a reactions, combined with a multifunctional core-shell structure as the SERS reporter, enabling highly sensitive one-pot determination of S. aureus. Compared with traditional two-step and one-pot analysis methods, this strategy offers superior sensitivity and can successfully identify real samples contaminated with S. aureus. The platform utilizes light-controlled CHA and CRISPR/Cas12a reactions, effectively preventing interference between different reaction systems. Therefore, the photoactivated one-pot CHA/Cas12a strategy provides a simple, rapid, highly sensitive, specific, and cost-effective method for one-pot determination of S. aureus in clinical samples.},
}

*Staphylococcus aureus/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*Gold/chemistry
*Spectrum Analysis, Raman/methods
Metal Nanoparticles/chemistry
Limit of Detection
Aptamers, Nucleotide/chemistry
Humans
CRISPR-Associated Proteins/genetics
DNA/chemistry
Endodeoxyribonucleases/chemistry
Photochemical Processes
Bacterial Proteins

@article {pmid40100800,
year = {2025},
author = {Wu, Y and Shao, Y and Li, W and Yu, Y and Rao, X and Li, J and Waterfield, NR and Yang, G},
title = {Establishment of an RPA-CRISPR/Cas12a combined diagnostic system for Pneumocystis jirovecii pneumonia.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {3},
pages = {e0012922},
pmid = {40100800},
issn = {1935-2735},
mesh = {Humans ; *Pneumocystis carinii/genetics/isolation & purification ; *Pneumonia, Pneumocystis/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Pneumocystis jirovecii causes severe pneumonia in immunocompromised individuals, leading to high mortality and an economic burden. There is a need for early detection methods suitable for low-resource settings and rapid point-of-care diagnostics. This study developed a detection method using Recombinase Polymerase Amplification (RPA) followed by CRISPR/Cas12a with fluorescence detection. The RPA primers and CRISPR-derived RNAs (crRNAs) were specifically designed to target the mitochondrial small subunit rRNA (mtSSU rRNA) gene of P. jirovecii. A total of 83 clinical samples were tested using this method, including 39 confirmed and 44 suspected cases of P. jirovecii infection. The combination of crRNA5 and crRNA6 demonstrated higher sensitivity compared to the current real-time PCR detection method, with a limit of detection (LOD) of 1 copy per reaction and showed no cross-reactions with other respiratory pathogens. The concordance of this method was validated with both infected and non-infected patients. In conclusion, the method developed in this study potentially provides a highly sensitive and rapid tool suitable for the early and on-site detection of P. jirovecii pneumonia. Furthermore, this method holds potential applications for the detection of other human pathogens, representing a significant advancement in diagnostic capabilities for low-resource settings.},
}

Humans
*Pneumocystis carinii/genetics/isolation & purification
*Pneumonia, Pneumocystis/diagnosis/microbiology
*CRISPR-Cas Systems
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods

@article {pmid40100209,
year = {2025},
author = {Cheng, L},
title = {Chemical Strategies to Modulate and Manipulate RNA Epigenetic Modifications.},
journal = {Accounts of chemical research},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.accounts.4c00844},
pmid = {40100209},
issn = {1520-4898},
abstract = {ConspectusRNA epigenetics has rapidly emerged as a key frontier in chemical biology, revealing that modifications to RNA bases and riboses can fine-tune essential cellular processes such as gene expression, translation, and metabolic homeostasis. Traditionally, researchers have relied on manipulating the "writers," "erasers," and "readers" of RNA modifications─i.e., protein cofactors─to alter and study these marks. Those enzyme-centric strategies, including small molecule inhibitors and CRISPR/Cas-based genetic perturbations, have been highly effective and are advancing in clinical applications. However, purely chemical approaches for installing, removing, or transforming RNA modifications without enzyme disturbance have offered distinct advantages, such as temporal control, reversibility, and bypassing compensatory biological feedback mechanisms that often arise with genetic or enzymatic inhibition. Every chemist should be concerned about RNA modifications, because they represent a striking intersection of molecular recognition, organic transformation, and cellular function. The ability to direct chemical reactivity at specific nucleosides in RNA can illuminate how individual modifications impact the overall gene regulation. Further, since improper RNA modification and damage patterns are implicated in cancer, metabolic disorders, and neurodegeneration, these chemical repair tools have potential as diagnostic and therapeutic interventions. Beyond medicine, agriculture also stands to benefit from chemical control of nucleoside-based plant hormones, possibly leading to improved crop productivity and resilience.In this Account, we outline several innovative chemical strategies tailored to different classes of RNA modifications. Flavin-based bioorthogonal chemistry has enabled demethylation of N[6]-methyladenosine (m[6]A) independent of endogenous demethylases, while oxidative bioorthogonal reactions can convert 5-methylcytidine (m[5]C) into distinct formyl derivatives for labeling and sequencing. Nitrogen-oxide and photochemical routes provided access for the selective removal of the side chain of N[6]-isopentenyladenosine (i[6]A), offering insights for both cell biology and plant hormone research. We also showcase how rationally designed small molecules can rewire complex RNA damage repair pathways, facilitating selective correction of vinyl-adduct lesions otherwise resistant to enzymatic repair. These purely chemical methods bypass the constraints of enzyme dependence, affording temporal precision (e.g., via light activation) and site-selective modification or labeling of RNA. By strategically engineering reactivity, we have uncovered new epitranscriptomic phenomena, such as in situ generation of non-native RNA modification, that offer fresh capabilities for cell imaging or targeted manipulation of plant callus development. Together, these discoveries signal a paradigm shift: chemical tools can complement or even surpass conventional enzyme-based methods for investigating, editing, and repairing RNA modifications. The ramifications are broad. Chemists can leverage these new reactivities to dissect the molecular underpinnings of diseases linked to epitranscriptomic dysregulation and to engineer next-generation therapeutic, diagnostic, and sequencing platforms. Plant biologists can apply the same chemical strategies to hone agronomic traits, from seed vigor to stress resilience. Ultimately, as we have deepened the mechanistic insights and refined reaction design for increased biocompatibility, purely chemical control of the RNA epigenome is poised to become one of the mainstream approaches across fields spanning chemistry, biology, and medicine─fostering deeper understanding of RNA's role in health and disease and opening new avenues for precise interventions.},
}

@article {pmid40099180,
year = {2025},
author = {Pan, Z and Xu, L and Fan, Z and Ren, F},
title = {CRIPSR-Cas for hepatitis virus: a systematic review and meta-analysis of diagnostic test accuracy studies.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1509890},
pmid = {40099180},
issn = {1664-302X},
abstract = {BACKGROUND AND AIMS: Hepatitis viruses pose a significant global health challenge, necessitating accurate and efficient diagnostic methods. The CRISPR-Cas system, renowned for gene editing, shows potential tool in virus detection. This systematic review and meta-analysis aims to evaluate the diagnostic accuracy of CRISPR-Cas-based tests for hepatitis viruses, aiming to provide evidence for their effectiveness in clinical settings.

METHODS: Studies from Web of Science, PubMed, and CNKI were analyzed. A bivariate random-effects model was employed to compute pooled estimates for sensitivity, specificity, and the area under the summary receiver operating characteristic (SROC) curve. Additionally, the methodological quality of the studies was evaluated using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tool.

RESULTS: Following a rigorous screening process, 14 studies meeting our inclusion criteria were selected from an initial pool of 657 studies. The pooled sensitivity and specificity of the CRISPR-Cas system in hepatitis virus detection showed high sensitivity (0.99, 95% CI: 0.95-1.00) and specificity (0.99, 95% CI: 0.93-1.00) with SROC area 1.00 (95% CI: 0.99-1.00). However, considering the notable heterogeneity among the included studies, subgroup analyses and meta-regression were conducted. These analyses revealed that the type of hepatitis virus detected and the format of the final result presentation could be potential sources of this heterogeneity.

CONCLUSION: This systematic review and meta-analysis demonstrates the high diagnostic accuracy of CRISPR-Cas system in detecting hepatitis viruses. However, conclusions are limited by study number and quality. Therefore, more high-quality data are still needed to support this conclusion.},
}

@article {pmid40098357,
year = {2025},
author = {Chong, CSC and Lau, YY and Michels, PAM and Lim, CSY},
title = {Insights into biofilm-mediated mechanisms driving last-resort antibiotic resistance in clinical ESKAPE pathogens.},
journal = {Critical reviews in microbiology},
volume = {},
number = {},
pages = {1-26},
doi = {10.1080/1040841X.2025.2473332},
pmid = {40098357},
issn = {1549-7828},
abstract = {The rise of antibiotic-resistant bacteria poses a grave threat to global health, with the ESKAPE pathogens, which comprise Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. being among the most notorious. The World Health Organization has reserved a group of last-resort antibiotics for treating multidrug-resistant bacterial infections, including those caused by ESKAPE pathogens. This situation calls for a comprehensive understanding of the resistance mechanisms as it threatens public health and hinder progress toward the Sustainable Development Goal (SDG) 3: Good Health and Well-being. The present article reviews resistance mechanisms, focusing on emerging resistance mutations in multidrug-resistant ESKAPE pathogens, particularly against last-resort antibiotics, and describes the role of biofilm formation in multidrug-resistant ESKAPE pathogens. It discusses the latest therapeutic advances, including the use of antimicrobial peptides and CRISPR-Cas systems, and the modulation of quorum sensing and iron homeostasis, which offer promising strategies for countering resistance. The integration of CRISPR-based tools and biofilm-targeted approaches provides a potential framework for managing ESKAPE infections. By highlighting the spread of current resistance mutations and biofilm-targeted approaches, the review aims to contribute significantly to advancing our understanding and strategies in combatting this pressing global health challenge.},
}

@article {pmid40097588,
year = {2025},
author = {Liu, J and Zhang, J and Zhao, T and Zhao, M and Su, M and Chen, Y and Huang, Z and Wang, Y and Zhong, C and Hu, Z and Zhou, P and Tian, R and He, D},
title = {SunTag-PE: a modular prime editing system enables versatile and efficient genome editing.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {452},
pmid = {40097588},
issn = {2399-3642},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Dependovirus/genetics ; },
abstract = {Prime editing (PE) holds tremendous potential in the treatment of genetic diseases because it can install any desired base substitution or local insertion/deletion. However, the full-length PE effector size (6.3-kb) is beyond the packaging capacity of adeno-associated virus (AAV), hindering its clinical translation. Various splitting strategies have been used to improve its delivery, but always accompanied by compromised PE efficiency. Here, we developed a modular and efficient SunTag-PE system that splits PE effectors into GCN4-nCas9 and single-chain variable fragment (scFv) tethered reverse transcriptase (RT). We observed that SunTag-PEs with 1×GCN4 in the N terminus of nCas9 was the most efficient configuration rather than multiple copies of GCN4. This SunTag-PE strategy achieved editing levels comparable to canonical fused-PE (nCas9 and RT are linked together) and higher than other split-PE strategies (including sPE and MS2-PE) in both PE2 and PE3 forms with no increase in insertion and deletion (indel) byproducts. Moreover, we successfully validated the modularity of SunTag-PE system in the Cas9 orthologs of SauCas9 and FrCas9. Finally, we employed dual AAVs to deliver SunTag-ePE3 and efficiently corrected the pathogenic mutation in HBB mutant cell line. Collectively, our SunTag-PE system provides an efficient modular splitting strategy for prime editing and further facilitate its transformation in clinics.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
HEK293 Cells
CRISPR-Associated Protein 9/genetics/metabolism
Dependovirus/genetics

@article {pmid40097290,
year = {2025},
author = {Zhong, J and Li, J and Chen, S and Xu, Y and Mao, X and Xu, M and Luo, S and Yang, Y and Zhou, J and Yuan, J and Su, L and Wang, G and Zhang, X and Li, X},
title = {Rapid and Efficient CRISPR-Based Detection of Dengue Virus in a Single-Tube.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxaf070},
pmid = {40097290},
issn = {1365-2672},
abstract = {BACKGROUND: Dengue Virus (DENV) is prevalent in tropical and subtropical regions. With the projected climate change, traditional detection methods face limitations, and there is an urgent need for more accurate and efficient diagnostic techniques.

OBJECTIVE: The aim is to integrate Recombinase-aided Amplification (recombinase-aided Amplification, RAA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to establish a special CRISPR-Cas system for rapid, convenient, high sensitivity, and high specificity typing detection of DENV.

METHODS: CRISPR RNA (crRNA) and RAA primers were designed based on the whole genome of four DENV serotypes. A single-tube assay combining RAA with CRISPR-Cas13a technology was developed after optimizing reagent concentrations.

RESULTS: The limit of detection (LoD) of DENV types 1-4 was 10³ copies·mL -1. No cross-reaction was observed between any of the DENV serotypes and the other three flaviviruses (Zika, West Nile, and Murray Valley encephalitis). The average sensitivity of One-step method was 95.8%, and the average specificity was 96.6%. Fluorescent signal intensities demonstrated a clear dose-dependent response, with the signal increasing as the sample concentration rose. This system can effectively distinguish non-target substances. Among them, One-step method has advantages in timeliness, ease of operation, and contamination control because it runs efficiently inside a tube and does not require the lid to be removed, but its sensitivity is relatively low. The Two-steps method performs well in sensitivity.

CONCLUSION: In this study, we developed a novel method for rapid typing and detection of DENV using RAA and CRISPR-Cas13a in a single-tube homogeneous system.},
}

@article {pmid40053823,
year = {2025},
author = {Liu, Y and Li, F and Lyu, Y and Wang, F and Lee, LTO and He, S and Guo, Z and Li, J},
title = {A Semiconducting Polymer NanoCRISPR for Near-Infrared Photoactivatable Gene Editing and Cancer Gene Therapy.},
journal = {Nano letters},
volume = {25},
number = {11},
pages = {4518-4525},
doi = {10.1021/acs.nanolett.5c00285},
pmid = {40053823},
issn = {1530-6992},
mesh = {Humans ; *Gene Editing/methods ; *Polymers/chemistry ; *Semiconductors ; *Neoplasms/therapy/genetics ; *Genetic Therapy/methods ; CRISPR-Cas Systems/genetics ; Infrared Rays ; Polo-Like Kinase 1 ; RNA, Guide, CRISPR-Cas Systems/genetics ; Nanoparticles/chemistry ; Green Fluorescent Proteins/genetics ; DNA, Single-Stranded/chemistry/genetics ; Cell Line, Tumor ; Animals ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) gene editing has poor efficacy and off-target side effect concerns. We herein report a semiconducting polymer (SP)-based nanoCRISPR system to improve CRISPR delivery efficacy and allow for near-infrared (NIR) photoactivatable gene editing for cancer therapy. An amphiphilic SP acts as a photothermal converter, and its backbone is grafted with single-stranded deoxyribonucleic acid (DNA), which enables hybridization with single guide ribonucleic acid (sgRNA) via complementary base pairing to form sgRNA/SP-DNA. This sgRNA/SP-DNA nanosystem (nanoCRISPR) can effectively deliver sgRNA into cells and generate heat under NIR laser irradiation via the photothermal effect. The localized heat triggers the dissociation of single-stranded DNA and sgRNA to control the release of sgRNA, thereby achieving precise regulation of CRISPR activity. This NIR photoactivatable gene editing technology is able to precisely regulate the expression of green fluorescent protein (GFP) and polo-like kinase 1 (PLK1) gene for precision gene therapy.},
}

Humans
*Gene Editing/methods
*Polymers/chemistry
*Semiconductors
*Neoplasms/therapy/genetics
*Genetic Therapy/methods
CRISPR-Cas Systems/genetics
Infrared Rays
Polo-Like Kinase 1
RNA, Guide, CRISPR-Cas Systems/genetics
Nanoparticles/chemistry
Green Fluorescent Proteins/genetics
DNA, Single-Stranded/chemistry/genetics
Cell Line, Tumor
Animals

@article {pmid38769424,
year = {2025},
author = {Khan, MA and Herring, G and Zhu, JY and Oliva, M and Fourie, E and Johnston, B and Zhang, Z and Potter, J and Pineda, L and Pflueger, J and Swain, T and Pflueger, C and Lloyd, JPB and Secco, D and Small, I and Kidd, BN and Lister, R},
title = {CRISPRi-based circuits to control gene expression in plants.},
journal = {Nature biotechnology},
volume = {43},
number = {3},
pages = {416-430},
pmid = {38769424},
issn = {1546-1696},
support = {DP210103954//Department of Education and Training | Australian Research Council (ARC)/ ; CE140100008//Department of Education and Training | Australian Research Council (ARC)/ ; CE230100015//Department of Education and Training | Australian Research Council (ARC)/ ; DE150100460//Department of Education and Training | Australian Research Council (ARC)/ ; GNT1178460//Department of Health | National Health and Medical Research Council (NHMRC)/ ; NA//Howard Hughes Medical Institute (HHMI)/ ; NA//Commonwealth Scientific and Industrial Research Organisation (CSIRO)/ ; },
mesh = {*Arabidopsis/genetics ; *Gene Expression Regulation, Plant ; Gene Regulatory Networks/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Promoter Regions, Genetic ; Bryopsida/genetics ; Plants, Genetically Modified/genetics ; Protoplasts/metabolism ; Brassica napus/genetics/metabolism ; },
abstract = {The construction of synthetic gene circuits in plants has been limited by a lack of orthogonal and modular parts. Here, we implement a CRISPR (clustered regularly interspaced short palindromic repeats) interference (CRISPRi)-based reversible gene circuit platform in plants. We create a toolkit of engineered repressible promoters of different strengths and construct NOT and NOR gates in Arabidopsis thaliana protoplasts. We determine the optimal processing system to express single guide RNAs from RNA Pol II promoters to introduce NOR gate programmability for interfacing with host regulatory sequences. The performance of a NOR gate in stably transformed Arabidopsis plants demonstrates the system's programmability and reversibility in a complex multicellular organism. Furthermore, cross-species activity of CRISPRi-based logic gates is shown in Physcomitrium patens, Triticum aestivum and Brassica napus protoplasts. Layering multiple NOR gates together creates OR, NIMPLY and AND logic functions, highlighting the modularity of our system. Our CRISPRi circuits are orthogonal, compact, reversible, programmable and modular and provide a platform for sophisticated spatiotemporal control of gene expression in plants.},
}

*Arabidopsis/genetics
*Gene Expression Regulation, Plant
Gene Regulatory Networks/genetics
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Promoter Regions, Genetic
Bryopsida/genetics
Plants, Genetically Modified/genetics
Protoplasts/metabolism
Brassica napus/genetics/metabolism

@article {pmid38760567,
year = {2025},
author = {Hsiung, CC and Wilson, CM and Sambold, NA and Dai, R and Chen, Q and Teyssier, N and Misiukiewicz, S and Arab, A and O'Loughlin, T and Cofsky, JC and Shi, J and Gilbert, LA},
title = {Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations.},
journal = {Nature biotechnology},
volume = {43},
number = {3},
pages = {369-383},
pmid = {38760567},
issn = {1546-1696},
support = {K01 HG012789/HG/NHGRI NIH HHS/United States ; R01HG012227//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; K01HG012789//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; DP2CA239597//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; UM1HG012660//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Chromatin/genetics/metabolism ; Humans ; CRISPR-Associated Proteins/genetics/metabolism ; Acidaminococcus/genetics ; Gene Editing/methods ; Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases ; },
abstract = {Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting one to three genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein-DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.},
}

*CRISPR-Cas Systems/genetics
*Chromatin/genetics/metabolism
Humans
CRISPR-Associated Proteins/genetics/metabolism
Acidaminococcus/genetics
Gene Editing/methods
Bacterial Proteins/genetics/metabolism
Endodeoxyribonucleases

@article {pmid38760566,
year = {2025},
author = {Pacalin, NM and Steinhart, Z and Shi, Q and Belk, JA and Dorovskyi, D and Kraft, K and Parker, KR and Shy, BR and Marson, A and Chang, HY},
title = {Bidirectional epigenetic editing reveals hierarchies in gene regulation.},
journal = {Nature biotechnology},
volume = {43},
number = {3},
pages = {355-368},
pmid = {38760566},
issn = {1546-1696},
support = {K08 CA273529/CA/NCI NIH HHS/United States ; T32 GM141819/GM/NIGMS NIH HHS/United States ; UM1 HG012660/HG/NHGRI NIH HHS/United States ; L30 TR002983/TR/NCATS NIH HHS/United States ; Hanna Gray Fellow//Howard Hughes Medical Institute (HHMI)/ ; T32GM141819//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; GRFP//National Science Foundation (NSF)/ ; NA//Howard Hughes Medical Institute (HHMI)/ ; L30TR002983//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; NA//Scleroderma Research Foundation (SRF)/ ; K08CA273529//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; RM1 HG007735/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; Jurkat Cells ; *Epigenesis, Genetic ; *Gene Expression Regulation ; CRISPR-Cas Systems/genetics ; Interleukin-2/genetics ; GATA1 Transcription Factor/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; T-Lymphocytes/metabolism/immunology ; },
abstract = {CRISPR perturbation methods are limited in their ability to study non-coding elements and genetic interactions. In this study, we developed a system for bidirectional epigenetic editing, called CRISPRai, in which we apply activating (CRISPRa) and repressive (CRISPRi) perturbations to two loci simultaneously in the same cell. We developed CRISPRai Perturb-seq by coupling dual perturbation gRNA detection with single-cell RNA sequencing, enabling study of pooled perturbations in a mixed single-cell population. We applied this platform to study the genetic interaction between two hematopoietic lineage transcription factors, SPI1 and GATA1, and discovered novel characteristics of their co-regulation on downstream target genes, including differences in SPI1 and GATA1 occupancy at genes that are regulated through different modes. We also studied the regulatory landscape of IL2 (interleukin-2) in Jurkat T cells, primary T cells and chimeric antigen receptor (CAR) T cells and elucidated mechanisms of enhancer-mediated IL2 gene regulation. CRISPRai facilitates investigation of context-specific genetic interactions, provides new insights into gene regulation and will enable exploration of non-coding disease-associated variants.},
}

Humans
*Gene Editing/methods
Jurkat Cells
*Epigenesis, Genetic
*Gene Expression Regulation
CRISPR-Cas Systems/genetics
Interleukin-2/genetics
GATA1 Transcription Factor/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
T-Lymphocytes/metabolism/immunology

@article {pmid40096954,
year = {2025},
author = {Zhong, Y and Yuan, P and Dai, L and Yang, L and Xu, Z and Chen, D},
title = {First report of blaVEB-3 and blaKPC-2 coexistence with a novel blaKPC-2 transposon in Klebsiella michiganensis.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {},
number = {},
pages = {105740},
doi = {10.1016/j.meegid.2025.105740},
pmid = {40096954},
issn = {1567-7257},
abstract = {BACKGROUND: Klebsiella michiganensis, an emerging opportunistic pathogen, poses public health risks due to its increasing multidrug resistance (MDR), especially to carbapenems.

CASE AND METHOD: A 46-year-old man with pulmonary fibrosis was hospitalized in Guangzhou, China, for worsening pneumonia. A multidrug-resistant K. michiganensis strain (YK6) was isolated from his sputum before treatment. The strain was characterized using MALDI-TOF mass spectrometry, antimicrobial susceptibility testing (AST), and whole genome sequencing (WGS). Targeted therapy guided by AST successfully resolved the infection.

RESULTS: The YK6 strain exhibited resistance to carbapenems, β-lactam/β-lactamase inhibitors, cephalosporins, aminoglycosides, and quinolones, except colistin and tigecycline. Genomic analysis revealed a 41.9-kb MDR island and an intact I-E CRISPR-Cas system on the chromosome, along with two plasmids: IncFIA/IncFII plasmid pYK6-1 carrying blaKPC-2 and IncC plasmid pYK6-2 harboring blaVEB-3. A novel blaKPC-2-transposon in pYK6-1 was identified, consisting of a non-Tn4401 element (NTE)-like structure (Tn3-ISKpn27-blaKPC-2-ΔISKpn6-korC) flanked by inversely oriented ISKpn19-tnpM-tnpR elements and 31-bp inverted repeats never reported, a configuration did not reported previously. Furthermore, the blaVEB-3 genetic environment in pYK6-2 featured a unique cassette: IS26-IS6100-blaVEB-3-tnp-ISAs1-qacEΔ1-sul1-ISCR1. An additional ISAs1 insertion between the tnpF-like integrase and qacEΔ1 distinguishes it from similar blaVEB-3-harboring cassettes. The blaVEB-3 resistance region in pYK6-2 likely originated from homologous recombination mediated by IS26 and Tn5403, which flank the gene cassette.

CONCLUSIONS: To our knowledge, this is the first report of concurrent blaVEB-3 and blaKPC-2 in K. michiganensis, along with a novel blaKPC-2 transposon structure. These findings highlight the urgent need for enhanced surveillance of MDR K. michiganensis to prevent treatment failures.},
}

@article {pmid40096597,
year = {2025},
author = {Gu, B and Ferreira, LMR and Herrera, S and Brown, L and Lieberman, J and Sherwood, RI and Meissner, TB and Strominger, JL},
title = {The TEA domain transcription factors TEAD1 and TEAD3 and WNT signaling determine HLA-G expression in human extravillous trophoblasts.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {12},
pages = {e2425339122},
doi = {10.1073/pnas.2425339122},
pmid = {40096597},
issn = {1091-6490},
support = {R01AI145862//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {Humans ; *Trophoblasts/metabolism/immunology ; *TEA Domain Transcription Factors/metabolism ; *Transcription Factors/metabolism/genetics ; *HLA-G Antigens/metabolism/genetics ; *Wnt Signaling Pathway ; *DNA-Binding Proteins/metabolism/genetics ; Female ; Pregnancy ; CRISPR-Cas Systems ; Gene Expression Regulation ; Immune Tolerance ; Extravillous Trophoblasts ; },
abstract = {Maternal-fetal immune tolerance guarantees a successful pregnancy throughout gestation. HLA-G, a nonclassical human leukocyte antigen (HLA) molecule exclusively expressed in extravillous trophoblasts (EVT), is a crucial factor in establishing maternal-fetal immune tolerance by interacting with inhibitory receptors on various maternal immune cells residing in the uterus. While trophoblast-specific cis-regulatory elements impacting HLA-G transcription have been described, the identity of trans-acting factors controlling HLA-G expression in EVT remains poorly understood. Utilizing a genome-wide CRISPR-Cas9 knockout screen, we find that the WNT signaling pathway negatively regulates HLA-G expression in EVT. In addition, we identified two trophoblast-specific transcription factors, TEAD1 and TEAD3, required for HLA-G transcription in EVT in a Yes-associated protein-independent manner. Altogether, we systematically elucidated essential genes and pathways underlying HLA-G expression in EVT, shedding light on the mechanisms of maternal-fetal tolerance and potentially providing insights into controlling HLA-G expression beyond EVT to protect allogeneic cells from immune rejection.},
}

Humans
*Trophoblasts/metabolism/immunology
*TEA Domain Transcription Factors/metabolism
*Transcription Factors/metabolism/genetics
*HLA-G Antigens/metabolism/genetics
*Wnt Signaling Pathway
*DNA-Binding Proteins/metabolism/genetics
Female
Pregnancy
CRISPR-Cas Systems
Gene Expression Regulation
Immune Tolerance
Extravillous Trophoblasts

@article {pmid40095971,
year = {2025},
author = {Pinaud, M and Zamborlini, A},
title = {Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {216},
pages = {},
doi = {10.3791/67469},
pmid = {40095971},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Electroporation/methods ; *Gene Knockout Techniques/methods ; THP-1 Cells ; Gene Editing/methods ; Single-Cell Analysis/methods ; },
abstract = {The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including their interplay with significant human pathogens such as the human immunodeficiency virus (HIV). Compared to other immortalized cell lines of myeloid origin, THP-1 cells retain many intact inflammatory signaling pathways and display phenotypic characteristics that more closely resemble those of primary monocytes, including the ability to differentiate into macrophages when treated with phorbol-12-myristate 13-acetate (PMA). The use of CRISPR-Cas9 technology to engineer THP-1 cells through targeted gene knockout (KO) provides a powerful approach to better characterize immune-related mechanisms, including virus-host interactions. This article describes a protocol for efficient CRISPR-Cas9-based engineering using electroporation to deliver pre-assembled Cas9:sgRNA ribonucleoproteins into the cell nucleus. Using multiple sgRNAs targeting the same locus at slightly different positions results in the deletion of large DNA fragments, thereby increasing editing efficiency, as assessed by the T7 endonuclease I assay. CRISPR-Cas9-mediated editing at the genetic level was validated by Sanger sequencing followed by Inference of CRISPR Edits (ICE) analysis. Protein depletion was confirmed by immunoblotting coupled with a functional assay. Using this protocol, up to 100% indels in the targeted locus and a decrease of over 95% in protein expression were achieved. The high editing efficiency makes it convenient to isolate single-cell clones by limiting dilution.},
}

Humans
*CRISPR-Cas Systems/genetics
*Electroporation/methods
*Gene Knockout Techniques/methods
THP-1 Cells
Gene Editing/methods
Single-Cell Analysis/methods

@article {pmid40094895,
year = {2025},
author = {Malik, S and Ahsan, O and Muhammad, K and Munawar, N and Waheed, Y},
title = {Phagetherapy updates: New frontiers against antibiotic resistance.},
journal = {European journal of microbiology & immunology},
volume = {},
number = {},
pages = {},
doi = {10.1556/1886.2024.00126},
pmid = {40094895},
issn = {2062-509X},
abstract = {Antibiotic resistance is a major problem in the healthcare industry, and it presents difficulties in managing bacterial diseases worldwide. The need to find alternative antibiotic-containing methods is thus a major area for the scientific community to work on. Bacteriophage therapy is an interesting alternative that has been used in scientific research for a long time to tackle antibiotic-resistant bacteria. The purpose of this review was to compile the latest data on bacteriophages, which are progressively being used as alternatives to antibiotics, and to identify the mechanisms associated with phage therapy. The results section delves into the growing challenges posed by antibiotics and explores the potential of bacteriophages as therapeutic alternatives. This study discusses how phages can decrease antibiotic resistance, highlighting their role in modulating microbiomes and addressing various complications. This study explored the intriguing question of whether bacteriophages can combat nonbacterial diseases and examined their indirect use in pest control. In addition, this study explores the application of the CRISPR-Cas system in combating antibiotic resistance and specifically addresses phage therapy for secondary bacterial infections in COVID-19. We will further discuss whether bacteriophages are a noteworthy alternative to antibiotics by considering the evolutionary trade-offs between phages and antibiotic resistance. This section concludes by outlining future perspectives and acknowledging limitations, particularly in the context of phage and CRISPR-Cas9-mediated phage therapy. The methodology adopted for this study is a comprehensive research strategy using the Google Scholar and PubMed databases, among others. In conclusion, phage therapy is a promising strategy for tackling antibiotic-resistant bacteria, contributing to improved food production and mitigating secondary health effects. However, effective regulation requires careful selection of phages in conjunction with antibiotics to ensure judicious control of the coevolutionary dynamics between phages and antibiotics.},
}

@article {pmid40093906,
year = {2025},
author = {Qin, X and Zhou, K and Dong, L and Yang, L and Li, W and Chen, Z and Shen, C and Han, L and Li, Y and Chan, AKN and Pokharel, SP and Qing, Y and Chen, M and Wang, K and Leung, K and Sau, L and Chen, CW and Deng, X and Su, R and Chen, J},
title = {CRISPR screening reveals ZNF217 as a vulnerability in high-risk B-cell acute lymphoblastic leukemia.},
journal = {Theranostics},
volume = {15},
number = {8},
pages = {3234-3256},
pmid = {40093906},
issn = {1838-7640},
mesh = {Humans ; Animals ; *CRISPR-Cas Systems/genetics ; *Trans-Activators/genetics/metabolism ; Mice ; Cell Line, Tumor ; *Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics/metabolism ; Cell Survival/genetics ; Myeloid-Lymphoid Leukemia Protein/genetics/metabolism ; Histone-Lysine N-Methyltransferase/genetics/metabolism ; },
abstract = {Rationale: Despite substantial advancement in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), it remains a leading cause of cancer mortality in children due to the high relapse rate. Moreover, the long-term survival rates for adult B-ALL patients are still less than 40%. The B-ALL patients carrying MLL rearrangements or BCR-ABL fusion represent high-risk B-ALL subtypes that face particularly dismal prognoses. This study aims to identify innovative therapeutic vulnerability for high-risk B-ALL. Methods: The CRISPR-Cas9 screen was conducted to pinpoint genes essential for high-risk B-ALL cell survival/growth. Both in vitro and in vivo models were then employed to investigate the pathological role of ZNF217 in high-risk B-ALL. To characterize the downstream functionally essential targets of ZNF217, we performed RNA-seq and CUT&RUN-seq, followed by integrative bioinformatics analysis and experimental validation. Results: Through the focused CRISPR-Cas9 screening, ZNF217 emerged as the most essential gene for the cell survival/growth of B-ALL driven by MLL rearrangement or BCR-ABL. Through in vitro gain- and loss-of-function assays, we demonstrated that ZNF217 is indeed required for B-ALL cell survival/growth. Moreover, we established the B-ALL xenograft model and patient-derived xenograft (PDX) model and demonstrated that ZNF217 depletion significantly suppressed B-ALL progression and substantially extended the survival of recipient mice. Through integrative multiple-omics analysis, we elucidated that ZNF217 exerts its oncogenic role in B-ALL through both CoREST-dependent and CoREST-independent mechanisms. Furthermore, we characterized FOS as a functionally essential downstream target of ZNF217, and ZNF217 inhibited FOS expression in a CoREST-independent manner. Conclusions: Our findings highlight ZNF217 as a promising therapeutic target for the treatment of high-risk B-ALL, such as those carrying MLL-rearrangements or BCR-ABL fusion.},
}

Humans
Animals
*CRISPR-Cas Systems/genetics
*Trans-Activators/genetics/metabolism
Mice
Cell Line, Tumor
*Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics/metabolism
Cell Survival/genetics
Myeloid-Lymphoid Leukemia Protein/genetics/metabolism
Histone-Lysine N-Methyltransferase/genetics/metabolism

@article {pmid40038872,
year = {2025},
author = {Duan, ZW and Wang, WT and Wang, Y and Wang, R and Hua, W and Shang, CY and Gao, R and Shen, HR and Li, Y and Wu, JZ and Yin, H and Wang, L and Li, JY and Xu, W and Liang, JH},
title = {SH3GL1-activated FTH1 inhibits ferroptosis and confers doxorubicin resistance in diffuse large B-cell lymphoma.},
journal = {Clinical and translational medicine},
volume = {15},
number = {3},
pages = {e70246},
pmid = {40038872},
issn = {2001-1326},
support = {82200887//National Natural Science Foundation of China/ ; 82370194//National Natural Science Foundation of China/ ; BK20220716//Jiangsu Science and Technology Department/ ; BE2023780//Jiangsu Science and Technology Department/ ; 2022M7114034//China Postdoctoral Science Foundation/ ; 2023M741463//China Postdoctoral Science Foundation/ ; },
mesh = {*Ferroptosis ; Doxorubicin/pharmacology ; *Drug Resistance, Neoplasm ; *Lymphoma, Large B-Cell, Diffuse/drug therapy/genetics/metabolism ; CRISPR-Cas Systems ; Immunohistochemistry ; Cell Proliferation ; Cell Survival ; Prognosis ; Humans ; Male ; Female ; Adult ; Middle Aged ; Aged ; Animals ; Mice ; Neoplasm Transplantation ; Gene Expression Regulation, Neoplastic ; Biomarkers, Tumor/biosynthesis ; },
abstract = {BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is predominant subtype of non-Hodgkin lymphoma and can be effectively treated. Nevertheless, a subset of patients experiences refractory or relapsed disease, highlighting the need for new therapeutic strategies.

METHODS: Depmap database based on CRISPR/Cas9 knock out analysis was employed to identify the essential gene SH3GL1, which encodes endophilin A2, as crucial for the proliferation and survival of DLBCL cells. Immunohistochemistry (IHC) staining was performed on the 126 paraffin-embedded clinical DLBCL samples to investigate the association between SH3GL1 expression levels and the prognosis. To investigate the specific mechanism modulated by SH3GL1 in the progression of DLBCL, an integrative approach was employed. This approach combined high-throughput sequencing technologies, such as Deep-DIA and LC-MS, with functional validation techniques, including CRISPR/Cas9 gene editing, xenograft models, and molecular pathway analyses.

RESULTS: Our study found that high expression levels of SH3GL1 correlate with poor prognosis in a cohort of 126 newly diagnosed DLBCL patients, underscoring its significance in disease progression. Mechanistically, we found that SH3GL1 deficiency triggers ferritin heavy chain 1 (FTH1)-mediated ferroptosis, specifically ferritinophagy-induced ferroptosis, in DLBCL cells. Additionally, high expression of SH3GL1 suppresses doxorubicin-induced ferroptosis. Cancer cells' resistance to conventional therapies is associated with increased sensitivity to ferroptosis.

CONCLUSIONS: These findings emphasise SH3GL1 as a promising prognostic biomarker and a potential therapeutic target in DLBCL, offering new avenues for treatment strategies aimed at overcoming drug resistance and improving patients' outcomes.

KEY POINTS: Elevated SH3GL1 expression in DLBCL patients was associated with a negative prognosis. SH3GL1 plays a crucial role in promoting DLBCL cell survival through the regulation of FTH1-mediated ferroptosis and doxorubicin resistance.},
}

*Ferroptosis
Doxorubicin/pharmacology
*Drug Resistance, Neoplasm
*Lymphoma, Large B-Cell, Diffuse/drug therapy/genetics/metabolism
CRISPR-Cas Systems
Immunohistochemistry
Cell Proliferation
Cell Survival
Prognosis
Humans
Male
Female
Adult
Middle Aged
Aged
Animals
Mice
Neoplasm Transplantation
Gene Expression Regulation, Neoplastic
Biomarkers, Tumor/biosynthesis

@article {pmid40036525,
year = {2025},
author = {Zheng, Z and Qian, Z and Huang, D and Weng, Z and Wang, J and Lin, C and Qiu, B and Lin, Z and Luo, F},
title = {Ultrasensitive Homogeneous Electrochemiluminescence Biosensor for N-Nitrosodimethylamine Detection Based on Vertically-Ordered Mesoporous Silica Film-Modified Electrode and CRISPR/Cas12a-Driven HRCA with Triple Signal Amplification.},
journal = {Analytical chemistry},
volume = {97},
number = {10},
pages = {5828-5835},
doi = {10.1021/acs.analchem.5c00555},
pmid = {40036525},
issn = {1520-6882},
mesh = {*Silicon Dioxide/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques ; *Luminescent Measurements ; *Electrodes ; *Dimethylnitrosamine/analysis/chemistry ; Porosity ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; Limit of Detection ; DNA/chemistry ; Humans ; Aptamers, Nucleotide/chemistry ; },
abstract = {Herein, we present an innovative electrochemiluminescence (ECL) biosensor for the ultrasensitive detection of N-nitrosodimethylamine (NDMA). The biosensor utilizes a triple signal amplification strategy, combining rolling circle amplification (RCA), CRISPR/Cas12a-driven hyperbranched rolling circle amplification (HRCA), and electrostatic repulsion with size exclusion effects from vertically ordered mesoporous silica film (VMSF)/indium tin oxide (ITO) on double-stranded DNA (dsDNA)-Ru(phen)3[2+] complexes. In this system, aptamers and circular DNA undergo RCA reactions, followed by the CRISPR/Cas12a-mediated HRCA process, producing abundant dsDNA. The electropositive ECL indicator, namely Ru(phen)3[2+], was subsequently adsorbed onto the electronegative dsDNA, forming dsDNA-Ru(phen)3[2+] complexes. These complexes are subjected to electrostatic repulsion and size exclusion by the VMSF-modified ITO electrode, resulting in a lower ECL intensity. Upon introducing NDMA, the aptamer preferentially binds to NDMA, thereby preventing the formation of long dsDNA. This process releases free Ru(phen)3[2+], which diffuses to the electrode surface through narrow mesoporous channels via electrostatic adsorption. Consequently, an enhanced and strong ECL signal is observed. The integration of VMSF enhances selectivity and sensitivity by excluding larger impurities and promoting the electrostatic repulsion of dsDNA-Ru(phen)3[2+] complexes near the electrode surface. Additionally, the CRISPR/Cas12a system eliminates the formation of primer dimers and reduces false positives through its unique cis- and trans-cleavage activities. The biosensor demonstrated excellent performance with a linear correlation between the ECL signal and NDMA concentration in the range spanning from 10 pg/mL to 10 μg/mL, achieving a low limit of detection of 5.33 pg/mL. This platform offers a reliable and robust solution for detecting NDMA in complex matrices, making it a promising tool for environmental monitoring, public health, and safety applications.},
}

*Silicon Dioxide/chemistry
*Biosensing Techniques/methods
*Electrochemical Techniques
*Luminescent Measurements
*Electrodes
*Dimethylnitrosamine/analysis/chemistry
Porosity
Nucleic Acid Amplification Techniques
CRISPR-Cas Systems
Limit of Detection
DNA/chemistry
Humans
Aptamers, Nucleotide/chemistry

@article {pmid39920943,
year = {2025},
author = {Li, H and Song, C and Li, Y and Zhang, T and Yang, X and Wang, H},
title = {Genome-wide CRISPR screen reveals host factors for gama- and delta-coronavirus infection in Huh7 cells.},
journal = {International journal of biological macromolecules},
volume = {304},
number = {Pt 1},
pages = {140728},
doi = {10.1016/j.ijbiomac.2025.140728},
pmid = {39920943},
issn = {1879-0003},
mesh = {Humans ; *CRISPR-Cas Systems ; Deltacoronavirus/genetics ; Host-Pathogen Interactions/genetics ; Coronavirus Infections/virology ; Animals ; Infectious bronchitis virus/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Virus Internalization ; Cell Line ; },
abstract = {Genome-wide CRISPR screening has emerged as a powerful tool for identifying novel host factors involved in viral infections. In recent years, host factors for several Alpha- and Beta-coronaviruses have been systematically screened and characterized. However, knowledge regarding Gamma- and Delta-coronavirus infections remains limited. In this study, we conducted genome-scale CRISPR knockout (KO) screening in Huh7 cells infected with infectious bronchitis virus (IBV), a Gamma-coronavirus, and porcine deltacoronavirus (PDCoV), a Delta-coronavirus. We identified known host factors for PDCoV, including APN and TMEM41B. We confirmed that human APN does not serve as a critical host factor for IBV. Notably, SPPL3 was identified as a key factor involved in viral particle entry and S protein-induced syncytium formation through the modulation of cellular N-glycosylation. Furthermore, we performed a meta-analysis integrating all Huh7 cell-based genome-wide CRISPR screens across the four genera of coronaviruses (Alpha-, Beta-, Gamma-, and Delta-coronaviruses). Our analysis highlighted conserved host pathways, particularly those related to proteoglycans, glycoproteins, and vesicle trafficking. TMEM41B, SCAP, and FAM98A emerged as the most frequently targeted host genes. These findings provide valuable insights into the life cycles of IBV and PDCoV infections and facilitate the development of host-directed therapeutic strategies.},
}

Humans
*CRISPR-Cas Systems
Deltacoronavirus/genetics
Host-Pathogen Interactions/genetics
Coronavirus Infections/virology
Animals
Infectious bronchitis virus/genetics/physiology
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Virus Internalization
Cell Line

@article {pmid39914548,
year = {2025},
author = {Liu, Y and Wang, J and Cui, G and Wang, X and Xiang, S and Huang, W and Liu, C},
title = {RNA aptamer-based CRISPR-Cas12a system for enhanced small molecule detection and point-of-care testing.},
journal = {International journal of biological macromolecules},
volume = {303},
number = {},
pages = {140675},
doi = {10.1016/j.ijbiomac.2025.140675},
pmid = {39914548},
issn = {1879-0003},
mesh = {*Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems ; Humans ; *Theophylline ; Point-of-Care Testing ; Biosensing Techniques/methods ; },
abstract = {The CRISPR-Cas12a system has emerged as a robust platform for small molecule detection. However, existing methodologies primarily emphasize DNA aptamer-based strategies. This study introduces an RNA aptamer-based CRISPR-Cas12a approach due to the fact that the majority of small molecules lack corresponding DNA aptamers. The approach employs theophylline RNA aptamer (TA) to regulate Cas12a activity through competitive inhibition of crRNA. The results demonstrate that this system effectively detects theophylline (TP) in various food, beverage, and human serum samples, exhibiting excellent selectivity and sensitivity. Additionally, a visual paper-based detection system showcases its applicability for real-time analysis in food matrices and human serum. The RNA aptamer-based CRISPR-Cas12a strategy holds significant potential for diverse biomedical applications, offering a versatile tool for future sensing applications through customized RNA aptamer designs for small molecules.},
}

*Aptamers, Nucleotide/chemistry
*CRISPR-Cas Systems
Humans
*Theophylline
Point-of-Care Testing
Biosensing Techniques/methods

@article {pmid39853276,
year = {2025},
author = {Shankar, S and Giraldo, D and Tauxe, GM and Spikol, ED and Li, M and Akbari, OS and Wohl, MP and McMeniman, CJ},
title = {Optimized genetic tools for neuroanatomical and functional mapping of the Aedes aegypti olfactory system.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {3},
pages = {},
doi = {10.1093/g3journal/jkae307},
pmid = {39853276},
issn = {2160-1836},
support = {R21AI139358/GF/NIH HHS/United States ; AID-OAA-F-16-00061//USAID/ ; 200-2017-93143/CC/CDC HHS/United States ; T32A1007417//Molecular and Cellular Basis of Infectious Diseases/ ; R01AI151004/GF/NIH HHS/United States ; S10OD016374/NH/NIH HHS/United States ; NS050274//JHU Center for Neuroscience Research Machine Shop/ ; 200-2017-93143/CC/CDC HHS/United States ; R21AI139358/NH/NIH HHS/United States ; },
mesh = {Animals ; *Aedes/genetics/physiology ; Olfactory Receptor Neurons/metabolism ; Animals, Genetically Modified ; Olfactory Pathways ; CRISPR-Cas Systems ; Transgenes ; Receptors, Odorant/genetics/metabolism ; },
abstract = {The mosquito Aedes aegypti is an emerging model insect for invertebrate neurobiology. We detail the application of a dual transgenesis marker system that reports the nature of transgene integration with circular donor template for CRISPR-Cas9-mediated homology-directed repair at target mosquito chemoreceptor genes. Employing this approach, we demonstrate the establishment of cell-type-specific T2A-QF2 driver lines for the A. aegypti olfactory co-receptor genes Ir8a and orco via canonical homology-directed repair and the CO2 receptor complex gene Gr1 via noncanonical homology-directed repair involving duplication of the intended T2A-QF2 integration cassette separated by intervening donor plasmid sequence. Using Gr1+ olfactory sensory neurons as an example, we show that introgression of such T2A-QF2 driver and QUAS responder transgenes into a yellow cuticular pigmentation mutant strain facilitates transcuticular calcium imaging of CO2-evoked neural activity on the maxillary palps with enhanced sensitivity relative to wild-type mosquitoes enveloped by dark melanized cuticle. We further apply Cre-loxP excision to derive marker-free T2A-QF2 in-frame fusions to clearly map axonal projection patterns from olfactory sensory neurons expressing these 3 chemoreceptors into the A. aegypti antennal lobe devoid of background interference from 3xP3-based fluorescent transgenesis markers. The marker-free Gr1 T2A-QF2 driver facilitates clear recording of CO2-evoked responses in this central brain region using the genetically encoded calcium indicators GCaMP6s and CaMPARI2. Systematic application of these optimized methods to different chemoreceptors stands to enable mapping A. aegypti olfactory circuits at peripheral and central levels of olfactory coding at high resolution.},
}

Animals
*Aedes/genetics/physiology
Olfactory Receptor Neurons/metabolism
Animals, Genetically Modified
Olfactory Pathways
CRISPR-Cas Systems
Transgenes
Receptors, Odorant/genetics/metabolism

@article {pmid39804955,
year = {2025},
author = {Weasner, BM and Weasner, BP and Cook, KR and Stinchfield, MJ and Kondo, S and Saito, K and Kumar, JP and Newfeld, SJ},
title = {A new Drosophila melanogaster research resource: CRISPR-induced mutations for clonal analysis of fourth chromosome genes.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {3},
pages = {},
doi = {10.1093/g3journal/jkaf006},
pmid = {39804955},
issn = {2160-1836},
support = {R24 OD028242/NH/NIH HHS/United States ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Mutation ; *CRISPR-Cas Systems ; Female ; Chromosomes, Insect/genetics ; Phenotype ; Male ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {As part of an ongoing effort to generate comprehensive resources for the experimental analysis of fourth chromosome genes in Drosophila melanogaster, the Fourth Chromosome Resource Project has used CRISPR mutagenesis with single guide RNAs to isolate mutations in 62 of the 80 fourth chromosome, protein-coding genes. These mutations were induced on a fourth chromosome bearing a basal FRT insertion to facilitate experimental approaches involving FLP recombinase-induced mitotic recombination. To permit straightforward comparisons among mutant stocks, most of the mutations were generated on isogenic fourth chromosomes, which were then crossed into a common genetic background. Of the 119 mutations, 84 are frameshift mutations likely to be null alleles, 29 are small, in-frame deletions, and 6 have yet to be characterized molecularly. The mutations were tested for recessive lethal, female-sterile, and visible phenotypes. Stable stocks for most of the mutations have been submitted to repositories in the United States and Japan for public distribution.},
}

Animals
*Drosophila melanogaster/genetics
*Mutation
*CRISPR-Cas Systems
Female
Chromosomes, Insect/genetics
Phenotype
Male
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40093900,
year = {2025},
author = {Bai, L and Pang, Y and Wang, T and Wang, S and Guo, K and Xuan, T and Zhang, Z and Liu, D and Qian, F and Zheng, Y and Jin, G and Wang, R},
title = {SPEAR: CRISPR-mediated ultrasensitive, specific and rapid one-pot detection strategy for cancer-related SNPs.},
journal = {Theranostics},
volume = {15},
number = {8},
pages = {3275-3288},
pmid = {40093900},
issn = {1838-7640},
mesh = {*Polymorphism, Single Nucleotide/genetics ; Humans ; *Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; *Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Point Mutation/genetics ; Early Detection of Cancer/methods ; Molecular Diagnostic Techniques/methods ; },
abstract = {Rationale: The ultrasensitive and accurate detection of driver mutations is critical for early cancer screening and precision medicine. Current methods face challenges in balancing sensitivity, specificity, and speed, which limits their clinical utility. Therefore, a rapid, sensitive, and specific method is essential for detecting cancer-related SNPs. Methods: This study introduces SPEAR (Specific Point mutation Evaluation via CRISPR-Cas Assisted Recognition), a novel methodology combining NEAR (Nicking Enzyme Amplification Reaction) isothermal amplification with SNP-specific recognition by Cas12b RNP in a one-pot configuration to detect cancer-related single nucleotide polymorphisms (SNPs). SPEAR leverages the power of NEAR isothermal amplification to efficiently amplify target DNA, followed by Cas12b RNP for SNP-specific recognition. This integrated approach ensures a rapid and precise mutation detection system in a single reaction. Results: The method was applied to blood samples for the detection of cancer-related mutations, with results obtained in approximately 30 min. The SPEAR enables detection of gene mutations at the single-molecule level and it can detect targets at a 0.1% ratio despite strong background interference. The method exhibits single-base resolution specificity, allowing for the detection of multiple SNPs in a single reaction. It outperforms first-generation sequencing (FGS) in both convenience and sensitivity, while remaining compatible with next-generation sequencing (NGS). Conclusion: SPEAR offers a rapid, sensitive, and convenient approach to detect cancer-related SNPs, with significant potential for clinical applications, including real-time detection and molecular diagnostics in precision medicine.},
}

*Polymorphism, Single Nucleotide/genetics
Humans
*Neoplasms/genetics/diagnosis
*CRISPR-Cas Systems/genetics
*Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
Point Mutation/genetics
Early Detection of Cancer/methods
Molecular Diagnostic Techniques/methods

@article {pmid40093809,
year = {2025},
author = {Li, B and Pan, Y and Wu, J and Miao, C and Wang, Z},
title = {Large-scale genomic-wide CRISPR screening revealed PRC1 as a tumor essential candidate in clear cell renal cell carcinoma.},
journal = {International journal of medical sciences},
volume = {22},
number = {7},
pages = {1658-1671},
pmid = {40093809},
issn = {1449-1907},
mesh = {Humans ; *Carcinoma, Renal Cell/genetics/pathology ; *Kidney Neoplasms/genetics/pathology/mortality ; *Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; *Cell Proliferation/genetics ; Biomarkers, Tumor/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Male ; Female ; Prognosis ; Middle Aged ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Protein Interaction Maps/genetics ; Cell Movement/genetics ; Cell Cycle Proteins ; },
abstract = {Background: Clear cell renal cell carcinoma (ccRCC) is a prevalent and aggressive subtype of kidney cancer, often associated with metastasis and recurrence. Identifying key genes involved in ccRCC progression is critical for improving treatment strategies and patient outcomes. Methods: We performed a large-scale genome-wide CRISPR screening to identify genes crucial to ccRCC progression using the DepMap database. For discovery and validation, we integrated multi-omics data from The Cancer Genome Atlas (TCGA), GEO, and the NJMU-ccRCC clinical cohort. Bioinformatics analyses, including differential expression, pathway enrichment, and protein-protein interaction network analysis, were conducted to elucidate the biological functions. To validate our findings, we employed immunohistochemistry, qRT-PCR, and various cellular assays to investigate the role of PRC1 in ccRCC. Results: CRISPR screening identified PRC1 as a key gene significantly overexpressed in ccRCC tissues from the DepMap database. Elevated PRC1 expression was associated with poor overall survival, disease-specific survival, and progression-free interval. Silencing PRC1 in ccRCC cell lines inhibited cell proliferation, migration, and colony formation. Functional enrichment analyses revealed that PRC1 is involved in essential processes such as cell cycle regulation, mitosis, and cytokinesis. Additionally, PRC1 expression was correlated with the activation of the Wnt/β-catenin pathway, suggesting that PRC1 plays a pivotal role in tumor progression. Conclusion: PRC1 emerges as a promising biomarker and therapeutic target for ccRCC. Elevated PRC1 expression is associated with poor prognosis, and its inhibition suppresses ccRCC cell proliferation and migration. Our findings underscore the crucial role of PRC1 in ccRCC progression and highlight the need for further investigation into its molecular mechanisms and therapeutic potential.},
}

Humans
*Carcinoma, Renal Cell/genetics/pathology
*Kidney Neoplasms/genetics/pathology/mortality
*Gene Expression Regulation, Neoplastic
Cell Line, Tumor
*Cell Proliferation/genetics
Biomarkers, Tumor/genetics/metabolism
CRISPR-Cas Systems/genetics
Male
Female
Prognosis
Middle Aged
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Protein Interaction Maps/genetics
Cell Movement/genetics
Cell Cycle Proteins

@article {pmid40093536,
year = {2025},
author = {Hershan, AA},
title = {Virology, epidemiology, transmissions, diagnostic tests, prophylaxis and treatments of human Mpox: Saudi Arabia perspective.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1530900},
pmid = {40093536},
issn = {2235-2988},
mesh = {Humans ; Saudi Arabia/epidemiology ; Animals ; *Mpox, Monkeypox/epidemiology/diagnosis ; *Monkeypox virus/genetics/isolation & purification ; Diagnostic Tests, Routine ; Antiviral Agents/therapeutic use/pharmacology ; },
abstract = {Mpox (Monkeypox) is a highly contagious viral disease that can be transmitted from animal-to-human or human-to-human through intimate contact, Mpox is caused by the monkeypox virus (MPXV), which is an enveloped double-stranded DNA that belongs to the genus Orthopoxvirus, Poxviridae family, and subfamily Chordopoxvirinae. Mpox cases were previously only reported in West and Central Africa, however in recent times non-endemic countries including Saudi Arabia (SA) also reported confirmed Mpox cases. The first laboratory-confirmed human Mpox case in SA was reported on 14 July 2022, since then a number of confirmed Mpox cases have been reported by WHO in SA. These confirmed Mpox cases in SA were observed among individuals with a history of visiting European Union countries. SA is not only at risk of importation of Mpox cases owing to travel to such countries, but also there are various other risk factors including geographic proximity to the African continent, trade in exotic animals, and massive inflow of tourists. Therefore, government health authorities of SA should continue to collaborate with various international health organizations including WHO to prevent, manage or monitor potential health risks at most of the entry points in SA including highways, seaports, and airports by ensuring adherence to hygiene protocols, vaccinations, and health screenings. There are a range of diagnostic tests are currently available that can be used in SA to confirm Mpox infections, including real-time PCR, loop-mediated isothermal amplification, serological testing, clustered regularly interspaced short palindromic repeat-CRISPR-associated protein (CRISPR-Cas)-based systems, whole-genome sequencing, electron microscopy, and virus isolation and culture. There is no approved treatment specifically for Mpox, however multiple approved antiviral agents for smallpox treatment were found to be useful in Mpox treatment and in the management of Mpox outbreaks, such as- trifluridine, brincidofovir, tecovirimat, and cidofovir. The aim of this review is to provide valuable insights regarding virology, pathogenesis, epidemiology, transmissions, clinical presentation, diagnostic tests, prophylactic measures and therapeutic options of Mpox from SA perspective. Moreover, a side-by-side discussion on the global trend and scenarios of Mpox has been provided for comparison and further improvement in measures against Mpox in SA.},
}

Humans
Saudi Arabia/epidemiology
Animals
*Mpox, Monkeypox/epidemiology/diagnosis
*Monkeypox virus/genetics/isolation & purification
Diagnostic Tests, Routine
Antiviral Agents/therapeutic use/pharmacology

@article {pmid40092787,
year = {2025},
author = {Smith, RL and Davenport, PW and Lakin, MR},
title = {A Study of CRISPR Ribonucleoprotein Displacement in Cell-Free Systems.},
journal = {ACS omega},
volume = {10},
number = {9},
pages = {9154-9164},
pmid = {40092787},
issn = {2470-1343},
abstract = {CRISPR/Cas-based transcription factors are a powerful tool for controlling gene expression in living cells and cell-free systems, as their programmable DNA-binding activity makes them a powerful tool for building and scaling up engineered genetic networks. The use of guide RNAs for targeting Cas proteins to desired binding sites opens up the possibility of using RNA engineering techniques to achieve programmable and dynamic control of CRISPR/Cas-based transcription factor activity and hence of gene expression. In this work, we investigate the use of RNA strand displacement systems to remove bound CRISPR/Cas ribonucleoprotein complexes from target DNA in cell-free systems. The binding of catalytically inactive dCas9 is monitored by using CRISPR interference to repress the expression of a reporter protein. We express an antisense RNA complementary to an extended toehold on an engineered guide RNA in an E. coli-based cell-free expression system with the goal of rapidly removing bound CRISPR/Cas ribonucleoproteins via strand displacement. We find that dCas9 appears to be surprisingly resistant to removal via this mechanism, which indicates that other strategies for dynamic removal of bound Cas proteins may prove to be more effective.},
}

@article {pmid40092730,
year = {2025},
author = {Deng, Y and Xu, J and Yang, M and Huang, Y and Yang, Y},
title = {Rapid detection of the GJB2 c.235delC mutation based on CRISPR-Cas13a combined with lateral flow dipstick.},
journal = {Open life sciences},
volume = {20},
number = {1},
pages = {20251064},
pmid = {40092730},
issn = {2391-5412},
abstract = {Hereditary hearing loss, an auditory neuropathy disorder, is characterized by its high prevalence and significant impact on the quality of life of those affected. In Chinese populations, the most prevalent gap junction beta-2 (GJB2) mutation hotspot is c.235delC. Currently available genetic tests require expensive instruments and specialized technicians or have long testing cycles and high costs, and therefore cannot meet point-of-care testing (POCT) requirements. The objective of this study was to evaluate the viability of a POCT kit. In only 42 min, we successfully identified the GJB2 mutation site c.235delC by integrating CRISPR-Cas nucleic acid detection with recombinase-aided amplification (RAA) and a lateral flow dipstick (LFD) method. This method has the capacity to detect low-abundance nucleic acids (as low as 10[2] copies/μL) and low mutation frequency (20%), in addition to accurately distinguishing wild-type, homozygous, and heterozygous mutation. This approach was utilized to assess blood samples from a total of 31 deaf patients and 5 healthy volunteers. All results were subsequently confirmed through the implementation of Sanger sequencing. Our detection results were consistent with Sanger sequencing results. The diagnostic sensitivity and specificity were 100%. The combination of CRISPR-Cas13a and LFD may be a promising method for POCT of deafness genes.},
}

@article {pmid40092158,
year = {2025},
author = {Sun, Y and Wu, G and Wang, Y and Jiang, J and Wang, H and Liu, F and Lu, F and Zhang, H},
title = {Application of multiple genomic-editing technologies in Streptomyces fungicidicus for improved enduracidin yield.},
journal = {Synthetic and systems biotechnology},
volume = {10},
number = {2},
pages = {564-573},
pmid = {40092158},
issn = {2405-805X},
abstract = {Streptomyces fungicidicus, an industrial strain for enduracidin production, shows significant potential as a cellular chassis for the synthesis of novel small peptides. Targeted deletion of secondary metabolite gene clusters offers a promising strategy to enhance strain performance, but is often hampered by the lack of efficient gene editing tools. In this study, we optimized the traditional homologous recombination method by integrating selection and counter-selection markers to streamline the gene editing process, and successfully deleted gene clusters of up to 54.4 kb. Recognizing the significant potential of CRISPR/Cas-based systems in Streptomyces, we evaluated the base editing efficiency of the CRISPR/cBEST system in S. fungicidicus, which enabled stop codon insertions in the targeted gene with different mutation rates depending on the applied sgRNA. Additionally, we established a CRISPR/Cas9 system in S. fungicidicus while incorporating a counter-selection marker for efficient screening, which greatly shortened the gene editing cycle. The resulting mutants with single and cumulative gene cluster deletions exhibited improved growth characteristics, including a prolonged logarithmic phase and increased biomass. Although cumulative deletions did not result in consistent yield improvements, the mutants with improved growth characteristics show potential for further strain optimization in the future. The optimized gene editing systems developed in this study provide a valuable foundation for engineering other Streptomyces species.},
}

@article {pmid40091120,
year = {2025},
author = {Moreno-Sánchez, I and Hernández-Huertas, L and Nahón-Cano, D and Martínez-García, PM and Treichel, AJ and Gómez-Marin, C and Tomás-Gallardo, L and da Silva Pescador, G and Kushawah, G and Egidy, R and Perera, A and Díaz-Moscoso, A and Cano-Ruiz, A and Walker, JA and Muñoz, MJ and Holden, K and Galcerán, J and Nieto, MÁ and Bazzini, AA and Moreno-Mateos, MA},
title = {Enhanced RNA-targeting CRISPR-Cas technology in zebrafish.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {2591},
pmid = {40091120},
issn = {2041-1723},
mesh = {*Zebrafish/genetics ; Animals ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; RNA, Messenger/genetics/metabolism ; Embryo, Nonmammalian/metabolism ; RNA/genetics/metabolism ; },
abstract = {CRISPR-Cas13 RNA-targeting systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its competence could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) use chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improve nuclear RNA targeting, and iii) compare different computational models and determine the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrate that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implement alternative RNA-targeting CRISPR-Cas systems such as CRISPR-Cas7-11 and CRISPR-DjCas13d. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.},
}

*Zebrafish/genetics
Animals
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods
RNA, Messenger/genetics/metabolism
Embryo, Nonmammalian/metabolism
RNA/genetics/metabolism

@article {pmid40088462,
year = {2025},
author = {Qing, M and Huang, C and Li, Y and Yu, Q and Hu, Q and Zhou, J and Yuan, R and Bai, L},
title = {Dithiothreitol Facilitates LbCas12a with Expanded PAM Preference for Ultrasensitive Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c00280},
pmid = {40088462},
issn = {1520-6882},
abstract = {Clustered regularly interspaced short palindromic repeats-associated (CRISPR/Cas) proteins have been used for a growing class of in vitro molecular diagnostics due to their modularity and high specificity in targeting nucleic acid. However, the requirement of a protospacer adjacent motif (PAM) for Cas protein-catalyzed trans-cleavage poses a challenge for random nucleic acid detection. Here, we demonstrate that dithiothreitol (DTT) enables LbCas12a to adopt a relaxed preference for PAM base pairing, thereby expanding the target sequence space. Accordingly, we propose a DTT-mediated CRISPR/Cas12a toolbox (DTT-deCRISPR) that exhibits relaxed PAM specificity and is readily compatible with nucleic acid amplification techniques including recombinase polymerase amplification (RPA) and polymerase chain reaction (PCR). As a proof of concept, we integrate DTT-deCRISPR with frequently used PCR for sensitively and selectively detecting high-risk human papillomavirus (HPV) 16 and 18. The platform demonstrates the ability to detect synthesized HPV 16 and 18 plasmids down to 1 aM within 60 min. Based on the receiver operating characteristic curve analysis, the clinical sensitivities of the developed method for detecting HPV 16 and 18 are 93.75% and 80.00%, respectively. We further incorporate it into a lateral flow assay (LFA) for point-of-care detection, and the HPV 16 and HPV 18 abundances determined by LFA for clinical samples are consistent with the fluorescence analysis results. Together, this work uncovers an unexpected connection between DTT and PAM preferences of LbCas12a, promoting the universality and flexibility of CRISPR technology in molecular diagnostics.},
}

@article {pmid40087886,
year = {2025},
author = {Wu, G and Taylor, E and Youmans, DT and Arnoult, N and Cech, TR},
title = {Rapid dynamics allow the low-abundance RTEL1 helicase to promote telomere replication.},
journal = {Nucleic acids research},
volume = {53},
number = {5},
pages = {},
pmid = {40087886},
issn = {1362-4962},
support = {R35GM143108/NH/NIH HHS/United States ; DGE 2040434//GRFP/ ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Humans ; *DNA Helicases/metabolism/genetics ; *Telomere/metabolism ; *DNA Replication ; *Telomeric Repeat Binding Protein 2/metabolism/genetics ; S Phase/genetics ; Single Molecule Imaging ; Chromatin/metabolism ; Cell Nucleus/metabolism/genetics ; CRISPR-Cas Systems ; Protein Binding ; HeLa Cells ; G-Quadruplexes ; },
abstract = {Regulator of telomere length 1 (RTEL1) helicase facilitates telomere replication by disassembling DNA secondary structures, such as G-quadruplexes and telomeric loops (t-loops), at the ends of the chromosomes. The recruitment of RTEL1 to telomeres occurs during the S-phase of the cell cycle, but the dynamics of the process has not been studied. Here, we utilized CRISPR genome editing and single-molecule imaging to monitor RTEL1 movement within human cell nuclei. RTEL1 utilizes rapid three-dimensional diffusion to search for telomeres and other nuclear targets. Only 5% of the chromatin-bound RTEL1 is associated with telomeres at any time in the S-phase, but the telomere-bound RTEL1 has much more extended associations. This binding is enhanced by the interaction between RTEL1 and the telomeric protein TRF2 but is largely independent of RTEL1 ATPase activity. The absence of RTEL1 catalytic activity leads to severe defects in cell proliferation, slow progression out of S-phase, and chromosome end-to-end fusion events. We propose that the rapid diffusion of RTEL1 allows this low-abundance protein to explore the nucleus, bind TRF2, and be recruited to telomeres.},
}

Humans
*DNA Helicases/metabolism/genetics
*Telomere/metabolism
*DNA Replication
*Telomeric Repeat Binding Protein 2/metabolism/genetics
S Phase/genetics
Single Molecule Imaging
Chromatin/metabolism
Cell Nucleus/metabolism/genetics
CRISPR-Cas Systems
Protein Binding
HeLa Cells
G-Quadruplexes

@article {pmid40087793,
year = {2025},
author = {Barraza-Flores, P and Moghadaszadeh, B and Lee, W and Isaac, B and Sun, L and Hickey, ET and Rockowitz, S and Sliz, P and Beggs, AH},
title = {Zebrafish and cellular models of SELENON-Congenital myopathy exhibit novel embryonic and metabolic phenotypes.},
journal = {Skeletal muscle},
volume = {15},
number = {1},
pages = {7},
pmid = {40087793},
issn = {2044-5040},
support = {P50HD105351//Eunice Kennedy Shriver National Institute of Child Health and Human Development/ ; T32NS007473/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Phenotype ; *Selenoproteins/genetics/metabolism ; *Disease Models, Animal ; *Zebrafish Proteins/genetics/metabolism ; Myoblasts/metabolism ; Muscular Diseases/genetics/metabolism/pathology ; Glutathione/metabolism ; Animals, Genetically Modified ; Reactive Oxygen Species/metabolism ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: SELENON-Congenital Myopathy (SELENON-CM) is a rare congenital myopathy caused by mutations of the SELENON gene characterized by axial muscle weakness and progressive respiratory insufficiency. Muscle histopathology may be non-specific, but commonly includes multiminicores or a dystrophic pattern. The SELENON gene encodes selenoprotein N (SelN), a selenocysteine-containing redox enzyme located in the endo/sarcoplasmic reticulum membrane where it colocalizes with mitochondria-associated membranes. However, the molecular mechanism(s) by which SelN deficiency cause SELENON-CM remain poorly understood. A hurdle is the lack of cellular and animal models that show easily assayable phenotypes.

METHODS: Using CRISPR-Cas9 we generated three zebrafish models of SELENON-CM, which were then studied by spontaneous coiling, hatching, and activity assays. We also performed selenon coexpression analysis using a single cell RNAseq zebrafish embryo-atlas. SelN-deficient myoblasts were generated and assayed for glutathione, reactive oxygen species, carbonylation, and nytrosylation levels. Finally, we tested Selenon-deficient myoblasts' metabolism using a Seahorse cell respirometer.

RESULTS: We report deep-phenotyping of SelN-deficient zebrafish and muscle cells. SelN-deficient zebrafish exhibit changes in embryonic muscle function and swimming activity in larvae. Analysis of single cell RNAseq data in a zebrafish embryo-atlas revealed coexpression of selenon and genes involved in the glutathione redox pathway. SelN-deficient zebrafish and mouse myoblasts exhibit altered glutathione and redox homeostasis, as well as abnormal patterns of energy metabolism, suggesting roles for SelN in these functions.

CONCLUSIONS: These data demonstrate a role for SelN in zebrafish early development and myoblast metabolism and provide a basis for cellular and animal model assays for SELENON-CM.},
}

Animals
*Zebrafish/genetics/metabolism
*Phenotype
*Selenoproteins/genetics/metabolism
*Disease Models, Animal
*Zebrafish Proteins/genetics/metabolism
Myoblasts/metabolism
Muscular Diseases/genetics/metabolism/pathology
Glutathione/metabolism
Animals, Genetically Modified
Reactive Oxygen Species/metabolism
Embryo, Nonmammalian/metabolism
CRISPR-Cas Systems

@article {pmid40087758,
year = {2025},
author = {Peng, W and Shi, M and Hu, B and Jia, J and Li, X and Wang, N and Man, S and Ye, S and Ma, L},
title = {Nanotechnology-leveraged CRISPR/Cas systems: icebreaking in trace cancer-related nucleic acids biosensing.},
journal = {Molecular cancer},
volume = {24},
number = {1},
pages = {78},
pmid = {40087758},
issn = {1476-4598},
support = {32072309//National Natural Science Foundation of China/ ; 19JCYBJC27800//Tianjin Municipal Science and Technology Committee/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Neoplasms/diagnosis/genetics ; *Nanotechnology/methods ; *Biomarkers, Tumor ; Animals ; Nucleic Acids/genetics ; },
abstract = {As promising noninvasive biomarkers, nucleic acids provide great potential to innovate cancer early detection methods and promote subsequent diagnosis to improve the survival rates of patient. Accurate, straightforward and sensitive detection of such nucleic acid-based cancer biomarkers in complex biological samples holds significant clinical importance. However, the low abundance creates huge challenges for their routine detection. As the next-generation diagnostic tool, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) with their high programmability, sensitivity, fidelity, single-base resolution, and precise nucleic acid positioning capabilities are extremely attractive for trace nucleic acid-based cancer biomarkers (NABCBs), permitting rapid, ultra-sensitive and specific detection. More importantly, by combing with nanotechnology, it can solve the long-lasting problems of poor sensitivity, accuracy and simplicity, as well as to achieve integrated miniaturization and portable point-of-care testing (POCT) detection. However, existing literature lacks specific emphasis on this topic. Thus, we intend to propose a timely one for the readers. This review will bridge this gap by providing insights for CRISPR/Cas-based nano-biosensing development and highlighting the current state-of-art, challenges, and prospects. We expect that it can provide better understanding and valuable insights for trace NABCBs detection, thereby facilitating advancements in early cancer screening/detection/diagnostics and win practical applications in the foreseeable future.},
}

Humans
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*Neoplasms/diagnosis/genetics
*Nanotechnology/methods
*Biomarkers, Tumor
Animals
Nucleic Acids/genetics

@article {pmid40087215,
year = {2025},
author = {Ding, Q and Cui, Z and Shi, Q and Zhang, Y and He, N and Guo, R and Tian, Y and Cao, S and Zhong, J and Wang, H},
title = {An advanced cytosine base editor enabled the generation of cattle with a stop codon in the β-lactoglobulin gene.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {14},
pmid = {40087215},
issn = {1573-9368},
mesh = {Animals ; Cattle/genetics ; *Lactoglobulins/genetics ; *Gene Editing/methods ; *Codon, Terminator/genetics ; *Cytosine/metabolism ; Milk/metabolism ; CRISPR-Cas Systems ; Female ; Animals, Genetically Modified/genetics ; Epithelial Cells/metabolism ; Mammary Glands, Animal/metabolism ; },
abstract = {β-Lactoglobulin (BLG) is an allergen present in milk that can induce an acute immune response in certain individuals. The successful use of cytosine base editors (CBEs) can introduce stop codons into premature mRNA, thereby generating animals with disrupted genes that negatively regulate target traits. In this study, we employed a CBE system to target the major milk allergen BLG in bovine embryos, mammary epithelial cells, and live cattle. First, the precise single-base editing of the BLG gene in bovine embryos was achieved by designing an effective sgRNA to induce a c.61C > T substitution in the coding region, converting codon 21Gln (p.21Gln) to a premature stop codon. Sanger sequencing revealed an editing efficiency of 83.3% (20 out of 24 embryos), including two homozygous edits. Second, a bovine mammary epithelial cell line harboring BLG edits was constructed using the same CBE system. Sequencing showed that the designed sgRNA1 enabled the simultaneous conversion of three consecutive cytosines (c.59-61CCC > TTT) to thymines. At position c.61, single-cell clones exhibited monoallelic or biallelic editing (BLG[c.61C > T]), with monoallelic edits at positions c.59 and c.60 (CC > TT). Gene expression analysis confirmed that the BLG[c.61C > T] mutation effectively suppressed BLG expression at both the mRNA and protein levels, even in monoallelically edited cells. Finally, we successfully generated a heterozygous BLG[c.61C > T] single-base-edited dairy cow that despite its heterozygosity, showed significantly reduced BLG expression in the mammary epithelial cells and milk. Collectively, this study demonstrates the feasibility of using CBEs to disrupt BLG expression in dairy cows and provides a foundation for application in generating hypoallergenic dairy products.},
}

Animals
Cattle/genetics
*Lactoglobulins/genetics
*Gene Editing/methods
*Codon, Terminator/genetics
*Cytosine/metabolism
Milk/metabolism
CRISPR-Cas Systems
Female
Animals, Genetically Modified/genetics
Epithelial Cells/metabolism
Mammary Glands, Animal/metabolism

@article {pmid40087133,
year = {2025},
author = {Della Valle, S and Orsi, E and Creutzburg, SCA and Jansen, LFM and Pentari, EN and Beisel, CL and Steel, H and Nikel, PI and Staals, RHJ and Claassens, NJ and van der Oost, J and Huang, WE and Patinios, C},
title = {Streamlined and efficient genome editing in Cupriavidus necator H16 using an optimised SIBR-Cas system.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.02.006},
pmid = {40087133},
issn = {1879-3096},
abstract = {Cupriavidus necator H16 is a promising microbial platform strain for CO2 valorisation. While C. necator is amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition to industrial applications. In this study, we simplified and accelerated the genome editing pipeline for C. necator by harnessing the Self-splicing Intron-Based Riboswitch (SIBR) system. We used SIBR to tightly control and delay Cas9-based counterselection, achieving >80% editing efficiency at two genomic loci within 48 h after electroporation. To further increase the versatility of the genome editing toolbox, we upgraded SIBR to SIBR2.0 and used it to regulate the expression of Cas12a. SIBR2.0-Cas12a could mediate gene deletion in C. necator with ~70% editing efficiency. Overall, we streamlined the genome editing pipeline for C. necator, facilitating its potential role in the transition to a bio-based economy.},
}

@article {pmid40063648,
year = {2025},
author = {Lavi, I and Bhattacharya, S and Awase, A and Orgil, O and Avital, N and Journo, G and Gurevich, V and Shamay, M},
title = {Unidirectional recruitment between MeCP2 and KSHV-encoded LANA revealed by CRISPR/Cas9 recruitment assay.},
journal = {PLoS pathogens},
volume = {21},
number = {3},
pages = {e1012972},
doi = {10.1371/journal.ppat.1012972},
pmid = {40063648},
issn = {1553-7374},
mesh = {*Methyl-CpG-Binding Protein 2/metabolism/genetics ; *Herpesvirus 8, Human/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Antigens, Viral/metabolism/genetics ; *Nuclear Proteins/metabolism/genetics ; HEK293 Cells ; },
abstract = {Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is associated with several human malignancies. During latency, the viral genomes reside in the nucleus of infected cells as large non-integrated plasmids, known as episomes. To ensure episome maintenance, the latency protein LANA tethers the viral episomes to the cell chromosomes during cell division. Directional recruitment of protein complexes is critical for the proper function of many nuclear processes. To test for recruitment directionality between LANA and cellular proteins, we directed LANA via catalytically inactive Cas9 (dCas9) to a repeat sequence to obtain easily detectable dots. Then, the recruitment of nuclear proteins to these dots can be evaluated. We termed this assay CRISPR-PITA for Protein Interaction and Telomere Recruitment Assay. Using this protein recruitment assay, we found that LANA recruits its known interactors ORC2 and SIN3A. Interestingly, LANA was unable to recruit MeCP2, but MeCP2 recruited LANA. Both LANA and histone deacetylase 1 (HDAC1) interact with the transcriptional-repression domain (TRD) and the methyl-CpG-binding domain (MBD) of MeCP2. Similar to LANA, HDAC1 was unable to recruit MeCP2. While heterochromatin protein 1 (HP1), which interacts with the N-terminal of MeCP2, can recruit MeCP2. We propose that available interacting domains force this recruitment directionality. We hypothesized that the tandem repeats in the SunTag may force MeCP2 dimerization and mimic the form of DNA-bound MeCP2. Indeed, providing only the tandem epitopes of SunTag allows LANA to recruit MeCP2 in infected cells. Therefore, CRISPR-PITA revealed the rules of unidirectional recruitment and allowed us to break this directionality.},
}

*Methyl-CpG-Binding Protein 2/metabolism/genetics
*Herpesvirus 8, Human/genetics/metabolism
Humans
*CRISPR-Cas Systems
*Antigens, Viral/metabolism/genetics
*Nuclear Proteins/metabolism/genetics
HEK293 Cells

@article {pmid40048486,
year = {2025},
author = {Roussis, IM and Pearton, DJ and Niazi, U and Tsaknakis, G and Papadopoulos, GL and Cook, R and Saqi, M and Ragoussis, J and Strouboulis, J},
title = {A novel role for Friend of GATA1 (FOG-1) in regulating cholesterol transport in murine erythropoiesis.},
journal = {PLoS genetics},
volume = {21},
number = {3},
pages = {e1011617},
doi = {10.1371/journal.pgen.1011617},
pmid = {40048486},
issn = {1553-7404},
mesh = {Animals ; *Erythropoiesis/genetics ; Mice ; *Cholesterol/metabolism ; *Transcription Factors/genetics/metabolism ; *Cell Differentiation ; Mice, Knockout ; Biological Transport ; GATA1 Transcription Factor/metabolism/genetics ; DNA-Binding Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Leukemia, Erythroblastic, Acute/genetics/metabolism/pathology ; ATP Binding Cassette Transporter 1/metabolism/genetics ; },
abstract = {Friend of GATA1 (FOG-1) is an essential transcriptional co-factor of the master erythroid transcription factor GATA1. The knockout of the Zfpm1 gene, coding for FOG-1, results in early embryonic lethality due to anemia in mice, similar to the embryonic lethal phenotype of the Gata1 gene knockout. However, a detailed molecular analysis of the Zfpm1 knockout phenotype in erythropoiesis is presently incomplete. To this end, we used CRISPR/Cas9 to knockout Zfpm1 in mouse erythroleukemic (MEL) cells. Phenotypic characterization of DMSO-induced terminal erythroid differentiation showed that the Zfpm1 knockout MEL cells did not progress past the proerythroblast stage of differentiation. Expression profiling of the Zfpm1 knockout MEL cells by RNAseq showed a lack of up-regulation of erythroid-related gene expression profiles. Bioinformatic analysis highlighted cholesterol transport as a pathway affected in the Zfpm1 knockout cells. Moreover, we show that the cholesterol transporters Abca1 and Ldlr fail to be repressed during erythroid differentiation in Zfpm1 knockout cells, resulting in higher intracellular lipid levels and higher membrane fluidity. We also show that in FOG-1 knockout cells, the nuclear levels of SREBP2, a key transcriptional regulator of cholesterol biosynthesis and transport, are markedly increased. On the basis of these findings we propose that FOG-1 (and, potentially, GATA1) regulate cholesterol homeostasis during erythroid differentiation directly through the down regulation of cholesterol transport genes and indirectly, through the repression of the SREBP2 transcriptional activator of cholesterol homeostasis. Taken together, our work provides a molecular basis for understanding FOG-1 functions in erythropoiesis and reveals a novel role for FOG-1 in cholesterol transport.},
}

Animals
*Erythropoiesis/genetics
Mice
*Cholesterol/metabolism
*Transcription Factors/genetics/metabolism
*Cell Differentiation
Mice, Knockout
Biological Transport
GATA1 Transcription Factor/metabolism/genetics
DNA-Binding Proteins/genetics/metabolism
CRISPR-Cas Systems
Leukemia, Erythroblastic, Acute/genetics/metabolism/pathology
ATP Binding Cassette Transporter 1/metabolism/genetics

@article {pmid40037377,
year = {2025},
author = {Emrich-Mills, TZ and Proctor, MS and Degen, GE and Jackson, PJ and Richardson, KH and Hawkings, FR and Buchert, F and Hitchcock, A and Hunter, CN and Mackinder, LCM and Hippler, M and Johnson, MP},
title = {Tethering ferredoxin-NADP+ reductase to photosystem I promotes photosynthetic cyclic electron transfer.},
journal = {The Plant cell},
volume = {37},
number = {3},
pages = {},
doi = {10.1093/plcell/koaf042},
pmid = {40037377},
issn = {1532-298X},
support = {RPG-2019-045//Leverhulme Trust/ ; BB/V006630/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //BBSRC White Rose DTP studentship in Mechanistic Biology/ ; //Diamond PhD studentship/ ; 854126//European Research Council Synergy/ ; HI 739/13-3//DFG/ ; HI 739/25-1//GoPMF/ ; },
mesh = {*Photosystem I Protein Complex/metabolism/genetics ; *Photosynthesis/genetics ; *Ferredoxin-NADP Reductase/metabolism/genetics ; Electron Transport ; *Chlamydomonas reinhardtii/genetics/metabolism ; CRISPR-Cas Systems ; NADP/metabolism ; },
abstract = {Fixing CO2 via photosynthesis requires ATP and NADPH, which can be generated through linear electron transfer (LET). However, depending on the environmental conditions, additional ATP may be required to fix CO2, which can be generated by cyclic electron transfer (CET). How the balance between LET and CET is determined remains largely unknown. Ferredoxin-NADP+ reductase (FNR) may act as the switch between LET and CET, channeling photosynthetic electrons to LET when it is bound to photosystem I (PSI) or to CET when it is bound to cytochrome b6f. The essential role of FNR in LET precludes the use of a direct gene knock-out to test this hypothesis. Nevertheless, we circumvented this problem using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing in Chlamydomonas reinhardtii. Through this approach, we created a chimeric form of FNR tethered to PSI via PSAF. Chimeric FNR mutants exhibited impaired photosynthetic growth and LET along with enhanced PSI acceptor side limitation relative to the wild type due to slower NADPH reduction. However, the chimeric FNR mutants also showed enhanced ΔpH production and NPQ resulting from increased CET. Overall, our results suggest that rather than promoting LET, tethering FNR to PSI promotes CET at the expense of LET and CO2 fixation.},
}

*Photosystem I Protein Complex/metabolism/genetics
*Photosynthesis/genetics
*Ferredoxin-NADP Reductase/metabolism/genetics
Electron Transport
*Chlamydomonas reinhardtii/genetics/metabolism
CRISPR-Cas Systems
NADP/metabolism

@article {pmid39950370,
year = {2025},
author = {Xiong, K and Wang, X and Feng, C and Zhang, K and Chen, D and Yang, S},
title = {Vectors in CRISPR Gene Editing for Neurological Disorders: Challenges and Opportunities.},
journal = {Advanced biology},
volume = {9},
number = {3},
pages = {e2400374},
doi = {10.1002/adbi.202400374},
pmid = {39950370},
issn = {2701-0198},
support = {81874304//National Natural Science Foundation of China/ ; 22122409//National Natural Science Foundation of China/ ; 22A350017//Key Scientific Research Projects/ ; 21HASTIT043//Programs for Science &Technology Innovation Talents in Universities of Henan Province/ ; ZYQR201912191//Outstanding Young Talents in Henan Province/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *Nervous System Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Dependovirus/genetics ; Animals ; Gene Transfer Techniques ; },
abstract = {Diseases of the nervous system are recognized as the second leading cause of death worldwide. The global prevalence of neurological diseases, such as Huntington's disease, Alzheimer's disease, and Parkinson's disease has seen a significant rise due to the increasing proportion of the aging population. The discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) genome editing technique has paved way for universal neurological diseases treatment. However, finding a safe and effective method to deliver CRISPR gene-editing tools remains a main challenge for genome editing therapies in vivo. Adeno-associated virus (AAV) is currently one of the most commonly used vector systems, but some issues remain unresolved, including capsid immunogenicity, off-target mutations, and potential genotoxicity. To address these concerns, researchers are actively encouraging the development of new delivery systems, like virus-like particles and nanoparticles. These novel systems have the potential to enhance targeting efficiency, thereby offering possible solutions to the current challenges. This article reviews CRISPR delivery vectors for neurological disorders treatment and explores potential solutions to overcome limitations in vector systems. Additionally, the delivery strategies of CRISPR systems are highlighted as valuable tools for studying neurological diseases, and the challenges and opportunities that these vectors present.},
}

Humans
*Gene Editing/methods
*Genetic Vectors/genetics
*Nervous System Diseases/therapy/genetics
*CRISPR-Cas Systems/genetics
*Genetic Therapy/methods
Dependovirus/genetics
Animals
Gene Transfer Techniques

@article {pmid39912332,
year = {2025},
author = {Bellchambers, HM and Padua, MB and Ware, SM},
title = {A CRISPR mis-insertion in the Zic3 5'UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin.},
journal = {Biology open},
volume = {14},
number = {3},
pages = {},
doi = {10.1242/bio.061677},
pmid = {39912332},
issn = {2046-6390},
support = {P01 HL 134599/HL/NHLBI NIH HHS/United States ; //Indiana University/ ; P01 HL 134599/NH/NIH HHS/United States ; //Indiana University School of Medicine/ ; },
mesh = {Animals ; Mice ; *5' Untranslated Regions ; *Transcription Factors/genetics/metabolism ; *Homeodomain Proteins/genetics/metabolism ; *RNA, Messenger/genetics/metabolism ; *Nucleic Acid Conformation ; CRISPR-Cas Systems ; Inverted Repeat Sequences ; Gene Editing ; Protein Biosynthesis ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mutagenesis, Insertional ; Phenotype ; },
abstract = {Zic3 loss of function is associated with a range of congenital defects, including heterotaxy and isolated heart defects in humans, as well as neural tube defects, situs anomalies, and tail kinks in model organisms. Here, we describe a novel Zic3ins5V mouse line generated due to a mis-insertion during the CRISPR genome editing process, which altered the Zic3 5'UTR structure. Mice with this insertion developed similar phenotypes to Zic3LacZ null mice, including heterotaxy, isolated heart defects, neural tube defects and tail kinks. Surprisingly, gene expression analysis revealed that the novel Zic3ins5V line displays higher levels of Zic3 mRNA, but western blot analysis confirmed that levels of ZIC3 were greatly reduced in vivo. RNAfold, an RNA secondary structure prediction tool, showed that this mis-insertion may cause the formation of a large stem-loop hairpin incorporating some of the 5'UTR and first exon of Zic3, and the insertion of similar hairpins in a cell-based assay caused the loss of ZIC3 expression. Thus, this mouse line displays a loss of ZIC3 protein consistent with the inhibitory effects of 5'UTR stem-loop hairpin structures.},
}

Animals
Mice
*5' Untranslated Regions
*Transcription Factors/genetics/metabolism
*Homeodomain Proteins/genetics/metabolism
*RNA, Messenger/genetics/metabolism
*Nucleic Acid Conformation
CRISPR-Cas Systems
Inverted Repeat Sequences
Gene Editing
Protein Biosynthesis
Clustered Regularly Interspaced Short Palindromic Repeats
Mutagenesis, Insertional
Phenotype

@article {pmid39756427,
year = {2025},
author = {Speckhart, SL and Pollock, AB and Alward, KJ and Farrell, K and Oliver, MA and Lee, K and Biase, FH and Ealy, AD},
title = {The interleukin-6 signal transducer receptor subunit is required for optimal in vitro bovine embryo development†.},
journal = {Biology of reproduction},
volume = {112},
number = {3},
pages = {434-446},
doi = {10.1093/biolre/ioaf006},
pmid = {39756427},
issn = {1529-7268},
support = {R21-OD026516-01/NH/NIH HHS/United States ; 2021-67034-35149//USDA National Institute of Food and Agriculture (NIFA)/ ; },
mesh = {Cattle/embryology ; Animals ; *Embryonic Development/physiology ; Embryo Culture Techniques ; Blastocyst/metabolism ; Signal Transduction ; Fertilization in Vitro ; Female ; Interleukin-6/metabolism/genetics/pharmacology ; Receptors, Interleukin-6/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {This work explored whether bovine embryo development relies on signaling from the interleukin-6 (IL6) cytokine family. This was accomplished by interrupting IL6 signal transducer (IL6ST), the common beta-subunit receptor used by the IL6 family. One series of studies cultured in vitro-produced embryos with SC144, a pharmacological IL6ST inhibitor. Providing the inhibitor at a concentration that partially diminished IL6ST signaling reduced development to the 16-cell and blastocyst stages and reduced inner-cell-mass cell numbers. Inhibitor concentrations that completely blocked IL6ST signaling prevented blastocyst development. Another series of studies used CRISPR-Cas9 to disrupt IL6ST. Two electroporation approaches were used to introduce guide RNAs and Cas9 protein into one-cell in vitro-produced embryos. Editing efficiency was ≥82%. Targeting IL6ST did not affect cleavage but reduced development to the 16-cell and blastocyst stages. A reduction in inner-cell-mass cell numbers was detected, and disorganization of the inner cell mass was observed in approximately one-half of the IL6ST-targeted blastocysts. These observations indicate that embryo-derived IL6 family members that signal through IL6ST are needed to support normal in vitro bovine embryo development. These signals are needed by the 16-cell stage and for inner-cell-mass cell development at the blastocyst stage. There is also evidence that these signals support the overall cellular organization of the blastocyst.},
}

Cattle/embryology
Animals
*Embryonic Development/physiology
Embryo Culture Techniques
Blastocyst/metabolism
Signal Transduction
Fertilization in Vitro
Female
Interleukin-6/metabolism/genetics/pharmacology
Receptors, Interleukin-6/metabolism/genetics
CRISPR-Cas Systems

@article {pmid39708832,
year = {2025},
author = {Kurashina, M and Snow, AW and Mizumoto, K},
title = {A modular system to label endogenous presynaptic proteins using split fluorophores in Caenorhabditis elegans.},
journal = {Genetics},
volume = {229},
number = {3},
pages = {},
doi = {10.1093/genetics/iyae214},
pmid = {39708832},
issn = {1943-2631},
support = {PJT-180563//This study was supported by CIHR/ ; RGPIN-2015-04022//NSERC/ ; },
mesh = {Animals ; *Caenorhabditis elegans/metabolism/genetics ; *Caenorhabditis elegans Proteins/metabolism/genetics ; *Luminescent Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Green Fluorescent Proteins/metabolism/genetics ; Presynaptic Terminals/metabolism ; Red Fluorescent Protein ; Nerve Tissue Proteins/metabolism/genetics ; Neurons/metabolism ; },
abstract = {Visualizing the subcellular localization of presynaptic proteins with fluorescent proteins is a powerful tool to dissect the genetic and molecular mechanisms underlying synapse formation and patterning in live animals. Here, we utilize split green and red fluorescent proteins to visualize the localization of endogenously expressed presynaptic proteins at a single-neuron resolution in Caenorhabditis elegans. By using CRISPR/Cas9 genome editing, we generated a collection of C. elegans strains in which endogenously expressed presynaptic proteins (RAB-3/Rab3, SNG-1/Synaptogyrin, CLA-1/Piccolo, SYD-2/Liprin-α, UNC-10/RIM, RIMB-1/RIM-BP, and ELKS-1/ELKS) are tagged with tandem repeats of GFP11 and/or wrmScarlet11. We show that the expression of GFP1-10 and wrmScarlet1-10 under neuron-specific promoters can robustly label presynaptic proteins in different neuron types. We believe that the combination of our knock-in strains and GFP1-10 and wrmScarlet1-10 plasmids is a versatile modular system useful for neuroscientists to examine the localization of endogenous presynaptic proteins in any neuron type in C. elegans.},
}

Animals
*Caenorhabditis elegans/metabolism/genetics
*Caenorhabditis elegans Proteins/metabolism/genetics
*Luminescent Proteins/metabolism/genetics
CRISPR-Cas Systems
Green Fluorescent Proteins/metabolism/genetics
Presynaptic Terminals/metabolism
Red Fluorescent Protein
Nerve Tissue Proteins/metabolism/genetics
Neurons/metabolism

@article {pmid40086675,
year = {2025},
author = {Li, XH and Lu, HZ and Yao, JB and Zhang, C and Shi, TQ and Huang, H},
title = {Recent advances in the application of CRISPR/Cas-based gene editing technology in Filamentous Fungi.},
journal = {Biotechnology advances},
volume = {81},
number = {},
pages = {108561},
doi = {10.1016/j.biotechadv.2025.108561},
pmid = {40086675},
issn = {1873-1899},
abstract = {Filamentous fungi are essential industrial microorganisms that can serve as sources of enzymes, organic acids, terpenoids, and other bioactive compounds with significant applications in food, medicine, and agriculture. However, the underdevelopment of gene editing tools limits the full exploitation of filamentous fungi, which still present numerous untapped potential applications. In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats) system, a versatile genome-editing tool, has advanced significantly and been widely applied in filamentous fungi, showcasing considerable research potential. This review examines the development and mechanisms of genome-editing tools in filamentous fungi, and contrasts the CRISPR/Cas9 and CRISPR/Cpf1 systems. The transformation and delivery strategies of the CRISPR/Cas system in filamentous fungi are also examined. Additionally, recent applications of CRISPR/Cas systems in filamentous fungi are summarized, such as gene disruption, base editing, and gene regulation. Strategies to enhance editing efficiency and reduce off-target effects are also highlighted, with the aim of providing insights for the future construction and optimization of CRISPR/Cas systems in filamentous fungi.},
}

@article {pmid40086107,
year = {2025},
author = {Yadav, G and Sharma, A and Hathi, UPS and Gaind, R and Singh, R},
title = {Development and optimization of multiplex PCR for rapid detection of type I-F1 and type I-F2 Cas cluster genes in Acinetobacter baumannii.},
journal = {Biologicals : journal of the International Association of Biological Standardization},
volume = {90},
number = {},
pages = {101824},
doi = {10.1016/j.biologicals.2025.101824},
pmid = {40086107},
issn = {1095-8320},
abstract = {Polymerase chain reaction (PCR), especially the multiplex PCR assay, enables simultaneous detection of multiple genes and is highly effective for diagnostic applications. The CRISPR-associated (Cas) system consists of several genes, and complete gene clusters are essential for its activity; multiplex PCR is an excellent method for detecting these multiple genes. This study focuses on the development and validation of a multiplex PCR protocol for the specific detection of CRISPR-Cas subtypes I-F1 and I-F2 found in A. baumannii, which is classified as a critical ESKAPE pathogen. The multiplex PCR method achieved a 100 % detection rate for isolates containing Cas subtypes I-F1 and I-F2 in clinical A. baumannii isolates. Testing across various genera and Acinetobacter species confirmed the high specificity of the assay, with no false positives, establishing it as a reliable tool for large-scale clinical applications. Of the 96 clinical A. baumannii isolates analysed, 29.167 % (n = 28) were multiplex PCR positive for a CRISPR-Cas system. Among these, 71.43 % (n = 20) had subtype I-F1, while 28.57 % (n = 8) had subtype I-F2. No clear association was found between Cas subtypes and resistance to the tested antibiotics or carbapenem genes. This study provides a valuable tool for monitoring CRISPR-Cas systems and can aid in various experimental and novel strategies to manage multidrug-resistant A. baumannii.},
}

@article {pmid40084131,
year = {2025},
author = {Zhang, Y and Yang, S and Zheng, X and Tan, X},
title = {Cyanobacterial type I CRISPR-Cas systems: distribution, mechanisms, and genome editing applications.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {13},
number = {},
pages = {1552030},
pmid = {40084131},
issn = {2296-4185},
abstract = {Cyanobacteria, renowned for their photosynthetic capabilities, serve as efficient microbial chassis capable of converting carbon dioxide into a spectrum of bio-chemicals. However, conventional genetic manipulation strategies have proven incompatible with the precise and systematic modifications required in the field of cyanobacterial synthetic biology. Here, we present an in-depth analysis of endogenous CRISPR-Cas systems within cyanobacterial genomes, with a particular focus on the Type I systems, which are the most widely distributed. We provide a comprehensive summary of the reported DNA defense mechanisms mediated by cyanobacterial Type I CRISPR-Cas systems and their current applications in genome editing. Furthermore, we offer insights into the future applications of these systems in the context of cyanobacterial genome editing, underscoring their potential to revolutionize synthetic biology approaches.},
}

@article {pmid40083940,
year = {2025},
author = {Zhu, X and Wang, S and Xue, Y and Wang, X and Hu, S and Liang, T and Liu, W},
title = {Coupling CRISPR-Cas and a personal glucose meter with an enzymatic reporter for portable detection of human papillomavirus in biological samples.},
journal = {Theranostics},
volume = {15},
number = {7},
pages = {2870-2882},
pmid = {40083940},
issn = {1838-7640},
mesh = {*CRISPR-Cas Systems ; Humans ; *Papillomavirus Infections/diagnosis/virology ; Point-of-Care Testing ; beta-Fructofuranosidase/metabolism/genetics ; Papillomaviridae/genetics/isolation & purification ; Biosensing Techniques/methods ; Female ; Glucose/analysis/metabolism ; Point-of-Care Systems ; Blood Glucose Self-Monitoring/methods/instrumentation ; Human papillomavirus 16/genetics/isolation & purification ; HeLa Cells ; Human Papillomavirus Viruses ; },
abstract = {Significant efforts and resources have been dedicated to developing CRISPR-Cas based point-of-care testing (POCT) and self-diagnosis methods for nucleic acid pathogens, aiming to complement the gold standard quantitative PCR tests, particularly in settings where centralized facilities, trained personnel, or resource-intensive equipment are unavailable. However, the reliance on stationary, high-cost readout machinery hinders their full deployment at the point of care. We aimed to develop a solid-phase invertase-labeled reporter (ILR) system that enables convenient readout of CRISPR-Cas reactions, facilitate HPV detection in a POCT-compatible manner. Methods: Through multiple chemical couplings, invertase is immobilized onto magnetic microbeads via a nucleic acid linker that responds to target nucleic acid-induced CRISPR-Cas activation. This activation releases active invertase, which then converts sucrose to glucose in proportion to the target's abundance. Enzymatic signal amplification by Cas12a/Cas13a and invertase compensates for the moderate sensitivity of personal glucose meters (PGMs). Results: When applied to human papillomavirus detection, the HPV18-targeted LAMP-Cas12a/ILR/PGM system can detect as few as 7 HPV18-positive HeLa cells out of 7,000, achieving 95.8% sensitivity and 100% specificity in cervical cell samples. Furthermore, minimal reagent adjustments allow for the rapid establishment of HPV16 and HPV52-targeted LAMP-Cas12a/ILR/PGM systems, offering satisfactory sensitivity, specificity, and cross-species detection. Conclusion: These findings demonstrate a highly efficient testing platform for a range of nucleic acid pathogens, suitable for both point-of-care and household use.},
}

*CRISPR-Cas Systems
Humans
*Papillomavirus Infections/diagnosis/virology
Point-of-Care Testing
beta-Fructofuranosidase/metabolism/genetics
Papillomaviridae/genetics/isolation & purification
Biosensing Techniques/methods
Female
Glucose/analysis/metabolism
Point-of-Care Systems
Blood Glucose Self-Monitoring/methods/instrumentation
Human papillomavirus 16/genetics/isolation & purification
HeLa Cells
Human Papillomavirus Viruses

@article {pmid40082762,
year = {2025},
author = {Haddad, A and Golan-Lev, T and Benvenisty, N and Goldberg, M},
title = {Genome-wide screening in human embryonic stem cells identifies genes and pathways involved in the p53 pathway.},
journal = {Molecular medicine (Cambridge, Mass.)},
volume = {31},
number = {1},
pages = {97},
pmid = {40082762},
issn = {1528-3658},
support = {2054/22//The Israel Science Foundation/ ; 3605/21//The ISF-Israel Precision Medicine Partnership (IPMP) Program/ ; 20230941//The Israel Cancer Association - Robin and David Gray donation/ ; },
mesh = {Humans ; *Tumor Suppressor Protein p53/metabolism/genetics ; *Signal Transduction ; *Human Embryonic Stem Cells/metabolism ; CRISPR-Cas Systems ; Computational Biology/methods ; Apoptosis/genetics ; Proto-Oncogene Proteins c-mdm2/genetics/metabolism ; Piperazines/pharmacology ; Genome-Wide Association Study ; Cell Line ; Genome, Human ; Imidazoles ; },
abstract = {BACKGROUND: The tumor suppressor protein, p53, which is mutated in half of human tumors, plays a critical role in cellular responses to DNA damage and maintenance of genome stability. Therefore, increasing our understanding of the p53 pathway is essential for improving cancer treatment and diagnosis.

METHODS: This study, which aimed to identify genes and pathways that mediate resistance to p53 upregulation, used genome-wide CRISPR-Cas9 loss-of-function screening done with Nutlin-3a, which inhibits p53-MDM2 interaction, resulting in p53 accumulation and apoptotic cell death. We used bioinformatics analysis for the identification of genes and pathways that are involved in the p53 pathway and cell survival assays to validate specific genes. In addition, we used RNA-seq to identify differentially expressed p53 target genes in gene knockout (KO) cell lines.

RESULTS: Our screen revealed three significantly enriched pathways: The heparan sulfate glycosaminoglycan biosynthesis, diphthamide biosynthesis and Hippo pathway. Notably, TRIP12 was significantly enriched in our screen. We found that TRIP12 is required for the p53-dependent transcription of several pro-apoptotic genes.

CONCLUSION: Our study has identified two novel pathways that play a role in p53-mediated growth restriction. Moreover, we have highlighted the interaction between the Hippo and the p53 pathways. Interestingly, we have shown that TRIP12 plays an important function in the p53 pathway by selectively affecting its role as a transcription factor.},
}

Humans
*Tumor Suppressor Protein p53/metabolism/genetics
*Signal Transduction
*Human Embryonic Stem Cells/metabolism
CRISPR-Cas Systems
Computational Biology/methods
Apoptosis/genetics
Proto-Oncogene Proteins c-mdm2/genetics/metabolism
Piperazines/pharmacology
Genome-Wide Association Study
Cell Line
Genome, Human
Imidazoles