@article {pmid40992249,
year = {2025},
author = {Greisle, T and Kunze, I and Wang, X and Malinowski, AR and Böttcher, A and Lickert, H and Burtscher, I},
title = {Generation of a Flattop-T2A-H2B-Venus x C-peptide-mCherry double reporter human iPSC line to monitor WNT/Planar cell polarity pathway activity.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103838},
doi = {10.1016/j.scr.2025.103838},
pmid = {40992249},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cell Polarity ; *Wnt Signaling Pathway ; Cell Line ; CRISPR-Cas Systems ; Insulin-Secreting Cells/metabolism/cytology ; Genes, Reporter ; C-Peptide/metabolism/genetics ; Cell Differentiation ; },
abstract = {Deriving functional β-cells from human induced pluripotent stem cells (hiPSCs) holds potential for cell replacement therapy, disease modeling, and drug testing in diabetes research. Wnt/Planar cell polarity (PCP) signaling is crucial for endocrine cell development and β-cell maturation in murine models and can be tracked by the expression of the tissue-specific effector gene Flattop. Here, we report the generation of a human fluorescent FLTP/CFAP126 (Flattop-T2A-H2B-Venus) and FLTP-Insulin (Flattop-T2A-H2B-Venus x C-peptide-mCherry) double reporter by CRISPR/Cas9 gene editing. These hiPSC reporter lines allow monitoring of WNT/PCP signaling during endocrine cell formation and studying its role in β-cells in a human model system.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Cell Polarity
*Wnt Signaling Pathway
Cell Line
CRISPR-Cas Systems
Insulin-Secreting Cells/metabolism/cytology
Genes, Reporter
C-Peptide/metabolism/genetics
Cell Differentiation

@article {pmid40983220,
year = {2025},
author = {Madhusudhan, K and Padmanaban, A and Parvathi, VD},
title = {Early detection of Parkinson's disease via aptamer-CRISPR platform.},
journal = {Neuroscience},
volume = {586},
number = {},
pages = {163-195},
doi = {10.1016/j.neuroscience.2025.09.027},
pmid = {40983220},
issn = {1873-7544},
mesh = {Humans ; *Parkinson Disease/diagnosis/genetics ; *Aptamers, Nucleotide ; Early Diagnosis ; *CRISPR-Cas Systems ; Biomarkers ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Parkinson's disease (PD) is a neurodegenerative disorder with a worldwide prevalence of around 9.4 million that is expected to double by 2040. It's extended prodromal phase allows irreversible neuronal loss to occur before manifestation of symptoms. Current diagnostic approaches, primarily based on clinical assessment and neuroimaging, are often delayed and lack sensitivity in the early stages, highlighting the need for an early, conclusive, and minimally invasive test. This review focuses on the integration of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) diagnostics with aptamers to detect PD-associated biomarkers. CRISPR systems utilising Cas12 and Cas13 enzymes offer high specificity and collateral cleavage activity that can be harnessed for signal amplification. Aptamers are short, single-stranded oligonucleotides that can be designed to identify nucleic and non-nucleic acid targets. Their fusion with CRISPR may enable the sensitive detection of key PD biomarkers such as α-Syn, dopa decarboxylase, glial fibrillary acidic protein, and neurofilament light chain in biological fluids like blood, CSF, urine, saliva, and sweat. We explore various strategies for aptamer-CRISPR integration, detection, and multiplexing techniques for parallel biomarker detection. We also examine existing diagnostic platforms and discuss barriers to clinical translation. Ultimately, aptamer-CRISPR diagnostics could represent a powerful, next-generation approach for early PD detection.},
}

Humans
*Parkinson Disease/diagnosis/genetics
*Aptamers, Nucleotide
Early Diagnosis
*CRISPR-Cas Systems
Biomarkers
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40953792,
year = {2025},
author = {Watts, JL and Willeke, L and Stottmann, RW},
title = {Mouse variants in Taf1c result in reduced survival to birth.},
journal = {Developmental biology},
volume = {528},
number = {},
pages = {143-151},
doi = {10.1016/j.ydbio.2025.09.011},
pmid = {40953792},
issn = {1095-564X},
mesh = {Animals ; Mice ; *TATA-Binding Protein Associated Factors/genetics/metabolism ; Humans ; *Transcription Factor TFIID/genetics ; Mutation, Missense ; Female ; Craniofacial Abnormalities/genetics ; Phenotype ; Ribosomes/metabolism/genetics ; Male ; CRISPR-Cas Systems ; },
abstract = {Ribosome biogenesis is a key cellular function and disruptions in this process can lead to congenital anomalies or "ribosomopathies" with varying phenotypes including craniofacial malformations and neurodevelopment symptoms. Classically, the mouse is a robust model to understand the molecular mechanisms underlying ribosomopathies to further elucidate human pathogenesis. We identified novel compound heterozygous missense variants in the TATA-box binding protein associated factor, RNA polymerase I subunit C (TAF1C) locus in a patient with some phenotypes consistent with ribosomopathies. TAF1C encodes a subunit of the SL1 complex which is critical for the RNA PolI complex to initiate ribosomal RNA transcription. We hypothesized that functional TAF1C is required at developmental stages critical for craniofacial and neurodevelopment. To test this hypothesis, we created mouse Taf1c variants orthologous to the human variants using CRISPR-CAS9 technology (Taf1c[R202Q] and Taf1c[S428A]). We also created an 11bp deletion to complement the missense variants (Taf1c[11bpdel]). We created multiple allelic combinations to determine the roles for Taf1c in survival and craniofacial development. Homozygous mice for any of these novel variants were underrepresented at organogenesis stages. We did not observe craniofacial anomalies in any surviving mice. Our results suggest that these specific TAF1C variants are not the cause of any human phenotype present in the patient motivating the study. However, we showed that Taf1c is required for embryonic survival and our studies contribute to knowledge about the role of ribosome biogenesis machinery throughout organogenesis.},
}

Animals
Mice
*TATA-Binding Protein Associated Factors/genetics/metabolism
Humans
*Transcription Factor TFIID/genetics
Mutation, Missense
Female
Craniofacial Abnormalities/genetics
Phenotype
Ribosomes/metabolism/genetics
Male
CRISPR-Cas Systems

@article {pmid40929753,
year = {2025},
author = {Herbrich, S and Ramachandran, H and Seibt, A and Tolle, I and Zink, A and Prigione, A and Rossi, A and Distelmaier, F},
title = {CRISPR/Cas9-mediated editing of COQ4 in induced pluripotent stem cells: A model for investigating COQ4-associated human coenzyme Q10 deficiency.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103825},
doi = {10.1016/j.scr.2025.103825},
pmid = {40929753},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *CRISPR-Cas Systems/genetics ; *Ubiquinone/deficiency/analogs & derivatives/genetics/metabolism ; *Gene Editing/methods ; *Mitochondrial Diseases/genetics/pathology/metabolism ; *Ataxia/genetics/pathology/metabolism ; *Mitochondrial Proteins/genetics/metabolism ; *Muscle Weakness/genetics/pathology/metabolism ; Cell Line ; },
abstract = {Pathogenic variants in the gene COQ4 cause primary coenzyme Q10 deficiency, which is associated with symptoms ranging from early epileptic encephalopathy up to adult-onset ataxia-spasticity spectrum disease. We genetically modified commercially available wild-type iPS cells by using a CRISPR/Cas9 approach to create heterozygous and homozygous isogenic cell lines carrying the disease-causing COQ4 variants c.458C > T, p.Ala153Val and c.437T > G, p.Phe146Cys, respectively. All iPSCs lines exhibited a normal cell morphology, expression of pluripotency markers, and the ability to differentiate into the three primary germ layers. The COQ4-deficient cell lines will provide a helpful tool to investigate the disease mechanism and to develop therapeutic strategies.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*CRISPR-Cas Systems/genetics
*Ubiquinone/deficiency/analogs & derivatives/genetics/metabolism
*Gene Editing/methods
*Mitochondrial Diseases/genetics/pathology/metabolism
*Ataxia/genetics/pathology/metabolism
*Mitochondrial Proteins/genetics/metabolism
*Muscle Weakness/genetics/pathology/metabolism
Cell Line

@article {pmid40925290,
year = {2025},
author = {Raabe, J and Lewandowski, V and Fuchs, S and Hammerschmidt, A and Piasecki, A and Orthey, E and Krämer, E and Ehler, E and Cuello, F},
title = {Generation of a biallelic NRAP-knockout mutant from a human iPSC line.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103829},
doi = {10.1016/j.scr.2025.103829},
pmid = {40925290},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Cell Line ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Alleles ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Myocytes, Cardiac/metabolism/cytology ; Mutation ; },
abstract = {Cardiomyopathies, a leading cause of mortality, are associated with dysfunctional intercalated discs, which connect neighbouring cardiomyocytes and ensure proper contractility. In human cardiac diseases, loss-of-function mutations of the intercalated disc-associated protein Nebulin-Related Anchoring Protein (NRAP) have been reported. NRAP plays a crucial role in myofibril assembly and mechanotransduction, however, its regulatory functions remain unclear. To investigate the effects of NRAP loss-of-function in cardiac disease, a human induced pluripotent stem cell (hiPSC) line was generated carrying a biallelic NRAP-knockout (KO) using the CRISPR-Cas9 technology. Control and mutant cell lines were assessed for karyotype integrity, pluripotency, off-target effects, mycoplasma contamination, and differentiation into ectoderm, mesoderm, and endoderm. This hiPSC line provides a valuable tool to study how NRAP modulates cardiac function and contributes to disease progression.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
Cell Differentiation
Cell Line
CRISPR-Cas Systems
Gene Knockout Techniques
Alleles
*Adaptor Proteins, Signal Transducing/genetics/metabolism
Myocytes, Cardiac/metabolism/cytology
Mutation

@article {pmid40902326,
year = {2025},
author = {Ran, Y and Ruan, J and Wang, Y and Feng, X and Tan, P and Guan, Y and Guo, X},
title = {Generation of a PHF19 knockout human embryonic stem cell line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103824},
doi = {10.1016/j.scr.2025.103824},
pmid = {40902326},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Transcription Factors/genetics/metabolism/deficiency ; Cell Line ; *Gene Knockout Techniques ; *DNA-Binding Proteins/genetics/metabolism/deficiency ; },
abstract = {PHD finger protein 19 (PHF19) is a polycomb protein that promoted cardiac hypertrophy via epigenetic targeting SIRT2. To determine the role of PHF19 in myocardial hypertrophy, we established a large fragment knockout model of PHF19 gene in human embryonic stem cells (hESCs-H7) using the CRISPR/Cas9 system based on a vector. This PHF19-KO cell line has a normal karyotype, classical human pluripotent stem cell morphology, strong pluripotency, and significantly reduced PHF19 gene expression, which will become a useful tool for further in-depth research on the pathogenesis of PHF19 gene deficiency induced myocardial hypertrophy.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*Transcription Factors/genetics/metabolism/deficiency
Cell Line
*Gene Knockout Techniques
*DNA-Binding Proteins/genetics/metabolism/deficiency

@article {pmid40850232,
year = {2025},
author = {Kim, JW and Jo, S and Kang, EH and Ryu, JH and Noh, H and Park, HJ and Kim, H},
title = {Generation of human embryonic stem cell line expressing dCas9-TET1 fusion protein for epigenetic editing.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103811},
doi = {10.1016/j.scr.2025.103811},
pmid = {40850232},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *Proto-Oncogene Proteins/genetics/metabolism ; *Mixed Function Oxygenases/genetics/metabolism ; *Epigenesis, Genetic ; CRISPR-Cas Systems ; Cell Line ; *CRISPR-Associated Protein 9/metabolism/genetics ; Epigenome Editing ; },
abstract = {CRISPR-based epigenome editing systems can induce site-specific transcriptional activation or repression of target genes. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) is a transcriptional activation effector involved in the cytosine demethylation of CpG dinucleotides in gene regulatory regions. In this study, we generated a human embryonic stem cell line that stably expresses catalytically dead Cas9 (dCas9) fused to the catalytic domain of TET1 via lentiviral transduction. This cell line can be used for locus-specific transcriptional activation in combination with single guide RNAs and serves as a valuable tool for epigenetic regulation in stem cell and organoid models.},
}

Humans
*Human Embryonic Stem Cells/metabolism/cytology
*Gene Editing/methods
*Proto-Oncogene Proteins/genetics/metabolism
*Mixed Function Oxygenases/genetics/metabolism
*Epigenesis, Genetic
CRISPR-Cas Systems
Cell Line
*CRISPR-Associated Protein 9/metabolism/genetics
Epigenome Editing

@article {pmid40848400,
year = {2025},
author = {Cota-Coronado, A and Manning, M and Kim, DH and Lee, J and Gibbons, A and Rosenbluh, J and Hill, RA and Sundram, S},
title = {Generation of two Betacellulin CRISPR-Cas9 knockout hiPSC lines to study the affected EGF system paradigm in schizophrenia.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103808},
doi = {10.1016/j.scr.2025.103808},
pmid = {40848400},
issn = {1876-7753},
mesh = {*Schizophrenia/genetics/metabolism/pathology ; Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Betacellulin/genetics/metabolism ; *Epidermal Growth Factor/metabolism/genetics ; Gene Knockout Techniques ; Cell Differentiation ; Cell Line ; },
abstract = {Several members of the epidermal growth factor (EGF) family have been implicated in the biology of schizophrenia (Ketharanathan et al., 2024). The EGF-related ligand, Betacellulin (BTC), plays an important role in the proliferation and differentiation of neural stem cells and our group found markedly reduced BTC levels in patients with schizophrenia. Nevertheless, the interplay of affected BTC and its participation in neural specification and neurodevelopment remains elusive. We generated Knockout (KO) - BTC clones from an existing hiPSC line through CRISPR/Cas9-mediated modification. Furthermore, we validated BTC-KO through genotyping/sequencing, FACS and Western Blot. Finally, we demonstrated trilineage differentiation potential in vitro.},
}

*Schizophrenia/genetics/metabolism/pathology
Humans
*CRISPR-Cas Systems/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
*Betacellulin/genetics/metabolism
*Epidermal Growth Factor/metabolism/genetics
Gene Knockout Techniques
Cell Differentiation
Cell Line

@article {pmid41073543,
year = {2025},
author = {Shi, Z and Cheng, TL},
title = {UGI relocation inside Cas9 reduces Cas9 dependent off target effects in cytosine base editors.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35518},
pmid = {41073543},
issn = {2045-2322},
support = {20ZR1403100//Shanghai Natural Science Foundation/ ; },
mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *Uracil-DNA Glycosidase/antagonists & inhibitors/metabolism ; Humans ; HEK293 Cells ; },
abstract = {Cytosine base editors (CBEs) achieve precise C-to-T conversions by addition of uracil DNA glycosylase inhibitor (UGI) with Cas9 nickase (nCas9) and cytidine deaminase, and the conventional fusion at the nCas9 carboxyl terminus effectively inhibits uracil excision repair to enhance editing efficiency. However, despite potent on-target activity, classical CBEs exhibit significant Cas9-dependent DNA off-target effects that necessitate optimization for future applications. Here we present a strategic UGI relocation through internal fusion within the nCas9 architecture. This spatial reorganization maintains comparable on-target editing efficiency while substantially reducing Cas9-dependent DNA off-target activity. Our findings establish an alternative engineering paradigm to develop high-fidelity CBEs, offering an improved platform for widespread genome editing applications.},
}

*Gene Editing/methods
*Cytosine/metabolism
*CRISPR-Associated Protein 9/metabolism/genetics/chemistry
*CRISPR-Cas Systems
*Uracil-DNA Glycosidase/antagonists & inhibitors/metabolism
Humans
HEK293 Cells

@article {pmid41073413,
year = {2025},
author = {Zhang, H and Li, M and Wang, G and Zhu, K and Guo, G and Fu, H and Hu, C and Chu, Z and Hu, J and Wu, Q and Chen, Y and Qiu, D and Xie, J and Li, D and Li, B and Li, W and Dong, L and Hou, Y and Cui, X and Huang, B and Liu, Y and Li, Y and Li, H and Yuan, C and Dong, L and Liu, Z and Lu, P},
title = {Paired NLRs originated from Triticum dicoccoides coordinately confer resistance to powdery mildew in wheat.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9040},
pmid = {41073413},
issn = {2041-1723},
mesh = {*Triticum/genetics/microbiology/immunology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Ascomycota/physiology/pathogenicity ; *Plant Proteins/genetics/metabolism ; *NLR Proteins/genetics/metabolism ; Haplotypes ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Wheat has evolved diverse resistance genes against powdery mildew, typically controlled by single-gene-encoded proteins. Here, we report the map-based cloning of PmWR183, a resistance locus encoding two adjacent NLR proteins (PmWR183-NLR1 and PmWR183-NLR2) from wild emmer wheat. Stable transformation and CRISPR/Cas9 knockout experiments demonstrate that the two NLRs function cooperatively: neither gene alone confers resistance, but their co-expression restores immunity, while disruption of either gene abolishes resistance. PmWR183 mediates a developmental stage-dependent response, with susceptibility at the seedling stage and strong resistance at the adult stage. Protein interaction assays reveal constitutive association of PmWR183-NLR1 and PmWR183-NLR2, supporting their cooperative role. Geographical and haplotype analyses show the locus originates from wild emmer and is rare in cultivated wheat, exhibiting at least nine haplotypes. Together, our findings uncover a rare NLR gene pair conferring effective resistance to powdery mildew, providing valuable resources for wheat breeding.},
}

*Triticum/genetics/microbiology/immunology
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics/immunology
*Ascomycota/physiology/pathogenicity
*Plant Proteins/genetics/metabolism
*NLR Proteins/genetics/metabolism
Haplotypes
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid41073016,
year = {2025},
author = {Nie, Y and Wang, W and Wang, N and Yuan, M and Huang, L and Sun, Y and Li, K and Liu, Z and Mu, Y},
title = {PCR-CRISPR/Cas12a-based fluorescence and lateral flow dipstick platforms for efficient screening of CD71 biallelic mutants.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344622},
doi = {10.1016/j.aca.2025.344622},
pmid = {41073016},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Receptors, Transferrin/genetics ; *Polymerase Chain Reaction/methods ; *Mutation ; *Antigens, CD/genetics ; *Alleles ; Fluorescence ; *CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas9 technology plays a pivotal role in gene editing and has been extensively utilized in gene function studies, disease modeling, and gene therapy. However, efficient and accurate detection of CRISPR/Cas9-induced mutants remains a challenge due to the complexity, time-consuming nature, and high cost of existing detection methods. Meanwhile, CRISPR/Cas12a systems have been widely applied in molecular diagnostics due to the non-specific trans-cleavage activity of Cas12a, yet their application in detecting CRISPR/Cas9-induced mutations remains limited. In this study, we developed a PCR-CRISPR/Cas12a-based method to enable the rapid and accurate screening of CD71 biallelic mutants. The detection system was mainly composed of CRISPR RNA specific to the CD71 gene-editing site, Cas12a protein, target DNA, and ssDNA probes for fluorescence or lateral flow dipstick assays. The system demonstrated high specificity in distinguishing CD71 biallelic mutants, with validation through TA cloning confirming its accuracy. Additionally, the method exhibited high sensitivity, establishing it as an efficient tool for biallelic mutated cell clone screening. These findings underscore the potential of PCR-CRISPR/Cas12a as a rapid, sensitive, and cost-effective approach for the precise identification of biallelic mutants, contributing to advancements in gene-editing research and molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
Humans
*Receptors, Transferrin/genetics
*Polymerase Chain Reaction/methods
*Mutation
*Antigens, CD/genetics
*Alleles
Fluorescence
*CRISPR-Associated Proteins/genetics/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41073014,
year = {2025},
author = {Cao, R and Wang, S and Guo, Q and Xie, H and Wang, Y and Li, T and Chang, F and Shi, H and Ding, S and Min, X and Duan, X},
title = {DNAzyme-driven SDA reaction regulates CRISPR/Cas12a for highly sensitive and selective analysis of underexpressed miRNA.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344620},
doi = {10.1016/j.aca.2025.344620},
pmid = {41073014},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics/metabolism ; *DNA, Catalytic/metabolism/chemistry ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Male ; Prostatic Neoplasms/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Underexpressed microRNA (miRNA) exerts a pivotal influence across a spectrum of physiological and pathological processes, with their role being particularly pronounced in the incipient stages of tumorigenesis. However, there are several challenges to analyzing these underexpressed miRNAs for their lower abundance and relative decreases in some cancers. Here, we developed a novel fluorescence biosensor based on the DNAzyme-driven strand displacement amplification (SDA) regulating CRISPR/Cas12a for the sensitive and selective detection of underexpressed miRNA, using prostate cancer-associated miR-222 as a proof-of-concept. This study innovatively expanded the application of DNAzyme substrates, designed as templates to trigger SDA and CRISPR/Cas12a reaction, which could effectively generate a positive signal output for detecting underexpressed miRNA. In the absence of miR-222, DNAzyme formation was blocked, allowing the complete substrate to activate SDA, which generated ssDNA that triggered CRISPR/Cas12a trans-cleavage activity to produce a strong fluorescent signal. In contrast, intact DNAzymes (in the presence of miR-222) cleaved the substrates into short DNA fragments, preventing SDA and CRISPR/Cas12a activation, thereby maintaining the sensor in a low fluorescent state. The biosensor demonstrated a linear detection range spanning from 0.1 pmol/L to 1 nmol/L, accompanied by a detection limit of 33.5 fmol/L. Moreover, it exhibited excellent specificity and anti-interference capacity, enabling the successful detection of miR-222 in blood samples. This "DNAzyme-SDA-CRISPR" fluorescence strategy offers a effective, programmability and scalable solution for detecting underexpressed miRNAs in early cancer screening, which is expected to become a powerful tool in early tumor diagnostics and precision therapy.},
}

*MicroRNAs/analysis/genetics/metabolism
*DNA, Catalytic/metabolism/chemistry
*CRISPR-Cas Systems
Humans
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Male
Prostatic Neoplasms/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41043311,
year = {2026},
author = {Hu, M and Zhou, C and Li, M and Zhao, J},
title = {From 3D culture to clinical decision-making: Systematic innovations in breast cancer organoids.},
journal = {Biomaterials advances},
volume = {179},
number = {},
pages = {214528},
doi = {10.1016/j.bioadv.2025.214528},
pmid = {41043311},
issn = {2772-9508},
mesh = {Humans ; *Organoids/pathology ; *Breast Neoplasms/pathology/therapy/genetics ; Female ; *Clinical Decision-Making ; Animals ; CRISPR-Cas Systems ; *Cell Culture Techniques, Three Dimensional/methods ; Tumor Microenvironment ; Gene Editing ; Precision Medicine ; },
abstract = {Breast cancer is a malignant tumour with high heterogeneity. Traditional research models rely mainly on 2D cell culture and patient-derived tumour xenografts (PDXs). However, these models have limited use in clinical trials because of their shortcomings in mimicking the tumour microenvironment and preserving the genetic background. In recent years, organoids, emerging models capable of self-organizing to form 3D structures in vitro, have become key tools for overcoming the traditional dilemma and are promising alternatives for breast cancer research. This review integrates cutting-edge technologies such as organ-on-a-chip and CRISPR/Cas9 gene editing to summarize the multidimensional generation strategy of breast cancer organoids and discusses the clinical value of translation from diagnosis to therapy. Compared with existing studies, this review provides a systematic solution from "model generation" to "precision medicine" for breast cancer research, and the hope is that this review will pave the way for the further development of organoids.},
}

Humans
*Organoids/pathology
*Breast Neoplasms/pathology/therapy/genetics
Female
*Clinical Decision-Making
Animals
CRISPR-Cas Systems
*Cell Culture Techniques, Three Dimensional/methods
Tumor Microenvironment
Gene Editing
Precision Medicine

@article {pmid41015100,
year = {2025},
author = {Deng, D and Yi, X and Wen, W and He, L and Peng, W},
title = {The role of the transformer gene in sex determination and its employment in CRISPR/Cas9-based homing gene drive in the global fruit pest Drosophila suzukii.},
journal = {Insect biochemistry and molecular biology},
volume = {184},
number = {},
pages = {104406},
doi = {10.1016/j.ibmb.2025.104406},
pmid = {41015100},
issn = {1879-0240},
mesh = {Animals ; *Sex Determination Processes/genetics ; CRISPR-Cas Systems ; Female ; Male ; *Drosophila Proteins/genetics/metabolism ; *Drosophila/genetics/growth & development ; *Gene Drive Technology ; Nuclear Proteins ; },
abstract = {Sex determination of Diptera is established by the cascade genes such as transformer (tra), though the primary signals for sex determination differ among different insects. Here, we report the isolation, expression and function of tra gene in an invasive pest, Drosophila suzukii, and study the potential use of the D. suzukii tra (Dstra) gene in CRISPR/Cas9-based homing gene drive for genetic-based pest management. The Dstra gene is highly conserved in structure and has a sex-specific transcript. To test the function of this gene in sex determination, Dstra dsRNA was injected into embryos. Almost all XX embryos developed into masculinized phenotypic male adults with intersex morphology. Abnormal ovaries were revealed in XX pseudomales upon dissection. Based on the necessary role of Dstra for female development, we developed and evaluated a homing gene drive that targets Dstra in D. suzukii. The drive component consisting of multiplex Dstra single guide RNAs and Cas9 with Dsvasa promoter was introduced into the Dstra locus. Abnormal development of both the external genitalia and gonads was observed in G0 and G1 chromosomal female adults that expressed the male-specific doublesex (dsx) transcript. Interestingly, knocking out Dstra led to significantly reduced fertility in adults of corresponding sex and moderate transmission rates of the DsRed gene (63.54 %) were observed. Our results not only confirm the conserved function of the Dstra gene in sex determination, but also highlight the potential of sex conversion-based suppression gene-drive strategy targeting the Dstra gene in controlling of D. suzukii populations.},
}

Animals
*Sex Determination Processes/genetics
CRISPR-Cas Systems
Female
Male
*Drosophila Proteins/genetics/metabolism
*Drosophila/genetics/growth & development
*Gene Drive Technology
Nuclear Proteins

@article {pmid41072992,
year = {2025},
author = {Shi, S and Qin, F and Wu, J and Yang, J and Zhang, X and Wang, S and Wen, W and Wu, Z},
title = {Ultrasensitive single-particle collision electrochemical platform employing CRISPR/Cas12a for ctDNA biosensing.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344590},
doi = {10.1016/j.aca.2025.344590},
pmid = {41072992},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Circulating Tumor DNA/blood/analysis/genetics ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Silver/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) is a characteristic tumor biomarker used for cancer diagnosis, treatment, and prognosis. However, the low concentration of ctDNA in peripheral blood and the interference of complex matrices with signals make the detection of ctDNA extremely challenging. Single-particle collision electrochemistry (SPCE) has been widely used in bioanalysis due to its advantages such as high throughput, simple operation, high sensitivity, and low detection limit. In this work, a novel SPCE biosensor for the rapid detection of ctDNA was developed by combining CRISPR/Cas12a with excellent cleavage activity and magnetic beads (MBs) with good separation and enrichment capabilities. The trans-cleavage ability of CRISPR/Cas12a can only be triggered in the presence of target ctDNA to effectively cleave the ssDNA2 on the surface of Ag NPs-ssDNA2 within 1 h, thereby activating the collision activity of silver nanoparticles (Ag NPs). ctDNA was quantified by the collision frequency of Ag NPs. The detection limit of the developed SPCE biosensor for ctDNA was as low as 4.2 fM, and the linear range was 10 fM-1 nM. In addition, MBs allow the biosensor to detect ctDNA in complex samples by directly sampling from complex matrices, with excellent sensitivity and specificity, demonstrating the great potential of the developed SPCE biosensor in the detection of patient samples.},
}

*Biosensing Techniques/methods
*Circulating Tumor DNA/blood/analysis/genetics
*Electrochemical Techniques/methods
Humans
*CRISPR-Cas Systems
Silver/chemistry
Metal Nanoparticles/chemistry
Limit of Detection

@article {pmid41072860,
year = {2025},
author = {Patel, RR and Arun, PP and Singh, SK and Singh, M},
title = {Overcoming Antimicrobial Resistance: Phage Therapy as a Promising Solution to Combat ESKAPE Pathogens.},
journal = {International journal of antimicrobial agents},
volume = {},
number = {},
pages = {107640},
doi = {10.1016/j.ijantimicag.2025.107640},
pmid = {41072860},
issn = {1872-7913},
abstract = {The global escalation of antimicrobial resistance (AMR) has intensified the search for alternative therapies, with bacteriophage (phage) therapy re-emerging as a promising solution. This review critically examines the therapeutic potential of phage therapy against multidrug-resistant (MDR) ESKAPE pathogens which are among the leading causes of hospital-acquired infections. The review discusses the distinct antibacterial strategies of phage namely, targeted lysis, enzymatic biofilm disruption, and synergy with antibiotics. It also explores the molecular regulation of phage life cycles, highlighting the therapeutic importance of the lytic-lysogenic switch. A central focus is the interplay between advanced delivery systems such as liposomes, hydrogels, nanofibers, and nanoemulsions, and specific administration routes including oral, topical, intravenous, intranasal, and intravesical approaches. These delivery strategies are essential for overcoming key physiological barriers such as gastric acidity, enzymatic degradation, and immune clearance, thereby enhancing phage stability, retention, and therapeutic efficacy. Recent innovations in phage engineering are also explored, particularly the use of CRISPR-Cas systems, synthetic biology, and continuous evolution platforms to broaden host range and optimize lytic function. The review further evaluates emerging clinical evidence, including outcomes from compassionate use cases and early-phase trials, which emphasize both the safety and therapeutic potential of phage therapy in real-world settings. Despite these advances, significant challenges persist, including bacterial resistance to phages, the need for regulatory clarity, and scalability of personalized treatments. With the integration of microbiology, nanotechnology, and clinical practice, phage therapy bridges the gap between ecological solutions and modern medicine, positioning itself as a versatile, sustainable pillar in the post-antibiotic era.},
}

@article {pmid41072406,
year = {2025},
author = {Henriques, WS and Bowman, J and Hall, LN and Gauvin, CC and Wei, H and Kuang, H and Zimanyi, CM and Eng, ET and Santiago-Frangos, A and Wiedenheft, B},
title = {Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2025.09.007},
pmid = {41072406},
issn = {1878-4186},
abstract = {Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify and determine structures of Cas1 and the Cas2/3 fusion proteins from Pseudomonas aeruginosa at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.},
}

@article {pmid41071884,
year = {2025},
author = {Guo, Y and Xu, M and Xue, H and Ding, X and Wong, AM and Lin, N and Pu, D and Wong, AM and Wang, X and Zhao, H and Wong, N},
title = {Genome-wide CRISPR screen identifies splicing factor SF3B4 in driving hepatocellular carcinoma.},
journal = {Science advances},
volume = {11},
number = {41},
pages = {eadw7181},
doi = {10.1126/sciadv.adw7181},
pmid = {41071884},
issn = {2375-2548},
mesh = {*Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy ; Humans ; *Liver Neoplasms/genetics/pathology/metabolism/drug therapy ; Animals ; Mice ; *RNA Splicing Factors/genetics/metabolism ; Ferroptosis/genetics ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Phenylurea Compounds/pharmacology ; Drug Resistance, Neoplasm/genetics ; T-Box Domain Proteins/genetics/metabolism ; Quinolines ; },
abstract = {Although genome sequencings have recognized many cancer-associated genes in hepatocellular carcinoma (HCC), distinguishing their functional effect remains challenging. Leveraging on a genome-wide CRISPR knockout (KO) screening, we uncovered spliceosome factors as major survival essential genes in HCC and up-regulations of ferroptosis suppressors [particularly glutamate-cysteine ligase catalytic subunit (GCLC)] in lenvatinib resistance. Our KO screen in patient-derived HCC organoid showed splicing factor 3b subunit 4 (SF3B4) to be top-ranked, conferring prosurvival signal in HCC organoid and driving tumorigenic potentials in both hepatic progenitor organoids and hydrodynamic tail vein injection HCC murine model. The combined RNA immunoprecipitation sequencing, long-read isoform sequencing, and transcriptome revealed characteristic splicing landscape regulated by SF3B4 and identified T-box transcription factor 3 (TBX3) variant TBX3+2a as a potent downstream effector. Our findings highlighted vital roles of SF3B4 in HCC cell survival and tumor progression, and the phenomenon of ferroptosis resistance in patients unresponsive to first-line agent lenvatinib.},
}

*Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy
Humans
*Liver Neoplasms/genetics/pathology/metabolism/drug therapy
Animals
Mice
*RNA Splicing Factors/genetics/metabolism
Ferroptosis/genetics
Gene Expression Regulation, Neoplastic
*CRISPR-Cas Systems
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats
Phenylurea Compounds/pharmacology
Drug Resistance, Neoplasm/genetics
T-Box Domain Proteins/genetics/metabolism
Quinolines

@article {pmid41070887,
year = {2025},
author = {Zhang, Y and Wu, Y and Guo, A and Liu, Y and Sun, Q and Zou, X and Sun, Z},
title = {Fluorescent biosensors for the detection of foodborne pathogenic bacteria in food: a comprehensive review.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay01025j},
pmid = {41070887},
issn = {1759-9679},
abstract = {Foodborne pathogenic bacterial contamination poses a major challenge to global food safety and public health, making the development of rapid, sensitive, and specific detection technologies critically important. Conventional methods are limited by their long turnaround time, complex operations, and reliance on large-scale instruments, making them unsuitable for on-site rapid detection. Fluorescent biosensors, which combine highly specific biological recognition elements with highly sensitive fluorescent signal output, demonstrate significant advantages in detecting foodborne pathogens. This review systematically summarizes recent advances in fluorescent biosensors for the detection of common foodborne pathogenic bacteria, with a focus on the application of signal amplification strategies such as functional nanomaterials, amplification techniques, CRISPR/Cas systems, and Argonaute proteins. Furthermore, it analyzes performance metrics including multiplex pathogen detection, real-time quantification, anti-interference capability, and on-site applicability. Finally, future development trends and challenges are discussed, aiming to provide insights for the innovation of food safety monitoring technologies.},
}

@article {pmid41069986,
year = {2025},
author = {Pandey, V and Sharma, S and Pokharel, YR},
title = {Exploring CRISPR-Cas: The transformative impact of gene editing in molecular biology.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102717},
pmid = {41069986},
issn = {2162-2531},
abstract = {This review traces the evolution of clustered regularly interspaced short palindromic repeats (CRISPR) technology from a prokaryotic immune mechanism to a versatile tool for precise genome engineering. We compare CRISPR with traditional gene-editing methods like RNA interference (RNAi), zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), emphasizing its advantages in target specificity, multiplexing, and ease of design. We examine various Cas enzyme classes, engineered variants, and their applications in dissecting genetic alterations at the cellular level. The review further explores CRISPR's expanding role in developing disease models using tissues, organoids, and animal systems, enhancing our understanding of disease mechanisms. Finally, we discuss CRISPR's emerging applications in diagnostics and its transformative impact on immunotherapy and cell-based cancer treatments.},
}

@article {pmid41069170,
year = {2025},
author = {Zhu, Z and Xue, J and Cao, J and Zhang, Z and Gu, T and Sun, Y and Wang, H},
title = {One-pot assay for rapid detection of heterozygous herbicide resistance in Digitaria ciliaris var. chrysoblephara by combining CRISPR/Cas and LAMP.},
journal = {Pest management science},
volume = {},
number = {},
pages = {},
doi = {10.1002/ps.70279},
pmid = {41069170},
issn = {1526-4998},
support = {CX(22)5003//Jiangsu Agricultural Science and Technology Innovation Fund/ ; 2023YFD1401100//National Key R&D Program of China/ ; },
abstract = {BACKGROUND: Resistance to the acetyl-CoA carboxylase (ACCase) inhibitor herbicide cyhalofop-butyl in Digitaria ciliaris var. chrysoblephara is mainly caused by a mutation at the W2027C or W2027S site; however, the existing methods for this mutation site have insufficient detection performance and are difficult to achieve integrated detection in the field.

RESULTS: In this work, we have developed and optimized a One-Pot single-nucleotide polymorphism (SNP) detection for herbicide resistance based on CRISPR/Cas recognition coupled with the loop-mediated isothermal amplification (LAMP), named OpCas-LAMP. By designing specific CRISPR/Cas guide RNAs and LAMP primers, the OpCas-LAMP can accurately identify with 1% heterozygous mutants of the W2027S or W2027C mutations ACCase gene in D.ciliaris var. chrysoblephara. The optimized reaction system exhibits optimal amplification efficiency at 65°C, effectively distinguishing within 60 min (30-min LAMP detection after 30 min CRISPR/Cas pre-cleavage) between homozygous mutant (HM), heterozygous mutant (HT) and wild-type (WT).

CONCLUSION: This method enables real-time one-pot field detection by integrated with miniaturized detection devices, significantly enhancing its practicality and potential for widespread application. This work provides a novel technical approach for detecting herbicide resistance for global weed resistance monitoring and management. © 2025 Society of Chemical Industry.},
}

@article {pmid41068986,
year = {2025},
author = {Wu, J and Jang, H and Kwak, H and Son, M and Jiang, W and Hwang, HY and Jo, DH and Kim, D and Kim, HH and Kim, JH},
title = {Engineered virus-like particle-assembled Vegfa-targeting Cas9 ribonucleoprotein treatment alleviates neovascularization in wet age-related macular degeneration.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {346},
pmid = {41068986},
issn = {1474-760X},
support = {2022M3A9F3017506//National Research Foundation of Korea/ ; 2022M3A9E4017127//National Research Foundation of Korea/ ; 202200004004//Kun-hee Lee Child Cancer & Rare Disease Project/ ; 18-2023-0010//Seoul National University Hospital/ ; GTL24021-000//National Research Council of Science and Technology/ ; },
mesh = {Animals ; *Vascular Endothelial Growth Factor A/genetics/metabolism ; Mice ; Gene Editing/methods ; *Ribonucleoproteins/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Choroidal Neovascularization/therapy/genetics ; CRISPR-Cas Systems ; NIH 3T3 Cells ; Disease Models, Animal ; Humans ; *Wet Macular Degeneration/therapy/genetics ; Genetic Therapy ; *Macular Degeneration/therapy/genetics ; },
abstract = {BACKGROUND: Age-related macular degeneration, particularly the wet form, is a leading cause of vision loss, characterized by vascular endothelial growth factor A (VEGFA) overproduction. Engineered virus-like particles (eVLPs) combine the efficiency of viral systems with the transient nature of non-viral platforms to offer a potential solution for delivering VEGFA-targeting genome editing enzymes in a safe and efficient manner. Here, we investigate the therapeutic efficacy of eVLPs for transient delivery of Vegfa-targeting Cas9 ribonucleoprotein in a laser-induced choroidal neovascularization mouse model of wet age-related macular degeneration.

RESULTS: We find that Cas9-eVLPs enables efficient intracellular delivery in vitro, achieving up to 99% insertion and deletion frequency at Vegfa target locus and significant VEGFA protein downregulation in NIH/3T3 cells. A single subretinal injection of Cas9-eVLPs into the mouse retinal pigment epithelium effectively disrupts Vegfa expression, achieving an average indel efficiency of 16.7%. Compared to control groups, the laser-induced choroidal neovascularization mouse model exhibits significantly reduced choroidal neovascularization formation following Cas9-eVLPs intervention, and decreased VEGFA protein levels are detected in the retinal pigment epithelium. Furthermore, the retinal anatomical and functional toxicity are not affected after treatment.

CONCLUSIONS: eVLPs exhibit the potential as a safe and efficient delivery platform for Cas9 ribonucleoproteins, achieving precise Vegfa downregulation and significant reduction in choroidal neovascularization in a mouse model of wet age-related macular degeneration. With transient delivery of gene editing enzymes, high editing efficiency, and minimal risk of genomic integration, eVLPs present a promising alternative to conventional delivery systems for advancing genome editing therapies in retinal diseases.},
}

Animals
*Vascular Endothelial Growth Factor A/genetics/metabolism
Mice
Gene Editing/methods
*Ribonucleoproteins/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
*Choroidal Neovascularization/therapy/genetics
CRISPR-Cas Systems
NIH 3T3 Cells
Disease Models, Animal
Humans
*Wet Macular Degeneration/therapy/genetics
Genetic Therapy
*Macular Degeneration/therapy/genetics

@article {pmid41066575,
year = {2025},
author = {Groessl, S and Kalis, R and Snaebjornsson, MT and Wambach, L and Haider, J and Andersch, F and Schulze, A and Palm, W and Zuber, J},
title = {Acidosis orchestrates adaptations of energy metabolism in tumors.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6769},
pages = {eadp7603},
doi = {10.1126/science.adp7603},
pmid = {41066575},
issn = {1095-9203},
mesh = {*Energy Metabolism/genetics ; *Acidosis/metabolism/genetics ; Humans ; *Pancreatic Neoplasms/metabolism/genetics ; Mitochondria/metabolism ; Cell Line, Tumor ; *Adaptation, Physiological/genetics ; Animals ; Mice ; Stress, Physiological ; CRISPR-Cas Systems ; MAP Kinase Signaling System ; Mitochondrial Dynamics ; },
abstract = {Malignant tumors are characterized by diverse metabolic stresses, including nutrient shortages, hypoxia, and buildup of metabolic by-products. To understand how cancer cells adapt to such challenges, we conducted sequential CRISPR screens to identify genes that affect cellular fitness under specific metabolic stress conditions in cell culture and to then probe their relevance in pancreatic tumors. Comparative analyses of hundreds of fitness genes revealed that cancer metabolism in vivo was shaped by bioenergetic adaptations to tumor acidosis. Mechanistically, acidosis suppressed cytoplasmic activity of extracellular signal-regulated kinase (ERK), thereby preventing oncogene-induced mitochondrial fragmentation and promoting fused mitochondria. The resulting boost in mitochondrial respiration supported cancer cell adaptations to various metabolic stresses. Thus, acidosis is an environmental factor that alters energy metabolism to promote stress resilience in cancer.},
}

*Energy Metabolism/genetics
*Acidosis/metabolism/genetics
Humans
*Pancreatic Neoplasms/metabolism/genetics
Mitochondria/metabolism
Cell Line, Tumor
*Adaptation, Physiological/genetics
Animals
Mice
Stress, Physiological
CRISPR-Cas Systems
MAP Kinase Signaling System
Mitochondrial Dynamics

@article {pmid41065057,
year = {2025},
author = {Anjali, and Punetha, M and Kumar, A and Tripathi, MK and Kishor Kumar, DG and Khanna, S and Nanda, R and Yadav, P and Sharma, S and Maurya, VP and Singh, G and Chouhan, VS},
title = {Application of CRISPR/Cas9 for GDF9 Gene Editing in Caprine Granulosa Cells: Effects on Receptor Signalling and FGF2 Response.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {60},
number = {10},
pages = {e70128},
doi = {10.1111/rda.70128},
pmid = {41065057},
issn = {1439-0531},
support = {NASF/GTR-8004/2019-20//ICAR - National Agricultural Science Fund/ ; },
mesh = {Animals ; Female ; *Granulosa Cells/metabolism ; *Growth Differentiation Factor 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/veterinary ; *Goats/genetics ; *Fibroblast Growth Factor 2/pharmacology/metabolism ; Signal Transduction ; Receptors, FSH/metabolism/genetics ; },
abstract = {Fecundity-related genes, such as GDF9, play a critical role in regulating ovulation, fertilisation and early embryonic development. This study aimed to elucidate the functional role of GDF9 in caprine granulosa cells by employing CRISPR/Cas9-mediated gene editing. The CRISPR/Cas9 system, incorporating single guide RNA (sgRNA) and Cas9 endonuclease, was used to specifically disrupt the GDF9 gene. Successful GDF9 knockout was confirmed via the T7 Endonuclease I (T7E1) cleavage assay. Subsequent analyses assessed the impact of GDF9 disruption on the expression of GDF9 and its associated receptors-BMPR-1A, BMPR-1B and BMPR-II. Additionally, the study examined the modulatory effects of fibroblast growth factor 2 (FGF2) on receptor expression. FGF2 treatment led to increased mRNA expression of BMPR-1A, BMPR-1B and BMPR-II in wild-type granulosa cells. Furthermore, follicle-stimulating hormone receptor (FSHR) levels were significantly upregulated, whereas luteinising hormone receptor (LHR) expression decreased following FGF2 stimulation in wild-type cells. In contrast, GDF9-knockout cells showed elevated expression of both FSHR and LHR. The study also investigated the impact of GDF9 deletion on the expression of key steroidogenic genes, particularly StAR. The combined presence of GDF9 and FGF2 synergistically enhanced StAR expression. Cellular responses to FGF2 included a downregulation of CASPASE 3, indicating reduced apoptosis and an upregulation of PCNA, suggesting increased cell proliferation. In conclusion, this study provides novel insights into the regulatory role of GDF9 in ovarian granulosa cell function and highlights the utility of CRISPR/Cas9 technology for functional genomics in caprine species. The findings have significant implications for enhancing reproductive performance through targeted gene modulation.},
}

Animals
Female
*Granulosa Cells/metabolism
*Growth Differentiation Factor 9/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing/veterinary
*Goats/genetics
*Fibroblast Growth Factor 2/pharmacology/metabolism
Signal Transduction
Receptors, FSH/metabolism/genetics

@article {pmid41064997,
year = {2025},
author = {Chen, X and Ye, Q and Liang, Q and Li, J and Huang, Y and Xia, Q and Xiao, J and Liao, C and Lau, CH and Zhu, H},
title = {CRISPR-based platforms for detecting tumor-associated genetic materials in clinical samples.},
journal = {Bioanalysis},
volume = {},
number = {},
pages = {1-15},
doi = {10.1080/17576180.2025.2571023},
pmid = {41064997},
issn = {1757-6199},
abstract = {Tumor-associated genetic markers are useful for early cancer screening, diagnosis, and treatment monitoring. However, traditional detection methods are complex in operation procedures, time-consuming, and the equipment costs are expensive. CRISPR/Cas systems are becoming emerging detection tools for tumor detection due to their programmability, rapid reaction, high targeting specificity, and the ability to amplify the signals. CRISPR/Cas has made breakthroughs in the detection of tumor-associated genetic materials including gene mutations, DNA methylation, miRNA, lncRNA, and circRNA detection. Herein, we critically discuss these advancements and describe the key concepts of each CRISPR/Cas system for detecting tumor-associated genetic materials. The significance of these tumor-associated genetic materials in cancer diagnosis and prognosis is highlighted.},
}

@article {pmid41064763,
year = {2025},
author = {Ragulakollu, S and Loganathan, A and Swaminatham, M and Chellappan, G and Veeraswamy, R and Jegadeesan, R},
title = {Molecular breeding approaches for sustainable rice blast management: recent advances and challenges.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1551018},
pmid = {41064763},
issn = {1664-462X},
abstract = {Rice (Oryza sativa. L) is a staple crop globally, but blast disease caused by fungal pathogens Magnaporthe oryzae is one of the most devastating and results in severe economic losses in rice production worldwide. Recent technological advancements have opened new possibilities for developing blast resistance. The dynamic and highly adaptable nature of M. oryzae allows it to overcome plant defense mechanisms rapidly, posing a major threat to global food security and agricultural sustainability. While foundational to early resistance development, traditional breeding approaches have been limited by their time-consuming nature and reliance on phenotypic selection. These methods often require several generations to establish stable resistance traits. However, with the emergence of molecular breeding technologies, resistance breeding has experienced significant acceleration and precision. Tools such as marker-assisted selection (MAS), marker-assisted backcross breeding (MABB), and quantitative trait locus (QTL) mapping allow for the identification and introgression of resistance genes (R genes) more efficiently and accurately. Recent advances in genome engineering techniques, particularly CRISPR-Cas 9, have transformed the capability to manipulate resistance genes directly, enabling targeted editing and stacking of multiple genes (gene pyramiding) for durable resistance. Moreover, omics technologies-including genomics, transcriptomics, proteomics, and metabolomics-offer a comprehensive understanding of the molecular interactions between host and pathogen, facilitating the discovery of novel resistance mechanisms and regulatory pathways. The integration of allele mining with advanced biotechnological tools has further promoted the development of cisgenic and intragenic plants, where resistance genes from related cultivars or wild species are introduced without foreign DNA, thus addressing public concerns over transgenic crops. These strategies enhance resistance and help retain the desirable agronomic traits of elite rice varieties. Despite these advancements, the high mutation rate and genetic plasticity of M. oryzae enable it to evolve and overcome resistance provided by single R genes. Therefore, understanding host-pathogen interactions at the molecular and cellular levels remains essential. Emerging technologies such as nanotechnology show promise in developing targeted fungicide delivery systems and innovative diagnostic tools. Synthetic biology opens avenues for constructing synthetic resistance pathways or deploying plant biosensors. Additionally, machine learning and artificial intelligence (AI) algorithms are increasingly used to predict disease outbreaks, model gene interactions, and optimize breeding strategies based on large datasets. Thus, managing rice blast disease necessitates a holistic approach combining conventional breeding wisdom with modern molecular tools and emerging technologies. The synergy among these approaches holds promise to enhance resistance durability and protect global rice production against evolving fungal threats. This review emphasizes recent advancements in managing rice blast disease, offering valuable insights to sustain resilient breeding programs against this pathogen.},
}

@article {pmid41064460,
year = {2025},
author = {Rentz, L and Hellwig, L and Schneider, S and Schmitz, RA},
title = {Functional insights into Solo-Cas4 in Methanosarcina mazei Gö1.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf024},
pmid = {41064460},
issn = {2633-6693},
abstract = {Solo-Cas4 homologs are Cas4-family proteins found outside of canonical CRISPR-Cas operons. Here, we present the biochemical characterization of Solo-Cas4 from Methanosarcina mazei Gö1. We found significantly upregulated solo-cas4 transcript levels during stationary phase, while remaining constant under oxygen exposure, temperature shifts, high salt conditions or virus challenge. Heterologously expressed as a SUMO-fusion, the purified tag-free protein displays an absorption peak at 420 nm, indicative of a [4Fe-4S]-cluster . Size-exclusion-chromatography revealed that Solo-Cas4 forms a higher oligomeric complex, with an apparent molecular mass of 318 kDa. In vitro nuclease activity assays demonstrated that Solo-Cas4 cleaves metal-dependent linear dsDNA, with highest cleavage activity in the presence of Mn[2+], followed by Mg[2+], while Ca[2+] and Cu[2+] result in negligible cleavage. Isoleucine169 was identified to be crucial for catalysis, mutating it to alanine completely abolished nuclease activity . Mutating any of the four conserved cysteines-proposed to coordinate the [4Fe-4S]-cluster did not affect nuclease activity; however, it abolishes metal cluster binding. Supercoiled circular dsDNA was preferentially nicked by Solo-Cas4 in the presence of Mg[2+], whereas Mn[2+] also led to linearization followed by complete degradation. Besides, ssDNA was cleaved by Solo-Cas4 but with lower activity. In agreement, Microscale thermophoresis analysis revealed strong dsDNA binding with highest affinity to supercoiled circular DNA, and weak ssDNA binding. Overall, these findings indicate that M. mazei Solo-Cas4 is a high oligomeric Cas4-family nuclease that preferentially targets supercoiled dsDNA and is upregulated during stationary growth.},
}

@article {pmid40988554,
year = {2025},
author = {Li, H and Gui, P and Li, X and Lin, Y and Ma, Z and Yu, H and Ma, F},
title = {CRISPR/Cas9-Mediated Construction of a YPS Gene-Deficient Komagataella phaffii Strain for Enhanced Expression of BIAP Ⅱ.},
journal = {Yeast (Chichester, England)},
volume = {42},
number = {8-10},
pages = {195-205},
doi = {10.1002/yea.70002},
pmid = {40988554},
issn = {1097-0061},
support = {//This work was supported by Youth Innovation Promotion Association Fellowship Program, CAS (2022327), Shandong Provincial Laboratory Project (SYS202209), Key Technologies R&D Program of Guangdong Province (2022B1111050001), Natural Science Foundation of Shandong Province, China (ZR2021QB155)./ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Alkaline Phosphatase/genetics/metabolism ; *Saccharomycetales/genetics/metabolism/enzymology ; Cattle ; *Aspartic Acid Endopeptidases/genetics/metabolism ; Recombinant Proteins/genetics/metabolism ; Gene Editing ; Proteolysis ; Gene Expression ; },
abstract = {Multiple isoforms of bovine intestinal alkaline phosphatase (BIAP) have been identified, among which type Ⅱ (BIAP Ⅱ) exhibits the highest specific activity. While Komagataella phaffii has been successfully employed for the secretory expression of recombinant BIAP Ⅱ, substantial proteolytic degradation during the secretion and expression processes has been observed, leading to reduced protein yield and challenging purification procedures. Our investigation demonstrates that the proteolytic cleavage of BIAP Ⅱ is predominantly mediated by secretory pathway proteases, particularly the aspartic protease yapsin (Yps), with Yps1 playing a crucial role. Genetic disruption of the YPS1 gene resulted in a remarkable 2.5-fold increase in BIAP Ⅱ production yield compared to the parental strain, accompanied by significantly reduced proteolytic degradation. Through detailed analysis, we have identified the Yps1 cleavage site within the BIAP Ⅱ peptide chain, located between Lys137 and Lys138. To further minimize BIAP Ⅱ proteolysis, we developed a YPS multigene-deficient engineered strain using CRISPR/Cas9-mediated triple gene editing technology. Additionally, we have established a novel dual-color quantitative PCR (DC-qPCR) method that enables rapid and precise determination of target gene dosage, thereby enhancing screening efficiency while reducing experimental errors associated with repeated sample processing. The strategies and methodologies developed in this study may serve as a valuable reference for optimizing the expression of various secretory heterologous proteins in Komagataella phaffii.},
}

Animals
*CRISPR-Cas Systems
*Alkaline Phosphatase/genetics/metabolism
*Saccharomycetales/genetics/metabolism/enzymology
Cattle
*Aspartic Acid Endopeptidases/genetics/metabolism
Recombinant Proteins/genetics/metabolism
Gene Editing
Proteolysis
Gene Expression

@article {pmid40962170,
year = {2025},
author = {He, H and Huang, Z and Wen, F and Ge, N and Lin, X and Pan, J},
title = {PSPC1 knockout promotes radiosensitivity, inhibits EMT, and metastasis of nasopharyngeal carcinoma cells.},
journal = {Experimental cell research},
volume = {452},
number = {2},
pages = {114755},
doi = {10.1016/j.yexcr.2025.114755},
pmid = {40962170},
issn = {1090-2422},
mesh = {Humans ; *Radiation Tolerance/genetics ; *Nasopharyngeal Carcinoma/genetics/pathology/radiotherapy/metabolism ; Animals ; *Nasopharyngeal Neoplasms/pathology/genetics/radiotherapy ; Cell Line, Tumor ; *Epithelial-Mesenchymal Transition/genetics/radiation effects ; Cell Movement/genetics/radiation effects ; Apoptosis/genetics/radiation effects ; Mice ; Mice, Nude ; *RNA-Binding Proteins/genetics/metabolism ; Cell Proliferation/genetics ; *Nuclear Proteins/genetics/metabolism ; Gene Expression Regulation, Neoplastic/genetics ; Neoplasm Metastasis ; Mice, Inbred BALB C ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: Paraspeckle component 1 (PSPC1) is upregulated in numerous cancers and is associated with reduced patient survival rates. Our previous research indicated that elevated PSPC1 levels in nasopharyngeal carcinoma (NPC) are positively linked to radiation resistance and tumor metastasis, two primary clinical challenges in NPC treatment. However, the precise role of PSPC1 in radiation resistance and metastasis of NPC cells remains unclear. This study aimed to explore the molecular mechanisms by which PSPC1 influences radiation resistance and metastasis in NPC.

METHODS: Using the radiation-resistant R743 and radiosensitive CNE2 cell lines of NPC, we examined the impact of PSPC1 expression on post-radiation survival, cell cycle progression, apoptosis, migration, invasion, and tumor growth. CRISPR/Cas9 genome editing was employed to generate PSPC1 knockout (KO) lines in R743 cells, while PSPC1 overexpression (pcD-PSPC1) was achieved in CNE2 cells via pcDNA3.1(+)-PSPC1 plasmid transfection.

RESULTS: PSPC1 knockout converted R743 cells from radioresistant to radiosensitive, whereas PSPC1 overexpression decreased radiosensitivity in CNE2 cells. Cell cycle analysis revealed that PSPC1 KO arrested R743 cells in the G2/M phase post-irradiation, while PSPC1 overexpression prevented G2/M phase arrest in CNE2 cells. PSPC1 KO increased irradiation-induced apoptosis in R743 cells, whereas PSPC1 overexpression decreased it in CNE2 cells. Post-radiation, PSPC1 KO cells showed significantly reduced migration and invasion abilities. Bioinformatics analysis identified SFPQ as a PSPC1-interacting protein, with PSPC1 KO resulting in SFPQ downregulation. Additionally, PSPC1 KO enhanced the radiosensitivity of xenografted tumors in nude mice.

CONCLUSION: Our findings suggest that PSPC1 is a pivotal factor in enhancing the survival and spread of NPC cells post-radiation. Targeting PSPC1 or its downstream pathways could offer novel strategies to overcome radiation resistance and metastasis in NPC cells.},
}

Humans
*Radiation Tolerance/genetics
*Nasopharyngeal Carcinoma/genetics/pathology/radiotherapy/metabolism
Animals
*Nasopharyngeal Neoplasms/pathology/genetics/radiotherapy
Cell Line, Tumor
*Epithelial-Mesenchymal Transition/genetics/radiation effects
Cell Movement/genetics/radiation effects
Apoptosis/genetics/radiation effects
Mice
Mice, Nude
*RNA-Binding Proteins/genetics/metabolism
Cell Proliferation/genetics
*Nuclear Proteins/genetics/metabolism
Gene Expression Regulation, Neoplastic/genetics
Neoplasm Metastasis
Mice, Inbred BALB C
Gene Knockout Techniques
CRISPR-Cas Systems

@article {pmid40952014,
year = {2025},
author = {Llanga, T and Bush, K and Sun, Y and Yan, A and Zhou, J and Gorodkin, J and Sullenger, BA},
title = {Binding and Ligand Activation Driven Enrichment-Directed Evolution of SaCas9 gRNAs Improves Gene Editing Efficiency.},
journal = {Nucleic acid therapeutics},
volume = {35},
number = {5},
pages = {209-219},
doi = {10.1177/21593337251370553},
pmid = {40952014},
issn = {2159-3345},
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Staphylococcus aureus/genetics/enzymology ; *Directed Molecular Evolution/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Ligands ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats-based editing is inefficient at over two-thirds of genetic targets. A primary cause is ribonucleic acid (RNA) misfolding that can occur between the spacer and scaffold regions of the gRNA, which hinders the formation of functional Cas9 ribonucleoprotein (RNP) complexes. Here, we uncover hundreds of highly efficient gRNA variant scaffolds for Staphylococcus aureus (Sa)Cas9 utilizing an innovative binding and ligand activation driven enrichment (BLADE) methodology, which leverages asymmetrical product dissociation over rounds of evolution. SaBLADE-derived gRNA scaffolds contain 7%-42% of nucleotide variation relative to wild type. gRNA variants are able to improve gene editing efficiency at all targets tested, and they achieve their highest levels of editing improvement (>400%) at the most challenging DNA target sites for the wild-type SaCas9 gRNA. This arsenal of SaBLADE-derived gRNA variants showcases the power and flexibility of combinatorial chemistry and directed evolution to enable efficient gene editing at challenging, or previously intractable, genomic sites.},
}

*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Staphylococcus aureus/genetics/enzymology
*Directed Molecular Evolution/methods
*CRISPR-Associated Protein 9/genetics/metabolism
*CRISPR-Cas Systems/genetics
Ligands
Humans

@article {pmid40935921,
year = {2025},
author = {Roh, H and Shen, SP and Hu, Y and Kwok, HS and Siegenfeld, AP and Lee, C and Zepeda, MU and Guo, CJ and Roseman, SA and Comenho, C and Sankaran, VG and Buenrostro, JD and Liau, BB},
title = {Coupling CRISPR scanning with targeted chromatin accessibility profiling using a double-stranded DNA deaminase.},
journal = {Nature methods},
volume = {22},
number = {10},
pages = {2083-2093},
pmid = {40935921},
issn = {1548-7105},
support = {R35GM153476//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; F31HL174076//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01DK103794//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DGE1745303//National Science Foundation (NSF)/ ; },
mesh = {Humans ; *Chromatin/genetics/metabolism ; Hematopoietic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Cytidine Deaminase/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Genome editing enables sequence-function profiling of endogenous cis-regulatory elements, driving understanding of their mechanisms. However, these approaches lack direct, scalable readouts of chromatin accessibility across long single-molecule chromatin fibers. Here we leverage double-stranded DNA cytidine deaminases to profile chromatin accessibility at endogenous loci of interest through targeted PCR and long-read sequencing, a method we term targeted deaminase-accessible chromatin sequencing (TDAC-seq). With high sequence coverage at targeted loci, TDAC-seq can be integrated with CRISPR perturbations to link genetic edits and their effects on chromatin accessibility on the same single chromatin fiber at single-nucleotide resolution. We employed TDAC-seq to parse CRISPR edits that activate fetal hemoglobin in human CD34[+] hematopoietic stem and progenitor cells (HSPCs) during erythroid differentiation as well as in pooled CRISPR and base-editing screens tiling an enhancer controlling the globin locus. We further scaled the method to interrogate 947 variants in a GFI1B-linked enhancer associated with myeloproliferative neoplasm risk in a single pooled CRISPR experiment in CD34[+] HSPCs. Together, TDAC-seq enables high-resolution sequence-function mapping of single-molecule chromatin fibers by genome editing.},
}

Humans
*Chromatin/genetics/metabolism
Hematopoietic Stem Cells/metabolism/cytology
*Gene Editing/methods
*CRISPR-Cas Systems
*Cytidine Deaminase/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40921108,
year = {2026},
author = {Zhang, Z and Wang, J and Li, C and Sun, H and Bu, S and Jia, Q and Wan, Y and Zhao, Y and Zhou, H and Hao, Z and Li, N and Yu, S and Wang, L and Wan, J},
title = {An ultrasensitive biosensor for H1N1 virus coupled with 3D spherical DNA nanostructure and CRISPR-Cas12a.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {346},
number = {},
pages = {126905},
doi = {10.1016/j.saa.2025.126905},
pmid = {40921108},
issn = {1873-3557},
mesh = {*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Nanostructures/chemistry ; *CRISPR-Cas Systems ; Limit of Detection ; *DNA/chemistry ; Animals ; *CRISPR-Associated Proteins/metabolism/chemistry ; Humans ; Aptamers, Nucleotide/chemistry ; Cattle ; Milk/virology ; Chickens ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {To achieve ultrasensitive and real-time detection of the H1N1 influenza virus, this study designed a nucleic acid-free fluorescent biosensor based on 3D spherical DNA nanostructure and CRISPR/Cas12a (3D-SDNC). The biosensor constructs a rigid 3D nano-framework via self-assembly of six oligonucleotide chains, with H1N1-specific nucleic acid aptamers and Cas12a activator strands strategically positioned at multi-spined vertices for precise spatial coupling between viral recognition and signal transduction. Upon aptamer-virus binding, the induced conformational change liberates the activator strand, thereby activating the trans-cleavage activity of the Cas12a/crRNA complex to efficiently cleave the HEX/BHQ1 double-labeled fluorescent probe and initiate cascade signal amplification. Experimental results demonstrate a detection limit of 0.17 copies/μL (S/N = 3), achieving qPCR-comparable sensitivity, with spike recovery rates of 91.89 % to 104.03 % (RSD < 5.12 %) in chicken serum, bovine serum, and milk matrices. The innovative nucleic acid-free extraction design reduces the total detection time to 40 min; its efficiency is three times higher than qPCR. Notably, we not only discovered the ultra-high sensitivity of the sensor to H1N1 but also unexpectedly found that the rigid structure of the 3D spherical DNA nanostructure conferred enhanced stability under storage conditions. This work establishes a groundbreaking molecular engineering paradigm for rapid pathogen diagnosis, combining ultrahigh sensitivity, fast response, and clinical utility.},
}

*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics
*Biosensing Techniques/methods
*Nanostructures/chemistry
*CRISPR-Cas Systems
Limit of Detection
*DNA/chemistry
Animals
*CRISPR-Associated Proteins/metabolism/chemistry
Humans
Aptamers, Nucleotide/chemistry
Cattle
Milk/virology
Chickens
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40886669,
year = {2026},
author = {Liu, W and Xu, L and Lyu, Y and Yang, C},
title = {MXene-integrated CRISPR/Cas12a biosensor with Split activators for direct and rapid fluorescent detection of MicroRNA.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {346},
number = {},
pages = {126850},
doi = {10.1016/j.saa.2025.126850},
pmid = {40886669},
issn = {1873-3557},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; Limit of Detection ; Spectrometry, Fluorescence/methods ; *Endodeoxyribonucleases/metabolism ; DNA, Single-Stranded/chemistry ; Bacterial Proteins ; Nitrites ; Transition Elements ; CRISPR-Associated Proteins ; },
abstract = {Early and accurate cancer diagnosis is essential for reducing cancer-related mortality, and miRNA-21 has emerged as a critical biomarker for the early detection of various malignancies In this study, we developed a novel fluorescence biosensor, termed the MXene-SNA-Cas12a, that enables direct and amplification-free detection of miRNA-21 by integrating the CRISPR/Cas12a system with a chimeric split nucleic acid (SNA) activator and MXene-assisted fluorescence modulation. Specifically, a split activator comprising S12 ssDNA hybridized with miRNA-21 was employed to activate the trans-cleavage activity of Cas12a, effectively overcoming the system's inherent limitation in RNA recognition. Simultaneously, MXene nanosheets served as efficient quenchers by adsorbing FAM-labeled ssDNA reporters through non-covalent interactions and facilitating target-induced strand release, enabling a robust fluorescence "on/off" mechanism. This biosensor demonstrated excellent linearity over a miRNA-21 concentration range of 50 pM-25 nM, with a detection limit as low as 16 pM. It exhibited high specificity and strong resistance to interference, making it well-suited for complex biological environments. Moreover, the programmable nature of the split activator allows for easy adaptation to detect other RNA targets through rational sequence redesign, offering a versatile platform for CRISPR/Cas12a-based RNA diagnostics.},
}

*MicroRNAs/analysis/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Humans
Limit of Detection
Spectrometry, Fluorescence/methods
*Endodeoxyribonucleases/metabolism
DNA, Single-Stranded/chemistry
Bacterial Proteins
Nitrites
Transition Elements
CRISPR-Associated Proteins

@article {pmid40851311,
year = {2025},
author = {Kittock, CM and Karia, K and Kc, P and Evans, C and Wollman, J and Meyerink, BL and Pilaz, LJ},
title = {Modeling MPPH syndrome in vivo using Breasi-CRISPR.},
journal = {HGG advances},
volume = {6},
number = {4},
pages = {100497},
pmid = {40851311},
issn = {2666-2477},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Gene Editing/methods ; Humans ; *Hydrocephalus/genetics/pathology ; *Megalencephaly/genetics/pathology ; *Polydactyly/genetics/pathology ; Neural Stem Cells/metabolism ; },
abstract = {The increasing availability and affordability of genetic testing has resulted in the identification of numerous novel variants associated with neurodevelopmental disorders. There remains a need for methods to analyze the functional impact of these variants. Some methods, like expressing these variants in cell culture, may be rapid, but they lack physiologic context. Other methods, like making a whole-mouse model, may provide physiologic accuracy, but these are costly and time-consuming. We recently developed a technique, Breasi-CRISPR (Brain Easi-CRISPR), which results in efficient genome editing of neural precursor cells via electroporation of CRISPR-Cas9 reagents into developing mouse brains. Since Breasi-CRISPR is extremely rapid and enables the analysis of targeted genes in vivo, we wondered whether this technique would accelerate the study of monogenic neurodevelopmental disorders. Here, we use Breasi-CRISPR to model megalencephaly postaxial polydactyly polymicrogyria hydrocephalus (MPPH) syndrome. We found that 2 days after Breasi-CRISPR, we were able to see neurodevelopmental phenotypes known to be associated with MPPH syndrome, including increased cyclin D2 protein abundance and an increase in neural progenitor proliferation. Thus, Breasi-CRISPR can efficiently model MPPH syndrome and may be a powerful method to add to the toolbox of those investigating the functional impact of patient variants in neurodevelopmental disorders.},
}

Animals
Mice
*CRISPR-Cas Systems
Disease Models, Animal
*Gene Editing/methods
Humans
*Hydrocephalus/genetics/pathology
*Megalencephaly/genetics/pathology
*Polydactyly/genetics/pathology
Neural Stem Cells/metabolism

@article {pmid40706707,
year = {2025},
author = {Davydova, S and Meccariello, A},
title = {Engineering new clustered regularly interspaced short palindromic repeats-mediated population control for tephritid pests.},
journal = {Current opinion in insect science},
volume = {72},
number = {},
pages = {101415},
doi = {10.1016/j.cois.2025.101415},
pmid = {40706707},
issn = {2214-5753},
mesh = {Animals ; *Tephritidae/genetics ; *Pest Control, Biological/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Male ; Population Control ; },
abstract = {Tephritid fruit flies threaten the agricultural industry with a rising intensity on a worldwide scale. The application of clustered regularly interspaced short palindromic repeats (CRISPR) in insects has resulted in a current boost of CRISPR studies in tephritid pests. One of the primary pathways toward more efficient population management lies in genetic improvements to the Sterile Insect Technique. Herein, we review the pivotal advances in CRISPR application in non-model tephritid fruit flies in recent years. This consists of proof-of-principle studies to optimise CRISPR tools, applications for female elimination and male sterility, and the existing CRISPR-based systems for population control.},
}

Animals
*Tephritidae/genetics
*Pest Control, Biological/methods
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Female
Male
Population Control

@article {pmid40658346,
year = {2025},
author = {Uchigashima, M and Mikuni, T},
title = {Single-cell endogenous protein labeling via CRISPR-Cas9-mediated genome editing in the mouse brain.},
journal = {Anatomical science international},
volume = {100},
number = {4},
pages = {579-590},
pmid = {40658346},
issn = {1447-073X},
support = {20H05914//Japan Society for the Promotion of Science/ ; 20H05918//Japan Society for the Promotion of Science/ ; 23K18160//Japan Society for the Promotion of Science/ ; 24K02130//Japan Society for the Promotion of Science/ ; 22K21353//Japan Society for the Promotion of Science/ ; 23H04672//Japan Society for the Promotion of Science/ ; 23H02574//Japan Society for the Promotion of Science/ ; 23K27265//Japan Society for the Promotion of Science/ ; 24H01229//Japan Society for the Promotion of Science/ ; 24K22000//Japan Society for the Promotion of Science/ ; 25H02490//Japan Society for the Promotion of Science/ ; JP19dm0207080//Japan Agency for Medical Research and Development/ ; JP24wm0625117//Japan Agency for Medical Research and Development/ ; JPMJFR231M//Fusion Oriented REsearch for disruptive Science and Technology/ ; JPMJPR16F9//Precursory Research for Embryonic Science and Technology/ ; CDA00043/2019-C//Human Frontier Science Program/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Brain/metabolism/cytology ; *Single-Cell Analysis/methods ; Electroporation ; Somatosensory Cortex/cytology/metabolism ; Pyramidal Cells/metabolism ; },
abstract = {High-precision mapping of endogenous proteins is essential for understanding the molecular mechanism underlying neuronal functions in the brain. The SLENDR (single-cell labeling of endogenous proteins by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair) technique provides single-cell endogenous protein labeling with genetically encoded tags within the mammalian brain through precise genome editing via homology-directed repair (HDR). This technique is based on the introduction of HDR-mediated genome editing into neuronal progenitors in embryonic brains by in utero electroporation. Subsequent histological analyses enable high-resolution interrogation of the subcellular distribution of endogenous proteins within a single neuron using conventional fluorescent microscopy. Here, we describe a step-by-step protocol for the SLENDR technique to label endogenous proteins with genetically encoded tags in single pyramidal cells of the mouse primary somatosensory cortex. This protocol would be helpful to visualize the molecular organization underlying biological processes at single-neuron levels in the brain, such as signal processing from synaptic inputs to neuronal outputs across different scales.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Mice
*Brain/metabolism/cytology
*Single-Cell Analysis/methods
Electroporation
Somatosensory Cortex/cytology/metabolism
Pyramidal Cells/metabolism

@article {pmid41063348,
year = {2025},
author = {Newman, A and Saha, A and Starrs, L and Arantes, PR and Palermo, G and Burgio, G},
title = {CRISPR-Cas12a REC2-Nuc interactions drive target-strand cleavage and constrain trans cleavage.},
journal = {Nucleic acids research},
volume = {53},
number = {18},
pages = {},
doi = {10.1093/nar/gkaf988},
pmid = {41063348},
issn = {1362-4962},
support = {//National Health and Medical Research Council/ ; //The Gordon and Gretel Bootes/ ; //National Computing Infrastructure/ ; //NCRIS/ ; //Australian Government Research Training Program/ ; R01GM141329/NH/NIH HHS/United States ; CHE-2144823//National Science Foundation/ ; 2138259//National Science Foundation/ ; 2138286//National Science Foundation/ ; 2138307//National Science Foundation/ ; 2137603//National Science Foundation/ ; 2138296//National Science Foundation/ ; FG-2023-20431//Alfred P. Sloan Foundation/ ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA Cleavage ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Escherichia coli/genetics ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Gene Editing ; Molecular Dynamics Simulation ; DNA/metabolism/chemistry/genetics ; *Bacterial Proteins/genetics/metabolism/chemistry ; Kinetics ; DNA, Single-Stranded/metabolism/genetics ; Mutation ; },
abstract = {CRISPR-Cas12a mediates RNA-guided cleavage of double-stranded DNA in cis, after which it remains catalytically active and non-specifically cleaves single-stranded DNA in trans. Native host defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well-characterized Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in Escherichia coli, and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-Nuc interactions. We generated and tested REC2 and Nuc mutants, including a hitherto unstudied 'Nuc-loop', integrity of which is critical for the function of Cas12. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications.},
}

*CRISPR-Cas Systems
*DNA Cleavage
Humans
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Escherichia coli/genetics
*Endodeoxyribonucleases/metabolism/genetics/chemistry
Gene Editing
Molecular Dynamics Simulation
DNA/metabolism/chemistry/genetics
*Bacterial Proteins/genetics/metabolism/chemistry
Kinetics
DNA, Single-Stranded/metabolism/genetics
Mutation

@article {pmid41062879,
year = {2025},
author = {Paul, K and Raman K, V and Baaniya, M and Jadhav, I and Bhattacharjee, S and Tilgam, J and Saakre, M and Kumari, P and Das, S and Vijayan, J and Sreevathsa, R and Pattanayak, D},
title = {A novel recombinant CRISPR/Cas9 vector system for genome editing in plants.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {45},
pmid = {41062879},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genetic Vectors/genetics ; Solanum tuberosum/genetics ; *Plants, Genetically Modified/genetics/growth & development ; Genome, Plant ; Arabidopsis/genetics ; Nicotiana/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; },
abstract = {Genome editing employing CRISPR/Cas9 systems has found widespread applications for knocking out targeted genes. In spite of exponential applications in plants for trait improvement, low editing efficiency in plants is a major concern. We report construction of a pCAMBIA2300 based binary vector cassette (pCR) harbouring novel recombinant CRISPR/Cas9 system for efficient genome editing in plants. The Cas9 cDNA with sequence encoding nuclear localization signals at the N-terminal and C-terminal ends had been codon optimized for better expression in plants. Undesirable internal restriction sites were removed. Small stretch of 5' UTR sequence of Rubisco small subunit (rcbS) of potato, harbouring in between potato granule bound starch synthase (GBSS) intron, was added at the 5' end of the Cas9 cDNA to function as 5' UTR. The recombinant Cas9 gene (rdCas9) was placed under the transcriptional control of CaMV 35S promoter and NOS terminator. The single guide RNA cassette (sgRNA) was comprised of Arabidopsis U6 promoter, 20-21 nucleotide (nt) spacer sequence, sgRNA scaffold sequence and potato U6 RNA Pol-III termination sequence. The 20-21 nt sgRNA spacer sequence could be added to the sgRNA construct by AarI or PaqCI digestion. The sgRNA construct had been designed in such a way so that single or multiplexed sgRNA could be cloned into the pCR vector cassette in a single step. Moreover, modular nature of this vector system can help to derive different combination of promoter, terminator with Cas9 and sgRNA constructs. The efficacy of the pCR vector system had been validated in Nicotiana tabacum and Solanum tuberosum by knocking out phytoene desaturase gene (PDS), through Agrobacterium-mediated transformation. The pCR binary vector system can be utilized as a versatile tool box for efficient genome editing of plant to improve agriculturally important traits.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Genetic Vectors/genetics
Solanum tuberosum/genetics
*Plants, Genetically Modified/genetics/growth & development
Genome, Plant
Arabidopsis/genetics
Nicotiana/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Promoter Regions, Genetic

@article {pmid41062549,
year = {2025},
author = {Sofras, D and Carolus, H and Subotić, A and Lobo Romero, C and Ennis, CL and Cuypers, L and Lagrou, K and Hernday, AD and Nobile, CJ and Rybak, JM and Van Dijck, P},
title = {A systematic comparison of CRISPR-Cas9 allele editing in Candida auris demonstrates unreliable cassette integration and effective episomal plasmid-based editing.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35105},
pmid = {41062549},
issn = {2045-2322},
support = {11J8122N//Fonds Wetenschappelijk Onderzoek/ ; 11D7620N//Fonds Wetenschappelijk Onderzoek/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; PDMT2/23/032//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; R35GM124594/GM/NIGMS NIH HHS/United States ; R35GM124594/GM/NIGMS NIH HHS/United States ; F31DE028488/DE/NIDCR NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Candida auris/genetics ; Alleles ; Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Candidozyma (Candida) auris is an emergent fungal pathogen of significant interest for molecular research. A handful of CRISPR-Cas9 based allele editing tools have been optimized for C. auris. Nonetheless, allele editing in this species remains a significant challenge, and different systems have different advantages and disadvantages. In this work, we compare four systems to introduce the genetic elements necessary for the production of Cas9 and the guide RNA molecule in the genome of C. auris, replacing the ENO1, LEU2 and HIS1 loci respectively, while the fourth system makes use of an episomal plasmid. We observed that the editing efficiency of all four systems was significantly different and strain-dependent. However, we did not detect correct integration of linear CRISPR cassette constructs in integration-based systems, in over 4,900 screened transformants. Still, all transformants, whether correctly edited or not, grew on selective nourseothricin media, suggesting ectopic integration of the CRISPR cassette, which was confirmed by long-read whole genome sequencing. The plasmid-based system showed the highest editing efficiency with an average of 41.9% correct transformants, despite yielding fewer transformants compared to the other systems. Transformation of protoplasts or silencing the non-homologous end joining (NHEJ) DNA repair pathway, by deleting two main NHEJ factors, KU70 and LIG4, did not improve the editing efficiency. While our research highlights important challenges in precise genome editing of C. auris by quantitatively evaluating the editing and targeting efficiencies of different methods, it also clearly shows the safety and usefulness of plasmid-based systems like EPIC, which we recommend for molecular work in this enigmatic fungal pathogen.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Plasmids/genetics
*Candida auris/genetics
Alleles
Genome, Fungal
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41062528,
year = {2025},
author = {Smidler, AL and Marrogi, EA and Scott, S and Mameli, E and Abernathy, D and Akbari, OS and Church, GM and Catteruccia, F and Esvelt, K},
title = {Engineering gene drive docking sites in a haplolethal locus in Anopheles gambiae.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35074},
pmid = {41062528},
issn = {2045-2322},
support = {AI120480-02//Foundation for the National Institutes of Health/ ; R01 AI104956/AI/NIAID NIH HHS/United States ; R01 AI104956/AI/NIAID NIH HHS/United States ; R01AI151004//Foundation for the National Institutes of Health/ ; R01 AI104956/AI/NIAID NIH HHS/United States ; R00-DK102669-04//Foundation for the National Institutes of Health/ ; IRSA1016432//Burroughs Wellcome Fund/ ; IRSA1016432//Burroughs Wellcome Fund/ ; IRSA1016432//Burroughs Wellcome Fund/ ; OPP1158190/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Anopheles/genetics ; Animals ; *Gene Drive Technology/methods ; Proteasome Endopeptidase Complex/genetics ; *Genetic Engineering/methods ; Mosquito Vectors/genetics ; *Genetic Loci ; CRISPR-Cas Systems ; },
abstract = {Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing and functional r1 alleles permits the cell to survive. Here we outline the development and characterization of two ΦC31-Recombination mediated cassette exchange gene drive docking lines with these features in Anopheles gambiae, a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired putative haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants in the process reveal a major lethality and infertility hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for development of future gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.},
}

*Anopheles/genetics
Animals
*Gene Drive Technology/methods
Proteasome Endopeptidase Complex/genetics
*Genetic Engineering/methods
Mosquito Vectors/genetics
*Genetic Loci
CRISPR-Cas Systems

@article {pmid41062469,
year = {2025},
author = {Han, J and Min, Y and Hu, L and Chen, JJ and Zhang, SX and Li, XF and Cheng, ZH and Liu, DF and Yu, HQ},
title = {Tailoring Cas12a functionality with a user-friendly and versatile crRNA variant toolbox.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8939},
pmid = {41062469},
issn = {2041-1723},
support = {52322002//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Mutation ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Escherichia coli/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Cas12a, with its unique targeting and cleavage activity towards DNA, has been widely applied in gene editing and molecular diagnostics. However, there currently lacks an activity regulation strategy that combines flexibility and simplicity to adapt Cas12a to different demands across various application scenarios. In this study, we present a simple yet effective strategy, wherein we systematically mutate the crRNA direct repeat (DR) sequence to uncover a range of distinct crRNA mutants, which are then compiled into a crRNA toolbox to enable flexible regulation of Cas12a activity. By harnessing the complementarity and synergy between these mutants, we successfully enhance Cas12a performance across various application scenarios. Our crRNA toolbox enables fine-tuned control over expression levels, improves base editing accuracy, enhances transformation and editing efficiency in prokaryote homologous recombination-mediated gene editing, and facilitates rapid, accurate, one-pot, semi-quantitative nucleic acid diagnostics. In summary, the DR sequence mutation strategy provides simple, flexible, and diverse options for Cas12a activity regulation and functional optimization.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/genetics/metabolism
Mutation
*Endodeoxyribonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Escherichia coli/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41061057,
year = {2025},
author = {Oonuma, K and Kuroda, R and Uchida, T and Zhang, SM},
title = {CRISPR/Cas9-germline editing of Biomphalaria glabrata: A breakthrough in genetic modification of snails that transmit schistosomiasis.},
journal = {Science advances},
volume = {11},
number = {41},
pages = {eadx5889},
pmid = {41061057},
issn = {2375-2548},
mesh = {Animals ; *Biomphalaria/genetics/parasitology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Schistosoma mansoni ; *Schistosomiasis/transmission/parasitology ; Humans ; },
abstract = {Human schistosomiasis remains one of the most devastating parasitic diseases worldwide, and the development of genetically modified vector snails has long been a goal in the field. Here, we report the successful creation of genetically modified Biomphalaria glabrata, an important intermediate host, using CRISPR/Cas9 gene editing. We targeted the fibrinogen-related protein 3.1 (FREP3.1) gene, confirmed stable germline transmission of the mutated gene, and established two different homozygous FREP3.1-edited lines. Disruption of the FREP 3.1 gene did not alter snail susceptibility to Schistosoma mansoni infection, possibly due to a limited role of FREP3.1 in resistance or to functional redundancy and/or compensatory expression within the highly diverse FREP gene family. Our study demonstrates successful germline editing, effective ex ovo culture of decapsulated embryos, and the generation of viable, genetically modified B. glabrata snails, thereby establishing a foundation for future genetic strategies to control schistosomiasis.},
}

Animals
*Biomphalaria/genetics/parasitology
*CRISPR-Cas Systems
*Gene Editing/methods
Schistosoma mansoni
*Schistosomiasis/transmission/parasitology
Humans

@article {pmid41060691,
year = {2025},
author = {Gao, F and Colles, FM and Ko, S and Luo, J and Sheppard, SK and Chen, M},
title = {Genomic epidemiology and the evolution of erm(B)-mediated macrolide resistance in Campylobacter.},
journal = {Microbial genomics},
volume = {11},
number = {10},
pages = {},
doi = {10.1099/mgen.0.001528},
pmid = {41060691},
issn = {2057-5858},
mesh = {*Campylobacter/genetics/drug effects/isolation & purification/classification ; *Macrolides/pharmacology ; Humans ; *Anti-Bacterial Agents/pharmacology ; Phylogeny ; Animals ; *Campylobacter Infections/epidemiology/microbiology ; *Drug Resistance, Bacterial/genetics ; China/epidemiology ; *Methyltransferases/genetics ; Genome, Bacterial ; Microbial Sensitivity Tests ; *Bacterial Proteins/genetics ; Gene Transfer, Horizontal ; Poultry/microbiology ; Evolution, Molecular ; },
abstract = {Campylobacter is a major foodborne bacterial pathogen that has become increasingly resistant to clinically important antimicrobials. Of particular concern is the emergence of erm(B)-mediated macrolide resistance, which has been increasingly documented across Campylobacter isolates from diverse ecological reservoirs. In this study, we investigated the genomic characteristics and epidemiology of erm(B)-carrying clinical Campylobacter isolates from Shanghai, alongside a globally representative dataset of all publicly available strains. Among clinical isolates obtained from a diarrhoeal outpatient surveillance programme between 2020 and 2023 in Shanghai, China, 16% (80/500) were erythromycin-resistant, with 23.8% (19/80) testing positive for erm(B). The genomes of these isolates were sequenced to identify erm(B) gene alleles. Phylogenetic analyses, pairwise comparisons of core and accessory genomes and examination of shared alleles revealed horizontal gene transfer as the predominant mechanism driving the transmission of erm(B) between isolates from various sources. Poultry was identified as a key reservoir for human infections caused by erm(B)-positive Campylobacter isolates. Comparative pangenome analyses of erm(B)-positive and negative isolates identified multiple accessory elements associated with erm(B) acquisition, among which the IS607 family transposon-associated tnpB gene exhibited sequence and structural homology to functional progenitors of CRISPR-Cas nucleases. These findings expand our understanding of the epidemiology of erm(B)-mediated macrolide resistance in Campylobacter and underscore the urgent need for enhanced antimicrobial stewardship in poultry production and targeted surveillance programmes to curb the spread of resistance.},
}

*Campylobacter/genetics/drug effects/isolation & purification/classification
*Macrolides/pharmacology
Humans
*Anti-Bacterial Agents/pharmacology
Phylogeny
Animals
*Campylobacter Infections/epidemiology/microbiology
*Drug Resistance, Bacterial/genetics
China/epidemiology
*Methyltransferases/genetics
Genome, Bacterial
Microbial Sensitivity Tests
*Bacterial Proteins/genetics
Gene Transfer, Horizontal
Poultry/microbiology
Evolution, Molecular

@article {pmid41042795,
year = {2025},
author = {Shen, Y and Fan, K and Gökbağ, B and Sun, N and Yang, C and Cheng, L and Li, L},
title = {A multi-layer encoder prediction model for individual sample specific gene combination effect (MLEC-iGeneCombo).},
journal = {PLoS computational biology},
volume = {21},
number = {10},
pages = {e1013547},
doi = {10.1371/journal.pcbi.1013547},
pmid = {41042795},
issn = {1553-7358},
mesh = {Humans ; Gene Knockout Techniques ; CRISPR-Cas Systems/genetics ; Computational Biology/methods ; Deep Learning ; *Models, Genetic ; Systems Biology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Using data from gene combination double knockout (CDKO) experiments, top ranked synthetic lethal (SL) gene pairs were highly inconsistent among different SL scores. This leads to a significant concern that SL prediction models highly depend on SL scores. In this paper, we introduce a new gene combination effect (GCE) measurement, log-fold change of dual-gRNA expression before and after CRISPR-cas9 lentivirus transfection. We show it is a direct and highly consistent measurement of GCE in all CDKO experiments. We therefore develop a multi-layer encoder model for individual sample specific GCE prediction, MLEC-iGeneCombo. Under a deep learning framework, MLEC-iGeneCombo is a systems biology model that contains sample specific multi-omics encoder, network encoder and cell-line encoder. For the first time, MLEC-iGeneCombo predicts GCE for a new cell. Using data from 18 CDKO experiments, MLEC-iGeneCombo achieves an average GCE prediction performance, 71.9%. All three encoders significantly improve the model's prediction performance (p[Formula: see text]), and their combined use yields the best GCE prediction performance. Our source code is available at https://github.com/karenyun/MLEC-iGeneCombo.},
}

Humans
Gene Knockout Techniques
CRISPR-Cas Systems/genetics
Computational Biology/methods
Deep Learning
*Models, Genetic
Systems Biology/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40930101,
year = {2025},
author = {Vučićević, D and Hsu, CW and Lopez Zepeda, LS and Burkert, M and Hirsekorn, A and Bilić, I and Kastelić, N and Landthaler, M and Lacadie, SA and Ohler, U},
title = {Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation.},
journal = {Cell genomics},
volume = {5},
number = {10},
pages = {100984},
doi = {10.1016/j.xgen.2025.100984},
pmid = {40930101},
issn = {2666-979X},
mesh = {Humans ; *Single-Cell Analysis/methods ; Transcription Factors/genetics/metabolism ; Neuroblastoma/genetics ; Homeodomain Proteins/genetics ; Gene Regulatory Networks/genetics ; Enhancer Elements, Genetic/genetics ; CRISPR-Cas Systems/genetics ; *Genomics/methods ; Cell Line, Tumor ; },
abstract = {Enhancers are known to spatiotemporally regulate gene transcription, yet the identification of enhancers and their target genes is often indirect, low resolution, and/or assumptive. To identify and functionally perturb enhancers at their endogenous sites, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma, and found many CRISPRa-responsive elements (CaREs) that alter cellular growth. To determine CaRE target genes, we developed TESLA-seq (targeted single-cell activation), which combines CRISPRa screening with targeted single-cell RNA sequencing and enables the parallel readout of the effect of hundreds of enhancers on all genes in the locus. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements, we found many CaREs and target connections normally active only in other tissues. This highlights the power of TESLA-seq to reveal gene regulatory networks, including edges active outside of a given experimental system.},
}

Humans
*Single-Cell Analysis/methods
Transcription Factors/genetics/metabolism
Neuroblastoma/genetics
Homeodomain Proteins/genetics
Gene Regulatory Networks/genetics
Enhancer Elements, Genetic/genetics
CRISPR-Cas Systems/genetics
*Genomics/methods
Cell Line, Tumor

@article {pmid40888415,
year = {2025},
author = {Zhao, T and Yu, L and Yin, M and Huang, S and Tian, R and Zhong, C and Nan, F and Zhang, H and Tian, X and Hu, Z},
title = {Enhanced One-Pot Cas12a-Based Nucleic Acid Detection via Epitope Insertion and Recruitment of Rad51.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {40},
pages = {e02417},
doi = {10.1002/smll.202502417},
pmid = {40888415},
issn = {1613-6829},
support = {32171465//National Natural Science Foundation of China/ ; 32371541//National Natural Science Foundation of China/ ; 82102392//National Natural Science Foundation of China/ ; 82172584//National Natural Science Foundation of China/ ; 2023M734091//China Postdoctoral Science Foundation/ ; 2023M734090//China Postdoctoral Science Foundation/ ; 2023M744121//China Postdoctoral Science Foundation/ ; 2024BCB057//Key Technology R&D Program of Hubei/ ; 0820250//Guangdong Special Support Plan Young Top-notch Talent/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Epitopes/metabolism ; *Rad51 Recombinase/metabolism ; Humans ; *Nucleic Acids/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The CRISPR-Cas12a system has emerged as a promising tool for nucleic acid-based diagnostics. However, its multi-step workflow and limited sensitivity hinder its integration into point-of-care testing (POCT). Here, the ECOT system (Engineered Cas12a for One-pot Test), a novel approach that combines protein engineering with one-pot detection, offering high sensitivity, specificity, and rapid response is introduced. By introducing GCN4 epitope insertions into LtCas12a and LbCas12a variants, their cis-cleavage activity, promoting efficient accumulation of amplification products is reduced. Additionally, the inclusion of scFv-Rad51 (single-chain variable fragment-Rad51) enhances Cas12a's trans-cleavage activity, amplifying signal intensity. The ECOT-Lb system demonstrated superior sensitivity in detecting low-copy HPV DNA samples, outperforming traditional qPCR in clinical tests. Achieving detection limits as low as 3 copies in under 30 min, the ECOT-Lb system is well-suited for home-based self-testing and widespread clinical diagnostics. This work provides a versatile and scalable protein engineering strategy that enhances the performance of CRISPR-based diagnostic tools, offering a promising platform for rapid molecular detection in diverse applications.},
}

*CRISPR-Cas Systems/genetics
*Epitopes/metabolism
*Rad51 Recombinase/metabolism
Humans
*Nucleic Acids/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40811280,
year = {2025},
author = {Chen, H and Jiang, Z},
title = {CrisprDA: A Data Augmentation Method Enhancing the Efficiency of sgRNA on-Target Activity Prediction.},
journal = {IEEE transactions on computational biology and bioinformatics},
volume = {22},
number = {5},
pages = {2313-2319},
doi = {10.1109/TCBBIO.2025.3591871},
pmid = {40811280},
issn = {2998-4165},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Computational Biology/methods ; *Deep Learning ; Neural Networks, Computer ; },
abstract = {The CRISPR/Cas9 system has emerged as a revolutionary technology in genome editing, yet the efficiency of this system is often limited by the activity level of single-guide RNAs (sgRNAs). In recent years, deep learning models have been increasingly utilized to predict sgRNA targeting activity. Notably, data scarcity rather than model architecture has become the predominant bottleneck in accurately predicting sgRNA activity. To overcome this challenge and enhance the performance of deep learning models, we propose Automix, a straightforward yet effective data augmentation method grounded in autoencoder technology. This method is complemented by CNLC (Confidence-based Nearest Label Correction), a pseudo-label correction technique designed to improve both the quality and quantity of training data. Additionally, we develop CrisprDA, a novel parallel architecture that integrates convolutional neural networks (CNNs) with attention mechanisms, for the precise prediction of sgRNA activity. Comprehensive experiments conducted on nine high-throughput datasets and eight functional datasets demonstrate that CrisprDA outperforms five compared methods, showing its superior predictive ability. Moreover, the application of Automix and CNLC to other comparative methods in our experiments further validates the effectiveness and generalizability of the proposed data augmentation strategy.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Humans
*Computational Biology/methods
*Deep Learning
Neural Networks, Computer

@article {pmid41058711,
year = {2025},
author = {Barraclough, A and Bär, I and van Duijl, T and Fijnvandraat, K and Eikenboom, JCJ and Leebeek, FWG and Bierings, R and Voorberg, J and Trasanidou, D},
title = {Rewriting the script: gene therapy and genome editing for von Willebrand Disease.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1620438},
pmid = {41058711},
issn = {2673-3439},
abstract = {In recent years gene therapy has emerged as a powerful technology for treatment of a large variety of inherited disorders. With the FDA approval of in vivo gene therapy of hemophilia A and B using AAV-mediated transgene delivery to hepatocytes, the path towards a new treatment era seemed paved. Also, CRISPR-Cas based approaches have reached the clinic, as in the ex vivo treatment of hematopoietic stem cells for sickle cell disease and thalassemia patients. The question arises whether these innovative strategies will also be suitable for patients with von Willebrand Disease (VWD). Whilst in and ex vivo delivery to endothelial cells (ECs) has been demonstrated, and CRISPR-Cas9 gene editing has been successful in ECs, there are currently no gene therapy options available for VWD. The wide variety of pathogenic VWF mutations makes development of broadly applicable, cost-effective gene therapies challenging. While delivery of von Willebrand factor (VWF) as a therapeutic transgene would be optimal, the size of VWF challenges efficient delivery. Therefore, treatment of VWD requires targeted, personalized gene therapy; for instance by using the newest CRISPR-Cas technologies which can be tailored to facilitate alteration and restoration of various pathogenic VWD variants. This review describes the inherited bleeding disorder VWD and potential gene therapy approaches for management of the disease. Thereby we are exploring different CRISPR-Cas technologies and recent developments in the field. Moreover, we will discuss the ongoing advances of in vivo delivery systems, all with the scope on ECs.},
}

@article {pmid41057395,
year = {2025},
author = {Osgood, JA and Brown, AC and Burnham, KL and Mielczarek, O and Migliorini, G and Tay, C and Zhang, P and Palmer, MH and Davies, B and Cowley, SA and Knight, JC},
title = {Evidence for enhancer activity in intron 1 of TNFRSF1A using CRISPR/Cas9 in human induced pluripotent stem cell-derived macrophages.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {34885},
pmid = {41057395},
issn = {2045-2322},
support = {20773/VAC_/Versus Arthritis/United Kingdom ; 204969/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; 2018-I2M-2-002//Chinese Academy of Medical Sciences Innovation 537 Fund for Medical Science/ ; LC0910-004//Oxford Martin School, University of Oxford/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Macrophages/metabolism/cytology ; *Introns/genetics ; *Enhancer Elements, Genetic ; *Receptors, Tumor Necrosis Factor, Type I/genetics ; Gene Editing ; Cell Differentiation ; Genome-Wide Association Study ; Gene Expression Regulation ; },
abstract = {TNFα is a common drug target in the treatment of autoimmune diseases, with pro-inflammatory functions that are primarily mediated through its receptor, TNFRSF1A. TNFRSF1A has been genetically associated with many immune-mediated diseases including ankylosing spondylitis, multiple sclerosis, and inflammatory bowel disease. Many of the genetic variants within or near TNFRSF1A that have been associated with disease through genome-wide association studies (GWAS) lie in non-coding regions of the genome. Understanding the functional consequences of these genetic variants is limited by incomplete understanding of TNFRSF1A gene regulation, including for specific cellular contexts relevant to inflammation and immunity such as macrophages. This work used CRISPR/Cas9 in human induced pluripotent stem cells followed by differentiation into macrophages to investigate putative regulatory elements in the TNFRSF1A gene locus. Through gene editing, with functional genomic readouts including the assay for transposase-accessible chromatin using sequencing (ATAC-Seq), chromatin immunoprecipitation with sequencing (ChIP-Seq), and RNA-Seq to assess the consequences of these edits, we present evidence for an enhancer of TNFRSF1A contained within an intron of the gene. Understanding gene regulation and the genomic context in which GWAS variants lie could bring us closer to deconvoluting the genetic basis of common disease aetiology and uncover effective drug targets.},
}

Humans
*CRISPR-Cas Systems
*Induced Pluripotent Stem Cells/cytology/metabolism
*Macrophages/metabolism/cytology
*Introns/genetics
*Enhancer Elements, Genetic
*Receptors, Tumor Necrosis Factor, Type I/genetics
Gene Editing
Cell Differentiation
Genome-Wide Association Study
Gene Expression Regulation

@article {pmid41057263,
year = {2025},
author = {Yang, B and Wang, K},
title = {CRISPR-Cas-Directed Genome Editing in Maize.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.top108448},
pmid = {41057263},
issn = {1559-6095},
abstract = {Genetic engineering techniques are essential for both plant science and agricultural biotechnology, enabling functional genomics studies, dissection of complex traits, and targeted crop improvement. Among the various genetic tools currently in use, clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based genome editing has emerged as a transformative technology due to its precision, versatility, and ease of use. In particular, CRISPR-Cas9 has become the most widely adopted platform for genome manipulation in plant systems, including maize, owing to its high editing efficiency, multiplexing capabilities, and scalability for diverse applications. This review highlights the biological significance and technical considerations necessary to implement CRISPR-Cas9 in maize. We discuss critical components for successful editing, including the selection of strong and tissue-appropriate promoters for Cas gene and guide RNA expression, codon optimization of Cas nuclease genes, effective guide RNA design, and multiplexing strategies using RNA polymerase III (Pol III)- or Pol II-dependent promoter-driven polycistronic expression systems. Additionally, we provide insights into vector construction methodologies and reliable genotyping techniques to detect and validate genome edits. Together, these elements constitute a practical framework for deploying genome editing in maize research and breeding. By optimizing these parameters, researchers can enhance the efficiency and accuracy of CRISPR-mediated genome modifications, accelerating functional genomic discovery and the development of improved maize varieties tailored to meet future agricultural demands.},
}

@article {pmid41056201,
year = {2025},
author = {Weber, VJ and Reschigna, A and Gerhardt, MJ and Heigl, T and Hinrichsmeyer, KS and van den Engel, S and Otify, DY and Gavrilov, Z and Blaser, F and Meneau, I and Betz, C and Bolz, HJ and Biel, M and Michalakis, S and Becirovic, E},
title = {CRISPR/Cas-mediated activation of genes associated with inherited retinal dystrophies in human cells for diagnostic purposes.},
journal = {JCI insight},
volume = {},
number = {},
pages = {},
doi = {10.1172/jci.insight.189615},
pmid = {41056201},
issn = {2379-3708},
abstract = {Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of IRD patients and other inherited diseases.},
}

@article {pmid41055650,
year = {2025},
author = {Che, X and Wei, Y and Wang, X and Wang, X and Wu, Z and Deng, J and Ge, S and Liu, X and Cai, Z and Zhang, H and He, L and Xu, J},
title = {Lipoxygenase ZmLOX3 Enhances Salt Tolerance of Maize Under the Regulation of ZmNAC032.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c02349},
pmid = {41055650},
issn = {1520-5118},
abstract = {Lipoxygenase (LOX) plays a critical role in plant biotic and abiotic stress responses by mediating lipid peroxidation and the production of jasmonic acid (JA). In this study, maize ZmLOX3 was identified as a positive regulator in salt stress tolerance. Overexpression of ZmLOX3 enhanced the salt tolerance of Arabidopsis. When maize seedlings were subjected to salt stress, the ZmLOX3[OE] lines exhibited a better growth phenotype than the control (B104) and the zmlox3[CR] (CRISPR/Cas) knockout mutants. Overexpression of ZmLOX3 improved ROS scavenging, Na[+]/K[+] homeostasis, and cell membrane stability. Transcriptome analyses revealed that ZmLOX3[OE] triggered the expression of genes involved in both the JA synthesis and signaling pathways. A transcription factor ZmNAC032 was identified via Y1H screening and was able to bind to the C[A/G]CG[T/G] sequence in the ZmLOX3 promoter and activate its expression. These findings are helpful for deciphering the function and regulatory status of ZmLOX in improving salt tolerance.},
}

@article {pmid41054568,
year = {2025},
author = {Xia, B and Wang, Z and Fei, T and Ma, Y and Guo, Y and Fei, D and Shu, X and Zhao, G and Ma, M and Yuan, H},
title = {Development and application of a CRISPR/Cas12a-based reverse transcription-recombinase polymerase amplification assay with lateral flow dipstick and fluorescence detection for Getah virus.},
journal = {PeerJ},
volume = {13},
number = {},
pages = {e20119},
pmid = {41054568},
issn = {2167-8359},
mesh = {*CRISPR-Cas Systems ; *Alphavirus/isolation & purification/genetics ; Animals ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Fluorescence ; *Alphavirus Infections/diagnosis/virology/veterinary ; Swine ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Getah virus (GETV), a mosquito-borne alphavirus classified as a zoonotic disease, primarily infects livestock, particularly pigs and horses. In recent years, it has re-emerged in multiple Asian countries, posing a potential threat to animal husbandry and public health. In this study, we developed a rapid and sensitive GETV detection method based on reverse transcription-recombinase polymerase amplification (RT-RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system combined with a lateral flow dipstick (LFD) for visual readout. By leveraging sequence conservation in the GETV E2 envelope protein-coding regions, we engineered matched crRNA guides and amplification primers to develop a rapid CRISPR-Cas12a diagnostic workflow. The optimized platform combines RT-RPA (42 °C/20 min) with Cas12a's trans-nuclease activity, permitting multiplex detection via real-time fluorescence quantification or immunochromatographic strip visualization. Analytical evaluation demonstrated a detection capability of 10 copies/µL and exclusive specificity against four pathogen controls, including Japanese encephalitis virus and pseudorabies virus. Validation performed using simulated clinical samples revealed 100% concordance between the results of RT-RPA-CRISPR/Cas12a-LFD and quantitative polymerase chain reaction (PCR), while reducing the total detection time to 50 minutes. This approach eliminated the need for advanced instrumentation owing to its simplified operational design, enabling field-deployable rapid detection capabilities that establish essential technical infrastructure for initiating timely GETV containment measures. This approach has broad application potential in the fields of food safety, clinical diagnostics, and environmental science.},
}

*CRISPR-Cas Systems
*Alphavirus/isolation & purification/genetics
Animals
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Fluorescence
*Alphavirus Infections/diagnosis/virology/veterinary
Swine
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41026092,
year = {2025},
author = {Toyonishi, G and Nakazawa, T and Koshi, D and Horii, M and An, GH and Kawauchi, M and Honda, Y},
title = {CRISPR/Cas9-directed disruption of wc-2 leads to the absence of fruiting body development in Pleurotus ostreatus.},
journal = {FEMS microbiology letters},
volume = {372},
number = {},
pages = {},
doi = {10.1093/femsle/fnaf104},
pmid = {41026092},
issn = {1574-6968},
support = {18KK0178//Japan Society for the Promotion of Science/ ; 22H00380//Japan Society for the Promotion of Science/ ; 22KK0090//Japan Society for the Promotion of Science/ ; PJ0175072025//Rural Development Administration/ ; },
mesh = {*Pleurotus/genetics/growth & development/radiation effects ; *Fruiting Bodies, Fungal/growth & development/genetics/radiation effects ; *CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; Light ; },
abstract = {Light, particularly blue light, is a key environmental factor that induces fruiting in certain agaricomycetes. In this study, we characterized mutant strains of Pleurotus ostreatus with disrupted wc-2, which encodes one of the white-collar proteins, Wc-2, to investigate the role of light in fruiting in P. ostreatus. We introduced two different plasmids containing expression cassettes for Cas9 and two different gRNAs targeting wc-2 separately into the dikaryotic P. ostreatus strain PC9×#64. Among the 11 dikaryotic hygromycin-resistant transformants, six strains did not form fruiting bodies. Genomic PCR followed by sequencing analysis suggested that all six fruitless strains were dikaryotic wc-2 disruptants. Small aggregate structures were not observed in the dikaryotic wc-2 disruptants grown under light conditions, as in PC9×#64 grown in a red box. These results suggest that Wc-2 is essential for the initiation of blue light-induced fruiting in P. ostreatus.},
}

*Pleurotus/genetics/growth & development/radiation effects
*Fruiting Bodies, Fungal/growth & development/genetics/radiation effects
*CRISPR-Cas Systems
*Fungal Proteins/genetics/metabolism
Light

@article {pmid41002250,
year = {2025},
author = {Gillmore, JD and Gane, E and Täubel, J and Pilebro, B and Echaniz-Laguna, A and Kao, J and Litchy, W and Shahda, S and Haagensen, A and Walsh, L and Smith, D and Kachadourian, J and Ward, JH and Lebwohl, D and Zhu, P and Xu, Y and Leung, A and Sonderfan, A and Gutstein, DE and Manvelian, G and Adams, D},
title = {Nexiguran Ziclumeran Gene Editing in Hereditary ATTR with Polyneuropathy.},
journal = {The New England journal of medicine},
volume = {393},
number = {14},
pages = {1375-1386},
doi = {10.1056/NEJMoa2510209},
pmid = {41002250},
issn = {1533-4406},
mesh = {Humans ; *Amyloid Neuropathies, Familial/therapy/genetics ; Male ; Female ; Middle Aged ; *Prealbumin/genetics/metabolism/antagonists & inhibitors ; Adult ; Aged ; *Gene Editing ; *Genetic Therapy/adverse effects/methods ; CRISPR-Cas Systems ; Neurofilament Proteins/blood ; Body Mass Index ; },
abstract = {BACKGROUND: Hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN) is a rare, multisystem, progressive, debilitating, and fatal disease characterized by tissue deposition of misfolded transthyretin (TTR) in peripheral nerves. Nexiguran ziclumeran (nex-z) is an investigational in vivo therapy based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease) that is designed to reduce serum TTR levels through selective inactivation of TTR in the liver.

METHODS: In this phase 1, open-label study, we administered one infusion of nex-z to patients with ATTRv-PN. Primary objectives included assessment of the safety and pharmacodynamics of nex-z. Secondary end points included changes in the familial amyloid polyneuropathy stage, polyneuropathy disability score, serum neurofilament light chain (NfL) level, modified body-mass index (modified BMI, defined as the conventional BMI [weight in kilograms divided by square of height in meters] multiplied by the albumin level in grams per liter), and modified Neuropathy Impairment Score+7 (mNIS+7; range, 0 to 304, with higher scores indicating more impairment).

RESULTS: A total of 36 patients received nex-z; the mean follow-up was 27 months. The mean percent change from baseline in the serum TTR level was -90% at day 28, which was sustained through month 24 (-92%). Treatment-related adverse events included transient infusion-related reactions (in 21 patients), decreased thyroxine level without hypothyroidism or elevated thyrotropin level (in 8), and headache (in 4). One participant died from cardiac amyloidosis, and one withdrew owing to progressive decline in motor function. Serious adverse events were reported in 11 patients. At month 24, the familial amyloid polyneuropathy stage and polyneuropathy disability score remained stable in 29 and 27 patients, respectively; improved in 2 and 5, respectively; and worsened in 2 and 2, respectively. The mean change in the serum NfL level was -9.0 pg per milliliter, and the change in the modified BMI was 24.7. The mean change from baseline in the mNIS+7 was -8.5.

CONCLUSIONS: A single administration of nex-z in patients with ATTRv-PN was associated with rapid, deep, and durable reductions in serum TTR levels. The results support further investigation of nex-z to treat ATTRv-PN. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).},
}

Humans
*Amyloid Neuropathies, Familial/therapy/genetics
Male
Female
Middle Aged
*Prealbumin/genetics/metabolism/antagonists & inhibitors
Adult
Aged
*Gene Editing
*Genetic Therapy/adverse effects/methods
CRISPR-Cas Systems
Neurofilament Proteins/blood
Body Mass Index

@article {pmid40990297,
year = {2025},
author = {Sheng, J and Dong, Y and Sun, S and Zhang, Y and Li, C and Xu, X and Wang, H},
title = {Construction of a Sensing Platform Integrated with a CRISPR/Cas12a-Triggered Colorimetric Strategy for the Quantitative Detection of Meat Freshness.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {40},
pages = {25604-25614},
doi = {10.1021/acs.jafc.5c04851},
pmid = {40990297},
issn = {1520-5118},
mesh = {*Colorimetry/methods/instrumentation ; *Meat/analysis/microbiology ; CRISPR-Cas Systems ; Animals ; *Pseudomonas/genetics/isolation & purification/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Biosensing Techniques/instrumentation/methods ; *Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Associated Proteins ; },
abstract = {Monitoring microbial determinants, such as Pseudomonas spp., is thus essential for assessing meat freshness. Here, a novel colorimetric sensing platform based on magnetic enzyme-labeled nanoparticles combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a without nucleic acid molecule preamplification was developed for detecting meat freshness. Under optimal conditions, a high-specificity crRNA was systematically verified, and the colorimetric sensor could accurately quantify Pseudomonas spp. loads with levels ranging from 1 × 10[3.7] to 1 × 10[8.7] CFU/mL, with a color change from colorless to yellow. A smart colorimetric platform, including a self-designed image acquisition device and self-programmed image analysis software, was developed and applied to the integrated determination of meat freshness by using the B-value in the RGB channel. The platform has been applied to both consumers and producers and has been validated by 48 actual samples of chilled meat. These findings provide new insights into the exploration of reliable tools for monitoring meat freshness.},
}

*Colorimetry/methods/instrumentation
*Meat/analysis/microbiology
CRISPR-Cas Systems
Animals
*Pseudomonas/genetics/isolation & purification/metabolism
*Bacterial Proteins/genetics/metabolism
*Biosensing Techniques/instrumentation/methods
*Endodeoxyribonucleases/genetics/metabolism
CRISPR-Associated Proteins

@article {pmid40985907,
year = {2025},
author = {Fei, S and Zhang, C and Zhang, X and Xie, Y and Fu, S and Wu, J},
title = {An Ultrasensitive Immunocapture (IC)-RPA-CRISPR/Cas12a Assay with Three Readout Modes for Detecting Xanthomonas oryzae pv. oryzicola of Rice Bacterial Leaf Streak.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {40},
pages = {25664-25675},
doi = {10.1021/acs.jafc.5c04360},
pmid = {40985907},
issn = {1520-5118},
mesh = {*Oryza/microbiology ; *Xanthomonas/genetics/isolation & purification ; *Plant Diseases/microbiology ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Plant Leaves/microbiology ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Xanthomonas oryzae pv oryzicola (Xoc) is the causal agent of rice bacterial leaf streak (BLS) and causes enormous losses of rice yields in many countries every year. Development of sensitive diagnostic techniques is crucial for its prevention and control. Here, we developed an ultrasensitive IC-RPA-CRISPR/Cas12a assay with three readout modes [qPCR machine, UV lamp, and lateral flow strip (LFS)] for Xoc detection in rice, which combined advantages of immunocapture, recombinase polymerase amplification (RPA), and CRISPR/Cas12a-based cleavage. Especially, the immunocapture step allows to capture and enrich Xoc from samples, which minimizes the interference from rice debris to benefit nucleic acid release and amplification and enhances the specificity and sensitivity of this assay. The detection limits of its three readout modes for Xoc bacterial suspension is 2, 6, and 60 CFU/mL, respectively. Collectively, this study provides a specific, ultrasensitive, practical approach for quarantine and detection of Xoc that will benefit the prevention and control of BLS.},
}

*Oryza/microbiology
*Xanthomonas/genetics/isolation & purification
*Plant Diseases/microbiology
CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods/instrumentation
Plant Leaves/microbiology
Bacterial Proteins/genetics/metabolism

@article {pmid40970715,
year = {2025},
author = {Zhao, C and Li, G and Shen, C and Xie, Y and Chen, Y and Ying, X and Chen, Y and Zhang, C},
title = {An extraction-free and one-pot two-temperature CRISPR/Cas12b system for visual detection of Group B Streptococcus by integrating with RPA.},
journal = {Journal of clinical microbiology},
volume = {63},
number = {10},
pages = {e0081925},
doi = {10.1128/jcm.00819-25},
pmid = {40970715},
issn = {1098-660X},
support = {2024KY1444//Medical and Health Research Project of Zhejiang Province/ ; LTGC23H200004//Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {*Streptococcus agalactiae/isolation & purification/genetics ; Humans ; *Streptococcal Infections/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Female ; Recombinases/metabolism ; Temperature ; Vagina/microbiology ; },
abstract = {UNLABELLED: Group B Streptococcus (GBS) is a major cause of neonatal infections, and rapid detection is essential for timely clinical intervention. In this study, we developed an extraction-free, one-pot CRISPR/Cas12b assay for visual detection of GBS by combining with isothermal amplification, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). The results showed that LAMP-CRISPR/Cas12b outperformed RPA-CRISPR/Cas12b system across all template concentrations, especially in low-copy template (30 and 10 copies/test) detection. To enhance the detection performance of RPA-CRISPR/Cas12b, we introduced a two-temperature protocol, with RPA reaction at 39°C followed by Cas12b activation at 62°C. Through the two-temperature approach, the detection rate of RPA-CRISPR/Cas12b system was significantly improved even in low-copy samples, achieving a sensitivity of 10 copies/test (1 copy/μL). Clinical validation using 60 vaginal-rectal swab samples showed 96.7% and 98.3% of concordance when compared to culture and qPCR methods, respectively. This assay offers a rapid (<1 h), highly sensitive, and user-friendly solution without requiring nucleic acid extraction or sophisticated instruments. Its compatibility with visual signal detection makes it ideal for point-of-care testing, especially in low-resource or time-sensitive settings. The platform can be adapted for broader pathogen detection in future field diagnostics.

IMPORTANCE: This study presents a rapid, convenient, and highly accurate method for Group B Streptococcus (GBS) detection by integrating the CRISPR/Cas12b system with recombinase polymerase amplification, an isothermal nucleic acid amplification technique. To streamline the workflow, we established a one-pot, extraction-free assay that significantly reduces the detection time. Through the systematic optimization of the dual-temperature conditions, we enhanced the amplification efficiency of target DNA, thereby improving the sensitivity of the CRISPR/Cas12b system. Additionally, the incorporation of a UV-visible detection system enables visual readout, facilitating instrument-free testing suitable for point-of-care (POC) applications.},
}

*Streptococcus agalactiae/isolation & purification/genetics
Humans
*Streptococcal Infections/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Female
Recombinases/metabolism
Temperature
Vagina/microbiology

@article {pmid40899880,
year = {2025},
author = {Liu, P and Zhang, J and Gong, Y and Liu, W and Xiao, G and Liang, J and Wang, X and Bi, J and Zhang, G},
title = {Application of engineered CRISPR/Cas12a variants with altered protospacer adjacent motif specificities for the detection of isoniazid resistance mutations in Mycobacterium tuberculosis.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0016525},
pmid = {40899880},
issn = {2165-0497},
mesh = {*Isoniazid/pharmacology ; *Mycobacterium tuberculosis/genetics/drug effects/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Antitubercular Agents/pharmacology ; Bacterial Proteins/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; Mutation ; Tuberculosis, Multidrug-Resistant/microbiology/diagnosis ; *Endodeoxyribonucleases/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Catalase/genetics ; Sensitivity and Specificity ; CRISPR-Associated Proteins/genetics ; Microbial Sensitivity Tests ; },
abstract = {UNLABELLED: Drug-resistant tuberculosis (TB) is a major global public health concern. Although isoniazid is currently considered one of the most effective first-line drugs for TB treatment, its efficacy is limited by the emergence of resistance. Therefore, it is imperative to develop new methods for detecting drug-resistant TB. In this study, we developed a nucleic acid detection system based on the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a_RR protein. The system combines recombinase polymerase amplification with an engineered CRISPR/Cas12a_RR protein to enable rapid and specific detection of the katG G944C mutation in isoniazid-resistant Mycobacterium tuberculosis (Mtb). It could detect the target DNA at concentrations as low as 1% in a mixed sample. Compared with TaqMan quantitative polymerase chain reaction and DNA sequencing, the CRISPR/Cas12a_RR system demonstrated superior detection performance in terms of sensitivity, specificity, and cost-effectiveness. Furthermore, it effectively differentiated between drug-resistant Mtb strains from wild-type Mtb strains in clinically isolated samples, with the entire detection process completed in 60 min. In conclusion, the CRISPR/Cas12a_RR detection system offers a novel, rapid, simple, sensitive, and specific approach for identifying isoniazid-resistant Mtb, with significant potential for clinical application, particularly in resource-limited settings.

IMPORTANCE: This study presents a novel method for detecting isoniazid-resistant Mycobacterium tuberculosis (Mtb) using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a mutants, offering rapid detection, cost-effectiveness, and high specificity, and thereby providing a promising new avenue for detecting isoniazid-resistant Mtb.},
}

*Isoniazid/pharmacology
*Mycobacterium tuberculosis/genetics/drug effects/isolation & purification
*CRISPR-Cas Systems/genetics
*Antitubercular Agents/pharmacology
Bacterial Proteins/genetics
Humans
*Drug Resistance, Bacterial/genetics
Mutation
Tuberculosis, Multidrug-Resistant/microbiology/diagnosis
*Endodeoxyribonucleases/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Catalase/genetics
Sensitivity and Specificity
CRISPR-Associated Proteins/genetics
Microbial Sensitivity Tests

@article {pmid40866699,
year = {2025},
author = {Pacesa, M and Nickel, L and Schellhaas, C and Schmidt, J and Pyatova, E and Kissling, L and Barendse, P and Choudhury, J and Kapoor, S and Alcaraz-Serna, A and Cho, Y and Ghamary, KH and Vinué, L and Yachnin, BJ and Wollacott, AM and Buckley, S and Westphal, AH and Lindhoud, S and Georgeon, S and Goverde, CA and Hatzopoulos, GN and Gönczy, P and Muller, YD and Schwank, G and Swarts, DC and Vecchio, AJ and Schneider, BL and Ovchinnikov, S and Correia, BE},
title = {One-shot design of functional protein binders with BindCraft.},
journal = {Nature},
volume = {646},
number = {8084},
pages = {483-492},
pmid = {40866699},
issn = {1476-4687},
support = {R35 GM138368/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Gene Editing ; Protein Binding ; CRISPR-Cas Systems/genetics ; Allergens/immunology/metabolism/chemistry ; Immunoglobulin E/immunology/metabolism ; *Protein Engineering/methods ; *Proteins/metabolism/chemistry ; Receptors, Cell Surface/metabolism/chemistry ; Binding Sites ; Models, Molecular ; CRISPR-Associated Protein 9/metabolism ; Animals ; },
abstract = {Protein-protein interactions are at the core of all key biological processes. However, the complexity of the structural features that determine protein-protein interactions makes their design challenging. Here we present BindCraft, an open-source and automated pipeline for de novo protein binder design with experimental success rates of 10-100%. BindCraft leverages the weights of AlphaFold2 (ref. [1]) to generate binders with nanomolar affinity without the need for high-throughput screening or experimental optimization, even in the absence of known binding sites. We successfully designed binders against a diverse set of challenging targets, including cell-surface receptors, common allergens, de novo designed proteins and multi-domain nucleases, such as CRISPR-Cas9. We showcase the functional and therapeutic potential of designed binders by reducing IgE binding to birch allergen in patient-derived samples, modulating Cas9 gene editing activity and reducing the cytotoxicity of a foodborne bacterial enterotoxin. Last, we use cell-surface-receptor-specific binders to redirect adeno-associated virus capsids for targeted gene delivery. This work represents a significant advancement towards a 'one design-one binder' approach in computational design, with immense potential in therapeutics, diagnostics and biotechnology.},
}

Humans
Gene Editing
Protein Binding
CRISPR-Cas Systems/genetics
Allergens/immunology/metabolism/chemistry
Immunoglobulin E/immunology/metabolism
*Protein Engineering/methods
*Proteins/metabolism/chemistry
Receptors, Cell Surface/metabolism/chemistry
Binding Sites
Models, Molecular
CRISPR-Associated Protein 9/metabolism
Animals

@article {pmid40862592,
year = {2025},
author = {Shen, Y and Yi, C and Wang, H and Tang, Y and Li, J},
title = {Development of a rapid and sensitive RPA-CRISPR/Cas12a-based assay for the detection of Brucella melitensis.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0099825},
pmid = {40862592},
issn = {2165-0497},
mesh = {*Brucellosis/diagnosis/microbiology ; *Brucella melitensis/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/genetics/metabolism ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; Reproducibility of Results ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Brucellosis, a zoonotic disease caused by Brucella species, presents significant public health challenges due to its complex diagnosis and the limited availability of rapid detection methods. To address these challenges, we developed a novel detection method that integrates recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system, enabling dual readout through fluorescence (FL) and lateral flow strip (LFS) detection. The RPA-CRISPR/Cas12a-FL assay demonstrated an impressive detection limit of 1 copy/μL, which is 10 times more sensitive than quantitative polymerase chain reaction, while the RPA-CRISPR/Cas12a-LFS method achieved a detection limit of 10 copies/μL, comparable to nested PCR. Specificity testing confirmed the robustness of the assay, as it produced strong signals exclusively for Brucella without cross-reactivity with other bacterial species. Clinical validation using serum samples from 24 confirmed brucellosis patients and six healthy controls demonstrated a 100% concordance with serological results, underscoring the reliability of this method for clinical applications. This assay provides a rapid, sensitive, and specific tool for Brucella detection, suitable for both laboratory and field settings, and holds significant potential for enhancing the diagnosis and control of brucellosis.IMPORTANCEBrucellosis is a significant zoonotic disease, and rapid and accurate diagnosis is crucial for its treatment and control. To address this need, we developed a novel detection method that combines recombinant enzyme polymerase amplification with a CRISPR/Cas12a system, achieving dual readout through fluorescence and lateral flow strips. The test demonstrates excellent sensitivity and specificity, with clinical validation confirming complete concordance with serological results. This approach offers a fast, reliable, and field-deployable solution for brucellosis diagnosis, significantly enhancing disease management and public health outcomes.},
}

*Brucellosis/diagnosis/microbiology
*Brucella melitensis/genetics/isolation & purification
Humans
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Recombinases/genetics/metabolism
*Molecular Diagnostic Techniques/methods
Limit of Detection
Reproducibility of Results
Bacterial Proteins/genetics
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40856920,
year = {2025},
author = {Thiel, J and Sürün, D and Brändle, DC and Teichert, M and Künzel, SR and Friedrich, U and Dahl, A and Schubert, K and Rzagalinski, I and Shevchenko, A and Traikov, S and Mirtschink, P and Wagenführ, L and Buchholz, F and Hölig, K and Tonn, T and Kronstein-Wiedemann, R},
title = {Knock Out of miRNA-30a-5p and Reconstitution of the Actin Network Dynamics Partly Restores the Impaired Terminal Erythroid Differentiation during Blood Pharming.},
journal = {Stem cell reviews and reports},
volume = {21},
number = {8},
pages = {2637-2653},
pmid = {40856920},
issn = {2629-3277},
support = {530364326//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Cell Differentiation/genetics ; *Actins/metabolism ; *Erythroid Cells/metabolism/cytology ; *Erythropoiesis/genetics ; Proto-Oncogene Mas ; Hematopoietic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Erythrocytes/metabolism/cytology ; bcl-X Protein/metabolism/genetics ; Gene Knockout Techniques ; Cell Line ; },
abstract = {In vitro red blood cell (RBC) production offers a promising complement to conventional blood donation, particularly for patients with rare blood types. Previously, we developed imBMEP-A, the first erythroid cell line derived from reticulocyte progenitors, which maintains robust hemoglobin expression and erythroid differentiation in the presence of erythropoietin (EPO) despite its immortalized state. However, clinical translation remains hindered by the inability to scale up production due to impaired in vitro enucleation of RBC progenitor cell lines. Enhancing enucleation efficiency in imBMEP-A cells involved CRISPR/Cas9-mediated knockout (K.O.) of miR-30a-5p, a key enucleation inhibitor, moderately increasing rates to 3.3 ± 0.4%- 8.9 ± 1.7%. Further investigation of enucleation inefficiencies led to transcriptome and proteome comparisons between imBMEP-miR30a-K.O. cells and hematopoietic stem cells (HSCs). These analyses revealed altered gene expression and protein abundances linked to metabolic transitions, apoptosis promotion, and cytoskeletal regulation. Notably, forced expression of the proto-oncogene c-Myc, required for cell immortalization, emerged as a key driver of these physiological changes. Counteracting these effects required optimization of imBMEP-A cells by activating BCL-XL transcription and knocking out SCIN, which encodes the actin-severing protein scinderin. While BCL-XL is upregulated in normal erythropoiesis, it is downregulated in imBMEP-A. Conversely, SCIN, typically absent in erythroid cells, is highly expressed in imBMEP-A, disrupting actin organization. These interventions improved viability, restored actin network formation, and increased terminal erythropoiesis, yielding 22.1 ± 1.7% more orthochromatic erythroblasts. These findings establish a foundation for optimizing imBMEP-A cells for therapeutic use and advancing the understanding the pathophysiology of erythroleukemia.},
}

*MicroRNAs/genetics/metabolism
Humans
*Cell Differentiation/genetics
*Actins/metabolism
*Erythroid Cells/metabolism/cytology
*Erythropoiesis/genetics
Proto-Oncogene Mas
Hematopoietic Stem Cells/metabolism/cytology
CRISPR-Cas Systems
Erythrocytes/metabolism/cytology
bcl-X Protein/metabolism/genetics
Gene Knockout Techniques
Cell Line

@article {pmid40833103,
year = {2025},
author = {Omachi, R and Imai, K and Sato, A and Tanaka, M and Mizushina, H and Takeuchi, K and Maeda, T},
title = {Development and clinical evaluation of a novel SHERLOCK test for Mycoplasma genitalium.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0044525},
pmid = {40833103},
issn = {2165-0497},
support = {24K10540//KAKEN/ ; 2024 Research grant//Charitable Trust Laboratory Medicine Research Foundation of Japan/ ; Grant-in-Aid for Young Researchers//Saitama Medical University Hospital/ ; },
mesh = {*Mycoplasma genitalium/genetics/isolation & purification ; Humans ; *Mycoplasma Infections/diagnosis/microbiology/urine ; Male ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Urethritis/microbiology/diagnosis ; *Molecular Diagnostic Techniques/methods ; Japan ; Point-of-Care Testing ; DNA, Bacterial/genetics ; CRISPR-Cas Systems ; },
abstract = {Mycoplasma genitalium (MG) is a sexually transmitted pathogen associated with urethritis. Nucleic acid amplification tests are the gold standard for its diagnosis but often require specialized equipment, which limits their use in point-of-care testing. This study aimed to develop a rapid, sensitive detection method for MG using a specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) test, which combines isothermal recombinase polymerase amplification and a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a reaction. We developed a novel SHERLOCK test targeting the Mg219 gene in MG. The SHERLOCK method was evaluated using 128 first-void urine samples collected from male patients who were suspected of MG urethritis in Japan. The results of SHERLOCK were compared to those of the cobas TV/MG test and in-house quantitative PCR. SHERLOCK was optimized for use with crude DNA extracted from clinical urine samples. The results were detected via a lateral flow assay, allowing for visual interpretation within 60 min. The method demonstrated a limit of detection of 10 copies/reaction and showed no cross-reactivity with other pathogens. In clinical evaluations of 128 urine samples, SHERLOCK showed an overall agreement rate of 91.4% with the cobas TV/MG PCR test; the positive and negative agreement rates were 79.6 and 100%, respectively. SHERLOCK showed superior performance to quantitative PCR. This study demonstrates that the novel SHERLOCK assay for MG has potential as a point-of-care test in the clinical setting. Further evaluation in prospective studies is needed to confirm its clinical value.IMPORTANCEMycoplasma genitalium (MG) is a causative agent of sexually transmitted infections and is associated with urethritis and prostatitis in men. To prevent the transmission of MG, it is essential to identify infected individuals through diagnostic testing and provide appropriate treatment. Nucleic acid amplification tests are commonly used for MG diagnosis in the clinical setting, but the point-of-care testing (POCT) for MG remains limited. In this study, we developed a novel nucleic acid amplification test-specific high-sensitivity enzymatic reporter unlocking (SHERLOCK)-for MG, combining crude DNA extraction with a lateral flow assay. Our SHERLOCK assay successfully detected MG in approximately 1 h, with a detection limit of 10 copies/reaction. Clinical evaluations using urine samples showed a high agreement rate with the cobas TV/MG test. SHERLOCK is expected to be a useful tool for POCT for MG.},
}

*Mycoplasma genitalium/genetics/isolation & purification
Humans
*Mycoplasma Infections/diagnosis/microbiology/urine
Male
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Urethritis/microbiology/diagnosis
*Molecular Diagnostic Techniques/methods
Japan
Point-of-Care Testing
DNA, Bacterial/genetics
CRISPR-Cas Systems

@article {pmid40819286,
year = {2025},
author = {Beck, CW and Reily-Bell, M and Bicknell, LS},
title = {Unilateral loss of recql4 function in Xenopus laevis tadpoles leads to ipsilateral ablation of the forelimb, hypoplastic Meckel's cartilage, and vascular defects.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {10},
pages = {},
doi = {10.1093/g3journal/jkaf179},
pmid = {40819286},
issn = {2160-1836},
support = {PRG1732//Neurological Foundation of New Zealand/ ; },
mesh = {Animals ; *Xenopus laevis/genetics ; Larva/genetics ; *RecQ Helicases/genetics/metabolism ; *Forelimb/abnormalities ; *Cartilage/abnormalities/metabolism ; *Xenopus Proteins/genetics/metabolism ; Gene Knockdown Techniques ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {RECQL4 encodes a RecQ helicase, one of a family of DNA unwinding enzymes with roles in DNA replication, double-strand break repair, and genomic stability. Pathogenic variants in RECQL4 are clinically associated with 3 rare autosomal recessive conditions: Rothmund-Thomson syndrome type II, Baller-Gerold syndrome, and RAPADILINO syndrome. These 3 syndromes show overlapping growth retardation, low bone density, and skeletal defects affecting the arms and hands. Here, we take advantage of the ability to generate one-sided CRISPR knockdowns of recql4 in Xenopus laevis tadpoles. Tadpoles develop normally until feeding starts, after which growth slows on the edited side, leading to a curved posture, smaller eyes (microphthalmia), and reduced head size (microcephaly). Forelimb buds fail to develop, leading to complete absence of the forelimb on the edited side. Additionally, Meckel's cartilage (lower jaw) ossification is absent or reduced and the hyoid cartilage is smaller, but this is not due to deficiencies in cranial neural crest migration on the edited side. Knockdown of recql4 also results in hypoplastic vasculature, with reduced branching from the aorta on the edited side. Taken together, our results clearly show the utility of unilateral CRISPR editing in Xenopus for understanding the specific phenotypic developmental effects of mutations affecting cell proliferation.},
}

Animals
*Xenopus laevis/genetics
Larva/genetics
*RecQ Helicases/genetics/metabolism
*Forelimb/abnormalities
*Cartilage/abnormalities/metabolism
*Xenopus Proteins/genetics/metabolism
Gene Knockdown Techniques
Phenotype
CRISPR-Cas Systems

@article {pmid40794772,
year = {2025},
author = {Després, PC and Gervais, NC and Fogal, M and Rogers, RKJ and Cuomo, CA and Shapiro, RS},
title = {Targeted loss of heterozygosity in Candida albicans using CRISPR-Cas9 reveals the functional impact of allelic variation.},
journal = {Genetics},
volume = {231},
number = {2},
pages = {},
doi = {10.1093/genetics/iyaf154},
pmid = {40794772},
issn = {1943-2631},
support = {//FRQS/ ; //NSERC/ ; //N.C.G./ ; //M.F./ ; PJT 162195//CIHR/ ; //R.S.S./ ; //CIFAR/ ; //Fungal Kingdom/ ; //C.A.C./ ; U19 AI110818/AI/NIAID NIH HHS/United States ; //Canada Research Chair/ ; },
mesh = {*Candida albicans/genetics/drug effects ; *Loss of Heterozygosity ; *CRISPR-Cas Systems ; Alleles ; Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; Drug Resistance, Fungal/genetics ; Gene Editing ; Genetic Variation ; Antifungal Agents/pharmacology ; },
abstract = {The diploid genome of the fungal pathogen Candida albicans is highly heterozygous, with most allele pairs diverging at either the coding or regulatory level. When faced with selection pressure like antifungal exposure, this hidden genetic diversity can provide a reservoir of adaptive mutations through loss of heterozygosity (LOH) events. Validating the potential phenotypic impact of LOH events observed in clinical or experimentally evolved strains can be difficult due to the challenge of precisely targeting one allele over the other. Here, we show that a CRISPR-Cas9 system can be used to overcome this challenge. By designing allele-specific guide RNA sequences, we can induce targeted, directed LOH events, which we validate by whole-genome long-read sequencing. Using this approach, we efficiently recapitulate a recently described LOH event that increases resistance to the antifungal fluconazole. Additionally, we find that the recombination tracts of these induced LOH events have similar lengths to those observed naturally. To facilitate future use of this method, we provide a database of allele-specific sgRNA sequences for Cas9 that provide near genome-wide coverage of heterozygous sites through either direct or indirect targeting. This approach will be useful in probing the adaptive role of LOH events in this important human pathogen.},
}

*Candida albicans/genetics/drug effects
*Loss of Heterozygosity
*CRISPR-Cas Systems
Alleles
Genome, Fungal
RNA, Guide, CRISPR-Cas Systems/genetics
Drug Resistance, Fungal/genetics
Gene Editing
Genetic Variation
Antifungal Agents/pharmacology

@article {pmid40758833,
year = {2025},
author = {Stuecker, TN and Hood, SE and Molina Pineda, J and Lenaduwe, S and Winter, J and Sadhu, MJ and Lewis, JA},
title = {Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {10},
pages = {},
doi = {10.1093/g3journal/jkaf175},
pmid = {40758833},
issn = {2160-1836},
support = {P20 GM103429/GM/NIGMS NIH HHS/United States ; MCB-1941824//National Science Foundation/ ; /HG/NHGRI NIH HHS/United States ; 1ZIAHG200401/GF/NIH HHS/United States ; //Arkansas IDeA Network of Biomedical Research Excellence/ ; //Summer Research Fellowship/ ; P20 GM103429/GM/NIGMS NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Vectors/genetics ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Fungal ; Escherichia coli/genetics ; },
abstract = {In vivo site-directed mutagenesis is a powerful genetic tool for testing the effects of specific alleles in their normal genomic context. While the budding yeast Saccharomyces cerevisiae possesses classical tools for site-directed mutagenesis, more efficient recent CRISPR-based approaches use Cas "cutting" combined with homologous recombination of a "repair" template that introduces the desired edit. However, current approaches are limited for fully prototrophic yeast strains and rely on relatively low-efficiency cloning of short gRNAs. We were thus motivated to simplify the process by combining the gRNA and its cognate repair template in cis on a single oligonucleotide. Moreover, we wished to take advantage of a new approach that uses an Escherichia coli retron (EcRT) to amplify repair templates as multi-copy single-stranded (ms)DNA in vivo, which are more efficient templates for homologous recombination. To this end, we have created a set of plasmids that express Cas9-EcRT, allowing for co-transformation with the gRNA-repair template plasmid in a single step. Our suite of plasmids contains different antibiotic (Nat, Hyg, Kan) or auxotrophic (HIS3, URA3) selectable markers, allowing for editing of fully prototrophic wild yeast strains. In addition to classic galactose induction, we generated a β-estradiol-inducible version of each plasmid to facilitate editing in yeast strains that grow poorly on galactose. The plasmid-based system results in >95% editing efficiencies for point mutations and >50% efficiencies for markerless deletions, in a minimum number of steps and time. We provide a detailed step-by-step guide on how to use this system.},
}

*Saccharomyces cerevisiae/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Vectors/genetics
Plasmids/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Genome, Fungal
Escherichia coli/genetics

@article {pmid40495695,
year = {2025},
author = {Wang, H and Zhan, H and Pan, B and Zeng, L and Chen, Z and Liu, S and Zhang, Q and Hong, X and Lu, J and Lin, X and Zhao, X and Lai, J and Jie, K and Li, Y and Zhong, J and Peng, S and Chen, S and Chen, C and Zhong, W and Wu, S and Pan, Y and Lin, T and Chen, X},
title = {Engineering CRISPR System-Based Bacterial Outer Membrane Vesicle Potentiates T Cell Immunity for Enhanced Cancer Immunotherapy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {37},
number = {39},
pages = {e2501565},
doi = {10.1002/adma.202501565},
pmid = {40495695},
issn = {1521-4095},
support = {2018YFA0902800//National Key Research and Development Program of China/ ; 82322056//National Natural Science Foundation of China/ ; 82341018//National Natural Science Foundation of China/ ; 82072827//National Natural Science Foundation of China/ ; 82273421//National Natural Science Foundation of China/ ; 82303405//National Natural Science Foundation of China/ ; 82472137//National Natural Science Foundation of China/ ; 2023A03J0718//Science and Technology Program of Guangzhou/ ; 2024B03J1234//Science and Technology Program of Guangzhou/ ; 2024A04J6558//Science and Technology Program of Guangzhou/ ; //Fundamental Research Funds for the Central Universities/ ; 23ykbj002//Sun Yat-sen University/ ; 2020B1111170006//Guangdong Provincial Clinical Research Centre for Urological Diseases/ ; 2020B1212060018//Guangdong Science and Technology Department/ ; 2018B030317001//Guangdong Science and Technology Department/ ; 2017B030314026//Guangdong Science and Technology Department/ ; },
mesh = {Animals ; Mice ; *Immunotherapy/methods ; *T-Lymphocytes/immunology ; Humans ; Cell Line, Tumor ; *Bacterial Outer Membrane/metabolism ; *CRISPR-Cas Systems ; *Neoplasms/therapy/immunology ; Escherichia coli/genetics ; Interleukin-12/genetics ; Chemokine CXCL9/genetics ; Genetic Engineering ; },
abstract = {Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment but only benefits a subset of patients because of insufficient infiltration and inactivation of effector T cells. Bacterial outer membrane vesicles (OMVs) can activate immunity and deliver therapeutic agents for immunotherapy. However, efficiently targeting and packaging therapeutic molecules into OMVs remains challenging. Here, the engineered E. coli BL21-derived OMVs enable the packaging of multiple genes, resulting in a 7-fold increase in DNA enrichment efficiency and gene silencing in vitro. Moreover, the engineered OMVs carrying genes encoding CXCL9 and IL12 (OMV-C9I12) reprogram tumor cells to secrete these factors, significantly enhancing T-cell chemotaxis and activation. More importantly, this system markedly inhibits tumors, extends survival, and synergizes with anti-PD-1/PD-L1 therapy in murine MB49 and B16F10 tumor models. Single-cell RNA sequencing (scRNA-seq) further reveals significant upregulation of T-cell chemotaxis and activation-related pathways following OMV-C9I12 treatment. Finally, OMV-C9I12 potentiates T cell-mediated immunotherapy and suppresses the growth of bladder and breast cancer tumors in humanized mouse models. These findings highlight the potential of this engineered OMV platform for cancer gene therapy and provide novel strategies to overcome resistance to immunotherapy.},
}

Animals
Mice
*Immunotherapy/methods
*T-Lymphocytes/immunology
Humans
Cell Line, Tumor
*Bacterial Outer Membrane/metabolism
*CRISPR-Cas Systems
*Neoplasms/therapy/immunology
Escherichia coli/genetics
Interleukin-12/genetics
Chemokine CXCL9/genetics
Genetic Engineering

@article {pmid40436751,
year = {2025},
author = {Bold-Erdene, A and Miura, K and Yamasaki, N and Miura, S and Ogata, S and Sasatani, M and Yamamoto, T and Kaminuma, O},
title = {Effect of gamma-ray exposure on the genome-editing efficiency of improved genome-editing via oviductal nucleic acids delivery (i-GONAD).},
journal = {Experimental animals},
volume = {74},
number = {4},
pages = {457-462},
doi = {10.1538/expanim.25-0036},
pmid = {40436751},
issn = {1881-7122},
mesh = {Animals ; Female ; *Gamma Rays/adverse effects ; *Gene Editing/methods ; Pregnancy ; Mice ; *Oviducts ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded/radiation effects ; *Nucleic Acids/administration & dosage ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {DNA double-strand breaks (DSBs) are among the most hazardous cellular damages, potentially leading to cell death or oncogenesis if unrepaired. Genome editing methods, such as the CRISPR/Cas9 system, induce DSBs and utilize these repair pathways for gene knockout and knock-in. Although ionizing radiation also induces DSBs, it is not clear whether the efficiency of genome editing is affected by ionizing radiation. This study investigated the impact of gamma-ray exposure on the genome editing efficiency of the improved genome editing via oviductal nucleic acid delivery (i-GONAD) method. Gamma-rays were exposed to pregnant mice receiving i-GONAD targeting the Hr gene, whose mutation causes hair loss in mice. The exposure on the fertilization day (Day 0) decreased natural delivery rates and litter sizes, with notable effects at 0.3 Gy or higher. Although the proportions of hairless offspring obtained by i-GONAD differed greatly between single-guide RNAs (sgRNAs) used, total mutation rates, including hairless, mosaic, and indel, were equivalent. Gamma-ray exposure on Day 0 and the day after fertilization (Day 1) similarly and almost dose-dependently enhanced the genome editing efficiency evaluated by the total mutation rate. This study suggests the improvement of genome editing efficiency by gamma-ray exposure, at least in i-GONAD method, potentially facilitating the creation of diverse experimental animal models.},
}

Animals
Female
*Gamma Rays/adverse effects
*Gene Editing/methods
Pregnancy
Mice
*Oviducts
CRISPR-Cas Systems
DNA Breaks, Double-Stranded/radiation effects
*Nucleic Acids/administration & dosage
RNA, Guide, CRISPR-Cas Systems

@article {pmid40029006,
year = {2025},
author = {Mattoscio, D and Baeza, LA and Bai, H and Colangelo, T and Castagnozzi, S and Marzotto, M and Cufaro, MC and Lotti, V and Yuan, YC and Mucci, M and Si, L and Zuccarini, M and Tredicine, M and D'Orazio, S and Pieragostino, D and Del Boccio, P and Sorio, C and Trerotola, M and Romano, M and Plebani, R},
title = {Inflammation and epithelial-mesenchymal transition in a CFTR-depleted human bronchial epithelial cell line revealed by proteomics and human organ-on-a-chip.},
journal = {The FEBS journal},
volume = {292},
number = {19},
pages = {5086-5104},
doi = {10.1111/febs.70050},
pmid = {40029006},
issn = {1742-4658},
support = {AT2021 to Roberto Plebani//Italian Ministry of Health (fund ex60%)/ ; AT2022 to Roberto Plebani//Italian Ministry of Health (fund ex60%)/ ; AIRC; #19548 to Tommaso Colangelo//Associazione Italiana per la Ricerca sul Cancro/ ; MFAG 2022 - ID. 27060 to Domenico Mattoscio//Associazione Italiana per la Ricerca sul Cancro/ ; //Fondazione Umberto Veronesi/ ; GMSG#01/2023 to Roberto Plebani//Fondazione per la Ricerca sulla Fibrosi Cistica/ ; FFC#11/2022 to Domenico Mattoscio//Fondazione per la Ricerca sulla Fibrosi Cistica/ ; },
mesh = {Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; Proteomics/methods ; *Epithelial-Mesenchymal Transition/genetics ; *Epithelial Cells/metabolism/pathology ; *Inflammation/pathology/genetics/metabolism ; *Cystic Fibrosis/pathology/genetics/metabolism ; *Bronchi/pathology/metabolism/cytology ; Cell Line ; Cell Movement/genetics ; Lab-On-A-Chip Devices ; CRISPR-Cas Systems ; Neutrophils/metabolism/pathology ; Signal Transduction ; Microphysiological Systems ; },
abstract = {Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, leading to chronic, unresolved inflammation of the airways due to uncontrolled recruitment of polymorphonuclear leukocytes (PMNs). Evidence indicates that CFTR loss-of-function, in addition to promoting a pro-inflammatory phenotype, is associated with an increased risk of developing cancer, suggesting that CFTR can exert tumor-suppressor functions. Three-dimensional (3D) in vitro culture models, such as the CF lung airway-on-a-chip, can be suitable for studying PMN recruitment, as well as events of cancerogenesis, that is epithelial cell invasion and migration, in CF. To gather insight into the pathobiology of CFTR loss-of-function, we generated CFTR-knockout (KO) clones of the 16HBE14o- human bronchial cell line by CRISPR/Cas9 gene editing, and performed a comparative proteomic analysis of these clones with their wild-type (WT) counterparts. Systematic signaling pathway analysis of CFTR-KO clones revealed modulation of inflammation, PMN recruitment, epithelial cell migration, and epithelial-mesenchymal transition. Using a latest-generation organ-on-a-chip microfluidic platform, we confirmed that CFTR-KO enhanced PMN recruitment and epithelial cell invasion of the endothelial layer. Thus, a dysfunctional CFTR affects multiple pathways in the airway epithelium that ultimately contribute to sustained inflammation and cancerogenesis in CF.},
}

Humans
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism
Proteomics/methods
*Epithelial-Mesenchymal Transition/genetics
*Epithelial Cells/metabolism/pathology
*Inflammation/pathology/genetics/metabolism
*Cystic Fibrosis/pathology/genetics/metabolism
*Bronchi/pathology/metabolism/cytology
Cell Line
Cell Movement/genetics
Lab-On-A-Chip Devices
CRISPR-Cas Systems
Neutrophils/metabolism/pathology
Signal Transduction
Microphysiological Systems

@article {pmid41053552,
year = {2025},
author = {Qin, Z and Surnido, W and Mizuta, H and Uji, T},
title = {Stable transgene expression and CRISPR-mediated knock-in system of a bacteria-derived antibiotic selection gene in the green alga Ulva prolifera.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1323},
pmid = {41053552},
issn = {1471-2229},
support = {JPMJOP1851//Japan Science and Technology Agency/ ; },
mesh = {*Ulva/genetics/drug effects ; *Gene Knock-In Techniques/methods ; *Transgenes/genetics ; Kanamycin Kinase/genetics ; CRISPR-Cas Systems ; Hygromycin B/pharmacology ; Anti-Bacterial Agents/pharmacology ; Edible Seaweeds ; },
abstract = {Ulva prolifera is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of U. prolifera cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in U. prolifera based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene (aph7"). Integration of aph7" in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of aph7". The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of aph7" cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase (UpAPT). APT gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance U. prolifera as a genetic platform, enabling functional research to elucidate Ulva biology, and to bring forth biotechnological utilization of algal resources.},
}

*Ulva/genetics/drug effects
*Gene Knock-In Techniques/methods
*Transgenes/genetics
Kanamycin Kinase/genetics
CRISPR-Cas Systems
Hygromycin B/pharmacology
Anti-Bacterial Agents/pharmacology
Edible Seaweeds

@article {pmid41053297,
year = {2025},
author = {Vanderperre, B and Muraleedharan, A and Dorion, MF and Larroquette, F and Del Cid Pellitero, E and Rajakulendran, N and Chen, CX and Larivière, R and Michaud-Tardif, C and Goiran, T and Chidiac, R and Lipuma, D and MacLeod, G and Thomas, R and Wang, Z and Reintsch, WE and Luo, W and Shlaifer, I and Zhang, F and Xia, K and Steinhart, Z and Linhardt, RJ and Trempe, JF and Liu, J and Durcan, TM and Angers, S and Fon, EA},
title = {Novel regulators of heparan sulfate proteoglycans modulate cellular uptake of α-synuclein fibrils.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1426},
pmid = {41053297},
issn = {2399-3642},
support = {021129//Michael J. Fox Foundation for Parkinson's Research (Michael J. Fox Foundation)/ ; MFE-152571//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)/ ; Canada Research Chair (Tier 1) in Parkinson's disease//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)/ ; },
mesh = {*alpha-Synuclein/metabolism/genetics ; Humans ; *Heparan Sulfate Proteoglycans/metabolism ; Golgi Apparatus/metabolism ; Animals ; CRISPR-Cas Systems ; Cation Transport Proteins/genetics/metabolism ; Mice ; },
abstract = {Synucleinopathies are characterized by the accumulation and propagation of α-synuclein (α-syn) aggregates throughout the brain, leading to neuronal dysfunction and death. In this study, we used an unbiased FACS-based genome-wide CRISPR/Cas9 knockout screening to identify genes that regulate the entry and accumulation of α-syn preformed fibrils (PFFs) in cells. We identified key genes and pathways specifically implicated in α-syn PFFs intracellular accumulation, including heparan sulfate proteoglycans (HSPG) biosynthesis and Golgi trafficking. All confirmed hits affected heparan sulfate (HS), a post-translational modification known to act as a receptor for proteinaceous aggregates including α-syn and tau. Intriguingly, deletion of SLC39A9 and C3orf58 genes, encoding respectively a Golgi-localized exporter of Zn[2+], and the Golgi-localized putative kinase DIPK2A, specifically impaired the uptake of α-syn PFFs, by preventing the binding of PFFs to the cell surface. Mass spectrometry-based analysis of HS chains in SLC39A9[-/-] and C3orf58[-/-] cells indicated major defects in HS homeostasis. Additionally, Golgi accumulation of NDST1, a prime HSPG biosynthetic enzyme, was detected in C3orf58[-/-] cells. Interestingly, C3orf58[-/-] human iPSC-derived microglia and dopaminergic neurons exhibited a strong reduction in their ability to internalize α-syn PFFs. Altogether, our data identifies new modulators of HSPGs that regulate α-syn PFFs cell surface binding and uptake.},
}

*alpha-Synuclein/metabolism/genetics
Humans
*Heparan Sulfate Proteoglycans/metabolism
Golgi Apparatus/metabolism
Animals
CRISPR-Cas Systems
Cation Transport Proteins/genetics/metabolism
Mice

@article {pmid41051826,
year = {2025},
author = {McCallum, GE and Ho, SFS and Cummins, EA and Wildsmith, AJ and McInnes, RS and Weigel, C and Tong, LYS and Quick, J and van Schaik, W and Moran, RA},
title = {The Kocurious case of Noodlococcus: genomic insights into Kocuria rhizophila from characterisation of a laboratory contaminant.},
journal = {Microbial genomics},
volume = {11},
number = {10},
pages = {},
doi = {10.1099/mgen.0.001526},
pmid = {41051826},
issn = {2057-5858},
mesh = {*Genome, Bacterial ; Phylogeny ; Genomics ; *Micrococcaceae/genetics/classification/isolation & purification ; Whole Genome Sequencing ; Sequence Analysis, DNA ; },
abstract = {The laboratory contaminant strain Noodlococcus was named for its coccoid cells and unusual colony morphology, which resembled a pile of noodles. Along with laboratory characterisation and electron microscopy, we generated a complete Noodlococcus genome sequence using Illumina and Oxford Nanopore data. The genome consisted of a single, circular, 2,732,108 bp chromosome that shared 97.5% average nucleotide identity (ANI) with the Kocuria rhizophila type strain TA68. We identified genomic features involved in replication (oriC), carotenoid synthesis (crt) and genome defence (CRISPR-Cas) and discovered four novel mobile elements (ISKrh4-7). Despite its environmental ubiquity and relevance to food production, bioremediation and human medicine, there have been few genomic studies of the Kocuria genus. We conducted a comparative, phylogenetic and pangenomic examination of all 257 publicly available Kocuria genomes, with a particular focus on the 56 that were identified as K. rhizophila. We found that there are two phylogenetically distinct clades of K. rhizophila, with within-clade ANI values of 96.7-100.0% and between-clade values of 89.5-90.4%. The second clade, which we refer to as Kocuria pseudorhizophila, exhibited ANI values of <95% relative to TA68 and should constitute a separate species. Delineation of the two clades would be consistent with the rest of the genus, where all other species satisfy the 95% ANI threshold criteria. Differences in the K. rhizophila and K. pseudorhizophila pangenomes likely reflect phenotypic as well as evolutionary divergence. This distinction is relevant to clinical and industrial settings, as strains and genomes from both clades are currently used interchangeably, which may lead to reproducibility issues and phenotype-genotype discordance. Investigating an innocuous laboratory contaminant has therefore provided useful insights into the understudied species K. rhizophila, prompting an unexpected reassessment of its taxonomy.},
}

*Genome, Bacterial
Phylogeny
Genomics
*Micrococcaceae/genetics/classification/isolation & purification
Whole Genome Sequencing
Sequence Analysis, DNA

@article {pmid41051673,
year = {2026},
author = {Kumar, P and Verma, V and Irfan, M},
title = {Target-Specific Single Guide RNA (sgRNA) Design and In Vitro Validation of Target-Specific sgRNAs for CRISPR/Cas9-Mediated Editing of a Plant Stress Memory-Associated Gene.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2988},
number = {},
pages = {145-156},
pmid = {41051673},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Arabidopsis/genetics ; *Stress, Physiological/genetics ; Arabidopsis Proteins/genetics ; Transcription Factors/genetics ; },
abstract = {CRISPR/Cas9 genome editing has emerged as a transformative tool in plant biology, enabling precise manipulation of genes involved in stress responses. In the context of plant stress memory, where prior exposure to environmental stress enhances subsequent stress tolerance. CRISPR-based approaches offer a powerful means to dissect and engineer underlying regulatory genes. A critical factor determining the success of CRISPR/Cas9 editing is the careful design and validation of single guide RNAs (sgRNAs), which guide the Cas9 nuclease to specific genomic targets. This chapter provides a detailed, step-by-step protocol for the design, in vitro transcription, and in vitro cleavage assay to check efficiency of target-specific sgRNAs for plant genome editing applications. As a case study, we describe the design and validation of sgRNAs targeting the Arabidopsis thaliana DREB2A gene, a key transcription factor associated with drought stress memory. Emphasis is placed on strategies to maximize on-target efficiency, minimize off-target effects, and assess sgRNA functionality in vitro prior to in planta applications. This chapter serves as a practical guide for researchers aiming to functionally characterize stress memory-associated genes using CRISPR/Cas9 technology.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*Arabidopsis/genetics
*Stress, Physiological/genetics
Arabidopsis Proteins/genetics
Transcription Factors/genetics

@article {pmid41051363,
year = {2025},
author = {Zhytnik, L and Ventura, L and Sclocco, A and Verhage, M and Bakker, AD and Shim, JH and Beaino, W and Pereira, PM and Hoogeland, ME and Heine, VM and Maas, H and Jaspers, RT and Niehoff, A and Zaucke, F and de Waard, V and Eekhoff, EMW and Micha, D},
title = {New Lens On Congenital Mild Bone Fragility: a Novel Col1a1 Knockout Mouse Model for Osteogenesis Imperfecta Type 1.},
journal = {Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research},
volume = {},
number = {},
pages = {},
doi = {10.1093/jbmr/zjaf138},
pmid = {41051363},
issn = {1523-4681},
abstract = {Osteogenesis imperfecta (OI) is a genetic disorder characterized by bone fragility. It is one of the most prevalent rare skeletal dysplasias. The mildest form, OI type 1, predominantly results from collagen type I haploinsufficiency due to pathogenic variants in the COL1A1 gene, leading to reduced collagen type I. Despite OI type 1 representing approximately half of the OI population, the lack of an effective mouse model has hindered research and therapy development(1). To address this gap, we developed a genetically engineered mouse model harbouring a heterozygous deletion of the Col1a1 allele using the CRISPR/Cas system. The bone phenotype was characterised in 8- and 24-week-old mice, assessing transcriptomics and serum markers for bone formation (procollagen type I N-terminal propeptide) and resorption (tartrate-resistant acid phosphatase 5b). Bone volume, microarchitecture, and strength were evaluated by micro-computed tomography, histomorphometry and three-point bending test. We showed that the decreased Col1a1 to Col1a2 mRNA ratio determines reduced collagen type I production in OI mice bones as the underlying mechanism of haploinsufficient OI. This was supported by COL1A1 to COL1A2 mRNA ratio findings in human OI cell models, including fibroblasts and induced mesenchymal stem cells, as well as in induced pluripotent and mesenchymal stem cell models that were edited to carry a heterozygous COL1A1 allele. Our findings indicate for the first time that reduced bone volume and altered bone microarchitecture in haploinsufficient OI depends on the Col1a1 to Col1a2 mRNA ratio regulation. This novel mouse model faithfully recapitulates OI type 1 and provides a vital tool for investigating the disease mechanism and developing targeted therapeutic strategies for this large neglected OI patient population.},
}

@article {pmid41048399,
year = {2025},
author = {Huang, S and Qin, H and Dai, B and Liu, M and Shen, J},
title = {Establishment and evaluation of a circAdpgk-0001 knockdown method using CRISPR-Cas13d RNA-targeting technology.},
journal = {PeerJ},
volume = {13},
number = {},
pages = {e20123},
pmid = {41048399},
issn = {2167-8359},
mesh = {*RNA, Circular/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; RNA, Small Interfering/genetics ; Humans ; Cell Line ; Actins/genetics/metabolism ; },
abstract = {BACKGROUND: The small interfering RNA (siRNA) method has been used to knock down circular RNAs (circRNAs). However, issues such as low efficiency and off-target effects have become increasingly recognized. Recent studies have demonstrated that CRISPR-Cas13 can specifically target and cleave RNA. In this study, we established a CRISPR-Cas13d-based RNA-targeting method to specifically knock down circRNAs, such as circAdpgk-0001, and compared its performance with the siRNA method.

METHODS: Four clustered regularly interspaced short palindromic repeats (CRISPR) RNAs (crRNAs) of different nucleotide lengths spanning the back-splicing junction (BSJ) of circAdpgk-0001 were designed. A CRISPR-RfxCas13d plasmid capable of specifically cleaving circAdpgk-0001 was constructed and transfected into the JS-1 cell line. Knockdown efficiency was assessed using quantitative real-time PCR (qRT-PCR) and compared with that of the siRNA method. The expression of activation-related factors alpha-smooth muscle actin (α-SMA) and collagen I in JS-1 cells was further evaluated using qRT-PCR and Western blot.

RESULTS: CRISPR-Cas13d with a 24-nucleotide crRNA showed the highest knockdown efficiency (∼50%). After further optimization, the knockdown efficiency of CRISPR-Cas13d reached 70%, significantly higher than that of the siRNA method (40%). Knockdown of circAdpgk-0001 using Cas13d reduced the expression of collagen I and α-SMA by approximately 40%, which was greater than the reduction achieved by siRNA-mediated knockdown.

CONCLUSION: CRISPR-Cas13d demonstrated higher efficiency than the siRNA method in knocking down circRNAs, providing a promising tool for investigating circRNA functions.},
}

*RNA, Circular/genetics
*CRISPR-Cas Systems/genetics
*Gene Knockdown Techniques/methods
RNA, Small Interfering/genetics
Humans
Cell Line
Actins/genetics/metabolism

@article {pmid41020566,
year = {2025},
author = {Xue, J and Mao, K and Tang, Z and Hu, J and Zhang, H},
title = {Machine-Learning-Assisted CRISPR/Cas12a Biosensors for Monitoring Organophosphorus Pesticide Degradation.},
journal = {Analytical chemistry},
volume = {97},
number = {39},
pages = {21491-21501},
doi = {10.1021/acs.analchem.5c03596},
pmid = {41020566},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Pesticides/analysis/metabolism ; *CRISPR-Cas Systems ; *Organophosphorus Compounds/analysis/metabolism ; *Machine Learning ; *Water Pollutants, Chemical/analysis/metabolism ; Smartphone ; Acetylcholinesterase/metabolism ; Manganese Compounds/chemistry ; Oxides/chemistry ; Bacterial Proteins/metabolism/genetics ; Limit of Detection ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Owing to the severe environmental and health issues posed by organophosphorus pesticides (OPs), a dual-enzyme cascade biosensing platform based on manganese dioxide (MnO2) and CRISPR/Cas12a was developed in this study. Smartphones were innovatively integrated with a stacked ensemble learning (SEL) model for ultrasensitive detection and dynamic monitoring of OPs in environmental water samples. A dual-enzyme cascade signal amplification strategy was used to construct this sensing platform. Acetylcholinesterase (AChE) catalyzes the generation of thiocholine (TCh), which, in turn, regulates the degradation of MnO2 nanosheets, releasing Cas12a activators and generating a fluorescence signal. Owing to the irreversible inhibition of AChE activity by OPs, dichlorvos (DDVP) was successfully detected, with a detection limit as low as 4.62 pg/mL. Additionally, the SEL model, integrated into the smartphone biosensing platform and incorporating random forest (RF), XGBoost, and ridge regression algorithms, exhibited strong performance in detecting OPs after optimization (R[2] = 0.9985). In real water samples, the SEL model achieved a recovery rate of 93.1-103.1%, and the degradation kinetics of DDVP were successfully monitored over 24 h, revealing significant differences in DDVP degradation rates across various water matrices. This study is the first to report the integration of CRISPR/Cas12a biosensing technology with an SEL model-driven smartphone detection platform, providing a novel approach for sensitive, portable, and intelligent monitoring of OPs and offering new insights for water quality monitoring and early detection of environmental risks.},
}

*Biosensing Techniques/methods
*Pesticides/analysis/metabolism
*CRISPR-Cas Systems
*Organophosphorus Compounds/analysis/metabolism
*Machine Learning
*Water Pollutants, Chemical/analysis/metabolism
Smartphone
Acetylcholinesterase/metabolism
Manganese Compounds/chemistry
Oxides/chemistry
Bacterial Proteins/metabolism/genetics
Limit of Detection
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40923283,
year = {2025},
author = {Man, Y and Posey, RR and Bai, H and Jiang, A and Dosta, P and Ocampo-Alvarado, D and Plebani, R and Ji, J and Belgur, C and Artzi, N and Ingber, DE},
title = {Preclinical assessment of pan-influenza A virus CRISPR RNA therapeutics in a human lung alveolus chip.},
journal = {Lab on a chip},
volume = {25},
number = {20},
pages = {5240-5254},
doi = {10.1039/d5lc00156k},
pmid = {40923283},
issn = {1473-0189},
mesh = {Humans ; *RNA, Viral/genetics ; *Pulmonary Alveoli/virology/cytology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Influenza A virus/genetics ; *Influenza A Virus, H3N2 Subtype/genetics ; *Lab-On-A-Chip Devices ; Influenza, Human/therapy ; },
abstract = {CRISPR technology offers an entirely new approach to therapeutic development because it can target specific nucleotide sequences with high specificity, however, preclinical animal models are not useful for evaluation of their efficacy and potential off-target effects because of high gene sequence variations between animals and humans. Here, we explored the potential of using the CRISPR effector Cas13 to develop a new therapeutic approach for influenza A virus (IAV) infections based on its ability to specifically and robustly cleave single-strand viral RNA using a complementary CRISPR RNA (crRNA). We engineered crRNAs to target highly conserved regions in the IAV genome to create a potential pan-viral treatment strategy. A human lung alveolus chip (Lung Chip) lined by human primary alveolar epithelial cells interfaced with human primary pulmonary microvascular endothelial cells and infected with a pandemic IAV H3N2 strain was used to evaluate the on-target and off-target effects of these antiviral crRNA therapeutics. Our data show that the crRNAs targeting highly conserved regions in the IAV genome potently reduced viral replication in the alveolar airspace in the Lung Chip, and this was accompanied by suppression of the human host inflammatory response as indicated by a significant reduction in cytokine production and recruitment of immune cells. Importantly, only minimal off-target effects were observed based on transcriptomic analyses. As these crRNAs inhibit replication of influenza H1N1 and H3N2 in A549 cells as well as H3N2 in Lung Chips, these findings support use of CRISPR-Cas13 as a potentially viable approach to develop pan-IAV therapeutics for combating future influenza pandemics. The results also demonstrate that human Organ Chips be useful as more clinically relevant preclinical models for testing the efficacy and safety of crRNA therapeutics.},
}

Humans
*RNA, Viral/genetics
*Pulmonary Alveoli/virology/cytology
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Influenza A virus/genetics
*Influenza A Virus, H3N2 Subtype/genetics
*Lab-On-A-Chip Devices
Influenza, Human/therapy

@article {pmid40910953,
year = {2025},
author = {Feng, D and Guo, J and Yan, J and Chen, J and Ding, L and Zhu, X and Chen, Z and Hu, Y and Zhang, M and Liu, J and Zhu, C and Liu, M and Zhao, C and Zhang, X and Xu, J},
title = {COG6 is an essential host factor for influenza A virus infection.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0136225},
doi = {10.1128/spectrum.01362-25},
pmid = {40910953},
issn = {2165-0497},
support = {2022YFC2604100//National Key Research and Development Program of China/ ; 2023YFC2605602//National Key Research and Development Program of China/ ; 82072273//National Natural Science Foundation of China/ ; },
mesh = {*Influenza A virus/physiology/genetics ; Humans ; Virus Replication ; Golgi Apparatus/metabolism ; Lysosomes/metabolism ; Animals ; *Influenza, Human/virology/metabolism ; *Host-Pathogen Interactions ; *Adaptor Proteins, Vesicular Transport/metabolism/genetics ; Viral Proteins/metabolism ; CRISPR-Cas Systems ; Madin Darby Canine Kidney Cells ; Dogs ; HEK293 Cells ; A549 Cells ; Virus Internalization ; },
abstract = {Influenza A virus (IAV) relies on the host cellular machinery to support its replication. Understanding these host dependencies can inform the development of novel antiviral strategies. In this study, we identified conserved oligomeric Golgi complex subunit 6 (COG6) as a novel host factor critical for IAV replication through a genome-wide clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) knockout screen. Disruption of COG6 significantly impaired viral replication. Mechanistically, COG6 supports IAV replication via two distinct means. First, consistent with the role of the COG complex in Golgi homeostasis, COG6 is required for the proper presentation of surface sialic acids, the primary receptor for IAV entry. Second, COG6 deficiency unexpectedly led to lysosome-dependent degradation of viral proteins. Notably, lysosomal activity was also upregulated in IAV-infected wild-type cells, albeit to a lesser extent than in COG6-deficient cells. Treatment with lysosomal inhibitors rescued viral protein stability in COG6 knockout cells. Protein interaction analysis further demonstrated that COG6-mediated stabilization of viral proteins did not rely on viral protein-COG6 interaction, refuting the hypothesis that COG6 acts as a shield factor to protect viral protein from lysosomal degradation. Moreover, knockout of other COG subunits produced similar antiviral effects, suggesting that an intact COG complex is required for IAV replication. Together, these findings uncover a critical role of the COG complex in regulating IAV replication and highlight a previously unappreciated functional link between the Golgi and lysosomes that could be exploited for treating IAV infections.IMPORTANCEDespite advances in virology, numerous host determinants facilitating influenza A virus (IAV) pathogenesis remain uncharacterized. Our study establishes conserved oligomeric Golgi complex subunit 6 (COG6) as a critical host factor promoting IAV infection through complementary mechanisms: receptor modulation and viral protein stabilization. This represents the first demonstration that the COG complex regulates viral pathogenesis through proteostasis mechanisms, fundamentally expanding our understanding of host-virus interactions at the organelle interface. These findings not only provide new perspectives on viral exploitation of Golgi trafficking networks but also identify potential therapeutic targets against evolving influenza strains.},
}

*Influenza A virus/physiology/genetics
Humans
Virus Replication
Golgi Apparatus/metabolism
Lysosomes/metabolism
Animals
*Influenza, Human/virology/metabolism
*Host-Pathogen Interactions
*Adaptor Proteins, Vesicular Transport/metabolism/genetics
Viral Proteins/metabolism
CRISPR-Cas Systems
Madin Darby Canine Kidney Cells
Dogs
HEK293 Cells
A549 Cells
Virus Internalization

@article {pmid40865226,
year = {2025},
author = {Zhang, X and Zhao, J and Dong, Y and Zhang, C and Hu, X and Wang, S and Chen, Y},
title = {Magnetic relaxation switching biosensor based on CRISPR-mediated cascade reaction for the amplification-free detection of Salmonella.},
journal = {Journal of hazardous materials},
volume = {497},
number = {},
pages = {139664},
doi = {10.1016/j.jhazmat.2025.139664},
pmid = {40865226},
issn = {1873-3336},
mesh = {*Biosensing Techniques/methods ; *Salmonella typhimurium/isolation & purification/genetics ; Alkaline Phosphatase/chemistry ; *CRISPR-Cas Systems ; Food Microbiology ; Limit of Detection ; Magnetite Nanoparticles/chemistry ; DNA, Single-Stranded ; DNA, Bacterial/analysis ; Food Contamination/analysis ; },
abstract = {Sensitive and accurate detection of foodborne pathogens is crucial for preventing foodborne outbreaks and ensuring public health safety, but challenged by extremely low infectious doses of many foodborne pathogens and matrix interference effects. In this study, we developed a magnetic relaxation switching biosensor boosted by CRISPR/Cas12a-mediated enzymatic cascade reaction (CMCR-MRS) for amplification-free detection of Salmonella typhimurium (S. typhimurium). CRISPR/Cas12a was designed to precisely target the pathogen-specific DNA and efficiently cleaved single-stranded DNA (ssDNA) immobilized on the magnetic nanoparticle-alkaline phosphatase (MNP-ALP) probes through trans-cleavage activity. Subsequently, the released ALP facilitated the conversion of paramagnetic Mn(VII) to Mn(II), which resulted in the change of transverse relaxation time (T2), achieving a high signal-to-background ratio with superior biocompatibility and minimal background interference, making them highly advantageous for sensitive detection in complex biological samples. Our assay showed a broad dynamic range from 40 to 10[7] CFU/mL and a limit of detection (LOD) of 10 CFU/mL for S. typhimurium without amplification. Furthermore, it has been successfully validated in real food samples, demonstrating strong consistency (R[2] = 0.989) with the quantitative real-time polymerase chain reaction (qPCR) test. CMCR-MRS can serve as a highly effective and reliable strategy for achieving sensitive and accurate pathogen detection.},
}

*Biosensing Techniques/methods
*Salmonella typhimurium/isolation & purification/genetics
Alkaline Phosphatase/chemistry
*CRISPR-Cas Systems
Food Microbiology
Limit of Detection
Magnetite Nanoparticles/chemistry
DNA, Single-Stranded
DNA, Bacterial/analysis
Food Contamination/analysis

@article {pmid40843899,
year = {2025},
author = {Stuible, M and Alpuche-Lazcano, SP and Gervais, C and Ouimet, M and Lippens, J and Pagé, M and Morasse, A and Moraitis, AN and Durocher, Y},
title = {Endogenous Retrovirus-Like Particle-Deficient CHO Cells Can be Generated by CRISPR or shRNA and Enriched Based on Cell-Surface Expression of Retroviral Envelope Protein.},
journal = {Biotechnology and bioengineering},
volume = {122},
number = {11},
pages = {3192-3204},
doi = {10.1002/bit.70043},
pmid = {40843899},
issn = {1097-0290},
support = {//The authors received no specific funding for this work./ ; },
mesh = {CHO Cells ; Cricetulus ; Animals ; *Viral Envelope Proteins/genetics/metabolism ; *RNA, Small Interfering/genetics/metabolism ; *Endogenous Retroviruses/genetics ; *CRISPR-Cas Systems ; Cricetinae ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Despite evidence that they are not functional or infective, retrovirus-like particles (RVLPs), originating from endogenous proviral sequences in Chinese hamster ovary (CHO) cells, present a safety risk for biotherapeutics manufactured using this cell line due to their resemblance to other mammalian leukemia viruses. Here, we demonstrate that CRISPR- and shRNA-based cell engineering strategies can be used to disrupt RVLP production by targeting the RVLP nucleotide sequences. Additionally, specific antibodies were generated to monitor RVLP protein expression, including RVLP envelope (Env) protein localized on the surface of CHO cells, greatly facilitating selection of RVLP-deficient clones. These modified CHO cells showed reduced RVLP production while maintaining or enhancing the ability to produce recombinant virus-like particles (VLPs), highlighting their potential application in biomanufacturing, especially for complex biologics that are incompatible with standard RVLP mitigation procedures, namely viral inactivation and nanofiltration.},
}

CHO Cells
Cricetulus
Animals
*Viral Envelope Proteins/genetics/metabolism
*RNA, Small Interfering/genetics/metabolism
*Endogenous Retroviruses/genetics
*CRISPR-Cas Systems
Cricetinae
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40829398,
year = {2025},
author = {Song, D and Xu, C and Sang, P and Liu, Y and Huang, X},
title = {Rapid and contamination-free detection of cucumber green mottle mosaic virus as a viral indicator in wastewater via UDG-RT-LAMP combined with CRISPR/Cas12a.},
journal = {Journal of hazardous materials},
volume = {497},
number = {},
pages = {139571},
doi = {10.1016/j.jhazmat.2025.139571},
pmid = {40829398},
issn = {1873-3336},
mesh = {*Wastewater/virology ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Tobamovirus/isolation & purification/genetics ; Uracil-DNA Glycosidase/genetics ; Biosensing Techniques ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; Molecular Diagnostic Techniques ; },
abstract = {The removal of viruses by wastewater treatment plants plays a pivotal role in ensuring water environment safety, where precise evaluation of elimination efficiency is essential for controlling viral dissemination. Current bacterial indicators exhibit limited correlations with virological safety parameters, whereas conventional viral detection methods face practical constraints such as high instrumentation requirements and long detection cycles. To overcome these limitations, this study presents an on-site detection method for cucumber green mottle mosaic virus (CGMMV) in wastewater as a viral indicator, integrating uracil-DNA glycosylase (UDG)-reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12a (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 12a)-mediated biosensing system. The developed method achieved effective prevention of aerosol contamination from residual amplicons via the dUTP-UDG system, rapid amplification via RT-LAMP, and improved sensitivity and visualization by CRISPR/Cas12a-mediated biosensing system. The potential of CGMMV as a viral indicator in wastewater treatment process was demonstrated, and the rapid detection was realized using the proposed method. This integrated approach achieves sensitive CGMMV detection (limit of detection of 1.13 copies/μL) within 35 min, demonstrating field applicability through equipment independence, contamination resistance, and rapid operation. The proposed assay offers a promising tool for rapid viral monitoring in wastewater treatment system for resource-limited settings.},
}

*Wastewater/virology
CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Tobamovirus/isolation & purification/genetics
Uracil-DNA Glycosidase/genetics
Biosensing Techniques
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases
Molecular Diagnostic Techniques

@article {pmid40810567,
year = {2025},
author = {Ji, C and Ru, L and Han, T and Mai, G and Zheng, L and Jiang, Y},
title = {VGRCOT: a one-tube visual detection method for group B Streptococcus combining RPA and CRISPR/Cas12a for point-of-care testing in reproductive health.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0139525},
doi = {10.1128/spectrum.01395-25},
pmid = {40810567},
issn = {2165-0497},
support = {2025ZNSFSC1561//Natural Science Foundation of Sichuan Province/ ; 2024SZY001//Key Research and Developmet Guidance Projects of Deyang City/ ; },
mesh = {Humans ; *Streptococcal Infections/diagnosis/microbiology ; *Streptococcus agalactiae/genetics/isolation & purification ; *Point-of-Care Testing ; *CRISPR-Cas Systems ; Female ; Pregnancy ; Sensitivity and Specificity ; Pregnancy Complications, Infectious/diagnosis/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Group B Streptococcus (GBS) is a significant pathogen that causes perinatal infections, seriously threatening the health of pregnant women and newborns. Prophylactic antibiotic treatment for pregnant women who screen positive for GBS can notably reduce the incidence and fatality of neonatal infections. Herein, we developed a visual nucleic acid method for GBS that integrates RPA and CRISPR/Cas12a in a one-tube setup, termed VGRCOT. The VGRCOT method achieved one-tube detection by adding the appropriate reagents to the bottom and lid of the EP tube, respectively. By rigorous optimization of ssDNA-FQ reporter concentration, crRNA concentration, RPA reaction time, and CRISPR/Cas12a cleavage time, VGRCOT can exhibit fluorescence under ultraviolet light, enabling visual detection. Under optimal conditions, VGRCOT has a satisfactory selectivity, and the detection limit was determined as 10[1] copies/reaction. Finally, VGRCOT also showed good performance comparable to qPCR in the actual detection of clinical specimens. Due to its ease of operation and convenient signal acquisition, VGRCOT shows promise for point-of-care testing in reproductive health.IMPORTANCEThis study presents a convenient, sensitive, and accurate visual detection method (VGRCOT) for GBS, combining RPA and CRISPR/Cas12a in a single reaction vessel. Through optimization of experimental conditions, VGRCOT enables detection within 60 min, with a minimum detection limit of 10[1] copies per reaction. VGRCOT offers several advantages by adding the appropriate reagents to the bottom and lid of the EP tube. The one-tube visualization method effectively prevents aerosol contamination, simplifies procedures, and enables visual detection without complex instruments, making it ideal for resource-limited environments. Additionally, its editable crRNA and the use of commonly available laboratory reagents allow for easy reprogramming to detect various pathogens, supporting scalable and low-cost batch production.},
}

Humans
*Streptococcal Infections/diagnosis/microbiology
*Streptococcus agalactiae/genetics/isolation & purification
*Point-of-Care Testing
*CRISPR-Cas Systems
Female
Pregnancy
Sensitivity and Specificity
Pregnancy Complications, Infectious/diagnosis/microbiology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40645879,
year = {2025},
author = {Liang, Y and Tong, S and Zhang, J and Tan, GY and Zhang, L and Lee, SY and Tong, Y},
title = {Expanding horizons of CRISPR applications beyond genome editing.},
journal = {Trends in genetics : TIG},
volume = {41},
number = {10},
pages = {934-953},
doi = {10.1016/j.tig.2025.06.003},
pmid = {40645879},
issn = {0168-9525},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Biosensing Techniques ; Single-Cell Analysis ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technologies have rapidly evolved beyond genome editing, transforming fields such as molecular diagnostics, biosensing, transcriptional regulation, molecular imaging, protein interaction mapping, and single-cell analysis. Emerging CRISPR-based diagnostics harness the collateral cleavage activity of CRISPR-associated (Cas) enzymes for rapid nucleic acid detection. Advanced biosensors extend CRISPR's capabilities to detect ions, metabolites, and proteins by integrating synthetic biology components. Catalytically inactive Cas proteins enable precise gene regulation and live-cell imaging of nucleic acids, whereas CRISPR-guided proximity labeling has revolutionized the mapping of biomolecular interactions. Recent single-cell CRISPR screens provide unprecedented resolution of cellular heterogeneity. Future research will focus on overcoming current limitations. The integration of CRISPR technologies with artificial intelligence (AI), spatial omics, and microfluidics is expected to further amplify their impact.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Biosensing Techniques
Single-Cell Analysis

@article {pmid40032999,
year = {2025},
author = {Sahu, SU and Castro, M and Muldoon, JJ and Asija, K and Wyman, SK and Krishnappa, N and de Oñate, L and Eyquem, J and Nguyen, DN and Wilson, RC},
title = {Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.},
journal = {Nature protocols},
volume = {20},
number = {10},
pages = {2735-2770},
pmid = {40032999},
issn = {1750-2799},
support = {K08 AI153767/AI/NIAID NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; K08AI153767//U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; UG3AI150552//U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; L40AI140341//U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; },
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Peptides/chemistry ; T-Lymphocytes/metabolism ; Cells, Cultured ; },
abstract = {Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in and base editing. The protocol involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) homology-directed repair template (HDRT) DNA may be included. In contrast to electroporation, PERC is appealing because it needs no dedicated hardware and has less impact on cell phenotype and viability. Because of the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Editing efficiencies can surpass 90% when using either Cas9 or Cas12a in primary T cells or HSPCs. After 3 h dedicated to reagent preparation, the PERC delivery step can be completed in 1 h, with the associated cell culture steps taking 3-7 d total. Because the protocol calls for only three readily available reagents (protein, RNA and peptide) and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes the protocol appealing for rapid deployment in research and clinical settings.},
}

Humans
*Hematopoietic Stem Cells/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics/metabolism
*Peptides/chemistry
T-Lymphocytes/metabolism
Cells, Cultured

@article {pmid41046988,
year = {2025},
author = {Chen, Y and Qi, ZD and Ji, R and Shi, N and Chen, H and Wei, DX},
title = {Synthetic biology for scalable production of medical polyhydroxyalkanoates: Advances and applications.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108722},
doi = {10.1016/j.biotechadv.2025.108722},
pmid = {41046988},
issn = {1873-1899},
abstract = {Polyhydroxyalkanoates (PHAs), characterized by their biodegradability and biocompatibility, present a promising, sustainable alternative to conventional synthetic polymers for biomedical applications. This study highlights the diversity of PHA monomers and structures, controllable biodegradability, and excellent biocompatibility, emphasizing their suitability for tissue engineering (bone, skin, cardiovascular, oral), anti-hair loss treatments, and drug delivery systems. Significant advancements in synthetic biology, encompassing CRISPR/Cas genome editing, promoter engineering, ribosome binding site optimization, metabolic pathway fine-tuning, and morphology engineering, have led to substantial improvements in PHA production efficiency and a reduction in associated costs. The adoption of next-generation industrial biotechnology (NGIB) using halophiles further enhances economic viability and simplifies the production process. The current commercial landscape and the future prospects of medical-grade PHAs, poised to become mainstream biodegradable materials, are also critically discussed.},
}

@article {pmid41046905,
year = {2025},
author = {Jaballah, SA and Ali, LM and Jehad, MA and Akhlaq, S and Rizvi, TA and Mustafa, F},
title = {Retroviral Vector Technology for Gene Therapy: History, Current Landscape, and Future Prospects.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {169473},
doi = {10.1016/j.jmb.2025.169473},
pmid = {41046905},
issn = {1089-8638},
abstract = {The concept of gene therapy and its practice has been prevalent for over five decades. The first successful retroviral vector-based gene therapy trial took place ∼35 years ago, followed by several setbacks. However, recent years have seen a surge in successes, offering new hope to patients with genetic and other disorders once deemed untreatable. Over the past decade, rapid advancements in molecular biology have led to the development of safer and more effective gene therapy strategies with various gene delivery systems now in use. Among these, viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses are the most widely employed in both research and clinical settings. This is due to their natural efficiency in delivering genetic material into target cells. Among these viral vectors, retroviruses stand out for their unique ability to reverse-transcribe and integrate their genetic material into the host genome, ensuring stable and long-term gene expression. This review highlights advances in retroviral vector development, examining both their therapeutic potential and associated challenges. It also explores strategies for vector production, including transient and stable systems tailored to meet clinical and regulatory demands. Significant progress is discussed in mitigating insertional mutagenesis and vector silencing. As a result, next-generation retroviral vectors with improved safety and efficacy have made it past regulatory-approval and are commercially available. Current innovations include replication-competent, non-integrating, integration-re-targeted, and hybrid CRISPR/Cas-expressing retroviral vectors undergoing pre-clinical and clinical investigations. This reflects a new era in gene therapy, with retroviral vectors reimagined for greater precision, control, and therapeutic impact.},
}

@article {pmid41045024,
year = {2025},
author = {Thomson, G and Mermaz, B and Sagawa, CHD and Lin, CI and Tachev, M and Joly, V and Irish, VF and Jacob, Y},
title = {Enzymatic depletion of transposable elements in sequencing libraries and its application for genotyping multiplexed CRISPR-edited plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70501},
doi = {10.1111/tpj.70501},
pmid = {41045024},
issn = {1365-313X},
support = {2023-70029-41277//U.S. Department of Agriculture/ ; },
mesh = {*DNA Transposable Elements/genetics ; Zea mays/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; DNA Methylation ; *Citrus/genetics ; Gene Library ; CRISPR-Cas Systems/genetics ; *Genotyping Techniques/methods ; Genotype ; High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, DNA/methods ; },
abstract = {Whole-genome sequencing has become a common strategy to genotype individual plants of interest. Although a limited number of genomic regions usually need to be surveyed with this strategy, excess sequencing information is almost always generated at an appreciable financial cost. Repetitive sequences (e.g., transposons), which can account for more than 80% of the genome of some plants, are often not required in these genotyping projects. Therefore, strategies that enrich DNA coding for the protein-coding genes prior to sequencing can lower the cost to obtain sufficient sequence information. Here, we present the development and application of methylation-sensitive reduced representation sequencing (MsRR-Seq), which relies on the cytosine methylation-sensitive restriction enzyme MspJI to deplete constitutive heterochromatic DNA before library construction. By applying MsRR-Seq to citrus and maize, we show that protein-coding genes can be enriched in sequencing datasets. We then describe the application of MsRR-Seq to facilitate the identification of complex mutants from populations of citrus plants resulting from multiplex CRISPR/Cas9 editing of four genes. Overall, this work demonstrates an easy and low-cost method to enrich non-repetitive DNA in high-throughput sequencing libraries, an approach that is especially useful for large plant genomes with an excessively high proportion of methylated repetitive sequences.},
}

*DNA Transposable Elements/genetics
Zea mays/genetics
*Gene Editing/methods
Genome, Plant/genetics
DNA Methylation
*Citrus/genetics
Gene Library
CRISPR-Cas Systems/genetics
*Genotyping Techniques/methods
Genotype
High-Throughput Nucleotide Sequencing/methods
Sequence Analysis, DNA/methods

@article {pmid41044600,
year = {2025},
author = {Conery, M and Pippin, JA and Wagley, Y and Trang, K and Pahl, MC and Villani, DA and Favazzo, LJ and Ackert-Bicknell, CL and Zuscik, MJ and Katsevich, E and Wells, AD and Zemel, BS and Voight, BF and Hankenson, KD and Chesi, A and Grant, SFA},
title = {GWAS-informed data integration and non-coding CRISPRi screen illuminate genetic etiology of bone mineral density.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {331},
pmid = {41044600},
issn = {1474-760X},
support = {Gates Grubstake Award//University of Colorado/ ; DMS 2113072//National Science Foundation, United States/ ; R01DK122586//National Institute of Diabetes and Digestive and Kidney Diseases,United States/ ; UM1 DK126194/DK/NIDDK NIH HHS/United States ; R01AI154773//National Institute of Allergy and Infectious Diseases/ ; R01 HD100406/HD/NICHD NIH HHS/United States ; UL1 TR001878/TR/NCATS NIH HHS/United States ; Henry Ruppenthal Family Professorship for Bioengineering and Orthopaedic Surgery//University of Michigan/ ; R01 AG072705/AG/NIA NIH HHS/United States ; Daniel B. Burke Endowed Chair for Diabetes Research//Children's Hospital of Philadelphia/ ; },
mesh = {Humans ; *Bone Density/genetics ; *Genome-Wide Association Study ; Osteoblasts/metabolism ; *CRISPR-Cas Systems ; Single-Cell Analysis ; Quantitative Trait Loci ; },
abstract = {BACKGROUND: Over 1100 independent signals have been identified with genome-wide association studies (GWAS) for bone mineral density (BMD), a key risk factor for mortality-increasing fragility fractures; however, the effector gene(s) for most remain unknown.

RESULTS: We execute a CRISPRi screen in human fetal osteoblasts (hFOBs) with single-cell RNA-seq read-out for 89 non-coding elements predicted to regulate osteoblast gene expression at BMD GWAS loci. The BMD relevance of hFOBs is supported by heritability enrichment from stratified LD-score regression involving 98 cell types grouped into 15 tissues. Twenty-three genes show perturbation in the screen, with four (ARID5B, CC2D1B, EIF4G2, and NCOA3) exhibiting consistent effects upon siRNA knockdown on three measures of osteoblast maturation and mineralization. Lastly, additional heritability enrichments, genetic correlations, and multi-trait fine-mapping unexpectedly reveal that many BMD GWAS signals are pleiotropic and likely mediate their effects via non-bone tissues.

CONCLUSIONS: Our results provide a roadmap for how single-cell CRISPRi screens may be applied to the challenging task of resolving effector gene identities at all BMD GWAS loci. Extending our CRISPRi screening approach to other tissues could play a key role in fully elucidating the etiology of BMD.},
}

Humans
*Bone Density/genetics
*Genome-Wide Association Study
Osteoblasts/metabolism
*CRISPR-Cas Systems
Single-Cell Analysis
Quantitative Trait Loci

@article {pmid41041610,
year = {2025},
author = {de Mello Fiallos, N and Irfan, M and Solbiati, J and R Walker, A and Frias-Lopez, J and Gibson, FC},
title = {CRISPR cas7 influences the host-pathogen interaction of Porphyromonas gingivalis.},
journal = {Journal of oral microbiology},
volume = {17},
number = {1},
pages = {2561790},
pmid = {41041610},
issn = {2000-2297},
abstract = {INTRODUCTION: Porphyromonas gingivalis, a Gram-negative anaerobe, is a key contributor to periodontal disease. Emerging evidence suggests a role for the P. gingivalis CRISPR-Cas system in disease progression, although the specific roles of its components remain unclear.

OBJECTIVES: Here we investigate the role of cas7, a Class 1 type I-B CRISPR-Cas system component, in P. gingivalis physiology and host interaction.

METHODS: We compared P. gingivalis wild-type and ∆cas7 strains for growth, biofilm formation, oxidative stress resistance, and hemagglutination. Host interactions were assessed using THP-1 macrophage-like cells to evaluate intracellular survival and cytokine response. Dual RNA-seq enabled host and microbe transcriptomic profiling during cellular infection, and Galleria mellonella was used to assess virulence.

RESULTS: The ∆cas7 mutant showed similar planktonic growth and biofilm formation compared to wild-type but was more sensitive to oxidative stress and had reduced hemagglutination. Although intracellular survival was unaffected, ∆cas7 altered the host cytokine production profile. Transcriptomic analysis revealed differential gene expression linked to oxidative stress and disease progression. In vivo, ∆cas7 infection led to a trend of increased larval mortality.

CONCLUSION: These findings reveal a previously unrecognized role for cas7 in modulating P. gingivalis virulence, offering new insights into CRISPR-Cas system functions in bacterial pathogenesis.},
}

@article {pmid40974079,
year = {2025},
author = {Mariki, A and Kohlmeier, KA and Mousavi, SM and Shabani, M},
title = {CRISPR and Myelin regeneration: a systematic review of applications in demyelinating CNS Disorders, with a focus on MS.},
journal = {Regenerative medicine},
volume = {20},
number = {9},
pages = {431-443},
doi = {10.1080/17460751.2025.2561451},
pmid = {40974079},
issn = {1746-076X},
mesh = {Humans ; Animals ; *Myelin Sheath/metabolism ; *Multiple Sclerosis/therapy/genetics ; *CRISPR-Cas Systems ; Gene Editing ; *Demyelinating Diseases/therapy/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Regeneration/genetics ; },
abstract = {AIMS: Current treatments for demyelinating disorders focus on slowing progression but fail to repair damaged myelin. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -based technology has the potential to address key challenges in myelin repair by targeting genetic dysfunctions, modulating immune responses, and promoting oligodendrocyte differentiation. This systematic review aimed to evaluate CRISPR applications for myelin regeneration.

METHODS: A comprehensive search of PubMed, Scopus, and other databases identified 48 studies. The included studies employed CRISPR in diverse experimental models, targeting genes associated with immune regulation and astrocyte activity, as well as correcting RNA splicing dysfunctions linked to neurodegeneration.

RESULTS: CRISPR-edited stem cells showed significant potential in promoting myelin regeneration, with enhanced functional recovery in animal models of multiple sclerosis (MS). While most research focused on MS, promising applications were also observed in neuromyelitis optica spectrum disorder (NMOSD), such as reducing astrocytic damage via AQP4 targeting, and in progressive multifocal leukoencephalopathy (PML), where CRISPR disrupted JC polyomavirus replication.

CONCLUSIONS: Despite its promise, challenges remain. Future research should prioritize optimizing CRISPR delivery systems, expanding applications to underexplored disorders, and conducting long-term safety assessments. Early results are encouraging, but further studies are essential to translate preclinical success into clinical therapies.},
}

Humans
Animals
*Myelin Sheath/metabolism
*Multiple Sclerosis/therapy/genetics
*CRISPR-Cas Systems
Gene Editing
*Demyelinating Diseases/therapy/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Regeneration/genetics

@article {pmid40972450,
year = {2026},
author = {Eom, KH and Yum, SY and Gim, GM and Kim, YC and Moon, B and Jang, G},
title = {SpCas9-mediated gene editing in bovine embryo via single adeno-associated virus infection using a novel micro-sized promoter.},
journal = {Theriogenology},
volume = {249},
number = {},
pages = {117676},
doi = {10.1016/j.theriogenology.2025.117676},
pmid = {40972450},
issn = {1879-3231},
mesh = {Animals ; Cattle/embryology/genetics ; *Gene Editing/veterinary/methods ; *Promoter Regions, Genetic ; *Dependovirus/genetics ; Genetic Vectors ; Embryo, Mammalian ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Fertilization in Vitro/veterinary ; },
abstract = {Genome editing in livestock offers practical solutions to address challenges related to land use, climate change, and food production. However, conventional delivery methods such as electroporation and microinjection impose physical stress on embryos, limiting scalability. This study aimed to develop a simplified, non-invasive, and scalable genome editing system for bovine embryos by designing an all-in-one adeno-associated virus (AAV) vector. A novel micro-sized promoter (50 bp), derived from the core regulatory region upstream of the bovine MSTN gene, was constructed to enable expression of Streptococcus pyogenes Cas9 (spCas9) within the AAV packaging limit (∼4.7 kb). This promoter was incorporated into an AAV cassette containing spCas9, a polyadenylation signal, a U6 promoter, and a single-guide RNA (sgRNA) targeting the bovine ALB gene. After confirming editing activity in bovine fibroblasts, the AAV6 vector was added directly to in vitro fertilization (IVF) cultures without physical manipulation. Genome editing was successfully induced, with insertion/deletion (indel) mutations detected in 33.8 ± 23.2 % of the blastocysts. Although blastocyst development was moderately reduced, gene editing was achieved without invasive techniques. These results demonstrate that a micro-promoter-based AAV system can support spCas9-mediated genome editing in bovine embryos through a single-vector infection strategy. The system presents a promising platform for producing gene-edited livestock and may contribute to more efficient and less labor-intensive applications in animal biotechnology.},
}

Animals
Cattle/embryology/genetics
*Gene Editing/veterinary/methods
*Promoter Regions, Genetic
*Dependovirus/genetics
Genetic Vectors
Embryo, Mammalian
*CRISPR-Associated Protein 9/genetics/metabolism
CRISPR-Cas Systems
Fertilization in Vitro/veterinary

@article {pmid40650655,
year = {2025},
author = {Li, Y and Zhang, Y and Li, C and Chen, G and Muhammad, P and Yao, Y and Gao, L and Liu, Z and Wang, Y},
title = {Advanced Cancer Immunotherapy via SMARCAL1 Blockade Using a Glucose-Responsive CRISPR Nanovaccine.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {37},
pages = {e02929},
doi = {10.1002/advs.202502929},
pmid = {40650655},
issn = {2198-3844},
support = {81901684//National Natural Science Foundation of China/ ; },
mesh = {*Immunotherapy/methods ; Animals ; Mice ; *Glucose/metabolism ; Humans ; *Cancer Vaccines/immunology/genetics ; *Neoplasms/therapy/immunology ; CRISPR-Cas Systems/genetics ; Glucose Oxidase ; Gene Editing/methods ; Membrane Proteins/metabolism ; Nanovaccines ; },
abstract = {Cancer immunotherapy that activates the stimulator of interferon genes (STING) signaling pathway to resist tumors has recently attracted considerable attention. However, STING activation can induce opposing interferon functions that contribute to T-cell exhaustion via programmed death-ligand 1 (PD-L1). In particular, effectively using the immune system to combat tumors remains a substantial challenge due to tumor immunosuppressive factors such as SMARCAL1. Here, a glucose-responsive CRISPR nanovaccine is developed for enhancing STING signaling while inhibiting interferon-mediated immunosuppressive feedback. The formulation encapsulates a bimetallic zeolitic imidazolate framework with glucose oxidase (GOx) and CRISPR-mediated SMARCAL1 gene-editing plasmids. The dual enzyme-driven cascade reactions of peroxidase and GOx generate reactive oxygen species (ROS) and gluconic acid, which release and activate the genome-editing system. The silencing of SMARCAL1 enhances STING activity and inhibits PD-L1 expression, resulting in the termination of PD-L1-mediated opposing functions of interferon. Zinc ions and double-stranded DNA formed via ROS further activate the STING pathway, effectively inducing dendritic cell maturation and immune system activation. This is a critical report of in situ CRISPR nanovaccination driven by dual enzymes. The work highlights the potential of glucose-responsive CRISPR nanovaccination in bolstering antitumor immunity and extends the implementation of gene editing in cancer immunotherapy.},
}

*Immunotherapy/methods
Animals
Mice
*Glucose/metabolism
Humans
*Cancer Vaccines/immunology/genetics
*Neoplasms/therapy/immunology
CRISPR-Cas Systems/genetics
Glucose Oxidase
Gene Editing/methods
Membrane Proteins/metabolism
Nanovaccines

@article {pmid40642954,
year = {2025},
author = {Fu, X and Wang, N and Li, L and Qiao, D and Qi, X and Liu, C and Gao, Z and Xie, C and Zhu, J},
title = {Development of cytosine and adenine base editors for maize precision breeding.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {10},
pages = {2731-2743},
doi = {10.1111/jipb.13964},
pmid = {40642954},
issn = {1744-7909},
support = {No. 2023YFD1202901//National Key Research and Development Program of China Stem Cell and Translational Research/ ; 241111112300//Henan Province key research and development project/ ; //Xinjiang "Leading the Charge with Open Competition" project/ ; No. 2022ZD04006//Science and Technology Innovation 2030 Major projects/ ; },
mesh = {*Zea mays/genetics/drug effects ; *Gene Editing/methods ; *Adenine/metabolism ; *Cytosine/metabolism ; *Plant Breeding/methods ; Plants, Genetically Modified ; Mutation/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Base editing technologies can improve crops, but their efficiency in maize remains suboptimal. This study attempts to overcome these limitations by examining optimized cytosine and adenine base editors (CBEs and ABEs), namely evoAPOBEC1, evoFERNY, evoCDA1, TadA8.20, and TadA8e, for precise genome editing in transient and stable expression maize cells. Employing a seed fluorescence reporter (SFR) system for rapid screening of BE transformants and transgene-free progenies, we enhanced editing efficiencies and heritability. Notably, TadA8.20 and evoCDA1 attained multiplexed editing efficiencies of up to 100.0% and 79.0% at the tested loci, respectively, with some homozygous and bi-allelic mutants exceeding 72.4% and 73.7%. Precise editing of ZmACC1/2 (acetyl-CoA carboxylase) improved herbicide resistance, with ZmACC2 mutants displaying improved performance. This study advances crop genetic engineering by facilitating robust, multi-locus modifications without altered agronomic performance, enhancing herbicide tolerance in maize. The successful utilization of these BE is a significant step forward in agricultural biotechnology and precision breeding.},
}

*Zea mays/genetics/drug effects
*Gene Editing/methods
*Adenine/metabolism
*Cytosine/metabolism
*Plant Breeding/methods
Plants, Genetically Modified
Mutation/genetics
CRISPR-Cas Systems/genetics

@article {pmid39994413,
year = {2025},
author = {Oliynyk, RT and Church, GM},
title = {Circular Vectors as an efficient, fully synthetic, cell-free approach for preparing small circular DNA as a plasmid substitute for guide RNA expression in CRISPR-Cas9 genome editing.},
journal = {Nature protocols},
volume = {20},
number = {10},
pages = {2942-2959},
pmid = {39994413},
issn = {1750-2799},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Plasmids/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *DNA, Circular/genetics ; Cell-Free System ; Humans ; },
abstract = {Robust expression of guide RNA (gRNA) is essential for successful implementation of CRISPR-Cas9 genome-editing methods. The gRNA components, such as an RNA polymerase promoter followed by the gRNA coding sequence and an RNA polymerase terminator sequence, and the Cas9 protein are expressed either via an all-in-one plasmid or separate dedicated plasmids. The preparation of such plasmids involves a laborious multi-day process of DNA assembly, bacterial cloning, validation, purification and sequencing. Our Circular Vector (CV) protocol introduces an efficient, fully synthetic, cell-free approach for preparing gRNA expression templates suitable for transfection, marking a significant advancement over traditional plasmid-based approaches. This protocol consists of the circularization and purification of linear double-stranded DNA (dsDNA) containing gRNA expression elements into compact, bacterial-backbone-free circular DNA expression vectors in as little as 3 h. We provide a guide to the design of the dsDNA template coding for gRNA elements for CRISPR-Cas9 base and prime editing, along with step-by-step instructions for the efficient preparation of gRNA-expressing CVs. In addition to rapid preparation, CVs created via this protocol offer several key advantages: a compact size, absence of a bacterial backbone, absence of bacterial endotoxins and no contamination by bacterial RNA or DNA fragments. These features make gRNA-expressing CVs a superior choice over plasmid-based gRNA expression templates.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
*Plasmids/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genetic Vectors/genetics
*DNA, Circular/genetics
Cell-Free System
Humans

@article {pmid41039221,
year = {2025},
author = {Ilmi, AFN and Kaewsapsak, P and Rotcheewaphan, S},
title = {Repression of mab_1999 impairs growth and alters cellular morphology of Mycobacterium abscessus.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {599},
pmid = {41039221},
issn = {1471-2180},
mesh = {*Mycobacterium abscessus/growth & development/genetics/drug effects/cytology/metabolism ; *Bacterial Proteins/genetics/metabolism ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Cell Division/genetics ; Gene Expression Regulation, Bacterial ; Mycobacterium smegmatis/genetics ; Gene Knockdown Techniques ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Cell division is essential for bacterial survival and represents a promising target for the development of novel antibiotics, particularly in mycobacteria. The role of the division protein FtsL in Mycobacterium abscessus remains poorly understood. This study investigated the effects of MAB_1999, a predicted homolog of FtsL, on the growth and cell division of M. abscessus.

METHOD: To investigate the function of mab_1999, a knockdown mutant was generated via CRISPR interference (CRISPRi). The phenotypic impact of mab_1999 suppression was evaluated, with a focus on its effects on M. abscessus growth, cellular morphology, and antibiotic susceptibility.

RESULTS: The putative homolog of FtsL in M. abscessus (MAB_1999) shares 54% amino acid sequence identity with FtsL from M. smegmatis (MSMEG_4234). CRISPRi-mediated repression of mab_1999 expression resulted in cell elongation and growth defects, although complete growth arrest was not observed. Furthermore, reduced mab_1999 expression increased the susceptibility of M. abscessus to β-lactam antibiotics, including ceftriaxone and imipenem.

CONCLUSIONS: Our findings suggest that mab_1999 is involved in cell division and cell wall integrity in M. abscessus. However, further investigation is necessary to confirm its identity as FtsL and to fully elucidate its role in the cell division process and cell wall synthesis.},
}

*Mycobacterium abscessus/growth & development/genetics/drug effects/cytology/metabolism
*Bacterial Proteins/genetics/metabolism
Anti-Bacterial Agents/pharmacology
Microbial Sensitivity Tests
Cell Division/genetics
Gene Expression Regulation, Bacterial
Mycobacterium smegmatis/genetics
Gene Knockdown Techniques
CRISPR-Cas Systems

@article {pmid41039127,
year = {2025},
author = {Safenkova, IV and Kamionskaya, MV and Ivanov, AV and Zherdev, AV and Dzantiev, BB},
title = {A novel tripod probe and lateral flow test to improve CRISPR/Cas12a assay: benefits of branched probe based on trebler phosphoramidite modification.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {711},
pmid = {41039127},
issn = {1436-5073},
support = {23-46-10011//Russian Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA Probes/chemistry ; *Biosensing Techniques/methods ; *Organophosphorus Compounds/chemistry ; Fluorescent Dyes/chemistry ; Limit of Detection ; Gold/chemistry ; *CRISPR-Associated Proteins/metabolism ; Metal Nanoparticles/chemistry ; *Bacterial Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; },
abstract = {CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of the probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 min. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin-streptavidin capture within 5 min. The CRISPR/Cas12a-tripod-LFT strategy demonstrated excellent sensitivity without preamplification, with a detection Limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.},
}

*CRISPR-Cas Systems
*DNA Probes/chemistry
*Biosensing Techniques/methods
*Organophosphorus Compounds/chemistry
Fluorescent Dyes/chemistry
Limit of Detection
Gold/chemistry
*CRISPR-Associated Proteins/metabolism
Metal Nanoparticles/chemistry
*Bacterial Proteins/metabolism
*Endodeoxyribonucleases/metabolism

@article {pmid40847019,
year = {2025},
author = {Ramani, B and Rose, IVL and Teyssier, N and Pan, A and Danner-Bocks, S and Sanghal, T and Yadanar, L and Tian, R and Ma, K and Palop, JJ and Kampmann, M},
title = {CRISPR screening by AAV episome-sequencing (CrAAVe-seq): a scalable cell-type-specific in vivo platform uncovers neuronal essential genes.},
journal = {Nature neuroscience},
volume = {28},
number = {10},
pages = {2129-2140},
pmid = {40847019},
issn = {1546-1726},
support = {R01 AG082141/AG/NIA NIH HHS/United States ; K99 AG062776/AG/NIA NIH HHS/United States ; RF1 AG062234/AG/NIA NIH HHS/United States ; R25 NS070680/NS/NINDS NIH HHS/United States ; K08 NS133300/NS/NINDS NIH HHS/United States ; T32 NS115706/NS/NINDS NIH HHS/United States ; EDUC4-12812//California Institute for Regenerative Medicine (CIRM)/ ; UCSF Hillblom/BARI Graduate Fellowship Award//Larry L. Hillblom Foundation (Larry L. Hillblom Foundation, Inc.)/ ; R01 AG082141/AG/NIA NIH HHS/United States ; K99 AG062776/AG/NIA NIH HHS/United States ; RF1 AG062234/AG/NIA NIH HHS/United States ; R25 NS070680/NS/NINDS NIH HHS/United States ; K08 NS133300/NS/NINDS NIH HHS/United States ; T32 NS115706/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Dependovirus/genetics ; *Neurons/metabolism ; Mice ; *CRISPR-Cas Systems/genetics ; *Genes, Essential/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice, Inbred C57BL ; *Genetic Testing/methods ; Humans ; },
abstract = {There is a substantial need for scalable CRISPR-based genetic screening methods that can be applied in mammalian tissues in vivo while enabling cell-type-specific analysis. Here we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe-seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in mouse tissues. We used this approach to screen two large sgRNA libraries, which collectively target over 5,000 genes, in mouse brains and uncovered genes essential for neuronal survival, of which we validated Rabggta and Hspa5. We highlight the reproducibility and scalability of the platform and show that it is sufficiently sensitive for screening in a restricted subset of neurons. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and coinfection rate on screen performance to establish general guidelines for large-scale in vivo screens.},
}

Animals
*Dependovirus/genetics
*Neurons/metabolism
Mice
*CRISPR-Cas Systems/genetics
*Genes, Essential/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mice, Inbred C57BL
*Genetic Testing/methods
Humans

@article {pmid41038958,
year = {2025},
author = {Chen, J and Huang, H and Chen, C and Xia, G and Huang, H and Xiong, Y and Luo, P and Chen, Y and Li, J and Wen, L and Li, L and Lin, J and Xu, G and Ji, C and Tian, W and Zhou, J and Wei, P and Shen, C and Wang, X},
title = {ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR expression in the liver.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1414},
pmid = {41038958},
issn = {2399-3642},
support = {8217051361//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Receptors, LDL/metabolism/genetics ; Animals ; *Liver/metabolism ; Mice ; *Cholesterol, LDL/blood/metabolism ; *Multidrug Resistance-Associated Proteins/metabolism/genetics ; Humans ; Male ; Proprotein Convertase 9/metabolism/genetics ; Hepatocytes/metabolism ; Mice, Inbred C57BL ; Hypercholesterolemia/metabolism/genetics ; CRISPR-Cas Systems ; Signal Transduction ; Hep G2 Cells ; },
abstract = {Low expression level of low-density lipoprotein receptor (LDLR) in hepatocytes leads to hypercholesterolemia and eventually contributes to atherosclerotic cardiovascular disease (ASCVD). Here, we report that inhibition of hepatocyte ABCC4, identified as a top hit from large-scale CRISPR/Cas9 screens, significantly increases hepatic LDLR abundance and enhances LDL cholesterol clearance. As a hepatic transporter for cAMP efflux, ABCC4 silencing alters its intracellular distribution and activates the downstream Epac2/Rap1a signaling pathway, which ultimately blocks PCSK9 protein expression, thereby preventing lysosomal degradation of LDLR. Furthermore, in both male mice and cell models, we demonstrate that liver-specific disruption and pharmacological inhibition of ABCC4 elevate hepatic plasma membrane LDLR levels and reduce plasma LDL cholesterol through ABCC4-cAMP-PCSK9 pathway. Collectively, our genome-wide CRISPR screening offers a valuable resource for identifying LDLR modifiers, providing potential insights for therapeutic strategies in hypercholesterolemia and atherosclerosis.},
}

*Receptors, LDL/metabolism/genetics
Animals
*Liver/metabolism
Mice
*Cholesterol, LDL/blood/metabolism
*Multidrug Resistance-Associated Proteins/metabolism/genetics
Humans
Male
Proprotein Convertase 9/metabolism/genetics
Hepatocytes/metabolism
Mice, Inbred C57BL
Hypercholesterolemia/metabolism/genetics
CRISPR-Cas Systems
Signal Transduction
Hep G2 Cells

@article {pmid41038616,
year = {2025},
author = {Aliciaslan, M and Erbasan, E and Erendor, F and Sanlioglu, S},
title = {Prime Editing: The Next Frontier in Precision Gene Therapy.},
journal = {The journal of gene medicine},
volume = {27},
number = {10},
pages = {e70040},
doi = {10.1002/jgm.70040},
pmid = {41038616},
issn = {1521-2254},
support = {//Akdeniz Üniversitesi/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *Precision Medicine/methods ; CRISPR-Cas Systems ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing (PE) represents a significant advancement in genome editing, offering high precision for diverse genetic modifications without inducing double-strand breaks or requiring exogenous donor DNA templates. This "search-and-replace" technology employs a Cas9 nickase-reverse transcriptase fusion protein, guided by a PE guide RNA (pegRNA), to directly install specified edits including all 12 base-to-base conversions and targeted insertions/deletions with high fidelity. Since its introduction, PE systems have undergone rapid evolution (e.g., PE2-PE6, PEmax), markedly improving editing efficiency, product purity, and targeting scope. Although PE efficacy is context dependent, influenced by pegRNA design, cellular milieu, and DNA repair pathway engagement, ongoing research focuses on comprehensive system optimization. These efforts include engineering the Cas9 nickase and reverse transcriptase components for enhanced performance and processivity, alongside developing improved pegRNA architectures and chemical modifications to increase their stability and editing efficiency. Furthermore, strategies to modulate the cellular environment, such as transiently altering DNA repair pathway activities, particularly mismatch repair, are being explored to boost the accuracy and yield of precise edits. PE holds substantial promise for basic research, including precise disease modeling, and has demonstrated successful correction of pathogenic mutations in preclinical models of various genetic disorders like sickle cell disease, cystic fibrosis, and inherited retinal diseases. A significant milestone was the US Food and Drug Administration's granting of Investigational New Drug (IND) clearance for the first clinical trial of PM359, a therapeutic based on PE. This agent employs an ex vivo strategy, correcting the NCF1 gene in patient-derived hematopoietic stem cells for the treatment of chronic granulomatous disease. Despite considerable progress, unlocking the complete therapeutic promise of PE requires overcoming significant hurdles, particularly in developing effective in vivo delivery systems for its sizable components, with ongoing research actively investigating diverse viral and nonviral approaches. The translation of this versatile platform into transformative precision gene therapies is critically dependent upon its continued responsible advancement under robust ethical and regulatory oversight.},
}

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

@article {pmid41037600,
year = {2025},
author = {de Alba, EL and Salguero, I and Giménez-Llorente, D and Montes-Torres, J and Fernández-Sanromán, Á and Casajús-Pelegay, E and Terrón-Bautista, J and Barroso-González, J and Bernal, JA and Macintyre, G and Fernández-Leiro, R and Losada, A and Cortés-Ledesma, F},
title = {A comprehensive genetic catalog of human double-strand break repair.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6768},
pages = {eadr5048},
doi = {10.1126/science.adr5048},
pmid = {41037600},
issn = {1095-9203},
mesh = {Humans ; *DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; *DNA Repair/genetics ; Von Hippel-Lindau Tumor Suppressor Protein/genetics ; INDEL Mutation ; Gene Editing ; DNA End-Joining Repair/genetics ; Kidney Neoplasms/genetics ; DNA-Binding Proteins/genetics ; Transcription Factors/genetics ; CRISPR-Associated Protein 9 ; Genome, Human ; Gene Knockout Techniques ; Carcinoma, Renal Cell/genetics ; },
abstract = {The analysis of DNA sequence outcomes provides molecular insights into double-strand break (DSB) repair mechanisms. Using parallel in-pool profiling of Cas9-induced insertions and deletions (indels) within a genome-wide knockout library, we present a comprehensive catalog that assesses the influence of nearly every human gene on DSB repair outcomes. This REPAIRome resource uncovers uncharacterized mechanisms, pathways, and factors involved in DSB repair, including opposing roles for XLF and PAXX, a molecular explanation for Cas9-induced multinucleotide insertions, HLTF functions in Cas9-induced DSB repair, the involvement of the SAGA complex in microhomology-mediated end joining, and an indel mutational signature linked to VHL loss, renal carcinoma, and hypoxia. These results exemplify the potential of REPAIRome to drive future discoveries in DSB repair, CRISPR-Cas gene editing and the etiology of cancer mutational signatures.},
}

Humans
*DNA Breaks, Double-Stranded
CRISPR-Cas Systems
*DNA Repair/genetics
Von Hippel-Lindau Tumor Suppressor Protein/genetics
INDEL Mutation
Gene Editing
DNA End-Joining Repair/genetics
Kidney Neoplasms/genetics
DNA-Binding Proteins/genetics
Transcription Factors/genetics
CRISPR-Associated Protein 9
Genome, Human
Gene Knockout Techniques
Carcinoma, Renal Cell/genetics