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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

@article {pmid40056601,
year = {2025},
author = {Wu, SR and Sharpe, J and Tolliver, J and Groth, AJ and Chen, R and Guerra García, ME and Valentine, V and Williams, NT and Jacob, S and Reitman, ZJ},
title = {Combining the RCAS/tv-a retrovirus and CRISPR/Cas9 gene editing systems to generate primary mouse models of diffuse midline glioma.},
journal = {Neoplasia (New York, N.Y.)},
volume = {62},
number = {},
pages = {101139},
doi = {10.1016/j.neo.2025.101139},
pmid = {40056601},
issn = {1476-5586},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Mice ; *Glioma/genetics/pathology/therapy ; *Disease Models, Animal ; *Brain Neoplasms/genetics/pathology/therapy ; Retroviridae/genetics ; Humans ; Tumor Suppressor Protein p53/genetics ; Mice, Transgenic ; Receptors, Virus ; Avian Proteins ; },
abstract = {Diffuse midline gliomas (DMGs) are lethal brain tumors that arise in children and young adults, resulting in a median survival of less than two years. Genetically engineered mouse models (GEMMs) are critical to studying tumorigenesis and tumor-immune interactions, which may inform new treatment approaches. However, current midline glioma GEMM approaches are limited in their ability to multiplex perturbations and/or target specific cell lineages in the brain for genetic manipulation. Here, we combined the RCAS/tv-a avian retrovirus system and CRISPR/Cas9 genetic engineering to drive midline glioma formation in mice. CRISPR/Cas9-based disruption of Trp53, a tumor suppressor that is frequently disrupted in midline gliomas, along with the oncogene PDGF-B resulted in high grade tumor formation with moderate latency (median time to tumor formation of 12 weeks). We confirmed CRISPR-mediated Trp53 disruption using next-generation sequencing (NGS) and immunohistochemistry (IHC). Next, we disrupted multiple midline glioma tumor suppressor genes (Trp53, Pten, Atm, Cdkn2a) in individual mouse brains. These mini-pooled in vivo experiments generated primary midline gliomas with decreased tumor latency (median time to tumor formation of 3.6 weeks, P < 0.0001, log-rank test compared to single-plex gRNA). Quantification of gRNA barcodes and CRISPR editing events revealed that all tumors contained cells with various disruptions of all target genes and suggested a multiclonal origin for the tumors as well as stronger selection for Trp53 disruption compared to disruption of the other genes. This mouse modeling approach will streamline midline glioma research and enable complex experiments to understand tumor evolution and therapeutics.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing
Mice
*Glioma/genetics/pathology/therapy
*Disease Models, Animal
*Brain Neoplasms/genetics/pathology/therapy
Retroviridae/genetics
Humans
Tumor Suppressor Protein p53/genetics
Mice, Transgenic
Receptors, Virus
Avian Proteins

@article {pmid40022904,
year = {2025},
author = {Setyono, ESA and Rogers, NK and Hofmann, A and Lickert, H and Burtscher, I},
title = {Generation of ARX-T2A-H2B-CFP x C-PEP-mCherry-hiPSC double reporter line for monitoring of pancreatic differentiation.},
journal = {Stem cell research},
volume = {84},
number = {},
pages = {103685},
doi = {10.1016/j.scr.2025.103685},
pmid = {40022904},
issn = {1876-7753},
mesh = {*Cell Differentiation ; Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Insulin-Secreting Cells/metabolism/cytology ; Genes, Reporter ; Cell Line ; Islets of Langerhans/metabolism/cytology ; CRISPR-Cas Systems ; Red Fluorescent Protein ; },
abstract = {Pancreatic islets consist of several different endocrine cell types that work in harmony. Aside from primary pancreatic islets, stem cell-derived pancreatic islets can be used as an alternative research and disease model. Here, we introduce a double reporter line of ARX-T2A-H2B-CFP x C-PEP-mCherry-hiPSC through CRISPR/Cas9-mediated insertion of mCherry in the C-terminus of C-Peptide in the previously published ARX-CFP hiPSC line. This reporter line allows live monitoring of stem cell-derived pancreatic alpha and beta cells throughout differentiation.},
}

*Cell Differentiation
Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
Insulin-Secreting Cells/metabolism/cytology
Genes, Reporter
Cell Line
Islets of Langerhans/metabolism/cytology
CRISPR-Cas Systems
Red Fluorescent Protein

@article {pmid40022903,
year = {2025},
author = {Chen, H and Liang, Y and Chen, Y and Liang, Y and Li, X and Duan, C and Cui, Z and Gu, J and Ding, C and Sun, X and Chen, J},
title = {Generation of a human iPSC line with heterozygous PRPF8 c.5792C > T, p. T1931M mutation to model retinitis pigmentosa using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {84},
number = {},
pages = {103689},
doi = {10.1016/j.scr.2025.103689},
pmid = {40022903},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Retinitis Pigmentosa/genetics/pathology ; *CRISPR-Cas Systems/genetics ; *Heterozygote ; Eye Proteins/genetics/metabolism ; Cell Line ; Mutation ; RNA-Binding Proteins ; },
abstract = {Mutations in the PRPF8 gene frequently result in retinitis pigmentosa (RP), an autosomal dominant inherited retinal disease that can lead to nyctalopia and progressive vision loss. Currently, no effective treatment is available. In this study, we used CRISPR/Cas9 technology to introduce a heterozygous point mutation inthe PRPF8 gene of a normal induced pluripotent stem cell (iPSC) line. This mutation mirrors that found in a previously reportedRP patient-derived iPSC line (CSUASOi006-A) from our group. Establishing the PRPF8 gene mutation cell line (CSUASOi012-A-2) provides a valuable cellular resource for studying the pathogenesis of RP.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism
*Retinitis Pigmentosa/genetics/pathology
*CRISPR-Cas Systems/genetics
*Heterozygote
Eye Proteins/genetics/metabolism
Cell Line
Mutation
RNA-Binding Proteins

@article {pmid39988206,
year = {2025},
author = {Rashid, MHO and Kayesh, MEH and Hashem, MA and Hifumi, T and Ogawa, S and Miyoshi, N and Tanaka, Y and Kohara, M and Tsukiyama-Kohara, K},
title = {Adeno-associated virus 2 CRISPR/Cas9-mediated targeting of hepatitis B virus in tree shrews.},
journal = {Virus research},
volume = {354},
number = {},
pages = {199550},
pmid = {39988206},
issn = {1872-7492},
mesh = {Animals ; *Dependovirus/genetics ; *Hepatitis B virus/genetics/physiology ; *CRISPR-Cas Systems ; *Tupaiidae/virology ; *Disease Models, Animal ; *Viral Load ; *Virus Replication ; Hepatitis B/virology/therapy ; DNA, Viral/genetics ; Liver/virology ; Genetic Vectors/genetics ; Tupaia/virology/genetics ; Genetic Therapy/methods ; Hepatitis B, Chronic/virology/therapy ; Humans ; Hepatocytes/virology ; Hepatitis B Core Antigens/genetics/immunology ; },
abstract = {Chronic hepatitis B virus (HBV) infection is a global health issue with limited therapeutic options given the persistence of viral episomal DNA (cccDNA). Previously, we investigated the effects of adeno-associated virus 2 (AAV2) vector-mediated delivery of three guide (g)RNAs/Cas9 selected from 16 gRNAs. AAV2/WJ11-Cas9 effectively suppressed HBV replication in vitro and in humanized chimeric mouse livers. In the present study, we examined the effect of AAV2/WJ11-Cas9 on the acute phase of HBV genotype F infection in an immunocompetent northern tree shrew (Tupaia belangeri; hereafter, "tupaia") model. AAV2/WJ11-Cas9 treatment significantly reduced the HBV viral load in serum at 1, 7, 10, and 14 days post-infection (dpi). HBV-F infection caused enlargement of hepatocytes and mild lymphocytic infiltration in the interlobular connective tissue. Thus, the virus damages hepatocytes and drives infection progression and HBV core antigen (HBcAg) accumulation, which were not observed in AAV2/WJ11-Cas9 treated and normal liver tissues. AAV2/WJ11-Cas9 treatment reduced HBV DNA and cccDNA in liver tissues, as well as serum levels of HBV surface antigen and HBV core-related antigen (HBcrAg), including HBcAg and HBeAg at 14 dpi. Anti-HBc, anti-HBs, and anti-AAV Abs production was also detected. AAV2/WJ11-Cas9 treatment suppressed inflammatory cytokines and TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, and TLR9 mRNA levels. Thus, WJ11/Cas9 delivered by AAV2 vectors may provide a new therapeutic approach for inhibiting HBV infection in immunocompetent animal models, which could be developed for use in humans through further translational research.},
}

Animals
*Dependovirus/genetics
*Hepatitis B virus/genetics/physiology
*CRISPR-Cas Systems
*Tupaiidae/virology
*Disease Models, Animal
*Viral Load
*Virus Replication
Hepatitis B/virology/therapy
DNA, Viral/genetics
Liver/virology
Genetic Vectors/genetics
Tupaia/virology/genetics
Genetic Therapy/methods
Hepatitis B, Chronic/virology/therapy
Humans
Hepatocytes/virology
Hepatitis B Core Antigens/genetics/immunology

@article {pmid39987590,
year = {2025},
author = {Zhang, L and Zhang, F and Yao, M},
title = {CRISPR/Cas9-mediated generation of a homozygous CRB2 knockout H1 human embryonic stem cell line.},
journal = {Stem cell research},
volume = {84},
number = {},
pages = {103677},
doi = {10.1016/j.scr.2025.103677},
pmid = {39987590},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; *Membrane Proteins/genetics/metabolism ; *Homozygote ; Gene Knockout Techniques ; Cell Line ; Cell Differentiation ; Carrier Proteins ; },
abstract = {Mutations in the Crumbs homolog 2 (CRB2) gene cause various autosomal recessive genetic diseases, such as leber congenital amaurosis, retinitis pigmentosa and ventriculomegaly with cystic kidney disease. However, the precise roles of CRB2 in cell fate determination remains unknown. Here, we generated a homozygous CRB2 knockout (CRB2[-/-]) H1 human embryonic stem cells (hESCs) using CRISPR/Cas9 system. This cell line maintained a normal morphology and karyotype, and expressed the pluripotency makers. Importantly, the cell line has the ability to differentiate into three germ layers. The CRB2[-/-] hESCs provide valuable resources for studying the mechanisms of genetic diseases caused by CRB2 mutations.},
}

Humans
*Human Embryonic Stem Cells/metabolism/cytology
*CRISPR-Cas Systems
*Membrane Proteins/genetics/metabolism
*Homozygote
Gene Knockout Techniques
Cell Line
Cell Differentiation
Carrier Proteins

@article {pmid39983483,
year = {2025},
author = {Aygar, S and Daheron, L},
title = {Generation of a human iPSC line with Notch3 R133C mutation by CRISPR/Cas9: A tool for investigating CADASIL and therapeutic targets.},
journal = {Stem cell research},
volume = {84},
number = {},
pages = {103678},
doi = {10.1016/j.scr.2025.103678},
pmid = {39983483},
issn = {1876-7753},
mesh = {Humans ; *Receptor, Notch3/genetics/metabolism ; *CADASIL/genetics/metabolism/pathology ; *Induced Pluripotent Stem Cells/metabolism ; *CRISPR-Cas Systems ; Mutation ; Cell Line ; },
abstract = {Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare neuro vascular disease that is caused by mutations in Notch3. Here, we developed an iPSC line harboring the R133C mutation in Notch3, which is among the most common mutations leading to CADASIL. This mutation alters the disulfide bonding pattern leading to Notch3 protein aggregation, granular osmiophilic material (GOM) formation and vascular changes. The iPSC line was generated using CRISPR/Cas9 and edits were confirmed by PCR and Sanger sequencing. This resource is a valuable tool for studying molecular mechanisms of CADASIL and enabling the development and screening of targeted therapies for Notch3-related pathologies.},
}

Humans
*Receptor, Notch3/genetics/metabolism
*CADASIL/genetics/metabolism/pathology
*Induced Pluripotent Stem Cells/metabolism
*CRISPR-Cas Systems
Mutation
Cell Line

@article {pmid39978205,
year = {2025},
author = {Pan, W and Yang, Y and Zhang, S and Liu, X and Hu, H and Qu, J and Liu, H},
title = {Generation of a homozygous CPAMD8 knockout human embryonic stem cell line (WAe009-A-2R) by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {84},
number = {},
pages = {103683},
doi = {10.1016/j.scr.2025.103683},
pmid = {39978205},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; Homozygote ; Gene Knockout Techniques ; Cell Line ; Cell Differentiation ; Retina/cytology/metabolism ; },
abstract = {CPAMD8, a constituent of the A2M/C3 (α-2-macroglobulin/complement 3) protein family, is strikingly expressed in the human fetal lens and distal neural retina. Mutations in CPAMD8 have been linked to anterior segment dysgenesis and primary congenital glaucoma. We utilized CRISPR/Cas9 technology to establish a homozygous CPAMD8 knockout human embryonic stem cell line for differentiating retinal organoids, with the intent of exploring the role of CPAMD8 in the early development of the human eye. The CPAMD8 knockout cell line exhibits normal morphology, pluripotency, and karyotype, serving as a valuable research tool for investigating the functions of CPAMD8 in ophthalmology.},
}

Humans
*CRISPR-Cas Systems
*Human Embryonic Stem Cells/metabolism/cytology
Homozygote
Gene Knockout Techniques
Cell Line
Cell Differentiation
Retina/cytology/metabolism

@article {pmid39961165,
year = {2025},
author = {Qi, X and Yao, F and Yongli, S and Xiaofen, Z},
title = {Generation of CNPY3 knock out cell line in the H1 (WA01) hESC background.},
journal = {Stem cell research},
volume = {84},
number = {},
pages = {103676},
doi = {10.1016/j.scr.2025.103676},
pmid = {39961165},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *Cell Differentiation ; Cell Line ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {The CNPY3 gene encodes a protein that interacts with members of the toll-like receptor (TLR) protein family and functions as a chaperone, aiding in the proper folding and export of these proteins. We generated a homozygous CNPY3 knockout human embryonic stem cell (hESC) line WAe001-A-2T (H1-CNPY3[-/-]), using CRISPR/Cas9 genome editing technology. The WAe001-A-2T cell line exhibited a normal karyotype and maintained the typical characteristics of undifferentiated hESCs, including pluripotent gene expression and differentiation potential in vivo. The CNPY3 knockout cell line serves as a valuable resource for investigating the role of the CNPY3 gene in embryonic development and lineage differentiation in vitro.},
}

Humans
*Human Embryonic Stem Cells/metabolism/cytology
*Cell Differentiation
Cell Line
Gene Knockout Techniques
CRISPR-Cas Systems

@article {pmid39954549,
year = {2025},
author = {Crossman, VG and Tiong, CF and Coles, CA and Bozaoglu, K and Forbes, R and Yiu, EM and Ruparelia, AA and Currie, PD and Vlahos, K and Howden, SE and North, KN and Lamandé, SR and Houweling, PJ},
title = {Generation of an iPSC line (with isogenic control) from the PBMCs of a COL6A1 (c.1056 + 2T > A) Bethlem myopathy patient.},
journal = {Stem cell research},
volume = {84},
number = {},
pages = {103673},
doi = {10.1016/j.scr.2025.103673},
pmid = {39954549},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Collagen Type VI/metabolism/genetics ; *Leukocytes, Mononuclear/metabolism ; *Contracture/pathology/genetics ; Cell Differentiation ; Muscular Dystrophies/genetics/pathology/congenital ; Cell Line ; CRISPR-Cas Systems ; Mutation ; },
abstract = {To produce an in vitro model of Bethlem myopathy, we reprogrammed the peripheral blood mononuclear cells (PBMCs) of a patient with a heterozygous COL6A1 c.1056 + 2T > A mutation at the exon/intron 14 boundary of the COL6A1 gene to induced pluripotent stem cells (iPSCs). Using CRISPR/Cas9 gene editing, we corrected the mutation to generate an isogenic control line. Both the patient and isogenic control iPSCs show a normal karyotype, express pluripotency markers and can differentiate into cell states that represent the three embryonic germ layers (endoderm, mesoderm and ectoderm). These cell lines will be differentiated and used to explore disease mechanisms and evaluate novel therapeutics for Bethlem myopathy.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism
*Collagen Type VI/metabolism/genetics
*Leukocytes, Mononuclear/metabolism
*Contracture/pathology/genetics
Cell Differentiation
Muscular Dystrophies/genetics/pathology/congenital
Cell Line
CRISPR-Cas Systems
Mutation

@article {pmid39952413,
year = {2025},
author = {Hamze, JG and Cambra, JM and Navarro-Serna, S and Martinez-Serrano, CA},
title = {Navigating gene editing in porcine embryos: Methods, challenges, and future perspectives.},
journal = {Genomics},
volume = {117},
number = {2},
pages = {111014},
doi = {10.1016/j.ygeno.2025.111014},
pmid = {39952413},
issn = {1089-8646},
mesh = {Animals ; *Gene Editing/methods ; Swine ; *CRISPR-Cas Systems ; Embryo, Mammalian/metabolism ; Nuclear Transfer Techniques ; Gene Transfer Techniques ; Animals, Genetically Modified ; },
abstract = {Gene editing technologies, particularly CRISPR/Cas9, have emerged as transformative tools in genetic modification, significantly advancing the use of porcine embryos in biomedical and agricultural research. This review comprehensively examines the various methodologies for gene editing and delivery methods, such as somatic cell nuclear transfer (SCNT), microinjection, electroporation, and lipofection. This review, focuses on the advantages or limitations of using different biological sources (in vivo- vs. in vitro oocytes/embryos). Male germ cell manipulation using sperm-mediated gene transfer (SMGT) and testis-mediated gene transfer (TMGT) represent innovative approaches for producing genetically modified animals. Although these technologies have revolutionized the genetic engineering field, all these strategies face challenges, including high rates of off-target events and mosaicism. This review emphasizes the need to refine these methods, with a focus on reducing mosaicism and improving editing accuracy. Further advancements are essential to unlocking the full potential of gene editing for both agricultural applications and biomedical innovations.},
}

Animals
*Gene Editing/methods
Swine
*CRISPR-Cas Systems
Embryo, Mammalian/metabolism
Nuclear Transfer Techniques
Gene Transfer Techniques
Animals, Genetically Modified

@article {pmid40082754,
year = {2025},
author = {Herr, S and Li, X and Wu, D and Hunter, CT and Magallanes-Lundback, M and Wood, JC and Kaczmar, N and Buell, CR and DellaPenna, D and Gore, MA},
title = {Total tocopherol levels in maize grain depend on chlorophyll biosynthesis within the embryo.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {328},
pmid = {40082754},
issn = {1471-2229},
support = {IOS-1546657//National Science Foundation, United States/ ; IOS-1546657//National Science Foundation, United States/ ; IOS-1546657//National Science Foundation, United States/ ; 1013641//U.S. Department of Agriculture/ ; },
mesh = {*Zea mays/genetics/metabolism ; *Tocopherols/metabolism ; *Chlorophyll/metabolism ; *Seeds/metabolism/genetics ; *Oxidoreductases Acting on CH-CH Group Donors/metabolism/genetics ; Plant Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Edible Grain/metabolism/genetics ; Biosynthetic Pathways ; },
abstract = {BACKGROUND: Tocopherols are a class of lipid-soluble compounds that have multiple functional roles in plants and exhibit vitamin E activity, an essential nutrient for human and animal health. The tocopherol biosynthetic pathway is conserved across the plant kingdom, but source of the key tocopherol pathway precursor, phytol, is unclear. Two protochlorophyllide reductases (POR1 and POR2) were previously identified as loci controlling the natural variation of total tocopherols in maize grain, a non-photosynthetic tissue. POR1 and POR2 are key genes in chlorophyll biosynthesis yet the contribution of the chlorophyll biosynthetic pathway to tocopherol biosynthesis is still not understood.

RESULTS: We took two approaches to alter the activity of these two POR genes within kernel tissue, physiological treatments and CRISPR/Cas9-mediated knockouts, to determine the role of chlorophyll biosynthesis for tocopherol content. Since light is required for POR enzymatic activity, we imposed a dark treatment on developing kernels, which reduced chlorophyll a and tocopherols levels in embryo tissue by 92-99% and 87-90%, respectively, compared to the light treatment. In CRISPR/Cas9-mediated knockouts, the levels of chlorophyll a and tocopherols in embryos of the por1 por2 double homozygous mutant were reduced by 98-100% and 76-83%, respectively, compared to WT.

CONCLUSION: These findings demonstrate that tocopherol synthesis in maize grain depends almost entirely on phytol derived from chlorophyll biosynthesis within the embryo. POR1 and POR2 activity play crucial roles in chlorophyll biosynthesis, underscoring the importance of POR alleles and their activity in the biofortification of vitamin E levels in non-photosynthetic grain of maize.},
}

*Zea mays/genetics/metabolism
*Tocopherols/metabolism
*Chlorophyll/metabolism
*Seeds/metabolism/genetics
*Oxidoreductases Acting on CH-CH Group Donors/metabolism/genetics
Plant Proteins/metabolism/genetics
CRISPR-Cas Systems
Edible Grain/metabolism/genetics
Biosynthetic Pathways

@article {pmid40082732,
year = {2025},
author = {Yook, S and Alper, HS},
title = {Recent advances in genetic engineering and chemical production in yeast species.},
journal = {FEMS yeast research},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsyr/foaf009},
pmid = {40082732},
issn = {1567-1364},
abstract = {Yeasts have emerged as well-suited microbial cell factory for the sustainable production of biofuels, organic acids, terpenoids, and specialty chemicals. This ability is bolstered by advances in genetic engineering tools, including CRISPR-Cas systems and modular cloning in both conventional (Saccharomyces cerevisiae) and non-conventional (Yarrowia lipolytica, Rhodotorula toruloides, Candida krusei) yeasts. Additionally, genome-scale metabolic models (GEMs) and machine learning approaches have accelerated efforts to create a broad range of compounds that help reduce dependency on fossil fuels, mitigate climate change, and offer sustainable alternatives to petrochemical-derived counterparts. In this review, we highlight the cutting-edge genetic tools driving yeast metabolic engineering and then explore the diverse applications of yeast-based platforms for producing value-added products. Collectively, this review underscores the pivotal role of yeast biotechnology in efforts to build a sustainable bioeconomy.},
}

@article {pmid40082551,
year = {2025},
author = {Khalili, S and Mohseninia, A and Liu, C and Banister, CE and Heine, P and Khazan, M and Morrison, SE and Gokare, P and Cowley, GS and Weir, BA and Pocalyko, D and Bachman, KE and Buckhaults, PJ},
title = {Comprehensive genomic dependency landscape of a human colon cancer organoid.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {436},
pmid = {40082551},
issn = {2399-3642},
mesh = {Humans ; *Colonic Neoplasms/genetics/pathology ; *Organoids/metabolism ; Genomics/methods ; CRISPR-Cas Systems ; Mutation ; Precision Medicine/methods ; },
abstract = {Identifying genetic dependencies in human colon cancer could help identify effective treatment strategies. Genome-wide CRISPR-Cas9 dropout screens have the potential to reveal genetic dependencies, some of which could be exploited as therapeutic targets using existing drugs. In this study, we comprehensively characterized genetic dependencies present in a colon cancer organoid avatar, and validated tumor-specific selectivity of select pharmacologic agents. We conducted a genome-wide CRISPR dropout screen to elucidate the genetic dependencies that interacted with select driver somatic mutations. We found distinct genetic dependencies that interacted with WNT, MAPK, PI3K, TP53, and mismatch repair pathways and validated targets that could be exploited as treatments for this specific subtype of colon cancer. These findings demonstrate the utility of functional genomic screening in the context of personalized medicine.},
}

Humans
*Colonic Neoplasms/genetics/pathology
*Organoids/metabolism
Genomics/methods
CRISPR-Cas Systems
Mutation
Precision Medicine/methods

@article {pmid40082411,
year = {2025},
author = {Liu, J and Aliaga Goltsman, DS and Alexander, LM and Khayi, KK and Hong, JH and Dunham, DT and Romano, CA and Temoche-Diaz, MM and Chadha, S and Fregoso Ocampo, R and Oki-O'Connell, J and Janson, OP and Turcios, K and Gonzalez-Osorio, L and Muysson, J and Rahman, J and Laperriere, SM and Devoto, AE and Castelle, CJ and Butterfield, CN and Cost, GJ and Brown, CT and Thomas, BC},
title = {Integration of therapeutic cargo into the human genome with programmable type V-K CAST.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {2427},
pmid = {40082411},
issn = {2041-1723},
mesh = {Humans ; *Gene Editing/methods ; *Genome, Human ; *CRISPR-Cas Systems ; Transposases/metabolism/genetics ; HEK293 Cells ; Transgenes ; DNA/genetics/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of programmable integration of large segments of DNA without creating double-strand breaks. Engineered Cascade CAST function in human cells but are challenging to deploy due to the complexity of the targeting components. Unlike Cascade, which require three Cas proteins, type V-K CAST require a single Cas12k effector for targeting. Here, we show that compact type V-K CAST from uncultivated microbes are repurposable for programmable DNA integration into the genome of human cells. Engineering for nuclear localization and function enables integration of a therapeutically relevant transgene at a safe-harbor site in multiple human cell types. Notably, off-targets are rare events reproducibly found in specific genomic regions. These CAST advancements are expected to accelerate applications of genome editing to therapeutic development, biotechnology, and synthetic biology.},
}

Humans
*Gene Editing/methods
*Genome, Human
*CRISPR-Cas Systems
Transposases/metabolism/genetics
HEK293 Cells
Transgenes
DNA/genetics/metabolism
CRISPR-Associated Proteins/metabolism/genetics

@article {pmid40081781,
year = {2025},
author = {Wu, X and Wan, X and Yu, H and Liu, H},
title = {Recent advances in CRISPR-Cas system for Saccharomyces cerevisiae engineering.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108557},
doi = {10.1016/j.biotechadv.2025.108557},
pmid = {40081781},
issn = {1873-1899},
abstract = {Yeast Saccharomyces cerevisiae (S. cerevisiae) is a crucial industrial platform for producing a wide range of chemicals, fuels, pharmaceuticals, and nutraceutical ingredients. It is also commonly used as a model organism for fundamental research. In recent years, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) system has become the preferred technology for genetic manipulation in S. cerevisiae owing to its high efficiency, precision, and user-friendliness. This system, along with its extensive toolbox, has significantly accelerated the construction of pathways, enzyme optimization, and metabolic engineering in S. cerevisiae. Furthermore, it has allowed researchers to accelerate phenotypic evolution and gain deeper insights into fundamental biological questions, such as genotype-phenotype relationships. In this review, we summarize the latest advancements in the CRISPR-Cas toolbox for S. cerevisiae and highlight its applications in yeast cell factory construction and optimization, enzyme and phenotypic evolution, genome-scale functional interrogation, gene drives, and the advancement of biotechnologies. Finally, we discuss the challenges and potential for further optimization and applications of the CRISPR-Cas system in S. cerevisiae.},
}

@article {pmid40080408,
year = {2025},
author = {Simwela, NV and Jaecklein, E and Sassetti, CM and Russell, DG},
title = {Impaired fatty acid import or catabolism in macrophages restricts intracellular growth of Mycobacterium tuberculosis.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
doi = {10.7554/eLife.102980},
pmid = {40080408},
issn = {2050-084X},
support = {AI155319//National Institute of Allergy and Infectious Diseases/ ; AI162598//National Institute of Allergy and Infectious Diseases/ ; OD032135//NIH Office of the Director/ ; T32AI007349//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Mycobacterium tuberculosis/metabolism/growth & development ; *Fatty Acids/metabolism ; *Macrophages/microbiology/metabolism ; Mice ; Tuberculosis/microbiology/metabolism ; Animals ; Lipid Metabolism ; Autophagy ; Humans ; CRISPR-Cas Systems ; },
abstract = {Mycobacterium tuberculosis (Mtb) infection of macrophages reprograms cellular metabolism to promote lipid retention. While it is clearly known that intracellular Mtb utilize host-derived lipids to maintain infection, the role of macrophage lipid processing on the bacteria's ability to access the intracellular lipid pool remains undefined. We utilized a CRISPR-Cas9 genetic approach to assess the impact of sequential steps in fatty acid metabolism on the growth of intracellular Mtb. Our analyses demonstrate that macrophages that cannot either import, store, or catabolize fatty acids restrict Mtb growth by both common and divergent antimicrobial mechanisms, including increased glycolysis, increased oxidative stress, production of pro-inflammatory cytokines, enhanced autophagy, and nutrient limitation. We also show that impaired macrophage lipid droplet biogenesis is restrictive to Mtb replication, but increased induction of the same fails to rescue Mtb growth. Our work expands our understanding of how host fatty acid homeostasis impacts Mtb growth in the macrophage.},
}

*Mycobacterium tuberculosis/metabolism/growth & development
*Fatty Acids/metabolism
*Macrophages/microbiology/metabolism
Mice
Tuberculosis/microbiology/metabolism
Animals
Lipid Metabolism
Autophagy
Humans
CRISPR-Cas Systems

@article {pmid40078539,
year = {2025},
author = {Sailer, AL and Brendel, J and Chernev, A and König, S and Bischler, T and Gräfenhan, T and Urlaub, H and Gophna, U and Marchfelder, A},
title = {Internal in-frame translation generates Cas11b, which is important for effective interference in an archaeal CRISPR-Cas system.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1543464},
pmid = {40078539},
issn = {1664-302X},
abstract = {CRISPR-Cas is a sophisticated defence system used by bacteria and archaea to fend off invaders. CRISPR-Cas systems vary in their Cas protein composition and have therefore been divided into different classes and types. Type I systems of bacteria have been shown to contain the small protein Cas11 as part of the interference complex known as Cascade. Here we show for the first time that an archaeal CRISPR-Cas type I-B system also contains a homolog of Cas11. The Cas11b protein, encoded by the cas8b gene in Haloferax volcanii, represents the first known case of an internal in-frame translation of an archaeal protein. Translation initiation at an internal methionine of the cas8b open reading frame results in synthesis of Cas11b. Cas11b is required for an effective CRISPR-Cas interference reaction, and in its absence fewer Cascade complexes are formed. Comparison of transcriptomes from wild type and a Cas11b-less strain shows that the depletion of Cas11b also results in differential transcript abundance of many genes, presumably affecting their regulation. Taken together, Cas11b is important for the defence reaction of the type I-B CRISPR-Cas system and seems to play an additional cellular role.},
}

@article {pmid40076976,
year = {2025},
author = {Song, B},
title = {Efforts to Downsize Base Editors for Clinical Applications.},
journal = {International journal of molecular sciences},
volume = {26},
number = {5},
pages = {},
pmid = {40076976},
issn = {1422-0067},
support = {20231291//Soonchunhyang University/ ; 2021R1I1A1A01056885//National Research Foundation of Korea/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Animals ; Dependovirus/genetics ; Genetic Vectors/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Since the advent of the clustered regularly interspaced short palindromic repeats (CRISPR) system in the gene editing field, diverse CRISPR-based gene editing tools have been developed for treating genetic diseases. Of these, base editors (BEs) are promising because they can carry out precise gene editing at single-nucleotide resolution without inducing DNA double-strand breaks (DSBs), which pose significant risks of genomic instability. Despite their outstanding advantages, the clinical application of BEs remains challenging due to their large size, which limits their efficient delivery, particularly in adeno-associated virus (AAV)-based systems. To address this issue, various strategies have been explored to reduce the size of BEs. These approaches include truncating the nonessential domains and replacing the bulky components with smaller substitutes without compromising the editing efficiency. In this review, we highlight the importance of downsizing BEs for therapeutic applications and introduce recent advances in size-reduction strategies. Additionally, we introduce the ongoing efforts to overcome other limitations of BEs, providing insights into their potential for improving in vivo gene editing.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
*Genetic Therapy/methods
Animals
Dependovirus/genetics
Genetic Vectors/genetics
DNA Breaks, Double-Stranded

@article {pmid40076953,
year = {2025},
author = {McCluskey, E and Velli, SK and Kaminski, R and Markward, T and Leming, H and Yu, D and Sajjan, U},
title = {HOXA1 Contributes to Bronchial Epithelial Cell Cycle Progression by Regulating p21/CDKN1A.},
journal = {International journal of molecular sciences},
volume = {26},
number = {5},
pages = {},
pmid = {40076953},
issn = {1422-0067},
support = {1R01HL157258-04A1/NH/NIH HHS/United States ; },
mesh = {*Cyclin-Dependent Kinase Inhibitor p21/metabolism/genetics ; *Homeodomain Proteins/genetics/metabolism ; *Epithelial Cells/metabolism ; *Cell Proliferation ; Humans ; *Bronchi/cytology/metabolism ; *Cell Movement/genetics ; Cell Cycle/genetics ; Transcription Factors/metabolism/genetics ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {Airway basal cells proliferate and regenerate airway epithelium after injury. The first step during airway epithelial repair is airway basal cell proliferation to close the wound. Previously, we demonstrated that homeobox (HOX) A1 expression is reduced in airway stem cells isolated from chronic obstructive pulmonary disease. HOXA1 is a developmental gene and plays a role in hematopoietic stem cell proliferation and differentiation, but its contribution to airway epithelial cell migration and proliferation is not known. In this study, we generated a HOXA1 knockout bronchial epithelial cell line using CRISPR/CAS9 technology followed by clonal expansion to investigate the role of HOXA1 in airway epithelial cell proliferation and migration. Compared to WT, HOXA1 knockout bronchial epithelial cells generated smaller spheroids than WT type cells, indicating a defect in cell proliferation. In the scratch assay, HOXA1 knockout cells showed substantial delay in migrating to the wounded area. By single-cell RNA sequencing and the clustering of cells based on HOXA1 expression, we identified a downregulation of genes involved in cell cycle progression. A cell cycle analysis by flow cytometry indicated partial cell cycle arrest at the G0/G1 phase in HOXA1 knockout cells. This was associated with a reduced expression of Cyclin E1 and an increased expression of the cyclin-dependent kinase inhibitor p21/CDKN1A. These results indicate that HOXA1 may contribute to cell proliferation by regulating cell cycle progression via p21/CDKN1A in airway epithelial cells.},
}

*Cyclin-Dependent Kinase Inhibitor p21/metabolism/genetics
*Homeodomain Proteins/genetics/metabolism
*Epithelial Cells/metabolism
*Cell Proliferation
Humans
*Bronchi/cytology/metabolism
*Cell Movement/genetics
Cell Cycle/genetics
Transcription Factors/metabolism/genetics
Cell Line
CRISPR-Cas Systems

@article {pmid40076923,
year = {2025},
author = {Ajayi, GO and Ma, A and Modarai, SR and Opdenaker, LM and Sims-Mourtada, J},
title = {CRISPR/Cas9 Targeting of Aldehyde Dehydrogenase 1A1 Reveals Heterogeneous Roles in Radiation Response and Redox Stress Across Clonal Lines in Triple-Negative Breast Cancer.},
journal = {International journal of molecular sciences},
volume = {26},
number = {5},
pages = {},
pmid = {40076923},
issn = {1422-0067},
support = {P20GM103446//NIH/NIGMS/ ; },
mesh = {*Triple Negative Breast Neoplasms/genetics/metabolism/pathology ; Humans ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Aldehyde Dehydrogenase 1 Family/genetics/metabolism ; Female ; *Retinal Dehydrogenase/metabolism/genetics ; Reactive Oxygen Species/metabolism ; Oxidation-Reduction ; Radiation Tolerance/genetics ; Oxidative Stress ; Cell Survival/radiation effects/genetics ; },
abstract = {The metabolic enzyme aldehyde dehydrogenase 1A1 (ALDH1A1), a cancer stem cell marker associated with poor outcomes in breast cancer, has emerged as a promising therapeutic target in TNBC. The aim of this study was to investigate the role of ALDH1A1 in radiation resistance and redox stress in triple negative breast cancer (TNBC). Functional knockouts of ALDH1A1 were generated by the CRISPR/Cas9-mediated deletion of ALDH1A1 in the SUM159 cell line, and three distinct clonal populations were isolated. Genetic targeting was confirmed by Sanger sequencing, and the loss of ALDH1A1 protein expression was validated by Western blotting. Functional assays assessed ALDEFLUOR activity, cell viability, self-renewal capacity, and reactive oxygen species (ROS) levels with or without radiation in both the bulk population and clonal lines. Interestingly, ALDEFLUOR activity was uniformly lost across all clonal lines; however, functional effects of ALDH1A1 loss on redox stress, survival, and radiation sensitivity were observed in only one clonal population. These findings highlight significant variability in the role of ALDH1A1 among clonal populations, reflecting the complexity of tumor heterogeneity. This underscores the importance of accounting for tumor heterogeneity when targeting ALDH1A1, as certain TNBC subpopulations may rely more heavily on ALDH1A1 function. These insights are critical for developing effective ALDH1A1-targeted therapies.},
}

*Triple Negative Breast Neoplasms/genetics/metabolism/pathology
Humans
*CRISPR-Cas Systems
Cell Line, Tumor
*Aldehyde Dehydrogenase 1 Family/genetics/metabolism
Female
*Retinal Dehydrogenase/metabolism/genetics
Reactive Oxygen Species/metabolism
Oxidation-Reduction
Radiation Tolerance/genetics
Oxidative Stress
Cell Survival/radiation effects/genetics

@article {pmid40076628,
year = {2025},
author = {Mosterd, C and Moineau, S},
title = {Insight into crRNA Processing in Streptococcus mutans P42S and Application of SmutCas9 in Genome Editing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {5},
pages = {},
pmid = {40076628},
issn = {1422-0067},
support = {NSERC Discovery//Natural Sciences and Engineering Research Council/ ; },
mesh = {*Streptococcus mutans/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Genome, Bacterial ; Bacterial Proteins/genetics/metabolism ; RNA, Bacterial/genetics/metabolism ; },
abstract = {CRISPR-Cas is an adaptive immune system found in bacteria and archaea that provides resistance against invading nucleic acids. Elements of this natural system have been harnessed to develop several genome editing tools, including CRISPR-Cas9. This technology relies on the ability of the nuclease Cas9 to cut DNA at specific locations directed by a guide RNA. In addition, the nuclease activity of Cas9 requires the presence of a short nucleotide motif (5'-NGG-3' for Cas9 from Streptococcus pyogenes) called PAM, flanking the targeted region. As the reliance on this PAM is typically strict, diverse Cas9 variants recognising different PAM motifs have been studied to target a broader range of genomic sites. In this study, we assessed the potential of Cas9 from Streptococcus mutans strain P42S (SmutCas9) in gene editing. SmutCas9 recognises the rarely targeted 5'-NAA-3' and 5'-NGAA-3' PAMs. To test its efficacy, two genes of the virulent lactococcal phage p2 were edited, thereby demonstrating the potential of SmutCas9 for gene editing purposes, particularly in AT-rich genomes. Sequencing of total RNA also revealed the RNA components of this system, allowing further molecular characterisation of the type II-A CRISPR-Cas system of S. mutans.},
}

*Streptococcus mutans/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
CRISPR-Associated Protein 9/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Genome, Bacterial
Bacterial Proteins/genetics/metabolism
RNA, Bacterial/genetics/metabolism

@article {pmid40015341,
year = {2025},
author = {Alok, A and Chauhan, H and Rout, B and Pandey, A and Singh, K},
title = {CRISPR/dCas9-KRAB mediated transcriptional suppression of NtbHLH47 enhances tolerance to iron stress and modulates iron content in tobacco.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {354},
number = {},
pages = {112449},
doi = {10.1016/j.plantsci.2025.112449},
pmid = {40015341},
issn = {1873-2259},
mesh = {*Nicotiana/genetics/metabolism ; *Iron/metabolism ; *Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics ; Homeostasis ; },
abstract = {Iron homeostasis is a multifaceted regulatory process that needs to be studied to elucidate iron distribution, uptake, and storage in plants. NtbHLH47, a homologue to AtbHLH47, is a negative regulator of iron. The current study deploys CRISPR interference-dCas9-KRAB (Krüppel-associated box) in the transcriptional suppression of NtbHLH47 and its effect on iron uptake by plants. The pHSN6I01 harbouring dCas9-KRAB and gRNA targeting NtbHHLH47 was constructed. Four gRNAs were designed, G1, G2, G3, and G4, located at + 19, + 111, + 232, and + 335 bp upstream from the ATG start codon in the promoter region of NtbHLH47. The NtbHLH47 was repressed in the developed transgenic lines of tobacco and the qRT-PCR analysis showed that target sites G1 and G2 suppressed NtbHLH47 effectively. The transgenic pHSN6I01 +G1 plants were tolerant to the elevated levels of iron, copper, zinc, and magnesium. The root Ferric chelate reductase activity of pHSN6I01 +G1 lines was reduced against wild type. The Perl staining showed high iron content in the roots of the pHSN6I01 +G1 plants. ICP-MS analysis showed increased Fe content in the roots of pHSN6I01 +G1 line suggesting that NtbHLH47 modulates it. The expression of NtbHLH38, NtbHLH100, NtbHLH101, and NtFIT was found to be upregulated in the pHSN6I01 +G1 line. This is the first report of using CRISPRi based on dCas9-KRAB in tobacco and its application in the functional validation of a gene. Using this, NtbHLH47 was transcriptionally suppressed and the generated lines expressed increased levels of iron in the roots of N. tabacum and gave insight in the iron homeostasis.},
}

*Nicotiana/genetics/metabolism
*Iron/metabolism
*Plants, Genetically Modified/genetics
CRISPR-Cas Systems
Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Stress, Physiological/genetics
Homeostasis

@article {pmid40007451,
year = {2025},
author = {Li, D and Cheng, W and Yin, F and Yao, Y and Wang, Z and Xiang, Y},
title = {A sensitive miRNA detection method based on a split-T7 switch modulating CRISPR/Cas12a system.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {23},
pages = {4555-4558},
doi = {10.1039/d5cc00170f},
pmid = {40007451},
issn = {1364-548X},
mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Promoter Regions, Genetic ; Cell Line, Tumor ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study presents a novel method for sensitive miRNA detection based on a split-T7 switch modulating CRISPR/Cas12a system. By integrating the split-T7 promoter-mediated transcription with the CRISPR/Cas12a system, this method can achieve femtomolar detection of the target miRNA within 1 h and successfully analyze miR-21 in samples from various cell lines, demonstrating its potential for clinical applications.},
}

*MicroRNAs/analysis/genetics
*CRISPR-Cas Systems/genetics
Humans
Promoter Regions, Genetic
Cell Line, Tumor
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39999849,
year = {2025},
author = {Fallon, TK and Knouse, KA},
title = {A roadmap toward genome-wide CRISPR screening throughout the organism.},
journal = {Cell genomics},
volume = {5},
number = {3},
pages = {100777},
doi = {10.1016/j.xgen.2025.100777},
pmid = {39999849},
issn = {2666-979X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome/genetics ; },
abstract = {Genome-wide CRISPR screening in the organism has tremendous potential to answer long-standing questions of mammalian physiology and disease. However, bringing this powerful technology in vivo presents unique challenges, including delivering a genome-wide sgRNA library to the appropriate cell type, achieving sufficient coverage of the library, and selecting for the phenotype of interest. In this review, we highlight recent advances in sgRNA delivery, library design, and phenotypic readout that can help overcome these technical challenges and thereby bring high-throughput genetic dissection to an increasing number of tissues and questions. We are excited about the potential for ongoing innovation in these areas to ultimately enable genome-wide CRISPR screening in any cell type of interest in the organism, allowing for unprecedented investigation into diverse questions of mammalian physiology and disease.},
}

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

@article {pmid39993709,
year = {2025},
author = {Pilz, RA and Skowronek, D and Bonde, LD and Kałużewski, T and Schamuhn, OJ and Busch, R and Gach, A and Rath, M and Steinhagen-Thiessen, E and Felbor, U},
title = {Oxford Nanopore long-read sequencing with CRISPR/Cas9-mediated target selection for accurate characterization of copy number variants in the LDLR gene.},
journal = {European journal of medical genetics},
volume = {74},
number = {},
pages = {105003},
doi = {10.1016/j.ejmg.2025.105003},
pmid = {39993709},
issn = {1878-0849},
mesh = {Humans ; *Receptors, LDL/genetics ; *DNA Copy Number Variations ; *CRISPR-Cas Systems ; *Hyperlipoproteinemia Type II/genetics ; *Nanopore Sequencing/methods ; Female ; Male ; Sequence Analysis, DNA/methods ; },
abstract = {INTRODUCTION: Familial hypercholesterolemia (FH) affects around 1 in 250 people. Most FH cases are caused by pathogenic LDLR variants, with copy number variations (CNVs) accounting for about 10 %. However, short-read gene panel sequencing and multiplex ligation-dependent probe amplification (MLPA) are limited in the specification of CNV breakpoints and the identification of complex structural variants (SVs).

MATERIALS AND METHODS: We designed crRNAs for Cas9-mediated target selection of LDLR and performed long-read sequencing (LRS) on an Oxford Nanopore MinION device using high-molecular-weight (HMW) DNA or DNA from standard purification. After establishing the LRS approach, we characterized two known LDLR CNVs and tested two individuals with strong clinical evidence of FH but no pathogenic variant in short-read gene panel sequencing.

RESULTS: Complete coverage of LDLR was achieved for both HMW DNA and DNA from standard purification. LRS allowed us to specify CNV breakpoints and showed that the known LDLR deletion is 19.2 kb in size encompassing exons 1-2 and the 5'-untranslated and promoter regions. Furthermore, LRS verified the in tandem localization of a large LDLR duplication covering exons 4-8. Both CNVs were classified as loss-of-function. Moreover, breakpoint information enabled confirmatory analysis by PCR and Sanger sequencing for both CNVs. No SVs were detected in two apparently mutation-negative FH probands using our approach.

CONCLUSIONS: Nanopore LRS with CRISPR/Cas9-mediated target selection allows for accurate characterization of CNVs and can therefore serve as a complementary method to short-read sequencing-based FH diagnostics by facilitating variant interpretation and enabling cost-effective PCR-based variant confirmation in subsequent familial analyses.},
}

Humans
*Receptors, LDL/genetics
*DNA Copy Number Variations
*CRISPR-Cas Systems
*Hyperlipoproteinemia Type II/genetics
*Nanopore Sequencing/methods
Female
Male
Sequence Analysis, DNA/methods

@article {pmid39964768,
year = {2025},
author = {Gibson, WT and Lengyell, TC and Korecki, AJ and Janssen, SM and Adair, BA and Gamu, D and Lorincz, MC and Simpson, EM},
title = {Minimally Humanized Ezh2 Exon-18 Mouse Cell Lines Validate Preclinical CRISPR/Cas9 Approach to Treat Weaver Syndrome.},
journal = {Human gene therapy},
volume = {36},
number = {5-6},
pages = {618-627},
doi = {10.1089/hum.2024.170},
pmid = {39964768},
issn = {1557-7422},
mesh = {*Enhancer of Zeste Homolog 2 Protein/genetics/metabolism ; Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Hand Deformities, Congenital/genetics/therapy ; *Abnormalities, Multiple/genetics/therapy ; Exons ; Cell Line ; Craniofacial Abnormalities/genetics/therapy ; Genetic Therapy/methods ; Congenital Hypothyroidism ; },
abstract = {Weaver syndrome is a rare neurodevelopmental disorder that encompasses macrocephaly, tall stature, obesity, brain anomalies, intellectual disability, and increased susceptibility to cancer. This dominant monogenic disorder is caused by germline variants in enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a key epigenetic writer. Unfortunately, there are no effective treatments for Weaver syndrome. However, preclinical results support the potential for therapeutic gains, despite the prenatal onset. Thus, for the first time, we tested whether CRISPR/Cas9 gene-editing strategies may be able to "correct" a Weaver syndrome variant at the DNA level. We initiated these preclinical studies by humanizing the region surrounding the most-common recurring patient-variant location in mouse embryonic stem cells (ESCs). Humanization ensures that DNA-binding strategies will be directly translatable to human cells and patients. We then introduced into ESCs the humanized region, but now carrying the Weaver syndrome EZH2 variant c.2035C>T p.Arg684Cys, and characterized the enzymatic properties of this missense variant. Our data showed a significant and dramatic reduction in EZH2-enzymatic activity, supporting previous cell-free studies of this variant as well as in vitro and in vivo mouse work by other teams. Intriguingly, this most-common variant does not create a complete loss-of-function, but rather is a hypomorphic allele. Together with prior reports describing hypomorphic effects of missense EZH2 variants, these results demonstrate that the etiology of Weaver syndrome does not require complete loss of EZH2 enzymatic activity. Toward therapy, we tested four CRISPR gene-editing strategies. We demonstrated that Streptococcus pyogenes Cas9 (SpCas9) showed the highest variant correction (70.5%), but unfortunately also the highest alteration of the nonvariant allele (21.1-26.2%), an important consideration for gene-editing treatment of a dominant syndrome. However, Staphylococcus aureus Cas9 (SaCas9) gave a variant correction (52.5%) that was not significantly different than SpCas9, and encouragingly the lowest alteration of the nonvariant allele (2.0%). Thus, the therapeutic strategy using the small SaCas9 enzyme, a size that allows flexibility in therapeutic delivery, was the most optimal for targeting the Weaver syndrome EZH2 variant c.2035C>T p.Arg684Cys.},
}

*Enhancer of Zeste Homolog 2 Protein/genetics/metabolism
Animals
Mice
*CRISPR-Cas Systems
*Gene Editing
Humans
*Hand Deformities, Congenital/genetics/therapy
*Abnormalities, Multiple/genetics/therapy
Exons
Cell Line
Craniofacial Abnormalities/genetics/therapy
Genetic Therapy/methods
Congenital Hypothyroidism

@article {pmid39893892,
year = {2025},
author = {Zheng, L and Zheng, C and Wang, W and Huang, F and Jiang, Y and Lu, J and Lou, Y},
title = {A CRISPR/Cas12a-based colorimetric AuNPs biosensor for naked-eye detection of pathogenic bacteria in clinical samples.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {250},
number = {},
pages = {114541},
doi = {10.1016/j.colsurfb.2025.114541},
pmid = {39893892},
issn = {1873-4367},
mesh = {*Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Colorimetry/methods ; *Biosensing Techniques/methods ; *Pseudomonas aeruginosa/isolation & purification/genetics ; Humans ; *CRISPR-Cas Systems ; DNA, Single-Stranded/chemistry ; Pseudomonas Infections/diagnosis/microbiology ; Limit of Detection ; },
abstract = {Pathogenic bacteria, such as Pseudomonas aeruginosa, pose significant threats to public health due to their multidrug resistance and association with severe infections. Rapid and reliable detection methods are crucial for timely treatment and effective infection control, especially in resource-limited settings. In this study, we developed a CRISPR/Cas12a-based colorimetric biosensor that leverages Cas12a's trans-cleavage activity to release left single-stranded DNA (lDNA). The released lDNA facilitates hybridization with clDNA-functionalized gold nanoparticles (AuNPs), resulting in a visible color change. The biosensor achieved a detection limit of 10[0] CFU/reaction for P. aeruginosa within 2 hours, with excellent specificity and robustness, as validated in spiked sputum and blood samples. Clinical testing using 32 blood samples (13 positive, 19 negative) confirmed its high diagnostic accuracy, achieving an AUC of 1 in ROC curve analysis. The platform's simplicity, robustness, and programmability suggest its broad potential for rapid infectious disease diagnostics, particularly in low-resource settings.},
}

*Gold/chemistry
*Metal Nanoparticles/chemistry
*Colorimetry/methods
*Biosensing Techniques/methods
*Pseudomonas aeruginosa/isolation & purification/genetics
Humans
*CRISPR-Cas Systems
DNA, Single-Stranded/chemistry
Pseudomonas Infections/diagnosis/microbiology
Limit of Detection

@article {pmid39611441,
year = {2025},
author = {Zhang, Q and Chen, H and Li, Z and Qiao, J and Liu, P and Zheng, C and Deng, Z and Li, X and Zhang, H},
title = {Bdyof is a Y-chromosome-specific gene required for male development in Bactrocera dorsalis.},
journal = {Pest management science},
volume = {81},
number = {4},
pages = {1785-1793},
doi = {10.1002/ps.8577},
pmid = {39611441},
issn = {1526-4998},
support = {32220103009//National Natural Science Foundation of China/ ; //China Agriculture Research System of MOF and MARA (CARS-26)/ ; },
mesh = {Animals ; Male ; *Tephritidae/genetics/growth & development ; *Y Chromosome/genetics ; *Insect Proteins/genetics/metabolism ; Testis/metabolism/growth & development ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: In many organisms, the Y chromosome contains important genes associated with sex determination and male reproductive development. However, there have been few studies of Y-chromosome-specific genes in non-model species due to the incomplete information of Y chromosome genome and difficulty in sequencing. Here, we screened 90 candidate Y-specific sequences in a constructed transcriptome assembly library by using the chromosome quotient method, among which 11 were unreported sequences associated with male reproductive development, including Bactrocera dorsalis Y-specific Oligozoospermia factor (Bdyof) with the highest expression in the testis.

RESULTS: CRISPR/Cas9-mediated knockout of Bdyof resulted in abnormal testis development, significantly reduced sperm count, and obviously lower egg hatching rate in homozygous mutant flies. In addition, Bdyof knockout decreased the expression of dsx-M.

CONCLUSION: This results provides new insights into the biological processes related to male reproductive development controlled by the Y-chromosome-specific gene Bdyof, thus providing a promising molecular target for the study of agricultural pests. © 2024 Society of Chemical Industry.},
}

Animals
Male
*Tephritidae/genetics/growth & development
*Y Chromosome/genetics
*Insect Proteins/genetics/metabolism
Testis/metabolism/growth & development
CRISPR-Cas Systems

@article {pmid40076433,
year = {2025},
author = {Lynch, CRH and Drummond, RSM and Jelley, L and Baker, L and Smit, E and Fleming, R and Billington, C},
title = {Optimization and Benchmarking of RT-LAMP-CRISPR-Cas12a for the Detection of SARS-CoV-2 in Saliva.},
journal = {International journal of molecular sciences},
volume = {26},
number = {5},
pages = {},
doi = {10.3390/ijms26051806},
pmid = {40076433},
issn = {1422-0067},
support = {C03X1904//Ministry of Business, Innovation and Employment/ ; ESRCBRF2022/1//Ministry of Business, Innovation and Employment/ ; },
mesh = {Humans ; *SARS-CoV-2/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Saliva/virology ; *COVID-19/virology/diagnosis ; *Molecular Diagnostic Techniques/methods/standards ; Sensitivity and Specificity ; Benchmarking ; RNA, Viral/genetics/analysis ; Endodeoxyribonucleases/genetics/metabolism ; COVID-19 Nucleic Acid Testing/methods ; Coronavirus Envelope Proteins/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Resource-limited settings and supply chain difficulties faced throughout the COVID-19 pandemic prompted the development of rapid and alternative methods of detecting SARS-CoV-2. These methods include reverse-transcription loop-mediated isothermal amplification (RT-LAMP), reverse-transcription recombinase polymerase amplification (RT-RPA), and CRISPR-Cas12a fluorescence detection. We describe RT-LAMP, RT-RPA, and CRISPR-Cas12a assays for the detection of the N and E-gene amplicons of SARS-CoV-2 and the optimization of various assay components, including incubation temperatures, Cas12a enzymes, reporter molecules, and the use of a lyophilized RT-LAMP master mix. We also describe the testing of a one-tube RT-LAMP-CRISPR-Cas12a assay. The one-tube assay showed promise in reducing hands-on time and improving time-to-result. We found no improvements in assay sensitivity with RT-RPA, but did achieve detection at a lower copy number with the lyophilized RT-LAMP master mix compared to liquid reagent (50 vs. 100 copies at 20 min). When used to detect the presence of SARS-CoV-2 RNA in clinical saliva samples from 75 infected patients, the discriminatory ability of the optimized RT-LAMP-CRISPR Cas12a assay was found to be comparable with RT-qPCR, with a minor reduction in sensitivity.},
}

Humans
*SARS-CoV-2/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
*Saliva/virology
*COVID-19/virology/diagnosis
*Molecular Diagnostic Techniques/methods/standards
Sensitivity and Specificity
Benchmarking
RNA, Viral/genetics/analysis
Endodeoxyribonucleases/genetics/metabolism
COVID-19 Nucleic Acid Testing/methods
Coronavirus Envelope Proteins/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40076283,
year = {2025},
author = {Hofacker, DT and Kalkuhl, S and Schmid, JF and Singh, S and Stafforst, T},
title = {A Simplified Guide RNA Synthesis Protocol for SNAP- and Halo-Tag-Based RNA Editing Tools.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {5},
pages = {},
doi = {10.3390/molecules30051049},
pmid = {40076283},
issn = {1420-3049},
mesh = {*RNA Editing ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Adenosine Deaminase/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {SNAP-tag and Halo-tag have been employed to achieve targeted RNA editing by directing the deaminase domain of human ADAR to specific sites in the transcriptome. This targeting is facilitated by short guide RNAs (gRNAs) complementary to the target transcript, which are chemically modified with benzylguanine or chloroalkane moieties to enable covalent binding to the respective self-labeling enzymes. However, broad application of this approach has been limited by challenges such as low scalability, the requirement for specialized chemical expertise and equipment, and labor-intensive protocols. In this study, we introduce streamlined, efficient protocols for the synthesis and purification of these linkers, suitable for SNAP-tag and Halo-tag applications, without the need for advanced chemical equipment. Our methods enable linker coupling in a kit-like manner and support the high-yield production of modified gRNAs. We demonstrate that the newly synthesized linkers and gRNA designs perform similarly to previously published constructs with regard to RNA editing efficiency. Moreover, large-scale production of modified gRNAs facilitates their use in studies involving cellular uptake and in vivo experiments.},
}

*RNA Editing
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
Adenosine Deaminase/metabolism/genetics
RNA-Binding Proteins/metabolism/genetics

@article {pmid40075056,
year = {2025},
author = {Yang, J and Wang, T and Huang, Y and Long, Z and Li, X and Zhang, S and Zhang, L and Liu, Z and Zhang, Q and Sun, H and Zhang, M and Yin, H and Liu, Z and Zhang, H},
title = {Insights into the compact CRISPR-Cas9d system.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {2462},
pmid = {40075056},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; *Cryoelectron Microscopy ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; Gene Editing/methods ; DNA/metabolism/chemistry/genetics ; Models, Molecular ; DNA Cleavage ; Humans ; },
abstract = {Cas9d, the smallest known member of the Cas9 family, employs a compact domain architecture for effective target cleavage. However, the underlying mechanism remains unclear. Here, we present the cryo-EM structures of the Cas9d-sgRNA complex in both target-free and target-bound states. Biochemical assays elucidated the PAM recognition and DNA cleavage mechanisms of Cas9d. Structural comparisons revealed that at least 17 base pairs in the guide-target heteroduplex is required for nuclease activity. Beyond its typical role as an adaptor between Cas9 enzymes and targets, the sgRNA also provides structural support and functional regulation for Cas9d. A segment of the sgRNA scaffold interacts with the REC domain to form a functional target recognition module. Upon target binding, this module undergoes a coordinated conformational rearrangement, enabling heteroduplex propagation and facilitating nuclease activity. This hybrid functional module precisely monitors heteroduplex complementarity, resulting in a lower mismatch tolerance compared to SpyCas9. Moreover, structure-guided engineering in both the sgRNA and Cas9d protein led to a more compact Cas9 system with well-maintained nuclease activity. Altogether, our findings provide insights into the target recognition and cleavage mechanisms of Cas9d and shed light on the development of high-fidelity mini-CRISPR tools.},
}

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

@article {pmid40073844,
year = {2025},
author = {Bi, K and Yates, KB},
title = {Parts and ICRAFTs: Finding new immunotherapy targets.},
journal = {Immunity},
volume = {58},
number = {3},
pages = {529-531},
doi = {10.1016/j.immuni.2025.02.016},
pmid = {40073844},
issn = {1097-4180},
mesh = {Humans ; *Immunotherapy/methods ; *Tumor Necrosis Factor alpha-Induced Protein 3/genetics ; *Neoplasms/immunology/therapy ; Animals ; CRISPR-Cas Systems ; },
abstract = {CRISPR screens are widely utilized to identify genes that regulate immune function or mediate sensitivity of cancer cells to immune attack. In this issue of Immunity, Zeng et al. present a computational framework for uncovering gene targets with dual function in both cancer and immune cells and nominate TNFAIP3 as a synergistic target whose ablation strongly elicits an antitumor response.},
}

Humans
*Immunotherapy/methods
*Tumor Necrosis Factor alpha-Induced Protein 3/genetics
*Neoplasms/immunology/therapy
Animals
CRISPR-Cas Systems

@article {pmid40073141,
year = {2025},
author = {Chiang, JC and Shang, Z and Rosales, T and Cai, L and Chen, WM and Cai, F and Vu, H and Minna, JD and Ni, M and Davis, AJ and Timmerman, RD and DeBerardinis, RJ and Zhang, Y},
title = {Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair.},
journal = {Science advances},
volume = {11},
number = {11},
pages = {eadt1241},
doi = {10.1126/sciadv.adt1241},
pmid = {40073141},
issn = {2375-2548},
mesh = {Humans ; *Lung Neoplasms/genetics/metabolism/pathology/drug therapy/radiotherapy ; *Lipoylation ; Cell Line, Tumor ; Animals ; *Recombinational DNA Repair/drug effects ; Mice ; DNA Damage ; Carcinoma, Non-Small-Cell Lung/genetics/metabolism/drug therapy/pathology/radiotherapy ; Ataxia Telangiectasia Mutated Proteins/metabolism/antagonists & inhibitors/genetics ; Lysine Acetyltransferase 5/metabolism/genetics ; Acyltransferases/antagonists & inhibitors/genetics/metabolism ; CRISPR-Cas Systems ; Xenograft Model Antitumor Assays ; },
abstract = {Lung cancer exhibits altered metabolism, influencing its response to radiation. To investigate the metabolic regulation of radiation response, we conducted a comprehensive, metabolic-wide CRISPR-Cas9 loss-of-function screen using radiation as selection pressure in human non-small cell lung cancer. Lipoylation emerged as a key metabolic target for radiosensitization, with lipoyltransferase 1 (LIPT1) identified as a top hit. LIPT1 covalently conjugates mitochondrial 2-ketoacid dehydrogenases with lipoic acid, facilitating enzymatic functions involved in the tricarboxylic acid cycle. Inhibiting lipoylation, either through genetic LIPT1 knockout or a lipoylation inhibitor (CPI-613), enhanced tumor control by radiation. Mechanistically, lipoylation inhibition increased 2-hydroxyglutarate, leading to H3K9 trimethylation, disrupting TIP60 recruitment and ataxia telangiectasia mutated (ATM)-mediated DNA damage repair signaling, impairing homologous recombination repair. In summary, our findings reveal a critical role of LIPT1 in regulating DNA damage and chromosome stability and may suggest a means to enhance therapeutic outcomes with DNA-damaging agents.},
}

Humans
*Lung Neoplasms/genetics/metabolism/pathology/drug therapy/radiotherapy
*Lipoylation
Cell Line, Tumor
Animals
*Recombinational DNA Repair/drug effects
Mice
DNA Damage
Carcinoma, Non-Small-Cell Lung/genetics/metabolism/drug therapy/pathology/radiotherapy
Ataxia Telangiectasia Mutated Proteins/metabolism/antagonists & inhibitors/genetics
Lysine Acetyltransferase 5/metabolism/genetics
Acyltransferases/antagonists & inhibitors/genetics/metabolism
CRISPR-Cas Systems
Xenograft Model Antitumor Assays

@article {pmid40073054,
year = {2025},
author = {Butterfield, GL and Rohm, D and Roberts, A and Nethery, MA and Rizzo, AJ and Morone, DJ and Garnier, L and Iglesias, N and Barrangou, R and Gersbach, CA},
title = {Characterization of diverse Cas9 orthologs for genome and epigenome editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {11},
pages = {e2417674122},
doi = {10.1073/pnas.2417674122},
pmid = {40073054},
issn = {1091-6490},
support = {U01AI146356//HHS | NIH (NIH)/ ; UM1HG012053//HHS | NIH (NIH)/ ; R01MH125236//HHS | NIH (NIH)/ ; RM1HG011123//HHS | NIH (NIH)/ ; EFMA-1830957//NSF (NSF)/ ; HR0011-19-2-0008//DOD | ARPA | Defense Sciences Office, DARPA (DSO)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Epigenome ; Streptococcus pyogenes/genetics ; Staphylococcus aureus/genetics ; HEK293 Cells ; Genome, Human ; },
abstract = {CRISPR-Cas9 systems have revolutionized biotechnology, creating diverse new opportunities for biomedical research and therapeutic genome and epigenome editing. Despite the abundance of bacterial CRISPR-Cas9 systems, relatively few are effective in human cells, limiting the overall potential of CRISPR technology. To expand the CRISPR-Cas toolbox, we characterized a set of type II CRISPR-Cas9 systems from select bacterial genera and species encoding diverse Cas9s. Four systems demonstrated robust and specific gene repression in human cells when used as nuclease-null dCas9s fused with a KRAB domain and were also highly active nucleases in human cells. These systems have distinct protospacer adjacent motifs (PAMs), including AT-rich motifs and sgRNA features orthogonal to the commonly used Staphylococcus aureus and Streptococcus pyogenes Cas9s. Additionally, we assessed gene activation when fused with the p300 catalytic domain. Notably, S. uberis Cas9 performed competitively against benchmarks with promising repression, activation, nuclease, and base editing activity. This study expands the CRISPR-Cas9 repertoire, enabling effective genome and epigenome editing for diverse applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans
*CRISPR-Associated Protein 9/metabolism/genetics
*Epigenome
Streptococcus pyogenes/genetics
Staphylococcus aureus/genetics
HEK293 Cells
Genome, Human

@article {pmid40072114,
year = {2025},
author = {Wu, J and Lu, K and Xie, R and Zhu, C and Luo, Q and Liang, XF},
title = {The Beneficial Role of the Thyroid Hormone Receptor Beta 2 (thrb2) in Facilitating the First Feeding and Subsequent Growth in Medaka as Fish Larval Model.},
journal = {Cells},
volume = {14},
number = {5},
pages = {},
doi = {10.3390/cells14050386},
pmid = {40072114},
issn = {2073-4409},
support = {2023YFD2400600//the National Key R&D Program of China/ ; 2024YFD2400102//the National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Thyroid Hormone Receptors beta/metabolism/genetics ; *Oryzias/metabolism/genetics/growth & development ; *Larva/metabolism/growth & development/genetics ; CRISPR-Cas Systems/genetics ; Feeding Behavior ; Retina/metabolism/growth & development ; Gene Knockout Techniques ; },
abstract = {During the early growth stages of fish larvae, there are significant challenges to their viability, so improving their visual environment is essential to promoting their growth and survival. Following the successful knockout of thyroid hormone receptor beta 2 (thrb2) using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology, there was an increase in the expression of UV opsin (short-wave-sensitive 1, sws1), while the expression of other cone opsins was significantly decreased. Further analysis of the retinal structure demonstrated that the thrb2 knockout resulted in an increased lens thickness and a decreased thickness of the ganglion cell layer (GCL), outer plexiform layer (OPL), and outer nuclear layer (ONL) in the retina. The slowing down of swimming speed under light conditions in thrb2[-/-] may be related to the decreased expression of phototransduction-related genes such as G protein-coupled receptor kinase 7a (grk7a), G protein-coupled receptor kinase 7b (grk7b), and phosphodiesterase 6c (pde6c). Notably, thrb2[-/-] larvae exhibited a significant increase in the amount and proportion of first feeding, and their growth rate significantly exceeded that of wild-type controls during the week after feeding. This observation suggests that although the development of the retina may be somewhat affected, thrb2[-/-] larvae show positive changes in feeding behaviour and growth rate, which may be related to their enhanced ability to adapt to their environment. These results provide novel insights into the function of the thrb2 gene in the visual system and behaviour and may have implications in areas such as fish farming and genetic improvement.},
}

Animals
*Thyroid Hormone Receptors beta/metabolism/genetics
*Oryzias/metabolism/genetics/growth & development
*Larva/metabolism/growth & development/genetics
CRISPR-Cas Systems/genetics
Feeding Behavior
Retina/metabolism/growth & development
Gene Knockout Techniques

@article {pmid39950863,
year = {2025},
author = {Sun, P and Wang, S and Yan, Q and Zeng, J and Wu, Z and Qi, X},
title = {Non-nuclear localization signal-guided CRISPR/Cas9 ribonucleoproteins for translocation and gene editing via apoferritin delivery vectors.},
journal = {Nanoscale},
volume = {17},
number = {11},
pages = {6660-6675},
doi = {10.1039/d4nr04762a},
pmid = {39950863},
issn = {2040-3372},
mesh = {*Apoferritins/chemistry ; Humans ; *CRISPR-Cas Systems ; *Gene Editing ; Animals ; *Nuclear Localization Signals/chemistry ; Ribonucleoproteins/chemistry/metabolism/genetics ; HeLa Cells ; Cell Line, Tumor ; Mice ; Doxorubicin/chemistry/pharmacology ; Mice, Nude ; Cell Nucleus/metabolism ; Nanoparticles/chemistry ; },
abstract = {Direct delivery of the Cas9/sgRNA ribonucleoprotein (RNP) via appropriate carriers has been proved to be an important advance for the in vivo translocation and gene editing of CRISPR/Cas9. These carriers often require the nuclear localization signal (NLS) to fuse with Cas9 or the NLS-bearing protein to form a complex with Cas9 to enter the nucleus. In this study, we introduced apoferritin nanocages as carriers and DOX as a nuclear trigger for the nuclear transport of the Cas9/sgRNA ribonucleoprotein without the NLS (RNP[-]). Our experiments showed that loading RNP[-] and DOX into 4L-FTH subunit-based apoferritin nanocages leads to efficient endocytosis and lysosomal escape. Specifically, when DOX was administered at a concentration of 1 μM, we observed the activation of cellular defense mechanisms, which effectively facilitated the translocation of 4L-HFn@RNP[-]/DOX nanoparticles into the nucleus, thereby enabling intranuclear RNP[-] delivery. This strategy has been empirically demonstrated to achieve gene editing efficiencies of approximately 33% for the Lcn2 gene in MDA-MB-231 cells and 17.9% for the copepod green fluorescent protein (copGFP) gene in HeLa.copGFP cells in vitro. Moreover, in vivo editing efficacy, as tested in a HeLa.copGFP nude mouse model, was confirmed to be 16%. This delivery system presents a novel therapeutic approach for the nuclear delivery of small molecules or nucleic acid drugs, potentially overcoming the challenges associated with nuclear entry barriers.},
}

*Apoferritins/chemistry
Humans
*CRISPR-Cas Systems
*Gene Editing
Animals
*Nuclear Localization Signals/chemistry
Ribonucleoproteins/chemistry/metabolism/genetics
HeLa Cells
Cell Line, Tumor
Mice
Doxorubicin/chemistry/pharmacology
Mice, Nude
Cell Nucleus/metabolism
Nanoparticles/chemistry

@article {pmid39870862,
year = {2025},
author = {Ramezani, M and Weisbart, E and Bauman, J and Singh, A and Yong, J and Lozada, M and Way, GP and Kavari, SL and Diaz, C and Leardini, E and Jetley, G and Pagnotta, J and Haghighi, M and Batista, TM and Pérez-Schindler, J and Claussnitzer, M and Singh, S and Cimini, BA and Blainey, PC and Carpenter, AE and Jan, CH and Neal, JT},
title = {A genome-wide atlas of human cell morphology.},
journal = {Nature methods},
volume = {22},
number = {3},
pages = {621-633},
pmid = {39870862},
issn = {1548-7105},
support = {NNF21SA0072102//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; 1DP2GM146252//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {Humans ; *Genome, Human ; CRISPR-Cas Systems ; Phenotype ; Genome-Wide Association Study/methods ; Genomics/methods ; Protein Interaction Maps ; Membrane Proteins/genetics/metabolism ; },
abstract = {A key challenge of the modern genomics era is developing empirical data-driven representations of gene function. Here we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-wide genotype-phenotype maps comprising CRISPR-Cas9-based knockouts of >20,000 genes in >30 million cells. Our optical pooled cell profiling platform (PERISCOPE) combines a destainable high-dimensional phenotyping panel (based on Cell Painting) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This perturbation atlas comprises high-dimensional phenotypic profiles of individual cells with sufficient resolution to cluster thousands of human genes, reconstruct known pathways and protein-protein interaction networks, interrogate subcellular processes and identify culture media-specific responses. Using this atlas, we identify the poorly characterized disease-associated TMEM251/LYSET as a Golgi-resident transmembrane protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes. In sum, this perturbation atlas and screening platform represents a rich and accessible resource for connecting genes to cellular functions at scale.},
}

Humans
*Genome, Human
CRISPR-Cas Systems
Phenotype
Genome-Wide Association Study/methods
Genomics/methods
Protein Interaction Maps
Membrane Proteins/genetics/metabolism

@article {pmid39838780,
year = {2025},
author = {Chen, S and Triki, M and Pinto Carneiro, S and Merkel, OM},
title = {A novel micelleplex for tumour-targeted delivery of CRISPR-Cas9 against KRAS-mutated lung cancer.},
journal = {Nanoscale},
volume = {17},
number = {11},
pages = {6604-6619},
pmid = {39838780},
issn = {2040-3372},
mesh = {Humans ; *Micelles ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *Lung Neoplasms/pathology/genetics/metabolism/therapy/drug therapy ; *CRISPR-Cas Systems ; A549 Cells ; Gene Editing ; Mutation ; RNA, Messenger/genetics/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/chemistry ; Polyethyleneimine/chemistry ; },
abstract = {CRISPR-Cas9 has emerged as a highly effective and customizable genome editing tool, holding significant promise for the treatment of KRAS mutations in lung cancer. In this study, we introduce a novel micelleplex, named C14-PEI, designed to co-deliver Cas9 mRNA and sgRNA efficiently to excise the mutated KRAS allele in lung cancer cells. C14-PEI is synthesised from 1,2-epoxytetradecane and branched PEI 600 Da via a ring-opening reaction. The resulting C14-PEI has a critical micelle concentration (CMC) of approximately 20.86 ± 0.15 mg L[-1], indicating its ability to form stable micelles at low concentrations. C14-PEI efficiently encapsulates mRNA into micelleplexes through electrostatic interactions. When the mass ratio is 8 (w/w 8), the C14-PEI formulation exhibits conducive properties, which showed encapsulation efficiency of eGFP mRNA at 99% and led to a 130-fold increase in eGFP expression in A549 cells compared to untreated cells, demonstrating the robust delivery and expression capability of the micelleplexes. Importantly, toxicity tests using intracellular reduction of a tetrazolium salt revealed no significant cytotoxicity, underscoring the biocompatibility of C14-PEI. C14-PEI also shows high efficiency in co-encapsulating Cas9 mRNA and sgRNA, as confirmed by agarose gel electrophoresis. At an sgRNA to Cas9 mRNA molar ratio of 10, the micelleplexes successfully mediate the cutting of mutated KRAS with an indel efficiency exceeding 60%, as determined by the T7 Endonuclease I (T7EI) assay. Droplet digital polymerase chain reaction (ddPCR) further demonstrates that the gene editing efficiency, measured by edited gene copies, is 48.5% in the w/w 4 group and 37.8% in the w/w 8 group. Treatment with C14-PEI micelleplexes containing Cas9 mRNA and sgRNA targeting the KRAS G12S mutation significantly impairs the migration capability of A549 cells and increases apoptosis rates. These findings suggest that C14-PEI effectively disrupts KRAS signalling pathways, leading to reduced tumor cell proliferation and enhanced cell death.},
}

Humans
*Micelles
*Proto-Oncogene Proteins p21(ras)/genetics/metabolism
*Lung Neoplasms/pathology/genetics/metabolism/therapy/drug therapy
*CRISPR-Cas Systems
A549 Cells
Gene Editing
Mutation
RNA, Messenger/genetics/metabolism/chemistry
RNA, Guide, CRISPR-Cas Systems/chemistry
Polyethyleneimine/chemistry

@article {pmid40071935,
year = {2025},
author = {Stagno, JR and Deme, JC and Dwivedi, V and Lee, YT and Lee, HK and Yu, P and Chen, SY and Fan, L and Degenhardt, MFS and Chari, R and Young, HA and Lea, SM and Wang, YX},
title = {Structural investigation of an RNA device that regulates PD-1 expression in mammalian cells.},
journal = {Nucleic acids research},
volume = {53},
number = {5},
pages = {},
doi = {10.1093/nar/gkaf156},
pmid = {40071935},
issn = {1362-4962},
support = {/RC/CCR NIH HHS/United States ; /CA/NCI NIH HHS/United States ; /NH/NIH HHS/United States ; HHS75N91019D00024/HH/HHS/United States ; P30GM133893/GM/NIGMS NIH HHS/United States ; KP1605010//Department of Energy's Office of Biological and Environmental Research/ ; DE-SC0012704//Department of Energy Basic Energy Sciences Program/ ; },
mesh = {*Programmed Cell Death 1 Receptor/metabolism/genetics ; Animals ; Mice ; RNA/metabolism/genetics ; Nucleic Acid Conformation ; Humans ; Tetracycline/pharmacology ; Gene Expression Regulation ; RNA, Messenger/genetics/metabolism ; CRISPR-Cas Systems ; Lymphocytes/metabolism ; },
abstract = {Synthetic RNA devices are engineered to control gene expression and offer great potential in both biotechnology and clinical applications. Here, we present multidisciplinary structural and biochemical data for a tetracycline (Tc)-responsive RNA device (D43) in both ligand-free and bound states, providing a structure-dynamical basis for signal transmission. Activation of self-cleavage is achieved via ligand-induced conformational and dynamical changes that stabilize the elongated bridging helix harboring the communication module, which drives proper coordination of the catalytic residues. We then show the utility of CRISPR-integrated D43 in EL4 lymphocytes to regulate programmed cell death protein 1 (PD-1), a key receptor of immune checkpoints. Treatment of these cells with Tc showed a dose-dependent reduction in PD-1 by immunostaining and a decrease in messenger RNA levels by quantitative PCR as compared with wild type. PD-1 expression was recoverable upon removal of Tc. These results provide mechanistic insight into RNA devices with potential for cancer immunotherapy or other applications.},
}

*Programmed Cell Death 1 Receptor/metabolism/genetics
Animals
Mice
RNA/metabolism/genetics
Nucleic Acid Conformation
Humans
Tetracycline/pharmacology
Gene Expression Regulation
RNA, Messenger/genetics/metabolism
CRISPR-Cas Systems
Lymphocytes/metabolism

@article {pmid40070114,
year = {2025},
author = {Bhargava, CN and Ashok, K and Pradhan, SK and Kumar, S and Manamohan, M and Rai, A and Asokan, R},
title = {CRISPR/Cas9 Mediated Editing of Bdtektin1 Gene Induces Sterility in Male Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae).},
journal = {Archives of insect biochemistry and physiology},
volume = {118},
number = {3},
pages = {e70043},
doi = {10.1002/arch.70043},
pmid = {40070114},
issn = {1520-6327},
support = {//The current research work has been carried out on the funding received from the ICAR-Network Project on Agricultural Bioinformatics and Computational Biology and is highly acknowledged./ ; },
mesh = {Animals ; *Tephritidae/genetics ; Male ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Insect Proteins/genetics/metabolism ; Infertility, Male/genetics ; Female ; },
abstract = {The Oriental fruit fly, Bactrocera dorsalis (B. dorsalis) is a highly invasive, widely distributed notorious pest restricting global fruit trade immensely. There are several approaches to managing this pest, still require newer approaches. In this regard, recently a novel approach called precision-guided sterile insect technique (pgSIT) is gaining momentum in inducing both female sex elimination or sex conversion and male sterility at one go. Developing a species-specific pgSIT system requires validation of targets such as sex determination and spermatogenesis genes. In this regard, B. dorsalis is highly amenable for area-wide pest management and in the present study, we have validated the loss-of-function of the spermatogenesis-related gene, tektin1 using the CRISPR/Cas9 ribonucleoprotein (RNP) complex. This gene was cloned from the local isolate of B. dorsalis and two promising single guide RNAs (sgRNAs) were designed and validated through in vitro restriction analysis. Injection of the RNP complex (sgRNA + Cas9 protein) into the G0 embryo resulted in three adult males carrying mutations at the target site. The phenotype of the mutants was determined through crossing studies, namely, △1♂ × WT ♀, △2♂ × WT ♀, △3♂ × WT ♀, and WT ♂ × WT ♀ and that showed hatching rates of 0%, 11.70%, 0%, and 45.12%, respectively. The mutant males had more nonviable sperm as compared to control. This study underscores the pivotal role of the Bdtektin1 gene for male fertility and is a promising candidate for further development of pgSIT system for B. dorsalis.},
}

Animals
*Tephritidae/genetics
Male
*CRISPR-Cas Systems
*Gene Editing/methods
*Insect Proteins/genetics/metabolism
Infertility, Male/genetics
Female

@article {pmid40070071,
year = {2025},
author = {Manchanda, D and Kumar, S and Makhija, M},
title = {Polysaccharide-Based Delivery Systems for CRISPR/Cas Gene Therapy: Overcoming Challenges and Advancing Pharmaceutical Solutions.},
journal = {Current gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.2174/0115665232369121250307075817},
pmid = {40070071},
issn = {1875-5631},
abstract = {The advent of CRISPR/Cas gene-editing technology has revolutionized molecular biology, offering unprecedented precision and potential in treating genetic disorders, cancers, and other complex diseases. However, for CRISPR/Cas to be truly effective in clinical settings, one of the most significant challenges lies in the delivery of the CRISPR components, including guide RNA (gRNA) and Cas protein, into specific cells or tissues. Safe, targeted, and efficient delivery remains a critical bottleneck. Viral vectors, lipid nanoparticles, and synthetic polymers have been explored, but they come with limitations, such as immunogenicity, toxicity, and limited delivery capacity. Polysaccharide-based delivery systems, with their natural origin, biocompatibility, and versatile chemical properties, offer a promising alternative that could address these delivery challenges while advancing the pharmaceutical applications of CRISPR/Cas gene therapy.},
}