@article {pmid39851560,
year = {2025},
author = {Bonowicz, K and Jerka, D and Piekarska, K and Olagbaju, J and Stapleton, L and Shobowale, M and Bartosiński, A and Łapot, M and Bai, Y and Gagat, M},
title = {CRISPR-Cas9 in Cardiovascular Medicine: Unlocking New Potential for Treatment.},
journal = {Cells},
volume = {14},
number = {2},
pages = {},
doi = {10.3390/cells14020131},
pmid = {39851560},
issn = {2073-4409},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Cardiovascular Diseases/genetics/therapy ; *Genetic Therapy/methods ; Animals ; },
abstract = {Cardiovascular diseases (CVDs) remain a significant global health challenge, with many current treatments addressing symptoms rather than the genetic roots of these conditions. The advent of CRISPR-Cas9 technology has revolutionized genome editing, offering a transformative approach to targeting disease-causing mutations directly. This article examines the potential of CRISPR-Cas9 in the treatment of various CVDs, including atherosclerosis, arrhythmias, cardiomyopathies, hypertension, and Duchenne muscular dystrophy (DMD). The technology's ability to correct single-gene mutations with high precision and efficiency positions it as a groundbreaking tool in cardiovascular therapy. Recent developments have extended the capabilities of CRISPR-Cas9 to include mitochondrial genome editing, a critical advancement for addressing mitochondrial dysfunctions often linked to cardiovascular disorders. Despite its promise, significant challenges remain, including off-target effects, ethical concerns, and limitations in delivery methods, which hinder its translation into clinical practice. This article also explores the ethical and regulatory considerations surrounding gene editing technologies, emphasizing the implications of somatic versus germline modifications. Future research efforts should aim to enhance the accuracy of CRISPR-Cas9, improve delivery systems for targeted tissues, and ensure the safety and efficacy of treatments in the long term. Overcoming these obstacles could enable CRISPR-Cas9 to not only treat but also potentially cure genetically driven cardiovascular diseases, heralding a new era in precision medicine for cardiovascular health.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Cardiovascular Diseases/genetics/therapy
*Genetic Therapy/methods
Animals

@article {pmid39849243,
year = {2025},
author = {Nawab, S and Ullah, MW and Shah, SB and Zhang, YF and Keerio, HA and Yong, YC},
title = {Recent advances in engineering non-native microorganisms for poly(3-hydroxybutyrate) production.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {2},
pages = {48},
pmid = {39849243},
issn = {1573-0972},
support = {2023M741421//China Postdoctoral Science Foundation/ ; 2022ZB650//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; 2023ZB399//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; BK20220003//Natural Science Foundation of Jiangsu Province/ ; },
mesh = {*Metabolic Engineering/methods ; *Hydroxybutyrates/metabolism ; *Polyesters/metabolism ; Escherichia coli/genetics/metabolism ; Biosynthetic Pathways/genetics ; Synthetic Biology/methods ; CRISPR-Cas Systems ; Bacteria/metabolism/genetics ; Metabolic Networks and Pathways/genetics ; Industrial Microbiology ; Polyhydroxybutyrates ; },
abstract = {Poly(3-hydroxybutyrate) (PHB) is a biodegradable polymer that belongs to a group of polymers called polyhydroxyalkanoates (PHAs). PHB can be synthesized from renewable resources, making it a promising alternative to petroleum-derived plastics. It is also considered non-toxic, biodegradable, and biocompatible, which makes it suitable for various applications in the medicine and biomedicine. Many microorganisms biosynthesize and accumulate PHB naturally. However, recent advancements in metabolic engineering and synthetic biology have allowed scientists to engineer non-native microorganisms to produce PHB. This review comprehensively summarizes all non-native microbial hosts used for PHB biosynthesis and discusses different metabolic engineering approaches used to enhance PHB production. These strategies include optimizing the biosynthesis pathway through cofactor engineering, metabolic pathway reconstruction, and cell morphology engineering. Moreover, the CRISPR/Cas9 approach is also used for manipulating the genome of non-host microorganisms to enable them produce PHB. Among non-native microbial hosts, Escherichia coli has been successfully used for industrial-scale PHB production. However, further genetic engineering approaches are needed to make non-native microbial hosts more suitable for large-scale PHB production.},
}

*Metabolic Engineering/methods
*Hydroxybutyrates/metabolism
*Polyesters/metabolism
Escherichia coli/genetics/metabolism
Biosynthetic Pathways/genetics
Synthetic Biology/methods
CRISPR-Cas Systems
Bacteria/metabolism/genetics
Metabolic Networks and Pathways/genetics
Industrial Microbiology
Polyhydroxybutyrates

@article {pmid39849074,
year = {2025},
author = {Das, S and Unhale, T and Marinach, C and Valeriano Alegria, BDC and Roux, C and Madry, H and Mohand Oumoussa, B and Amino, R and Iwanaga, S and Briquet, S and Silvie, O},
title = {Constitutive expression of Cas9 and rapamycin-inducible Cre recombinase facilitates conditional genome editing in Plasmodium berghei.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {2949},
pmid = {39849074},
issn = {2045-2322},
support = {ANR-11-LABX-0024//Agence Nationale de la Recherche/ ; EQU201903007823//Fondation pour la recherche médicale, France/ ; },
mesh = {*Plasmodium berghei/genetics ; *Integrases/metabolism/genetics ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; *Sirolimus/pharmacology ; Mice ; Malaria/parasitology ; },
abstract = {Malaria is caused by protozoan parasites of the genus Plasmodium and remains a global health concern. The parasite has a highly adaptable life cycle comprising successive rounds of asexual replication in a vertebrate host and sexual maturation in the mosquito vector Anopheles. Genetic manipulation of the parasite has been instrumental for deciphering the function of Plasmodium genes. Conventional reverse genetic tools cannot be used to study essential genes of the asexual blood stages, thereby necessitating the development of conditional strategies. Among various such strategies, the rapamycin-inducible dimerisable Cre (DiCre) recombinase system emerged as a powerful approach for conditional editing of essential genes in human-infecting P. falciparum and in the rodent malaria model parasite P. berghei. We previously generated a DiCre-expressing P. berghei line and validated it by conditionally deleting several essential asexual stage genes, revealing their important role also in sporozoites. Another potent tool is the CRISPR/Cas9 technology, which has enabled targeted genome editing with higher accuracy and specificity and greatly advanced genome engineering in Plasmodium spp. Here, we developed new P. berghei parasite lines by integrating the DiCre cassette and a fluorescent marker in parasites constitutively expressing Cas9. Owing to the dual integration of CRISPR/Cas9 and DiCre, these new lines allow unparalleled levels of gene modification and conditional regulation simultaneously. To illustrate the versatility of this new tool, we conditionally knocked out the essential gene encoding the claudin-like apicomplexan micronemal protein (CLAMP) in P. berghei and confirmed the role of CLAMP during invasion of erythrocytes.},
}

*Plasmodium berghei/genetics
*Integrases/metabolism/genetics
*Gene Editing/methods
Animals
*CRISPR-Cas Systems
*Sirolimus/pharmacology
Mice
Malaria/parasitology

@article {pmid39804945,
year = {2025},
author = {Huang, Y and Pei, S and Lv, X and Yang, F and Gong, X and Li, N and Guo, Y and Feng, Y and Xiao, L},
title = {Stage-specific expression and divergent functions of two insulinase-like proteases associated with host infectivity in Cryptosporidium.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {1},
pages = {e0012777},
doi = {10.1371/journal.pntd.0012777},
pmid = {39804945},
issn = {1935-2735},
mesh = {Animals ; *Cryptosporidiosis/parasitology ; Mice ; *Cryptosporidium parvum/genetics/enzymology/pathogenicity ; Mice, Knockout ; Insulysin/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; },
abstract = {BACKGROUND: The determinants of differences in host infectivity among Cryptosporidium species and subtypes are poorly understood. Results from recent comparative genomic studies suggest that gains and losses of multicopy subtelomeric genes encoding insulinase-like proteases (INS-19 and INS-20 in Cryptosporidium parvum and their orthologs in closely related species) may potentially contribute to these differences.

In this study, we investigated the expression and biological function of the INS-19 and INS-20 of C. parvum. CRISPR/Cas9 was used to endogenously tag both genes with the hemagglutinin epitope. Immunofluorescence analysis revealed that INS-19 and INS-20 are expressed at different developmental stages of the pathogen. Although knockout of either had no detectable effect on the in vitro growth of C. parvum, knockout of INS-20, deletion of its multiple domains, or mutation of the active motif in the functional domain reduced the intensity of C. parvum infection in IFN-γ knockout mice. Consistent with this, mice infected with the INS-20-deleted mutant had reduced intestinal damage and parasite burden.

CONCLUSIONS/SIGNIFICANCE: These results suggest that INS-19 and INS-20 have stage-specific expression with distinct biological functions, and that the presence of the INS-20 in zoonotic C. parvum contributes to its infectivity and fitness in mice.},
}

Animals
*Cryptosporidiosis/parasitology
Mice
*Cryptosporidium parvum/genetics/enzymology/pathogenicity
Mice, Knockout
Insulysin/genetics/metabolism
Protozoan Proteins/genetics/metabolism
Humans
CRISPR-Cas Systems
Mice, Inbred C57BL

@article {pmid39804925,
year = {2025},
author = {Ricou, A and Simon, M and Duflos, R and Azzopardi, M and Roux, F and Budar, F and Camilleri, C},
title = {Identification of novel genes responsible for a pollen killer present in local natural populations of Arabidopsis thaliana.},
journal = {PLoS genetics},
volume = {21},
number = {1},
pages = {e1011451},
doi = {10.1371/journal.pgen.1011451},
pmid = {39804925},
issn = {1553-7404},
mesh = {*Arabidopsis/genetics ; *Pollen/genetics ; *Arabidopsis Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Alleles ; DNA Copy Number Variations/genetics ; Mitochondria/genetics/metabolism ; Genes, Plant ; },
abstract = {Gamete killers are genetic loci that distort segregation in the progeny of hybrids because the killer allele promotes the elimination of the gametes that carry the sensitive allele. They are widely distributed in eukaryotes and are important for understanding genome evolution and speciation. We had previously identified a pollen killer in hybrids between two distant natural accessions of Arabidopsis thaliana. This pollen killer involves three genetically linked genes, and we previously reported the identification of the gene encoding the antidote that protects pollen grains from the killer activity. In this study, we identified the two other genes of the pollen killer by using CRISPR-Cas9 induced mutants. These two genes are necessary for the killer activity that we demonstrated to be specific to pollen. The cellular localization of the pollen killer encoded proteins suggests that the pollen killer activity involves the mitochondria. Sequence analyses reveal predicted domains from the same families in the killer proteins. In addition, the C-terminal half of one of the killer proteins is identical to the antidote, and one amino acid, crucial for the antidote activity, is also essential for the killer function. Investigating more than 700 worldwide accessions of A. thaliana, we confirmed that the locus is subject to important structural rearrangements and copy number variation. By exploiting available de novo genomic sequences, we propose a scenario for the emergence of this pollen killer in A. thaliana. Furthermore, we report the co-occurrence and behavior of killer and sensitive genotypes in several local populations, a prerequisite for studying gamete killer evolution in the wild. This highlights the potential of the Arabidopsis model not only for functional studies of gamete killers but also for investigating their evolutionary trajectories at complementary geographical scales.},
}

*Arabidopsis/genetics
*Pollen/genetics
*Arabidopsis Proteins/genetics/metabolism
CRISPR-Cas Systems
Alleles
DNA Copy Number Variations/genetics
Mitochondria/genetics/metabolism
Genes, Plant

@article {pmid39661534,
year = {2025},
author = {Dhinoja, S and Mary, J and Qaryoute, AA and De Maria, A and Jagadeeswaran, P},
title = {Generation and characterization of zebrafish f9l mutant confirmed that f9l is f10 like gene.},
journal = {Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis},
volume = {36},
number = {1},
pages = {26-33},
doi = {10.1097/MBC.0000000000001337},
pmid = {39661534},
issn = {1473-5733},
mesh = {Animals ; *Zebrafish/genetics ; Zebrafish Proteins/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {AIM: This study aimed to create an f9l mutant zebrafish using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and characterize its coagulation properties to investigate its functional similarity to human FX and explore the potential synergy between f9l and f10 .

METHODS: Three gRNAs targeting exon 8 encoded by the catalytic domain of the f9l gene were injected into 300 single-cell zebrafish embryos using CRISPR/Cas9 technology. DNA from the resulting adults was extracted from tail tips, and PCR was used to detect indels. The identified founder mutant was bred to homozygosity, and functional assays, kinetic Russel viper venom time, bleeding assay in adults, and venous laser injury on larvae were conducted to assess its hemostatic function. Additionally, f10 was knocked down in f9l homozygous embryos using f10 antisense morpholinos to study their interaction by monitoring its survival.

RESULTS: DNA from 60 adults was screened for indels, resulting in a fish with a heritable complex mutation involving one insertion and two deletions in exon 8. The f9l homozygous mutants exhibited impaired F10 activity, mild bleeding after mechanical injury, and developmental deformities in early larval stages. The caudal vein thrombosis assay showed variable occlusion times, indicating a bleeding phenotype with incomplete penetrance. Knocking down f10 in f9l homozygous embryos resulted in 50% mortality within five dpf, compared to f9l homozygous embryos injected with control morpholinos.

CONCLUSION: In summary, we generated f9l knockout and showed it is a paralog to f10. We also found a synergy between f9l and f10 genes, highlighting its importance in hemostasis.},
}

Animals
*Zebrafish/genetics
Zebrafish Proteins/genetics
Mutation
CRISPR-Cas Systems

@article {pmid39847570,
year = {2025},
author = {Al Sium, SM and Goswami, B and Chowdhury, SF and Naser, SR and Sarkar, MK and Faruq, MJ and Habib, MA and Akter, S and Banu, TA and Sarkar, MMH and Khan, MS},
title = {An insight into the genome-wide analysis of bacterial defense mechanisms in a uropathogenic Morganella morganii isolate from Bangladesh.},
journal = {PloS one},
volume = {20},
number = {1},
pages = {e0313141},
doi = {10.1371/journal.pone.0313141},
pmid = {39847570},
issn = {1932-6203},
mesh = {*Morganella morganii/genetics/isolation & purification ; Bangladesh ; *Urinary Tract Infections/microbiology ; *Genome, Bacterial ; Humans ; *Phylogeny ; Genomic Islands/genetics ; Anti-Bacterial Agents/pharmacology ; },
abstract = {The gram-negative, facultative anaerobic bacterium Morganella morganii is linked to a number of illnesses, including nosocomial infections and urinary tract infections (UTIs). A clinical isolate from a UTI patient in Bangladesh was subjected to high-throughput whole genome sequencing and extensive bioinformatics analysis in order to gather knowledge about the genomic basis of bacterial defenses and pathogenicity in M. morganii. With an average nucleotide identity (ANI) of more than 97% similarity to a reference genome and phylogenetic analysis verified the isolate as M. morganii. Genome annotation identified 3,718 protein-coding sequences, including genes for metabolism, protein processing, stress response, energy, and membrane transport. The presence of biosynthetic gene clusters points to the isolate's ability to create bioactive compounds, including antibiotics. Genomic islands contained genes for metal transporters, stress proteins, toxin proteins, and genes related to horizontal gene transfer. The beta-lactam resistance gene blaDHA was found using antimicrobial resistance (AMR) gene analysis across three databases. The virulence genes kdsA and cheY, which may be involved in chemotaxis and lipopolysaccharide production, were also available in the isolate, suggesting its high pathogenicity. The genome contained mobile genetic components and defense mechanisms, such as restriction modification and CRISPR-Cas systems, indicating the bacterium's ability to defend itself against viral attacks. This thorough investigation sheds important light on M. morganii's pathogenicity and adaptive tactics by revealing its genetic characteristics, AMR, virulence components, and defense mechanisms. For the development of targeted treatments and preventing the onset of resistance in clinical care, it is essential to comprehend these genetic fingerprints.},
}

*Morganella morganii/genetics/isolation & purification
Bangladesh
*Urinary Tract Infections/microbiology
*Genome, Bacterial
Humans
*Phylogeny
Genomic Islands/genetics
Anti-Bacterial Agents/pharmacology

@article {pmid39847105,
year = {2025},
author = {Karimi-Fard, A and Saidi, A and Tohidfar, M and Emami, SN},
title = {CRISPR-Cas-mediated adaptation of Thermus thermophilus HB8 to environmental stress conditions.},
journal = {Archives of microbiology},
volume = {207},
number = {2},
pages = {41},
pmid = {39847105},
issn = {1432-072X},
mesh = {*Thermus thermophilus/genetics/metabolism ; *CRISPR-Cas Systems ; *Stress, Physiological ; *Adaptation, Physiological/genetics ; *Bacterial Proteins/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; },
abstract = {Bacteria experience a continual array of environmental stresses, necessitating adaptive mechanisms crucial for their survival. Thermophilic bacteria, such as Thermus thermophilus, face constant environmental challenges, particularly high temperatures, which requires robust adaptive mechanisms for survival. Studying these extremophiles provides valuable insights into the intricate molecular and physiological processes used by extremophiles to adapt and survive in harsh environments. Through meta-analysis of microarray data, we revealed the key genes in T. thermophilus HB8 that respond to various environmental stresses. The analysis revealed 20 differentially expressed genes (DEGs), including 13 upregulated and seven downregulated genes, with a threshold of|log fold change| > 1 and an adjusted p-value < 0.05. Several genes identified as up-regulated in our analysis belonged to the CRISPR-associated protein (Cas) family. To validate these findings, we further evaluated the relative expression levels of TTHB188 (cas1/casA), TTHB189 (cas2/casB), TTHB190 (cas7/casC), TTHB191 (cas5/casD), TTHB192 (cas6/casE), and TTHB193 (cas1e) using RT-qPCR under H2O2 and salt stress conditions. The RT-qPCR analysis revealed significant up-regulation of transcripts, casA, casB, casC, casD, casE, and cas1e under salt stress. However, under H2O2 stress, only, casA, casB, and casC exhibited substantial increases in expression. Our findings may indicate that the CRISPR-associated proteins significantly impact the adaptive response of T. thermophilus HB8 to various environmental stresses, particularly salt stress, highlighting its significance in extremophile survival and adaptation. This research offers an important understanding of the complex strategies used by extremophiles to survive in challenging conditions.},
}

*Thermus thermophilus/genetics/metabolism
*CRISPR-Cas Systems
*Stress, Physiological
*Adaptation, Physiological/genetics
*Bacterial Proteins/genetics/metabolism
*Gene Expression Regulation, Bacterial

@article {pmid39845892,
year = {2024},
author = {Hu, D and Hu, L and Lu, Y and Dong, X and Cao, X and Bai, S and Zhang, L and Li, D and Sun, Y},
title = {Intein-mediated split SaCas9 for genome editing in plants.},
journal = {Frontiers in genome editing},
volume = {6},
number = {},
pages = {1506468},
pmid = {39845892},
issn = {2673-3439},
abstract = {Virus-induced genome editing (VIGE) technologies have been developed to address the limitations to plant genome editing, which heavily relies on genetic transformation and regeneration. However, the application of VIGE in plants is hampered by the challenge posed by the size of the commonly used gene editing nucleases, Cas9 and Cas12a. To overcome this challenge, we employed intein-mediated protein splicing to divide the SaCas9 transcript into two segments (Split-v1) and three segments (Split-v3). The Split-v1 system demonstrated genome editing efficiencies in transgenic plants comparable to those achieved with wild-type SaCas9, with efficiencies ranging from 70.2% to 96.1%. Additionally, we constructed barley stripe mosaic virus (BSMV)-based vectors to co-express Split-v1 SaCas9 and gRNAs targeting LcHRC, LcGW2, and LcTB1 in sheepgrass (Leymus chinensis), a Gramineae forage species known for its recalcitrance to genetic transformation. Infected leaves of sheepgrass exhibited genome editing efficiencies ranging from 10.40% to 37.03%. These results demonstrate the potential of intein-mediated split nuclease systems to broaden the applicability of VIGE in challenging plant species.},
}

@article {pmid39844455,
year = {2025},
author = {Jonsdottir, TK and Paoletta, MS and Ishizaki, T and Hernandez, S and Ivanova, M and Herrera Curbelo, A and Saiki, PA and Selinger, M and Das, D and Henriksson, J and Bushell, ESC},
title = {A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkaf005},
pmid = {39844455},
issn = {1362-4962},
support = {2021-06602//Swedish Research Council/ ; 2019.0178//Knut and Alice Wallenberg Foundation/ ; 23 3102 Pj//Swedish Cancer Society/ ; 202160312//Japan Society for the Promotion of Science/ ; //Swedish Universities/ ; },
mesh = {*Plasmodium berghei/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Animals ; Malaria/parasitology ; Genome, Protozoan/genetics ; Genetic Vectors/genetics ; },
abstract = {Many Plasmodium genes remain uncharacterized due to low genetic tractability. Previous large-scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called P. berghei high-throughput (PbHiT), which uses a single cloning step to generate targeting vectors with 100-bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide an online resource of knockout and tagging designs to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasite's biology at scale.},
}

*Plasmodium berghei/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Knockout Techniques
Animals
Malaria/parasitology
Genome, Protozoan/genetics
Genetic Vectors/genetics

@article {pmid39844454,
year = {2025},
author = {Garcia-Guerra, A and Sathyaprakash, C and de Jong, OG and Lim, WF and Vader, P and El Andaloussi, S and Bath, J and Reine, J and Aoki, Y and Turberfield, AJ and Wood, MJA and Rinaldi, C},
title = {Tissue-specific modulation of CRISPR activity by miRNA-sensing guide RNAs.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkaf016},
pmid = {39844454},
issn = {1362-4962},
support = {MR/Y009703/1/MRC_/Medical Research Council/United Kingdom ; //Kennedy's Disease Association/ ; 578222//Muscular Dystrophy Association/ ; MR/N024850/1/MRC_/Medical Research Council/United Kingdom ; 317110//Marie Curie Initial Training Network EScoDNA/ ; //xford University Press John Fell Fund/ ; LCF/BQ/EU16/11560044//La Caixa Foundation Fellowship/ ; //University of Oxford/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Animals ; *CRISPR-Cas Systems ; Argonaute Proteins/metabolism/genetics ; Mice ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Organ Specificity/genetics ; HEK293 Cells ; },
abstract = {Nucleic acid nanostructures offer unique opportunities for biomedical applications due to their sequence-programmable structures and functions, which enable the design of complex responses to molecular cues. Control of the biological activity of therapeutic cargoes based on endogenous molecular signatures holds the potential to overcome major hurdles in translational research: cell specificity and off-target effects. Endogenous microRNAs (miRNAs) can be used to profile cell type and cell state, and are ideal inputs for RNA nanodevices. Here, we present CRISPR MiRAGE (miRNA-activated genome editing), a tool comprising a dynamic single-guide RNA that senses miRNA complexed with Argonaute proteins and controls downstream CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) activity based on the detected miRNA signature. We study the operation of the miRNA-sensing single-guide RNA and attain muscle-specific activation of gene editing through CRISPR MiRAGE in models of Duchenne muscular dystrophy. By enabling RNA-controlled gene editing activity, this technology creates opportunities to advance tissue-specific CRISPR treatments for human diseases.},
}

*MicroRNAs/genetics/metabolism
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
Humans
*Muscular Dystrophy, Duchenne/genetics/therapy
Animals
*CRISPR-Cas Systems
Argonaute Proteins/metabolism/genetics
Mice
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Organ Specificity/genetics
HEK293 Cells

@article {pmid39844394,
year = {2025},
author = {Feng, S and Li, J and Yan, A and Zhu, X and Zhang, L and Tang, D and Liu, L},
title = {Application of Gene Editing in Triple-Negative Breast Cancer Research.},
journal = {Cell biochemistry and function},
volume = {43},
number = {1},
pages = {e70044},
doi = {10.1002/cbf.70044},
pmid = {39844394},
issn = {1099-0844},
support = {//This work was supported by the Scientific Research Project of Department of Education of Guangdong Province (Project Number:2023ZDZX2057) and GuangDong Basic and Applied Basic Research Foundation (Project Number:2023A1515140070)./ ; },
mesh = {Humans ; *Gene Editing ; *Triple Negative Breast Neoplasms/genetics/therapy ; Female ; Animals ; Precision Medicine ; },
abstract = {With the rapid development of gene editing technology, its application in breast cancer has gradually become the focus of research. This article reviews the application of gene editing technology in the treatment of breast cancer, and discusses its challenges and future development directions. The key application areas of gene editing technology in the treatment of breast cancer will be outlined, including the discovery of new therapeutic targets and the development of drugs related to the pathway. Gene editing technology has played an important role in the discovery of new therapeutic targets. Through the use of gene editing technology, breast cancer-related genes are systematically edited to regulate key regulatory factors on related pathways or key tumor suppressor genes such as FOXC1 and BRCA, and the results are analyzed in cell or animal experiments, and the target is obtained from the experimental results, which provides important clues for the development of new drugs. This approach provides an innovative way to find more effective treatment strategies and inhibit tumor growth. In addition, gene editing technology has also promoted the personalization of breast cancer treatment. By analyzing a patient's genomic information, researchers can pinpoint key genetic mutations in a patient's tumor and design personalized treatments. This personalized treatment approach is expected to improve the therapeutic effect and reduce adverse reactions. Finally, the application of gene editing technology also provides support for the development of breast cancer immunotherapy. By editing immune cells to make them more potent against tumors, researchers are trying to develop more effective immunotherapies to bring new treatment options to breast cancer patients.},
}

Humans
*Gene Editing
*Triple Negative Breast Neoplasms/genetics/therapy
Female
Animals
Precision Medicine

@article {pmid39843407,
year = {2025},
author = {Liu, X and Huang, L and Ye, Y and Wang, H and Tang, M and He, F and Xia, Z and Deng, S and Zhang, P and Dai, R and Liang, S},
title = {Staphylococcus aureus nt5 gene mutation through CRISPR RNA-guided base editing weakens bacterial virulence and immune evasion.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2451163},
doi = {10.1080/21505594.2025.2451163},
pmid = {39843407},
issn = {2150-5608},
mesh = {*Staphylococcus aureus/genetics/pathogenicity/drug effects ; Animals ; Mice ; *Staphylococcal Infections/microbiology ; *Immune Evasion ; Virulence/genetics ; *5'-Nucleotidase/genetics ; *Mutation ; Bacterial Proteins/genetics ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Bacterial/genetics ; Daptomycin/pharmacology ; Gene Editing/methods ; Female ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The resistance of commonly used clinical antibiotics, such as daptomycin (DAP), has become increasingly serious in the fight against Staphylococcus aureus (S. aureus) infection. It is essential to explore key pathogenicity-driven genes/proteins in bacterial infection and antibiotics resistance, which contributes to develop novel therapeutic strategies against S. aureus infections. The nt5 gene of S. aureus, encoding 5'-nucleotidase (NT5), is nearly unknown for its function in drug resistance and bacterial infection. Herein, to reveal nt5 gene role in drug resistance and infection ability of S. aureus, we performed nt5[C166T] gene mutation using a clustered regulatory interspaced short palindromic repeat ribonucleic acid (RNA)-guided base editing system to investigate the lose-of-function of NT5 protein. Subsequent transcriptome sequencing of the mutant strain revealed that nt5 inactivation caused changes in cell membrane integrity and inhibited nucleotide metabolism, suggesting the nt5 gene may be involved in bacterial drug resistance and virulence. The mutant strain exhibited enhanced tolerance to DAP treatment by attenuating cell membrane potential dissipation and slowing deoxyribonucleic acid release. Moreover, the nt5 mutation alleviated abscess degree of mouse kidneys caused by S. aureus infection byreducing the expression of IL-1β, IL-6, and IL-18. The nt5 mutant strain was easily swallowed by host immune cells, resulting in weak bacterial toxicity of the S. aureus mutant in the bacterial infection process. In summary, nt5 gene mutation confers tolerance to DAP and a lower bacterial capacity to form kidney abscesses through phagocytosis of host immune cells, which indicates the targeted inhibition of NT5 protein would offer a potential new therapeutic strategy against S. aureus infection.},
}

*Staphylococcus aureus/genetics/pathogenicity/drug effects
Animals
Mice
*Staphylococcal Infections/microbiology
*Immune Evasion
Virulence/genetics
*5'-Nucleotidase/genetics
*Mutation
Bacterial Proteins/genetics
Anti-Bacterial Agents/pharmacology
Drug Resistance, Bacterial/genetics
Daptomycin/pharmacology
Gene Editing/methods
Female
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid39841730,
year = {2025},
author = {Khan, H and Huang, X and Raj, V and Wang, H},
title = {A versatile site-directed gene trap strategy to manipulate gene activity and control gene expression in Caenorhabditis elegans.},
journal = {PLoS genetics},
volume = {21},
number = {1},
pages = {e1011541},
doi = {10.1371/journal.pgen.1011541},
pmid = {39841730},
issn = {1553-7404},
mesh = {*Caenorhabditis elegans/genetics ; Animals ; *CRISPR-Cas Systems ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Gene Expression Regulation/genetics ; Animals, Genetically Modified ; Transgenes ; Transcription Factors/genetics/metabolism ; Gene Editing/methods ; },
abstract = {The ability to manipulate gene activity and control transgene expression is essential to study gene function. While several genetic tools for modifying genes or controlling expression separately are available for Caenorhabditis elegans, there are no genetic approaches to generate mutations that simultaneously disrupt gene function and provide genetic access to the cells expressing the disrupted gene. To achieve this, we developed a versatile gene trap strategy based on cGAL, a GAL4-UAS bipartite expression system for C. elegans. We designed a cGAL gene trap cassette and used CRISPR/Cas9 to insert it into the target gene, creating a bicistronic operon that simultaneously expresses a truncated endogenous protein and the cGAL driver in the cells expressing the target gene. We demonstrate that our cGAL gene trap strategy robustly generated loss-of-function alleles. Combining the cGAL gene trap lines with different UAS effector strains allowed us to rescue the loss-of-function phenotype, observe the gene expression pattern, and manipulate cell activity spatiotemporally. We show that, by recombinase-mediated cassette exchange (RMCE) via microinjection or genetic crossing, the cGAL gene trap lines can be further engineered in vivo to easily swap cGAL with other bipartite expression systems' drivers, including QF/QF2, Tet-On/Tet-Off, and LexA, to generate new gene trap lines with different drivers at the same genomic locus. These drivers can be combined with their corresponding effectors for orthogonal transgenic control. Thus, our cGAL-based gene trap is versatile and represents a powerful genetic tool for gene function analysis in C. elegans, which will ultimately provide new insights into how genes in the genome control the biology of an organism.},
}

*Caenorhabditis elegans/genetics
Animals
*CRISPR-Cas Systems
*Caenorhabditis elegans Proteins/genetics/metabolism
Gene Expression Regulation/genetics
Animals, Genetically Modified
Transgenes
Transcription Factors/genetics/metabolism
Gene Editing/methods

@article {pmid39841239,
year = {2025},
author = {Bohra, A and Tiwari, A and Pareek, S and Joshi, R and Satheesh Naik, SJ and Kumari, K and Verma, RL and Parihar, AK and Patil, PG and Dixit, GP},
title = {Past and future of cytoplasmic male sterility and heterosis breeding in crop plants.},
journal = {Plant cell reports},
volume = {44},
number = {2},
pages = {33},
pmid = {39841239},
issn = {1432-203X},
mesh = {*Plant Breeding/methods ; *Crops, Agricultural/genetics ; *Plant Infertility/genetics ; *Hybrid Vigor/genetics ; *Pollen/genetics/physiology ; Gene Editing ; Cytoplasm/genetics ; },
abstract = {Plant breeding needs to embrace genetic innovations to ensure stability in crop yields under fluctuating climatic conditions. Development of commercial hybrid varieties has proven to be a sustainable and economical alternative to deliver superior yield, quality and resistance with uniformity in a number of food crops. Cytoplasmic male sterility (CMS), a maternally inherited inability to produce functional pollen, facilitates a three-line system for efficient hybrid seed production strategies in crops. The CMS system has illustrated its potential as a robust pollination control mechanism to support the billion-dollar seed industry. In plants, CMS arises due to a genomic conflict between mitochondrial open reading frames (orfs) and nuclear-encoding restoration-of-fertility (Rf) genes, leading to floral abnormalities and pollen sterility. Research on pollen sterility and fertility restoration provides deeper insights into cytoplasmic-nuclear interplay in plants and elucidates key molecular targets for hybrid breeding in crops. More recently, programmable gene editing (e.g., TALEN, CRISPR-Cas) has emerged as a promising tool to functionally validate CMS and Rf genes and obviate the need for pollen donors or Rf-genes for hybrid breeding. Modern genomic prediction models have allowed establishment of high-performing heterotic groups and patterns for sustaining long-term gain in hybrid breeding. This article reviews latest discoveries elucidating the molecular mechanisms behind CMS and fertility restoration in plants. We then present our perspective on how evolving genetic technologies are contributing to advance fundamental knowledge of the CMS-Rf genetic system for producing crop hybrids with high heterosis.},
}

*Plant Breeding/methods
*Crops, Agricultural/genetics
*Plant Infertility/genetics
*Hybrid Vigor/genetics
*Pollen/genetics/physiology
Gene Editing
Cytoplasm/genetics

@article {pmid39744795,
year = {2025},
author = {Li, X and Huang, Z and Lau, CH and Li, J and Zou, M and Wu, W and Chen, X and Li, J and Huang, Y and Wang, T and Li, Y and Xu, M and Huang, X and Zhu, H and Yang, C},
title = {One-pot isothermal CRISPR/Dx system for specific and sensitive detection of microRNA.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {4},
pages = {823-833},
doi = {10.1039/d4ay01695e},
pmid = {39744795},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis ; Humans ; *Pancreatic Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Sensitivity and Specificity ; },
abstract = {MicroRNA (miRNA) is a promising biomarker for the early diagnosis of pancreatic cancer. To enable sensitive and reliable miRNA detection, we have developed a one-pot isothermal CRISPR/Dx detection system by combining rolling circle amplification (RCA) and CRISPR/Cas12a. RCA and CRISPR/Cas12a reactions are carried out in a single closed tube, bypassing the transferring step. We demonstrate the feasibility of our one-pot CRISPR/Dx system in detecting pancreatic cancer by targeting miR-25, miR-191, miR-205, and miR-1246. When applied to fluorescence- and lateral flow strip paper-based detection platforms, our one-pot CRISPR/Dx system detects synthetic miR-25 at a LOD of 6.60 fM and 500 fM, respectively. It has high targeting specificity, as shown by its ability to discriminate miR-25 with a single-base mutation and highly homologous miRNA species. It is also successfully generalized to detect other pancreatic cancer-associated miRNAs, including miR-191, miR-205, and miR-1246. Importantly, our one-pot CRISPR/Dx system enables specific and sensitive detection of endogenous miR-25 in the human pancreatic cancer cell line PANC-1. We have successfully developed a one-pot isothermal CRISPR/Dx system for detecting miRNA with high specificity and sensitivity. It is highly flexible and economical, as a common crRNA can detect different miRNAs and only requires minor modifications to the locking padlock probe. Therefore, it can potentially be translated into clinical settings and POCT for the diagnosis of various human cancers.},
}

*MicroRNAs/genetics/analysis
Humans
*Pancreatic Neoplasms/genetics/diagnosis
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Sensitivity and Specificity

@article {pmid39732069,
year = {2025},
author = {Zhang, J and Zhou, Y and Qiao, J and Liu, Y},
title = {Recent advances in spatiotemporal control of the CRISPR/Cas9 system.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {248},
number = {},
pages = {114474},
doi = {10.1016/j.colsurfb.2024.114474},
pmid = {39732069},
issn = {1873-4367},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Animals ; },
abstract = {The CRISPR/Cas9 gene-editing technology, derived from the adaptive immune mechanisms of bacteria, has demonstrated remarkable advantages in fields such as gene function research and the treatment of genetic diseases due to its simplicity in design, precise targeting, and ease of use. Despite challenges such as off-target effects and cytotoxicity, effective spatiotemporal control strategies have been achieved for the CRISPR/Cas9 system through precise regulation of Cas9 protein activity as well as engineering of guide RNAs (gRNAs). This review provides a comprehensive analysis of the core components and functional mechanisms underlying the CRISPR/Cas9 system, highlights recent advancements in spatiotemporal control strategies, and discusses future directions for development.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Animals

@article {pmid39704119,
year = {2025},
author = {Lv, L and Liu, S and Fu, Y and Zhang, Y and Wang, M and Sun, J and Wang, Y and Lu, Y and Niu, G},
title = {A tunable and reversible thermo-inducible bio-switch for streptomycetes.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkae1236},
pmid = {39704119},
issn = {1362-4962},
support = {2023YFD1700700//National Key Research and Development Program of China/ ; 32370077//National Natural Science Foundation of China/ ; },
mesh = {*Streptomyces/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; *CRISPR-Cas Systems ; *Temperature ; Anti-Bacterial Agents/pharmacology/biosynthesis ; Bacterial Proteins/metabolism/genetics ; Anthraquinones/metabolism ; Gene Editing/methods ; Benzoisochromanequinones ; },
abstract = {Programmable control of bacterial gene expression holds great significance for both applied and academic research. This is particularly true for Streptomyces, a genus of Gram-positive bacteria and major producers of prodigious natural products. Despite that a few inducible regulatory systems have been developed for use in Streptomyces, there is an increasing pursuit to augment the toolkit of high-performance induction systems. We herein report a robust and reversible thermo-inducible bio-switch, designated as StrepT-switch. This bio-switch enables tunable and reversible control of gene expression using physiological temperatures as stimulation inputs. It has been proven successful in highly efficient CRISPR/Cas9-mediated genome engineering, as well as programmable control of antibiotic production and morphological differentiation. The versatility of the device is also demonstrated by thermal induction of a site-specific relaxase ZouA for overproduction of actinorhodin, a blue pigmented polyketide antibiotic. This study showcases the exploration a temperature-sensing module and exemplifies its versatility for programmable control of various target genes in Streptomyces species.},
}

*Streptomyces/genetics/metabolism
*Gene Expression Regulation, Bacterial
*CRISPR-Cas Systems
*Temperature
Anti-Bacterial Agents/pharmacology/biosynthesis
Bacterial Proteins/metabolism/genetics
Anthraquinones/metabolism
Gene Editing/methods
Benzoisochromanequinones

@article {pmid39663454,
year = {2025},
author = {Arce, MM and Umhoefer, JM and Arang, N and Kasinathan, S and Freimer, JW and Steinhart, Z and Shen, H and Pham, MTN and Ota, M and Wadhera, A and Dajani, R and Dorovskyi, D and Chen, YY and Liu, Q and Zhou, Y and Swaney, DL and Obernier, K and Shy, BR and Carnevale, J and Satpathy, AT and Krogan, NJ and Pritchard, JK and Marson, A},
title = {Central control of dynamic gene circuits governs T cell rest and activation.},
journal = {Nature},
volume = {637},
number = {8047},
pages = {930-939},
pmid = {39663454},
issn = {1476-4687},
mesh = {Humans ; *Lymphocyte Activation ; *Gene Regulatory Networks ; *CD4-Positive T-Lymphocytes/immunology/metabolism/cytology ; *Interleukin-2 Receptor alpha Subunit/metabolism/genetics ; *Single-Cell Analysis ; Kruppel-Like Transcription Factors/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Mediator Complex/metabolism/genetics ; Gene Expression Regulation ; T-Lymphocytes, Regulatory/immunology/metabolism/cytology ; },
abstract = {The ability of cells to maintain distinct identities and respond to transient environmental signals requires tightly controlled regulation of gene networks[1-3]. These dynamic regulatory circuits that respond to extracellular cues in primary human cells remain poorly defined. The need for context-dependent regulation is prominent in T cells, where distinct lineages must respond to diverse signals to mount effective immune responses and maintain homeostasis[4-8]. Here we performed CRISPR screens in multiple primary human CD4[+] T cell contexts to identify regulators that control expression of IL-2Rα, a canonical marker of T cell activation transiently expressed by pro-inflammatory effector T cells and constitutively expressed by anti-inflammatory regulatory T cells where it is required for fitness[9-11]. Approximately 90% of identified regulators of IL-2Rα had effects that varied across cell types and/or stimulation states, including a subset that even had opposite effects across conditions. Using single-cell transcriptomics after pooled perturbation of context-specific screen hits, we characterized specific factors as regulators of overall rest or activation and constructed state-specific regulatory networks. MED12 - a component of the Mediator complex - serves as a dynamic orchestrator of key regulators, controlling expression of distinct sets of regulators in different T cell contexts. Immunoprecipitation-mass spectrometry revealed that MED12 interacts with the histone methylating COMPASS complex. MED12 was required for histone methylation and expression of genes encoding key context-specific regulators, including the rest maintenance factor KLF2 and the versatile regulator MYC. CRISPR ablation of MED12 blunted the cell-state transitions between rest and activation and protected from activation-induced cell death. Overall, this work leverages CRISPR screens performed across conditions to define dynamic gene circuits required to establish resting and activated T cell states.},
}

Humans
*Lymphocyte Activation
*Gene Regulatory Networks
*CD4-Positive T-Lymphocytes/immunology/metabolism/cytology
*Interleukin-2 Receptor alpha Subunit/metabolism/genetics
*Single-Cell Analysis
Kruppel-Like Transcription Factors/metabolism/genetics
CRISPR-Cas Systems/genetics
Mediator Complex/metabolism/genetics
Gene Expression Regulation
T-Lymphocytes, Regulatory/immunology/metabolism/cytology

@article {pmid39660638,
year = {2025},
author = {Goh, CG and Bader, AS and Tran, TA and Belotserkovskaya, R and D'Alessandro, G and Jackson, SP},
title = {TDP1 splice-site mutation causes HAP1 cell hypersensitivity to topoisomerase I inhibition.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkae1163},
pmid = {39660638},
issn = {1362-4962},
support = {855741//ERC Synergy Award/ ; //A*STAR National Science Scholarship/ ; C17918/A28870//CRUK RadNet Cambridge Award/ ; 227014/Z/23/Z//Wellcome/ ; //University of Cambridge/ ; DRCPGM\100005/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Humans ; *Phosphoric Diester Hydrolases/genetics/metabolism ; *Topoisomerase I Inhibitors/pharmacology ; *DNA Topoisomerases, Type I/genetics/metabolism ; *Camptothecin/pharmacology ; Mutation ; Cell Line ; RNA Splice Sites/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {HAP1 is a near-haploid human cell line commonly used for mutagenesis and genome editing studies due to its hemizygous nature. We noticed an unusual hypersensitivity of HAP1 to camptothecin, an antineoplastic drug that stabilizes topoisomerase I cleavage complexes (TOP1ccs). We have attributed this hypersensitivity to a deficiency of TDP1, a key phosphodiesterase involved in resolving abortive TOP1ccs. Through whole-exome sequencing and subsequent restoration of TDP1 protein via CRISPR-Cas9 endogenous genome editing, we demonstrate that TDP1 deficiency and camptothecin hypersensitivity in HAP1 cells are a result of a splice-site mutation (TDP1 c.660-1G > A) that causes exon skipping and TDP1 loss of function. The lack of TDP1 in HAP1 cells should be considered when studying topoisomerase-associated DNA lesions and when generalizing mechanisms of DNA damage repair using HAP1 cells. Finally, we also report the generation of HAP1 STAR clones with restored TDP1 expression and function, which may be useful in further studies to probe cellular phenotypes relating to TOP1cc repair.},
}

Humans
*Phosphoric Diester Hydrolases/genetics/metabolism
*Topoisomerase I Inhibitors/pharmacology
*DNA Topoisomerases, Type I/genetics/metabolism
*Camptothecin/pharmacology
Mutation
Cell Line
RNA Splice Sites/genetics
CRISPR-Cas Systems
Gene Editing/methods

@article {pmid39658047,
year = {2025},
author = {Crawford, KD and Khan, AG and Lopez, SC and Goodarzi, H and Shipman, SL},
title = {High throughput variant libraries and machine learning yield design rules for retron gene editors.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkae1199},
pmid = {39658047},
issn = {1362-4962},
support = {MCB 2137692//National Science Foundation/ ; R21EB031393/EB/NIBIB NIH HHS/United States ; //W. M. Keck Foundation/ ; //Pew Biomedical Scholars Program/ ; //Gary and Eileen Morgenthaler Fund/ ; //UCSF Discovery Fellows Program/ ; R21EB031393/EB/NIBIB NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Gene Library ; *Machine Learning ; RNA-Directed DNA Polymerase/metabolism/genetics ; RNA, Untranslated/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The bacterial retron reverse transcriptase system has served as an intracellular factory for single-stranded DNA in many biotechnological applications. In these technologies, a natural retron non-coding RNA (ncRNA) is modified to encode a template for the production of custom DNA sequences by reverse transcription. The efficiency of reverse transcription is a major limiting step for retron technologies, but we lack systematic knowledge of how to improve or maintain reverse transcription efficiency while changing the retron sequence for custom DNA production. Here, we test thousands of different modifications to the Retron-Eco1 ncRNA and measure DNA production in pooled variant library experiments, identifying regions of the ncRNA that are tolerant and intolerant to modification. We apply this new information to a specific application: the use of the retron to produce a precise genome editing donor in combination with a CRISPR-Cas9 RNA-guided nuclease (an editron). We use high-throughput libraries in Saccharomyces cerevisiae to additionally define design rules for editrons. We extend our new knowledge of retron DNA production and editron design rules to human genome editing to achieve the highest efficiency Retron-Eco1 editrons to date.},
}

*Gene Editing/methods
*Saccharomyces cerevisiae/genetics
*CRISPR-Cas Systems
*Gene Library
*Machine Learning
RNA-Directed DNA Polymerase/metabolism/genetics
RNA, Untranslated/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39657782,
year = {2025},
author = {Li, Z and Wang, X and Janssen, JM and Liu, J and Tasca, F and Hoeben, RC and Gonçalves, MAFV},
title = {Precision genome editing using combinatorial viral vector delivery of CRISPR-Cas9 nucleases and donor DNA constructs.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkae1213},
pmid = {39657782},
issn = {1362-4962},
support = {//China Scholarship Council/ ; //Prinses Beatrix Spierfonds/ ; //Dutch Duchenne Parent/ ; //Horizon Europe/ ; /NWO_/Dutch Research Council/Netherlands ; },
mesh = {*Genetic Vectors/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Dependovirus/genetics ; Humans ; HEK293 Cells ; Transgenes ; Adenoviridae/genetics ; DNA/genetics/metabolism ; Homologous Recombination ; DNA End-Joining Repair/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Genome editing based on programmable nucleases and donor DNA constructs permits introducing specific base-pair changes and complete transgenes or live-cell reporter tags at predefined chromosomal positions. A crucial requirement for such versatile genome editing approaches is, however, the need to co-deliver in an effective, coordinated and non-cytotoxic manner all the required components into target cells. Here, adenoviral (AdV) and adeno-associated viral (AAV) vectors are investigated as delivery agents for, respectively, engineered CRISPR-Cas9 nucleases and donor DNA constructs prone to homologous recombination (HR) or homology-mediated end joining (HMEJ) processes. Specifically, canonical single-stranded and self-complementary double-stranded AAVs served as sources of ectopic HR and HMEJ substrates, whilst second- and third-generation AdVs provided for matched CRISPR-Cas9 nucleases. We report that combining single-stranded AAV delivery of HR donors with third-generation AdV transfer of CRISPR-Cas9 nucleases results in selection-free and precise whole transgene insertion in large fractions of target-cell populations (i.e. up to 93%) and disclose that programmable nuclease-induced chromosomal breaks promote AAV transduction. Finally, besides investigating relationships between distinct AAV structures and genome-editing performance endpoints, we further report that high-fidelity CRISPR-Cas9 nucleases are critical for mitigating off-target chromosomal insertion of defective AAV genomes known to be packaged in vector particles.},
}

*Genetic Vectors/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Dependovirus/genetics
Humans
HEK293 Cells
Transgenes
Adenoviridae/genetics
DNA/genetics/metabolism
Homologous Recombination
DNA End-Joining Repair/genetics
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid39633028,
year = {2025},
author = {Yin, JA and Frick, L and Scheidmann, MC and Liu, T and Trevisan, C and Dhingra, A and Spinelli, A and Wu, Y and Yao, L and Vena, DL and Knapp, B and Guo, J and De Cecco, E and Ging, K and Armani, A and Oakeley, EJ and Nigsch, F and Jenzer, J and Haegele, J and Pikusa, M and Täger, J and Rodriguez-Nieto, S and Bouris, V and Ribeiro, R and Baroni, F and Bedi, MS and Berry, S and Losa, M and Hornemann, S and Kampmann, M and Pelkmans, L and Hoepfner, D and Heutink, P and Aguzzi, A},
title = {Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes.},
journal = {Nature biomedical engineering},
volume = {9},
number = {1},
pages = {127-148},
pmid = {39633028},
issn = {2157-846X},
mesh = {Humans ; *Gene Silencing ; *CRISPR-Cas Systems/genetics ; *Genome, Human/genetics ; Gene Library ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Gene Deletion ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcription Factors/genetics/metabolism ; },
abstract = {Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wide ablation (19,936 plasmids) of human protein-coding genes and for their activation and epigenetic silencing (22,442 plasmids), with each plasmid encoding an array of four non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. The quadruple-sgRNA libraries yielded high perturbation efficacies in deletion (75-99%) and silencing (76-92%) experiments and substantial fold changes in activation experiments. Moreover, an arrayed activation screen of 1,634 human transcription factors uncovered 11 novel regulators of the cellular prion protein PrP[C], screening with a pooled version of the ablation library led to the identification of 5 novel modifiers of autophagy that otherwise went undetected, and 'post-pooling' individually produced lentiviruses eliminated template-switching artefacts and enhanced the performance of pooled screens for epigenetic silencing. Quadruple-sgRNA arrayed libraries are a powerful and versatile resource for targeted genome-wide perturbations.},
}

Humans
*Gene Silencing
*CRISPR-Cas Systems/genetics
*Genome, Human/genetics
Gene Library
RNA, Guide, CRISPR-Cas Systems/genetics
Plasmids/genetics
Gene Deletion
HEK293 Cells
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Transcription Factors/genetics/metabolism

@article {pmid39496933,
year = {2025},
author = {Hwang, GH and Lee, SH and Oh, M and Kim, S and Habib, O and Jang, HK and Kim, HS and Kim, Y and Kim, CH and Kim, S and Bae, S},
title = {Large DNA deletions occur during DNA repair at 20-fold lower frequency for base editors and prime editors than for Cas9 nucleases.},
journal = {Nature biomedical engineering},
volume = {9},
number = {1},
pages = {79-92},
pmid = {39496933},
issn = {2157-846X},
support = {2021M3A9H3015389//National Research Foundation of Korea (NRF)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; *DNA Repair/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; Sequence Deletion/genetics ; DNA/genetics ; Cell Line, Tumor ; DNA End-Joining Repair/genetics ; T-Lymphocytes ; Endonucleases/genetics/metabolism ; },
abstract = {When used to edit genomes, Cas9 nucleases produce targeted double-strand breaks in DNA. Subsequent DNA-repair pathways can induce large genomic deletions (larger than 100 bp), which constrains the applicability of genome editing. Here we show that Cas9-mediated double-strand breaks induce large deletions at varying frequencies in cancer cell lines, human embryonic stem cells and human primary T cells, and that most deletions are produced by two repair pathways: end resection and DNA-polymerase theta-mediated end joining. These findings required the optimization of long-range amplicon sequencing, the development of a k-mer alignment algorithm for the simultaneous analysis of large DNA deletions and small DNA alterations, and the use of CRISPR-interference screening. Despite leveraging mutated Cas9 nickases that produce single-strand breaks, base editors and prime editors also generated large deletions, yet at approximately 20-fold lower frequency than Cas9. We provide strategies for the mitigation of such deletions.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*DNA Breaks, Double-Stranded
*DNA Repair/genetics
*CRISPR-Associated Protein 9/metabolism/genetics
Sequence Deletion/genetics
DNA/genetics
Cell Line, Tumor
DNA End-Joining Repair/genetics
T-Lymphocytes
Endonucleases/genetics/metabolism

@article {pmid39428427,
year = {2025},
author = {Yang, F and Wei, N and Cai, S and Liu, J and Lan, Q and Zhang, H and Shang, L and Zheng, B and Wang, M and Liu, Y and Zhang, L and Fei, C and Tong, W and Liu, C and Kuang, E and Tong, G and Gu, F},
title = {Genome-wide CRISPR screens identify CLC-2 as a drug target for anti-herpesvirus therapy: tackling herpesvirus drug resistance.},
journal = {Science China. Life sciences},
volume = {68},
number = {2},
pages = {515-526},
pmid = {39428427},
issn = {1869-1889},
mesh = {*Chloride Channels/genetics/metabolism ; Animals ; *Antiviral Agents/pharmacology ; Mice ; *CLC-2 Chloride Channels ; *Drug Resistance, Viral/genetics ; *Herpesvirus 1, Suid/drug effects/genetics ; Humans ; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; Herpesviridae/drug effects/genetics ; Virus Replication/drug effects ; },
abstract = {The emergence of drug resistance to virus (i.e., acyclovir (ACV) to herpesviruses) has been termed one of the common clinical issues, emphasizing the discovery of new antiviral agents. To address it, a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening was performed in mouse haploid embryonic stem cells infected with pseudorabies virus (PRV), an α-herpesvirus causing human and pig diseases. The results demonstrated that type 2 voltage-gated chloride channels (CLC-2) encoded by one of the identified genes, CLCN2, is a potential drug target for anti-herpesvirus therapy. CLC-2 inhibitors, omeprazole (OME) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), can efficiently inhibit infection of multiple herpesviruses in cellulo (i.e., PRV, HSV and EBV), and effectively treat murine herpes simplex encephalitis (HSE). Additionally, DIDS was found to inhibit HSV-1 replication by blocking the PI3K/Akt pathway. Most importantly, both DIDS and OME were able to inhibit ACV-resistant HSV-1 strain infection. The study's findings suggest that targeting host-cell factors such as CLC-2 may be a promising approach to tackling herpesvirus drug resistance. The discovery of CLC-2 as a potential drug target for anti-herpesvirus therapy provides a new direction for the development of novel antiviral agents.},
}

*Chloride Channels/genetics/metabolism
Animals
*Antiviral Agents/pharmacology
Mice
*CLC-2 Chloride Channels
*Drug Resistance, Viral/genetics
*Herpesvirus 1, Suid/drug effects/genetics
Humans
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems/genetics
Herpesviridae/drug effects/genetics
Virus Replication/drug effects

@article {pmid39265628,
year = {2025},
author = {Crissey, MAS and Versace, A and Bhardwaj, M and Jain, V and Liu, S and Singh, A and Beer, LA and Tang, HY and Villanueva, J and Gimotty, PA and Xu, X and Amaravadi, RK},
title = {Divergent effects of acute and chronic PPT1 inhibition in melanoma.},
journal = {Autophagy},
volume = {21},
number = {2},
pages = {394-406},
doi = {10.1080/15548627.2024.2403152},
pmid = {39265628},
issn = {1554-8635},
support = {P01 CA114046/CA/NCI NIH HHS/United States ; P30 CA010815/CA/NCI NIH HHS/United States ; P50 CA261608/CA/NCI NIH HHS/United States ; R01 CA266404/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Melanoma/genetics/pathology/drug therapy/metabolism ; Humans ; Mice ; *Autophagy/drug effects/physiology/genetics ; *Thiolester Hydrolases/metabolism/genetics/antagonists & inhibitors ; *Mice, Knockout ; Proto-Oncogene Mas ; Cell Line, Tumor ; Lysosomes/metabolism/drug effects ; CRISPR-Cas Systems/genetics ; },
abstract = {Macroautophagy/autophagy-lysosome function promotes growth and survival of cancer cells, making them attractive targets for cancer therapy. One intriguing lysosomal target is PPT1 (palmitoyl-protein thioesterase 1). PPT1 inhibitors derived from chloroquine block autophagy, have significant antitumor activity in preclinical models and are being developed for clinical trials. However, the role of PPT1 in tumorigenesis remains poorly understood. Here we report that in melanoma cells, acute siRNA or pharmacological PPT1 inhibition led to increased ferroptosis sensitivity and significant loss of viability, whereas chronic PPT1 knockout using CRISPR-Cas9 produced blunted ferroptosis that led to sustained viability and growth. Each mode of PPT1 inhibition produced lysosome-autophagy inhibition but distinct proteomic changes, demonstrating the complexity of cellular adaptation mechanisms. To determine whether total genetic loss of Ppt1 would affect tumorigenesis in vivo, we developed a Ppt1 conditional knockout mouse model. We then crossed it into the BrafCA, PtenloxP, Tyr:CreERT2 melanoma mouse model to investigate the impact of Ppt1 loss on tumorigenesis. Loss of Ppt1 had no impact on melanoma histology, time to tumor initiation, or survival of tumor-bearing mice. These results suggest that chemical PPT1 inhibitors produce different adaptations than genetic PPT1 inhibition, and additional studies are warranted to fully understand the mechanism of chloroquine derivatives that target PPT1 in cancer.Abbreviations: 4-HT: 4-hydroxytamoxifen; BRAF: B-Raf proto-oncogene, serine/threonine kinase; cKO: conditional knockout; CRISPR-Cas9: clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9; DC661: A specific PPT1 inhibitor; DMSO: dimethyl sulfoxide; Dox; doxycycline hyclate; Easi-CRISPR: efficient additions with ssDNA inserts-CRISPR; GNS561/ezurpimtrostat: A PPT1 inhibitor; Hug: human guide; iCas: inducible CRISPR-Cas9; KO: knockout; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; LDLR: low density lipoprotein receptor; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NT: non-target; PTEN: phosphatase and tensin homolog; PPT1: palmitoyl-protein thioesterase 1; RSL3: RAS-selective lethal small molecule 3; SCRIB/SCRB1: scribble planar cell polarity protein; Tyr:CreERT2: tyrosinase-driven Cre recombinase fused with the tamoxifen-inducible mutant ligand binding domain of the human estrogen receptor; UGCG: UDP-glucose ceramide glucosyltransferase; WT: wild-type.},
}

Animals
*Melanoma/genetics/pathology/drug therapy/metabolism
Humans
Mice
*Autophagy/drug effects/physiology/genetics
*Thiolester Hydrolases/metabolism/genetics/antagonists & inhibitors
*Mice, Knockout
Proto-Oncogene Mas
Cell Line, Tumor
Lysosomes/metabolism/drug effects
CRISPR-Cas Systems/genetics

@article {pmid39187662,
year = {2025},
author = {Tong, M and Palmer, N and Dailamy, A and Kumar, A and Khaliq, H and Han, S and Finburgh, E and Wing, M and Hong, C and Xiang, Y and Miyasaki, K and Portell, A and Rainaldi, J and Suhardjo, A and Nourreddine, S and Chew, WL and Kwon, EJ and Mali, P},
title = {Robust genome and cell engineering via in vitro and in situ circularized RNAs.},
journal = {Nature biomedical engineering},
volume = {9},
number = {1},
pages = {109-126},
pmid = {39187662},
issn = {2157-846X},
support = {W81XWH-22-1-0401//U.S. Department of Defense (United States Department of Defense)/ ; R01 NS131560/NS/NINDS NIH HHS/United States ; AHA 916973//American Heart Association (American Heart Association, Inc.)/ ; U54CA274502//U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; U54 CA274502/CA/NCI NIH HHS/United States ; DP2NS111507//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; OT2OD032742//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*RNA, Circular/genetics ; *Cell Engineering/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Myocytes, Cardiac/metabolism/cytology ; Neurons/metabolism ; Mice ; HEK293 Cells ; Genome/genetics ; RNA/genetics ; Introns/genetics ; Gene Editing/methods ; Protein Engineering/methods ; },
abstract = {Circularization can improve RNA persistence, yet simple and scalable approaches to achieve this are lacking. Here we report two methods that facilitate the pursuit of circular RNAs (cRNAs): cRNAs developed via in vitro circularization using group II introns, and cRNAs developed via in-cell circularization by the ubiquitously expressed RtcB protein. We also report simple purification protocols that enable high cRNA yields (40-75%) while maintaining low immune responses. These methods and protocols facilitate a broad range of applications in stem cell engineering as well as robust genome and epigenome targeting via zinc finger proteins and CRISPR-Cas9. Notably, cRNAs bearing the encephalomyocarditis internal ribosome entry enabled robust expression and persistence compared with linear capped RNAs in cardiomyocytes and neurons, which highlights the utility of cRNAs in these non-dividing cells. We also describe genome targeting via deimmunized Cas9 delivered as cRNA and a long-range multiplexed protein engineering methodology for the combinatorial screening of deimmunized protein variants that enables compatibility between persistence of expression and immunogenicity in cRNA-delivered proteins. The cRNA toolset will aid research and the development of therapeutics.},
}

*RNA, Circular/genetics
*Cell Engineering/methods
Humans
*CRISPR-Cas Systems/genetics
Animals
Myocytes, Cardiac/metabolism/cytology
Neurons/metabolism
Mice
HEK293 Cells
Genome/genetics
RNA/genetics
Introns/genetics
Gene Editing/methods
Protein Engineering/methods

@article {pmid38987629,
year = {2025},
author = {Sousa, AA and Hemez, C and Lei, L and Traore, S and Kulhankova, K and Newby, GA and Doman, JL and Oye, K and Pandey, S and Karp, PH and McCray, PB and Liu, DR},
title = {Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells.},
journal = {Nature biomedical engineering},
volume = {9},
number = {1},
pages = {7-21},
pmid = {38987629},
issn = {2157-846X},
support = {P01HL152960//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; R35GM118062//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; Liu investigatorship//Howard Hughes Medical Institute (HHMI)/ ; T32 GM008313/GM/NIGMS NIH HHS/United States ; UH3 HL147366/HL/NHLBI NIH HHS/United States ; RM1HG009490//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Gene Editing/methods ; *Epithelial Cells/metabolism ; HEK293 Cells ; *Cystic Fibrosis/genetics/therapy ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bronchi/cytology ; },
abstract = {Prime editing (PE) enables precise and versatile genome editing without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human cystic fibrosis (CF) transmembrane conductance regulator (CFTR) F508del, a three-nucleotide deletion that is the predominant cause of CF. By combining six efficiency optimizations for PE-engineered PE guide RNAs, the PEmax architecture, the transient expression of a dominant-negative mismatch repair protein, strategic silent edits, PE6 variants and proximal 'dead' single-guide RNAs-we increased correction efficiencies for CFTR F508del from less than 0.5% in HEK293T cells to 58% in immortalized bronchial epithelial cells (a 140-fold improvement) and to 25% in patient-derived airway epithelial cells. The optimizations also resulted in minimal off-target editing, in edit-to-indel ratios 3.5-fold greater than those achieved by nuclease-mediated homology-directed repair, and in the functional restoration of CFTR ion channels to over 50% of wild-type levels (similar to those achieved via combination treatment with elexacaftor, tezacaftor and ivacaftor) in primary airway cells. Our findings support the feasibility of a durable one-time treatment for CF.},
}

Humans
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism
*Gene Editing/methods
*Epithelial Cells/metabolism
HEK293 Cells
*Cystic Fibrosis/genetics/therapy
CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Bronchi/cytology

@article {pmid39840369,
year = {2024},
author = {Moss, O and Li, X and Wang, ES and Kanagarajan, S and Guan, R and Ivarson, E and Zhu, LH},
title = {Knockout of BnaX.SGT.a caused significant sinapine reduction in transgene-free rapeseed mutants generated by protoplast-based CRISPR RNP editing.},
journal = {Frontiers in plant science},
volume = {15},
number = {},
pages = {1526941},
pmid = {39840369},
issn = {1664-462X},
abstract = {Rapeseed (Brassica napus L.) is known for its high-quality seed oil and protein content. However, its use in animal feed is restricted due to antinutritional factors present in the seedcake, with sinapine being one of the main compounds that reduces palatability. Attempts to develop rapeseed germplasm with lower sinapine levels through traditional breeding methods have shown limited progress. Genetic transformation methods could create new genotypes with reduced sinapine levels by silencing key genes involved in sinapine biosynthesis, though these methods often result in transgenic or genetically modified plants. The recent development of CRISPR-Cas technology provides a precise and efficient approach to crop improvement, with the potential to generate transgene-free mutants. In this study, we targeted the BnaX.SGT.a genes for knockout using CRISPR-Cas editing. By utilizing our newly established protoplast regeneration and transfection protocol for rapeseed, we demonstrated that DNA-free CRISPR editing via protoplast-based ribonucleoprotein (RNP) delivery was highly effective. We achieved successful knockout of the BnaX.SGT.a paralogues, with an average mutation efficiency of over 30%. Sequencing results revealed a variety of mutation types, from 1 bp insertions to 10 bp deletions, with most mutants exhibiting frameshift mutations that led to premature stop codons. The mutants displayed no visible phenotypic differences in growth patterns or flowering compared to the wild type. Importantly, sinapine content was significantly reduced in all T2 generation mutants analysed, while seed weight remained comparable between mutants and the wild type.},
}

@article {pmid39838465,
year = {2025},
author = {Cheng, J and Chen, J and Chen, D and Li, B and Wei, C and Liu, T and Wang, X and Wen, Z and Jin, Y and Sun, C and Yang, G},
title = {Development of a Komagataella phaffii cell factory for sustainable production of (+)-valencene.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {29},
pmid = {39838465},
issn = {1475-2859},
support = {2024SSYS0103//the major science and technology project of Zhejiang Province/ ; 2023C4S02002//the Start-up funds of Xianghu Laboratory/ ; },
mesh = {*Sesquiterpenes/metabolism ; *Metabolic Engineering/methods ; Saccharomycetales/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Sesquiterpene (+)-valencene is a characteristic aroma component from sweet orange fruit, which has a variety of biological activities and is widely used in industrial manufacturing of food, beverage and cosmetics industries. However, at present, the content in plant sources is low, and its yield and quality would be influenced by weather and land, which limit the supply of (+)-valencene. The rapid development of synthetic biology has accelerated the construction of microbial cell factories and provided an effective alternative method for the production of natural products.

RESULTS: In this study, we first introduced the ( +)-valencene synthase into Komagataella phaffii by CRISPR/Cas9 system, and successfully constructed a ( +)-valencene producer with the initial yield of 2.1 mg/L. Subsequently, the ( +)-valencene yield was increased to 8.2 mg/L by fusing farnesyl pyrophosphate synthase with ( +)-valencene synthase using the selected ligation linker. High expression of key genes IDI1, tHMG1, ERG12 and ERG19 enhanced metabolic flux of MVA pathway, and the yield of ( +)-valencene was further increased by 27%. Besides, in-situ deletion of the promoter of ERG9 increased the yield of ( +)-valencene to 48.1 mg/L. Finally, we optimized the copy number of farnesyl pyrophosphate synthase and ( +)-valencene synthase fusion protein, and when the copy number reached three, the yield of ( +)-valencene achieved 173.6 mg/L in shake flask level, which was 82-fold higher than that of the starting strain CaVAL1.

CONCLUSIONS: The results obtained here suggest that K. phaffii has the potential to efficiently synthesize other terpenoids.},
}

*Sesquiterpenes/metabolism
*Metabolic Engineering/methods
Saccharomycetales/metabolism/genetics
CRISPR-Cas Systems

@article {pmid39838422,
year = {2025},
author = {Hou, S and Yang, S and Bai, W},
title = {Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {28},
pmid = {39838422},
issn = {1475-2859},
support = {NO. XDC0120203//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; No. 306GJHZ2022010MI//CAS-NSTDA Joint Research Program/ ; TSBICIP-KJGG-020//Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Homologous Recombination ; Genome, Fungal ; },
abstract = {BACKGROUND: Ogataea polymorpha, a non-conventional methylotrophic yeast, has demonstrated significant potential for heterologous protein expression and the production of high-value chemicals and biopharmaceuticals. However, the lack of precise and efficient genome editing tools severely hinders the construction of cell factories. Although the CARISP-Cas9 system has been established in Ogataea polymorpha, the gene editing efficiency, especially for multiple genes edition, needs to be further improved.

RESULTS: In this study, we developed an efficient CRISPR-Cpf1-mediated genome editing system in O. polymorpha that exhibited high editing efficiency for single gene (98.1 ± 1.7%), duplex genes (93.9 ± 2.4%), and triplex genes (94.0 ± 6.0%). Additionally, by knocking out non-homologous end joining (NHEJ) related genes, homologous recombination (HR) efficiency was increased from less than 30% to 90 ~ 100%, significantly enhancing precise genome editing capabilities. The increased HR rates enabled over 90% integration efficiency of triplex genes, as well as over 90% deletion rates of large DNA fragments up to 20 kb. Furthermore, using this developed CRISPR-Cpf1 system, triple genes were precisely integrated into the genome by one-step, enabling lycopene production in O. polymorpha.

CONCLUSIONS: This novel multiplexed genome-editing tool mediated by CRISPR-Cpf1 can realize the deletion and integration of multiple genes, which holds great promise for accelerating engineering efforts on this non-conventional methylotrophic yeast for metabolic engineering and genomic evolution towards its application as an industrial cell factory.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Homologous Recombination
Genome, Fungal

@article {pmid39837388,
year = {2025},
author = {Wang, X and Li, Y and Friess, D and Yao, L and Wang, X and He, Z and He, W and Li, M and Wang, W},
title = {Guanidyl-rich highly branched poly(β-amino ester)s for the delivery of dual CRISPR ribonucleoprotein for efficient large DNA fragment deletion.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jconrel.2025.01.032},
pmid = {39837388},
issn = {1873-4995},
abstract = {Gene editing technologies, particularly clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, have revolutionized the ability to modify gene sequences in living cells for therapeutic purposes. Delivery of CRISPR/Cas ribonucleoprotein (RNP) is preferred over its DNA and RNA formats in terms of gene editing effectiveness and low risk of off-target events. However, the intracellular delivery of RNP poses significant challenges and necessitates the development of non-viral vectors. Our previous study has demonstrated that phenyl guanidine (PG) group modified linear poly(β-amino ester)s (PAEs) can facilitate CRISPR/Cas9 RNP mediated gene knockout in HeLa cells. Here, we further investigated the utilization of highly branched PAEs (HPAEs) with PG groups (HPAE-PG) for efficient delivery of cytosolic protein and CRISPR/Cas9 RNP complexes, while also examining the influence of branching units and branching ratios on the delivery process. The efficiency of HPAE-PG/RNP transfection for large DNA fragment deletion was assessed using a dual sgRNA-guided approach to delete exon 80 of the human COL7A1 gene, which harbors mutations associated with dystrophic epidermolysis bullosa (DEB). Our findings demonstrate that HPAE-PG/RNP successfully induced a deletion of 56 base pairs (exon 80) within COL7A1 in both HEK cells and keratinocytes derived from recessive DEB patients. This study highlights the potential of HPAE-PG as a non-viral vector for large DNA fragment deletion, emphasizing the importance of branching factors of HPAEs in optimizing CRISPR RNP delivery for therapeutic applications in genetic disorders.},
}

@article {pmid39791925,
year = {2025},
author = {Guo, W and Guo, Y and Xu, H and Li, C and Zhang, X and Zou, X and Sun, Z},
title = {Ultrasensitive "On-Off" Ratiometric Fluorescence Biosensor Based on RPA-CRISPR/Cas12a for Detection of Staphylococcus aureus.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {3},
pages = {2167-2173},
doi = {10.1021/acs.jafc.4c12202},
pmid = {39791925},
issn = {1520-5118},
mesh = {*Staphylococcus aureus/genetics/isolation & purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Fluorescence ; Bacterial Proteins/genetics/metabolism ; Food Contamination/analysis ; Limit of Detection ; CRISPR-Associated Proteins/genetics/metabolism ; Recombinases/metabolism/genetics ; Metal-Organic Frameworks/chemistry ; Endodeoxyribonucleases/genetics/metabolism ; },
abstract = {Staphylococcus aureus (S. aureus) is a major pathogenic bacterium responsible for bacterial foodborne diseases, making its rapid, specific, and accurate detection crucial. In this study, we develop a ratiometric biosensor based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats/CRISPR associated protein 12a (RPA-CRISPR/Cas12a) system and Eu-metal-organic framework (Eu-MOF) fluorescent nanomaterials for the high-sensitivity detection of S. aureus, combining with RPA for efficient isothermal amplification, this sensor enhances specificity and sensitivity by utilizing the target activation of CRISPR/Cas12a. The Eu-MOF serves a dual function, providing stable red fluorescence as a reference signal and adsorbing FAM-labeled probes for fluorescence quenching, forming a dual-signal system that significantly reduces background interference. This ratiometric design enables accurate and quantitative detection over a wide range (7.9 × 10[0] to 7.9 × 10[8] CFU/mL) with a low detection limit of 3 CFU/mL. Overall, with these merits of simplicity, rapid response, high sensitivity, and specificity, this dual-signal biosensor offers a promising method for accurately evaluating S. aureus contamination in food under complex substrate conditions.},
}

*Staphylococcus aureus/genetics/isolation & purification
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Fluorescence
Bacterial Proteins/genetics/metabolism
Food Contamination/analysis
Limit of Detection
CRISPR-Associated Proteins/genetics/metabolism
Recombinases/metabolism/genetics
Metal-Organic Frameworks/chemistry
Endodeoxyribonucleases/genetics/metabolism

@article {pmid39663988,
year = {2025},
author = {Xiao, X and Zhang, C and Zhang, L and Zuo, C and Wu, W and Cheng, F and Wu, D and Xie, G and Mao, X and Yang, Y},
title = {A phage amplification-assisted SEA-CRISPR/Cas12a system for viable bacteria detection.},
journal = {Journal of materials chemistry. B},
volume = {13},
number = {4},
pages = {1372-1382},
doi = {10.1039/d4tb02178a},
pmid = {39663988},
issn = {2050-7518},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Bacteriophages/genetics ; Urinary Tract Infections/diagnosis/microbiology ; Escherichia coli/isolation & purification/genetics ; Bacteria/isolation & purification/genetics/virology ; },
abstract = {Rapid and accurate detection of viable bacteria is essential for the clinical diagnosis of urinary tract infections (UTIs) and for making effective therapeutic decisions. However, most current molecular diagnostic techniques are unable to differentiate between viable and non-viable bacteria. In this study, we introduce a novel isothermal platform that integrates strand exchange amplification (SEA) with the CRISPR/Cas12a system, thereby enhancing both the sensitivity and specificity of the assay and achieving detection of phage DNA at concentrations as low as 4 × 10[2] copies per μL. Moreover, the incorporation of phages facilitates the specific recognition of viable bacteria and amplifies the initial signal through the inherent specificity and propagation properties of these phages. By employing the phage-assisted SEA-Cas12a approach, we successfully detected viable bacteria in human urine samples without the necessity of DNA extraction within 3.5 hours, achieving a detection limit of 10[3] CFU per mL. Considering its speed, accuracy, and independence from specialized equipment, this platform demonstrates significant potential as a robust tool for the rapid detection of various pathogens in resource-limited settings, thereby facilitating timely clinical management of UTI patients.},
}

*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*Bacteriophages/genetics
Urinary Tract Infections/diagnosis/microbiology
Escherichia coli/isolation & purification/genetics
Bacteria/isolation & purification/genetics/virology

@article {pmid39377274,
year = {2025},
author = {Tonsager, AJ and Stargell, LA},
title = {An undergraduate research experience in CRISPR-Cas9 mediated eukaryotic genome editing to teach fundamental biochemistry techniques.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {53},
number = {1},
pages = {33-45},
doi = {10.1002/bmb.21862},
pmid = {39377274},
issn = {1539-3429},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Students ; Humans ; *Saccharomyces cerevisiae/genetics ; *Biochemistry/education ; Universities ; Research/education ; Curriculum ; },
abstract = {CRISPR-Cas9 technology is an established, powerful tool for genome editing through the ability to target specific DNA sequences of interest for introduction of desired genetic modifications. CRISPR-Cas9 is utilized for a variety of purposes, ranging from a research molecular biology tool to treatment for human diseases. Due to its prominence across a variety of applications, it is critical that undergraduates in the life sciences are educated on CRISPR-Cas9 technology. To this end, we created an intensive eight-week long course-based undergraduate research experience (CURE) designed for students to understand CRISPR-Cas9 genome editing and perform it in Saccharomyces cerevisiae. Students enrolled in the CURE participate in 2, 3-h sessions a week and are engaged in the entire process of CRISPR-Cas9 genome editing, from preparation of genome editing reagents to characterization of mutant yeast strains. During the process, students master fundamental techniques in the life sciences, including sterile technique, Polymerase Chain Reaction (PCR), primer design, sequencing requirements, and data analysis. The course is developed with flexibility in the schedule for repetition of techniques in the event of a failed experiment, providing an authentic research experience for the students. Additionally, we have developed the course to be easily modified for the editing of any yeast gene, offering the potential to expand the course in research-driven classroom or laboratory settings.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Students
Humans
*Saccharomyces cerevisiae/genetics
*Biochemistry/education
Universities
Research/education
Curriculum

@article {pmid39039307,
year = {2024},
author = {Ferreira da Silva, J and Tou, CJ and King, EM and Eller, ML and Rufino-Ramos, D and Ma, L and Cromwell, CR and Metovic, J and Benning, FMC and Chao, LH and Eichler, FS and Kleinstiver, BP},
title = {Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {39039307},
issn = {1546-1696},
support = {R35GM142553//U.S. Department of Health Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35 GM142553/GM/NIGMS NIH HHS/United States ; UM1-HG012010//U.S. Department of Health Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; P180777//Swiss National Science Foundation | National Center of Competence in Research Quantum Science and Technology (NCCR QSIT - Quantum Science and Technology)/ ; DP2-CA281401//U.S. Department of Health Human Services | NIH | NIH Office of the Director (OD)/ ; ECOR FMD Fellowship//Massachusetts General Hospital (MGH)/ ; Kayden-Lambert MGH Research Scholar Award 2023-2028//Massachusetts General Hospital (MGH)/ ; P01-HL142494//U.S. Department of Health Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; FARA Fellowship//Friedreich's Ataxia Research Alliance (FARA)/ ; ALTF 750-2022//European Molecular Biology Organization (EMBO)/ ; 2020295403//National Science Foundation (NSF)/ ; U54 NS115052/NS/NINDS NIH HHS/United States ; Howard M. Goodman Fellowship//Massachusetts General Hospital (MGH)/ ; P01 HL142494/HL/NHLBI NIH HHS/United States ; UM1 HG012010/HG/NHGRI NIH HHS/United States ; DP2 CA281401/CA/NCI NIH HHS/United States ; },
abstract = {Genome editing technologies based on DNA-dependent polymerases (DDPs) could offer several benefits compared with other types of editors to install diverse edits. Here, we develop click editing, a genome writing platform that couples the advantageous properties of DDPs with RNA-programmable nickases to permit the installation of a range of edits, including substitutions, insertions and deletions. Click editors (CEs) leverage the 'click'-like bioconjugation ability of HUH endonucleases with single-stranded DNA substrates to covalently tether 'click DNA' (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs and their clkDNAs, we demonstrate the ability to install precise genome edits with minimal indels in diverse immortalized human cell types and primary fibroblasts with precise editing efficiencies of up to ~30%. Editing efficiency can be improved by rapidly screening clkDNA oligonucleotides with various modifications, including repair-evading substitutions. Click editing is a precise and versatile genome editing approach for diverse biological applications.},
}

@article {pmid37884392,
year = {2023},
author = {Liu, J and Lu, J and Yao, B and Zhang, Y and Huang, S and Chen, X and Shen, Y and Wang, X},
title = {Construction of humanized CYP1A2 rats using CRISPR/Cas9 to promote drug metabolism and pharmacokinetic research.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {52},
number = {1},
pages = {},
doi = {10.1124/dmd.123.001500},
pmid = {37884392},
issn = {1521-009X},
mesh = {Animals ; *Cytochrome P-450 CYP1A2/genetics/metabolism ; Humans ; Rats ; *CRISPR-Cas Systems ; *Microsomes, Liver/metabolism ; Male ; Clozapine/analogs & derivatives/pharmacokinetics/metabolism ; Gene Editing/methods ; Rats, Sprague-Dawley ; Theophylline/pharmacokinetics/metabolism/analogs & derivatives ; },
abstract = {Cytochrome P450 family 1 subfamily A member 2 (CYP1A2), performs an indispensable role in metabolism of both exogenous and endogenous substances. What is more, CYP1A2 functions in human diseases by regulating homeostasis of cholesterol. Despite the emergence of gene-editing animal models, genetically humanized animals that overcome species differences for further exploring the role of CYP1A2 in drug metabolism and human diseases have not yet been constructed. In this study, we inserted human CYP1A2 cDNA into the rat Cyp1a2 gene by using CRISPR/Cas9 technology. Results showed that human CYP1A2 was successfully expressed in humanized rat liver and there were no statistically significant differences of physiological symptoms compared with wild-type (WT) rats. In vitro incubation results indicated the different inhibition of furafylline on CYP1A2 activity in human liver microsomes, humanized CYP1A2 (hCYP1A2) rat liver microsomes, and WT rat liver microsomes, with IC50 values of 7.1 μM, 36.5 μM, and 285.8 μM, respectively. Meanwhile, pharmacokinetic characteristics of clozapine were conducted, and the results suggested that in hCYP1A2 rats, clozapine tended to be metabolized into norclozapine. Both the in vitro and in vivo results demonstrated the different metabolic functions of CYP1A2 in humanized and WT rats. We successfully constructed a novel humanized CYP1A2 rat model using the CRISPR/Cas9 system, providing a powerful tool for better predicting CYP1A2-mediated drug metabolism and pharmacokinetics. Significance Statement Human CYP1A2 takes active part both in the biotransformation of exogenous substances and endogenous substances. Meanwhile, it plays a regulatory role in human diseases, including hypercholesterolemia, hypertension as well as various malignant tumors. This study successfully constructed humanized CYP1A2 rat model by CRISPR/Cas9 technology, providing a powerful model for promoting drug development and safety evaluation, as well as further exploring the role of CYP1A2 in human diseases.},
}

Animals
*Cytochrome P-450 CYP1A2/genetics/metabolism
Humans
Rats
*CRISPR-Cas Systems
*Microsomes, Liver/metabolism
Male
Clozapine/analogs & derivatives/pharmacokinetics/metabolism
Gene Editing/methods
Rats, Sprague-Dawley
Theophylline/pharmacokinetics/metabolism/analogs & derivatives

@article {pmid39836103,
year = {2025},
author = {Mai, Z and Zhou, T and Lin, Z},
title = {Detecting CYP2C19 genes through an integrated CRISPR/Cas13a-assisted system.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4ay01930j},
pmid = {39836103},
issn = {1759-9679},
abstract = {CYP2C19 gene single nucleotide polymorphisms (SNPs) should be considered in the clinical use of clopidogrel as they have important guiding value for predicting the risk of bleeding and thrombosis after clopidogrel treatment. The CRISPR/Cas system is increasingly used for SNP detection owing to its single-nucleotide mismatch specificity. Simultaneous detection of multiple SNPs for rapid identification of the CYP2C19 genotype is important, but there is no method to detect a wide variety of CYP2C19 SNPs. This study proposes a new integrated system that integrates the PCR reaction and CRISPR/Cas detection of three CYP2C19 genes on a device, achieving rapid, sensitive, and specific detection. In our design, magnetic beads with three different sizes capture target nucleic acid from the sample, which are dragged through different areas by magnetic force, for PCR amplification reaction and CRISPR/Cas13a detection of CYP2C19*2, CYP2C19*3 and CYP2C19*17 genes. Note that magnetic beads were sorted via microporous PC membranes of different apertures. This study exhibits a broad clinical application prospect and provides a favorable tool for clinical clopidogrel administration.},
}

@article {pmid39834176,
year = {2025},
author = {Tang, M},
title = {Research Status of Clustered Regulary Interspaced Short Palindromic Repeats Technology in the Treatment of Human Papillomavirus (HPV) Infection Related Diseases.},
journal = {Cancer control : journal of the Moffitt Cancer Center},
volume = {32},
number = {},
pages = {10732748241300654},
doi = {10.1177/10732748241300654},
pmid = {39834176},
issn = {1526-2359},
mesh = {Humans ; *Papillomavirus Infections/therapy/genetics/virology ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Female ; Papillomaviridae/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Uterine Cervical Neoplasms/therapy/virology/genetics ; Gene Editing/methods ; Human Papillomavirus Viruses ; },
abstract = {Background: CRISPR/Cas9 technology has rapidly advanced as a pivotal tool in cancer research, particularly in the precision targeting required for both detecting and treating malignancies. Its high specificity and low off-target effects make it exceptionally effective in applications involving Human Papillomavirus (HPV) related diseases, most notably cervical cancer. This approach offers a refined methodology for the rapid detection of viral infections and provides a robust platform for the safe and effective treatment of diseases associated with viral infections through gene therapy.Purpose: Gene therapy, within this context, involves the strategic delivery of genetic material into target cells via a vector. This is followed by the meticulous modulation of gene expression, whether through correction, addition, or suppression, specifically honed to target tumor cells while sparing healthy cells. This dual capacity to diagnose and treat at such a precise level underscores the transformative potential of CRISPR/Cas9 in contemporary medical science, particularly in oncology and virology.Research Design: This article provides an overview of the advancements made in utilizing the CRISPR-Cas9 system as a research tool for HPV-related treatments while summarizing its application status in basic research, diagnosis, and treatment of HPV.Data Collection: Furthermore, it discusses the future prospects for this technology within emerging areas of HPV research and precision medicine in clinical practice, while highlighting technical challenges and potential directions for future development.},
}

Humans
*Papillomavirus Infections/therapy/genetics/virology
*CRISPR-Cas Systems/genetics
*Genetic Therapy/methods
Female
Papillomaviridae/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Uterine Cervical Neoplasms/therapy/virology/genetics
Gene Editing/methods
Human Papillomavirus Viruses

@article {pmid39833630,
year = {2025},
author = {Zhang, B and Li, J and Yu, W},
title = {Integration of CRISPR/dCas9-Based methylation editing with guide positioning sequencing identifies dynamic changes of mrDEGs in breast cancer progression.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {82},
number = {1},
pages = {46},
pmid = {39833630},
issn = {1420-9071},
support = {32270645//National Natural Science Foundation of China/ ; 18JC1411101//Major Special Projects of Basic Research of Shanghai Science and Technology Commission/ ; },
mesh = {Humans ; *Breast Neoplasms/genetics/pathology ; *DNA Methylation/genetics ; Female ; *CRISPR-Cas Systems/genetics ; *Disease Progression ; *Gene Editing/methods ; *Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Dynamic changes in DNA methylation are prevalent during the progression of breast cancer. However, critical alterations in aberrant methylation and gene expression patterns have not been thoroughly characterized. Here, we utilized guide positioning sequencing (GPS) to conduct whole-genome DNA methylation analysis in a unique human breast cancer progression model: MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and metastatic carcinoma). By integrating with mRNA-seq and matched clinical expression data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO), six representative methylation-related differentially expressed genes (mrDEGs) were identified, including CAVIN2, ARL4D, DUSP1, TENT5B, P3H2, and MMP28. To validate our findings, we independently developed and optimized the dCas9-DNMT3L-DNMT3A system, achieving a high efficiency with a 98% increase in methylation at specific sites. DNA methylation levels significantly increased for the six genes, with CAVIN2 at 67.75 ± 1.05%, ARL4D at 53.29 ± 6.32%, DUSP1 at 57.63 ± 8.46%, TENT5B at 44.00 ± 5.09%, P3H2 at 58.50 ± 3.90%, and MMP28 at 49.60 ± 5.84%. RT-qPCR confirmed an inverse correlation between increased DNA methylation and gene expression. Most importantly, we mimicked tumor progression in vitro, demonstrating that transcriptional silencing of the TENT5B promotes cell proliferation in MCF10A cells owing to the crosstalk between hypermethylation and histone deacetylation. This study unveils the practical implications of DNA methylation dynamics of mrDEGs in reshaping epigenomic features during breast cancer malignant progression through integrated data analysis of the methylome and transcriptome. The application of the CRISPR/dCas9-based methylation editing technique elucidates the regulatory mechanisms and functional roles of individual genes within the DNA methylation signature, providing valuable insights for understanding breast cancer pathogenesis and facilitating potential therapeutic approaches in epigenome editing for patients with breast cancer.},
}

Humans
*Breast Neoplasms/genetics/pathology
*DNA Methylation/genetics
Female
*CRISPR-Cas Systems/genetics
*Disease Progression
*Gene Editing/methods
*Gene Expression Regulation, Neoplastic
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39833501,
year = {2025},
author = {Gawlig, C and Hirschberger, R and Hanci, G and Schott, S and Marandi, S and Hesse, IR and Rühl, M},
title = {Full sequencing of 100mer sgRNA via tandem mass spectrometry by targeted RNase H digestion with customized probes.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {39833501},
issn = {1618-2650},
abstract = {The use of single-guide RNA (sgRNA) for gene editing using the CRISPR Cas9 system has become a powerful technique in various fields, especially with the growing interest in such molecules as therapeutic options in the last years. An important parameter for the use of these molecules is the verification of the correct sgRNA oligonucleotide sequence. Apart from next-generation sequencing protocols, mass spectrometry (MS) has been proven as a powerful technique for this purpose. The protocol and investigations presented in this work show an optimal digestion and 100% sequence coverage of sgRNA, while top-down approaches or other ribonuclease (RNase) digestion strategies obtain a sequence coverage of up to 80-90% utilizing multiple RNases. The results in this publication were obtained by utilizing DNA-RNA hybrid GAPmer-like probes and RNase H, an enzyme which specifically hydrolyzes RNA in DNA-RNA double strands. We assessed the optimal length of the DNA segment of these hybrid probes to maximize the specificity of the RNase H digestion and to achieve complete sequence confirmation by tandem MS analysis of the resulting digestion products. Furthermore, we showed that the approach is applicable for the identification of common synthesis-related impurities, like truncations and elongations. Despite the fact that the accessibility of this approach for highly modified molecules is limited to nucleotides which are not 2'-O-methylated, the optimized sequence coverage makes it a viable method.},
}

@article {pmid39833409,
year = {2025},
author = {Zhang, X and Qiu, M and Han, B and Liao, L and Peng, X and Lin, J and Zhang, N and Hai, L and Liang, L and Ma, Y and Li, W and Liu, M},
title = {Generation and propagation of high fecundity gene edited fine wool sheep by CRISPR/Cas9.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {2557},
pmid = {39833409},
issn = {2045-2322},
support = {2023SNGGGCC010//the Tianshan Talent Training Program-high-level talents for the advancement of modern agriculture and animal husbandry./ ; 2023TSYCTD0007//the Scientific and Technological Innovation Team Project of Xinjiang Uygur Autonomous Region./ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Fertility/genetics ; Sheep/genetics ; Female ; Wool ; Bone Morphogenetic Protein Receptors, Type I/genetics ; Litter Size/genetics ; Alleles ; },
abstract = {CRISPR/Cas9 technology has been widely utilized to enhance productive performance, increase disease resistance and generate medical models in livestock. The FecB allele in sheep is a mutation in the BMPRIB gene, recognized as the first major gene responsible for the high fecundity trait in sheep, leading to an increased ovulation rate in ewe. In this study, we employed CRISPR/Cas9-mediated homologous-directed repair (HDR) to introduce a defined point mutation (c.746 A > G) using single-stranded oligonucleotides (ssODN) and the ligase IV inhibitor (SCR7) into the BMPRIB gene of fine wool sheep. A total of nine gene-edited sheep were produced, six of which carried the targeted point mutation, with a precise base substitution efficiency (A > G) of 31.6%. Based on the six targeted founders (F0), we expanded the BMPRIB-targeted population, which included F1 heterozygous (B+) and F2 homozygous(BB) or heterozygous offspring. The average litter size of F1 ewes carrying the B + allele reached 170%, comparable to that of heterozygous native Australian Booroola sheep. Gene-edited ewes with B + and BB genotype produced 0.62 and 0.42 more lambs, respectively, compared to wide-type ewes (p < 0.01). Our results also indicated that the parity signification, our data demonstrate that highly efficient introduction of the intended base mutation into the sheep genome can be achieved by combining the CRISPR/Cas9 system with ssODN and SCR7. The offspring of BMPR/B edited sheep with the defined mutation exhibited high fecundity performance. Compared to conventional sheep breeding strategies, genetic improvement through gene editing offered significant advantages without compromising the fine wool traits of Merino sheep, which are often affected by routine cross-breeding methods.},
}

Animals
*Gene Editing/methods
*CRISPR-Cas Systems
*Fertility/genetics
Sheep/genetics
Female
Wool
Bone Morphogenetic Protein Receptors, Type I/genetics
Litter Size/genetics
Alleles

@article {pmid39833307,
year = {2025},
author = {Di Cristina, G and Dirksen, E and Altenhein, B and Büschges, A and Korsching, SI},
title = {Pioneering genome editing in parthenogenetic stick insects: CRISPR/Cas9-mediated gene knockout in Medauroidea extradentata.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {2584},
pmid = {39833307},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *Parthenogenesis/genetics ; *Insecta/genetics ; Phenotype ; Female ; Pigmentation/genetics ; },
abstract = {The parthenogenetic life cycle of the stick insect Medauroidea extradentata offers unique advantages for the generation of genome-edited strains, as an isogenic and stable mutant line can in principle be achieved already in the first generation (G0). However, genetic tools for the manipulation of their genes had not been developed until now. Here, we successfully implement CRISPR/Cas9 as a technique to modify the genome of the stick insect M. extradentata. As proof-of-concept we targeted two genes involved in the ommochrome pathway of eye pigmentation (cinnabar and white, second and first exon, respectively), to generate knockout (KO) mutants. Microinjections were performed within 24 h after oviposition, to focus on the mononuclear (and haploid) stage of development. The KOs generated resulted in distinct eye and cuticle colour phenotypes for cinnabar and white. Homozygous cinnabar mutants showed pale pigmentation of eyes and cuticle. They develop into adults capable of producing viable eggs. Homozygous white KO resulted in a completely unpigmented phenotype in developing embryos that were unable to hatch. In conclusion, we show that CRISPR/Cas9 can be successfully applied to the genome of M. extradentata by creating phenotypically different and viable insects. This powerful gene editing technique can now be employed to create stable genetically modified lines using a parthenogenetic non-model organism.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
*Gene Knockout Techniques/methods
*Parthenogenesis/genetics
*Insecta/genetics
Phenotype
Female
Pigmentation/genetics

@article {pmid39833279,
year = {2025},
author = {Bi, C and Yuan, B and Zhang, Y and Wang, M and Tian, Y and Li, M},
title = {Prevalent integration of genomic repetitive and regulatory elements and donor sequences at CRISPR-Cas9-induced breaks.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {94},
pmid = {39833279},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; Regulatory Sequences, Nucleic Acid ; },
abstract = {CRISPR-Cas9 genome editing has been extensively applied in both academia and clinical settings, but its genotoxic risks, including large insertions (LgIns), remain poorly studied due to methodological limitations. This study presents the first detailed report of unintended LgIns consistently induced by different Cas9 editing regimes using various types of donors across multiple gene loci. Among these insertions, retrotransposable elements (REs) and host genomic coding and regulatory sequences are prevalent. RE frequencies and 3D genome organization analysis suggest LgIns originate from randomly acquired genomic fragments by DNA repair mechanisms. Additionally, significant unintended full-length and concatemeric double-stranded DNA (dsDNA) donor integrations occur when donor DNA is present. We further demonstrate that phosphorylated dsDNA donors consistently reduce large insertions and deletions by almost two-fold without compromising homology-directed repair (HDR) efficiency. Taken together, our study addresses a ubiquitous and overlooked risk of unintended LgIns in Cas9 editing, contributing valuable insights for the safe use of Cas9 editing tools.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Humans
Regulatory Sequences, Nucleic Acid

@article {pmid39833194,
year = {2025},
author = {Kim, DG and Gu, B and Cha, Y and Ha, J and Lee, Y and Kim, G and Cho, BK and Oh, MK},
title = {Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {874},
pmid = {39833194},
issn = {2041-1723},
support = {RS-2024-00509338//National Research Foundation of Korea (NRF)/ ; RS-2024-00357320//National Research Foundation of Korea (NRF)/ ; RS-2021-NR056596//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Streptomyces/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Multigene Family ; *Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Secondary Metabolism/genetics ; Metabolic Engineering/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipulation using Cas9 within strains possessing high GC content genome. The Cas9-BD, a modified Cas9 with the addition of polyaspartate to its N- and C-termini, is developed with decreased off-target binding and cytotoxicity compared with wild-type Cas9. Cas9-BD and similarly modified dCas9-BD have been successfully employed for simultaneous biosynthetic gene cluster (BGC) refactoring, multiple BGC deletions, or multiplexed gene expression modulations in Streptomyces. We also demonstrate improved secondary metabolite production using multiplexed genome editing with multiple single guide RNA libraries in several Streptomyces strains. Cas9-BD is also used to capture large BGCs using a developed in vivo cloning method. The modified CRISPR-Cas9 system is successfully applied to many Streptomyces sp., providing versatile and efficient genome editing tools for strain engineering of actinomycetes with high GC content genome.},
}

*Streptomyces/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
*Multigene Family
*Genome, Bacterial
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Secondary Metabolism/genetics
Metabolic Engineering/methods
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid39833175,
year = {2025},
author = {Goold, HD and Kroukamp, H and Erpf, PE and Zhao, Y and Kelso, P and Calame, J and Timmins, JJB and Wightman, ELI and Peng, K and Carpenter, AC and Llorente, B and Hawthorne, C and Clay, S and van Wyk, N and Daniel, EL and Harrison, F and Meier, F and Willows, RD and Cai, Y and Walker, RSK and Xu, X and Espinosa, MI and Stracquadanio, G and Bader, JS and Mitchell, LA and Boeke, JD and Williams, TC and Paulsen, IT and Pretorius, IS},
title = {Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {841},
pmid = {39833175},
issn = {2041-1723},
support = {DP200100717//Department of Education and Training | Australian Research Council (ARC)/ ; CE200100029//Department of Education and Training | Australian Research Council (ARC)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Genome, Fungal ; Chromosomes, Artificial, Yeast/genetics ; Chromosomes, Fungal/genetics ; Synthetic Biology/methods ; CRISPR-Cas Systems ; Open Reading Frames/genetics ; RNA, Transfer/genetics/metabolism ; },
abstract = {The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5' UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms.},
}

*Saccharomyces cerevisiae/genetics/metabolism
Genome, Fungal
Chromosomes, Artificial, Yeast/genetics
Chromosomes, Fungal/genetics
Synthetic Biology/methods
CRISPR-Cas Systems
Open Reading Frames/genetics
RNA, Transfer/genetics/metabolism

@article {pmid39832868,
year = {2025},
author = {Alarcón-Iniesta, H and de Arana, G and López-Valls, M and Pardo, D and Escalona-Noguero, C and Rodríguez, C and Castellanos, M and Cobelo, S and Martínez-Ramírez, I and Camarero, J and Heras, SL and de Vicente, J and Valera, A and Smith, W and Bernardo-Gavito, R and Cantón, R and Galán, JC and Granados, D and Miranda, R and Guerrero, H and Sot, B},
title = {CRISPR-associated Plasmonic Colorimeter Method (Ca-PCM): A real-time RGB detection system for gold nanoparticles-based nucleic acid biosensors.},
journal = {Analytica chimica acta},
volume = {1338},
number = {},
pages = {343601},
doi = {10.1016/j.aca.2024.343601},
pmid = {39832868},
issn = {1873-4324},
mesh = {*Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Colorimetry ; *Biosensing Techniques/methods ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: The detection of genetic sequences represents the gold standard procedure for species discrimination, genetic characterisation of tumours, and identification of pathogens. The development of new molecular detection methods, accessible and cost effective, is of great relevance. Biosensors based on plasmonic nanoparticles, such as gold nanoparticles (AuNPs), provide a powerful and versatile platform for highly sensitive, economic, user-friendly and label-free sensing. However, the readout techniques typically employed with such sensors lack temporal and kinetic information, which hampers the ability to perform quantitative detection.

RESULTS: In this study, a novel methodology designated the 'CRISPR-associated Plasmonic Colorimeter Method' (Ca-PCM), has been developed. This method combines RNA target recognition by CRISPR LwaCas13a, AuNPs' aggregation, and real-time colorimetric Red-Green-Blue (RGB) analysis. The system registers the AuNP's plasmonic signatures in real-time using a RGB colour sensor with 3-channel silicon photodiodes having blue, green and red sensitivities. The acquired signals are automatically analysed by an algorithm designed to distinguish between positive and negative samples and to correlate the temporal spectral patterns of aggregation with dose-dependent molecular detection of the RNA target. In addition, the combination of Ca-PCM with a previous isothermal amplification allows the target efficient detection in real clinical applications.

SIGNIFICANCE: We have shown that the combination of RGB analysis and continuous temporal measurements is a novel and promising method to characterise the behaviour of gold nanoparticle-based biosensors and to achieve dose-dependent target detection. In addition, the simplicity and cost-effectiveness of this new approach expand the possibilities of other plasmonic-based biosensors and their applicability in low-resources clinical environments.},
}

*Gold/chemistry
*Metal Nanoparticles/chemistry
*Colorimetry
*Biosensing Techniques/methods
Humans
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems/genetics

@article {pmid39832863,
year = {2025},
author = {Zhang, T and Wang, X and Jiang, Y and Zhang, Y and Zhao, S and Hu, J and Hu, F and Peng, N},
title = {A miniaturized RPA-CRISPR/Cas12a-based nucleic acid diagnostic platform for rapid and simple self-testing of SARS-CoV-2.},
journal = {Analytica chimica acta},
volume = {1338},
number = {},
pages = {343593},
doi = {10.1016/j.aca.2024.343593},
pmid = {39832863},
issn = {1873-4324},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *COVID-19/diagnosis/virology ; CRISPR-Cas Systems/genetics ; Self-Testing ; COVID-19 Nucleic Acid Testing/methods/instrumentation ; RNA, Viral/analysis/genetics ; Limit of Detection ; Molecular Diagnostic Techniques/methods/instrumentation ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) RESULTS: In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30 min with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) SIGNIFICANCE: This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52).},
}

*SARS-CoV-2/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
Humans
*COVID-19/diagnosis/virology
CRISPR-Cas Systems/genetics
Self-Testing
COVID-19 Nucleic Acid Testing/methods/instrumentation
RNA, Viral/analysis/genetics
Limit of Detection
Molecular Diagnostic Techniques/methods/instrumentation
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39832801,
year = {2025},
author = {Debnath, A and Sengupta, A and Rudrapal, S and Kumar, A and Rani, M},
title = {In-silico study of molecular adaptations in halophilic Cas9.},
journal = {Letters in applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/lambio/ovaf006},
pmid = {39832801},
issn = {1472-765X},
abstract = {This study explores the structural adaptations of the CRISPR-Cas9 system in halophilic bacteria, focusing on Cas9 protein of halophilic bacterium Salicibibacter cibi. Protein sequences were analysed using different tools such as ExPASy ProtParam for different physicochemical properties, PONDR web server for disordered regions, and InterPro server and WebLogo for domains. Protein structures were generated using the AlphaFold database, and the quality of the modelled structure was checked through PROCHECK. The protein surface's amino acids and electrostatic potential were visualized using PyMOL, APBS server and UCSF chimera. Comparative analysis revealed that halophilic Cas9 proteins possess a higher abundance of acidic residues, resulting in enhanced stability and hydration in saline conditions; halophilic Cas9 proteins also shows higher intrinsically disordered regions. Electrostatic potential maps confirmed that S. cibi Cas9 proteins maintain a highly negative surface charge, crucial for adaptation to salt-rich environments. These findings provide insights into the molecular mechanisms driving the structural and functional adaptations of Cas9 in salty environment, highlighting its potential applications in genome editing-based biotechnological approaches in extreme conditions.},
}

@article {pmid39832424,
year = {2025},
author = {Choudhary, R and Ahmad, F and Kaya, C and Upadhyay, SK and Muneer, S and Kumar, V and Meena, M and Liu, H and Upadhyaya, H and Seth, CS},
title = {Decrypting proteomics, transcriptomics, genomics, and integrated omics for augmenting the abiotic, biotic, and climate change stress resilience in plants.},
journal = {Journal of plant physiology},
volume = {305},
number = {},
pages = {154430},
doi = {10.1016/j.jplph.2025.154430},
pmid = {39832424},
issn = {1618-1328},
abstract = {As our planet faces increasing environmental challenges, such as biotic pressures, abiotic stressors, and climate change, it is crucial to understand the complex mechanisms that underlie stress responses in crop plants. Over past few years, the integration of techniques of proteomics, transcriptomics, and genomics like LC-MS, IT-MS, MALDI-MS, DIGE, ESTs, SAGE, WGS, GWAS, GBS, 2D-PAGE, CRISPR-Cas, cDNA-AFLP, HLS, HRPF, MPSS, CAGE, MAS, IEF, MudPIT, SRM/MRM, SWATH-MS, ESI have significantly enhanced our ability to comprehend the molecular pathways and regulatory networks, involved in balancing the ecosystem/ecology stress adaptation. This review offers thorough synopsis of the current research on utilizing these multi-omics methods (including metabolomics, ionomics) for battling abiotic (salinity, temperature (chilling/freezing/cold/heat), flood (hypoxia), drought, heavy metals/loids), biotic (pathogens like fungi, bacteria, virus, pests, and insects (aphids, caterpillars, moths, mites, nematodes) and climate change stress (ozone, ultraviolet radiation, green house gases, carbon dioxide). These strategies can expedite crop improvement, and act as powerful tools with high throughput and instant database generation rates. They also provide a platform for interpreting intricate, systematic signalling pathways and knowing how different environmental stimuli cause phenotypic responses at cellular and molecular level by changing the expression of stress-responsive genes like RAB18, KIN1, RD29B, OsCIPK03, OsSTL, SIAGL, bZIP, SnRK, ABF. This review discusses various case studies that exemplify the successful implementation of these omics tools to enhance stress tolerance in plants. Finally, it highlights challenges and future prospects of utilizing these approaches in combating stress, emphasizing the need for interdisciplinary collaborations and bio-technological advancements for sustainable agriculture and food security.},
}

@article {pmid39832407,
year = {2025},
author = {Mao, Z and Chen, R and Huang, L and Ren, S and Liu, B and Gao, Z},
title = {CRISPR analysis based on Pt@MOF dual-modal signal for multichannel fluorescence and visual detection of norovirus.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117153},
doi = {10.1016/j.bios.2025.117153},
pmid = {39832407},
issn = {1873-4235},
abstract = {Norovirus is a globally prevalent pathogen that causes acute viral gastroenteritis across all age groups, characterized by its high infectivity and low infectious dose. Consequently, the development of rapid, sensitive, and accurate detection technologies for norovirus presents a significant challenge. In this study, we demonstrate a combination of CRISPR-Cas-based reactions with Pt@MOF-linked immunoassay-like assays. This methodology enables both qualitative analysis and colorimetric readouts of Cas12a-mediated DNA/RNA detection at room temperature, as well as the generation of fluorescent signal readout through base deprotonation-induced Pt@MOF cleavage of a fluorogenic substrate. Furthermore, the integration of RPA amplification with noncanonical PAM-designed CRISPR significantly enhances the sensitivity and flexibility of detection, facilitating the extension of this strategy to other targets. Ultimately, the strategy was validated in spiked food samples with a 100% accuracy rate, consistent with RT-qPCR results. Collectively, this work showcases a viable approach for a dual-functional Pt@MOF-based CRISPR biosensing platform for bioanalysis and a flexible, universal strategy based on noncanonical PAM-designed gRNAs.},
}

@article {pmid39764982,
year = {2025},
author = {Yue, Y and Liu, M and Ma, M and Xu, Z and Zhang, H and Wang, Q and Liu, R},
title = {CRISPR/Cas14a integrated with DNA walker based on magnetic self-assembly for human papillomavirus type 16 oncoprotein E7 ultrasensitive detection.},
journal = {Biosensors & bioelectronics},
volume = {272},
number = {},
pages = {117135},
doi = {10.1016/j.bios.2025.117135},
pmid = {39764982},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Papillomavirus E7 Proteins/genetics ; Animals ; Humans ; Mice ; *Human papillomavirus 16/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Limit of Detection ; Papillomavirus Infections/virology/diagnosis ; Electrochemical Techniques/methods ; Uterine Cervical Neoplasms/virology/diagnosis ; Female ; },
abstract = {To enhance the biomarker diagnostics sensitivity and selectivity of human papillomavirus type 16 oncoprotein E7 (HPV16 E7) in serum, a label/enzyme-free electrochemical detection platform was developed. This platform featured a type of "Super-turn-off" nanobiosensor monitored through differential pulse voltammetry (DPV). It integrated the magnetic self-assembly property of the α-Fe2O3/Fe3O4@Au/Sub/BSA signal transport nano-medium with the high specificity of CRISPR/Cas14a and the amplification capability of the bipedal walker (DNA walker composed of two ssDNA strands), resulting in the enhanced specificity and anti-interference performance while remaining stable at 4 °C for over 30 days. The results demonstrated that the combination of walker and CRISPR yielded superior sensitivity and analytical capability compared with using either technology alone, achieving a detection limit of 67.17 fg mL[-1], a quantification limit of 0.22 pg mL[-1], and serum sample recovery rates of 98.46%-115.78%. Additionally, this platform was applied to detect serum and tissue samples from mouse models at various stages of cervical cancer, significantly improving the accuracy and effectiveness of early diagnosis and prognostic evaluation. This novel approach held promise as an efficient tool for point-of-care clinical detection of HPV16 E7, potentially reducing cervical cancer mortality.},
}

*Biosensing Techniques/methods
*Papillomavirus E7 Proteins/genetics
Animals
Humans
Mice
*Human papillomavirus 16/genetics/isolation & purification
*CRISPR-Cas Systems
*Limit of Detection
Papillomavirus Infections/virology/diagnosis
Electrochemical Techniques/methods
Uterine Cervical Neoplasms/virology/diagnosis
Female

@article {pmid39740588,
year = {2025},
author = {Liu, H and Lv, MM and Li, X and Su, M and Nie, YG and Ying, ZM},
title = {Ligation-recognition triggered RPA-Cas12a cis-cleavage fluorogenic RNA aptamer for one-pot and label-free detection of MicroRNA in breast cancer.},
journal = {Biosensors & bioelectronics},
volume = {272},
number = {},
pages = {117106},
doi = {10.1016/j.bios.2024.117106},
pmid = {39740588},
issn = {1873-4235},
mesh = {*Breast Neoplasms/genetics/diagnosis/blood ; Humans ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; *MicroRNAs/blood/genetics/analysis ; Female ; Nucleic Acid Amplification Techniques/methods ; Fluorescent Dyes/chemistry ; CRISPR-Cas Systems ; Limit of Detection ; Bacterial Proteins/chemistry/genetics ; CRISPR-Associated Proteins/chemistry/genetics ; Endodeoxyribonucleases/chemistry ; },
abstract = {"One-pot" assays which combine amplification with CRISPR/Cas12a system are in constant attracted for biosensors development. Herein, we present a one-pot isothermal assay that Ligation-recognition triggered Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a cis-cleavage (LRPA-CRISPR) fluorescent biosensor for sensitive, specific, and label-free miRNA detection. Firstly, we reveal the programmed double-stranded DNA amplicons, which utilized the ligation-recognition and polymerization to form and amplified by the RPA system. Meanwhile, we enabled exponential ligation-recognition triggered recombinase polymerase amplification of miRNA-21 sequences and exploited the cis-cleavage mechanism of Cas12a with transcription to generate functional Mango RNA for signal output. This assay can be completed within 40 min and can allow a limit of detection of 3.43 aM for miRNA-21 detection, owing to the RPA with transcription amplification and enables to product the functional Mango RNA aptamer by in vitro transcription that binds to the TO1-Biotin fluorogenic dye. Moreover, our method exhibits the advantages of self-supply crRNA, label-free, excellent specificity, and universal detection platform via the design of one-pot detection in serum and cell samples, showing tremendous potential in biomarkers diagnostics of breast cancer.},
}

*Breast Neoplasms/genetics/diagnosis/blood
Humans
*Biosensing Techniques/methods
*Aptamers, Nucleotide/chemistry
*MicroRNAs/blood/genetics/analysis
Female
Nucleic Acid Amplification Techniques/methods
Fluorescent Dyes/chemistry
CRISPR-Cas Systems
Limit of Detection
Bacterial Proteins/chemistry/genetics
CRISPR-Associated Proteins/chemistry/genetics
Endodeoxyribonucleases/chemistry

@article {pmid39622311,
year = {2025},
author = {Kim, M and Hutchins, EJ},
title = {CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos.},
journal = {Developmental biology},
volume = {519},
number = {},
pages = {5-12},
doi = {10.1016/j.ydbio.2024.11.013},
pmid = {39622311},
issn = {1095-564X},
support = {R00 DE028592/DE/NIDCR NIH HHS/United States ; R35 GM150763/GM/NIGMS NIH HHS/United States ; T32 DE007306/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Chick Embryo ; *Gene Knockdown Techniques/methods ; *PAX7 Transcription Factor/genetics/metabolism ; Neural Crest/metabolism/embryology ; Gene Expression Regulation, Developmental/genetics ; Morpholinos/genetics ; },
abstract = {The chick embryo is a classical model system commonly used in developmental biology due to its amenability to gene perturbation experiments. Pairing this powerful model organism with cutting-edge technology can significantly expand the range of experiments that can be performed. Recently, the CRISPR-Cas13d system has been successfully adapted for use in zebrafish, medaka, killifish, and mouse embryos to achieve targeted gene expression knockdown. Despite its success in other animal models, no prior study has explored the potential of CRISPR-Cas13d in the chick. Here, we present an adaptation of the CRISPR-Cas13d system to achieve targeted gene expression knockdown in the chick embryo. As proof-of-principle, we demonstrate the knockdown of PAX7, an early neural crest marker. Application of this adapted CRISPR-Cas13d technique resulted in effective knockdown of PAX7 expression and function, comparable to knockdown achieved by translation-blocking morpholino. CRISPR-Cas13d complements preexisting knockdown tools such as CRISPR-Cas9 and morpholinos, thereby expanding the experimental potential and versatility of the chick model system.},
}

Animals
*CRISPR-Cas Systems/genetics
Chick Embryo
*Gene Knockdown Techniques/methods
*PAX7 Transcription Factor/genetics/metabolism
Neural Crest/metabolism/embryology
Gene Expression Regulation, Developmental/genetics
Morpholinos/genetics

@article {pmid39313516,
year = {2025},
author = {Liang, YL and Hu, YX and Li, FF and You, HM and Chen, J and Liang, C and Guo, ZF and Jing, Q},
title = {Adaptor protein Src-homology 2 domain containing E (SH2E) deficiency induces heart defect in zebrafish.},
journal = {Acta pharmacologica Sinica},
volume = {46},
number = {2},
pages = {404-415},
pmid = {39313516},
issn = {1745-7254},
mesh = {Animals ; *Zebrafish ; *Zebrafish Proteins/genetics/metabolism/deficiency ; Heart Defects, Congenital/genetics/metabolism ; NF-kappa B/metabolism ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; Signal Transduction ; CRISPR-Cas Systems ; },
abstract = {Adaptor proteins play crucial roles in signal transduction across diverse signaling pathways. Src-homology 2 domain-containing E (SH2E) is the adaptor protein highly expressed in vascular endothelial cells and myocardium during zebrafish embryogenesis. In this study we investigated the function and mechanisms of SH2E in cardiogenesis. We first analyzed the spatiotemporal expression of SH2E and then constructed zebrafish lines with SH2E deficiency using the CRISPR-Cas9 system. We showed that homozygous mutants developed progressive pericardial edema (PCE), dilated atrium, abnormal atrioventricular looping and thickened atrioventricular wall from 3 days post fertilization (dpf) until death; inducible overexpression of SH2E was able to partially rescue the PCE phenotype. Using transcriptome sequencing analysis, we demonstrated that the MAPK/ERK and NF-κB signaling pathways might be involved in SH2E-deficiency-caused PCE. This study underscores the pivotal role of SH2E in cardiogenesis, and might help to identify innovative diagnostic techniques and therapeutic strategies for congenital heart disease.},
}

Animals
*Zebrafish
*Zebrafish Proteins/genetics/metabolism/deficiency
Heart Defects, Congenital/genetics/metabolism
NF-kappa B/metabolism
Adaptor Proteins, Signal Transducing/genetics/metabolism
Signal Transduction
CRISPR-Cas Systems

@article {pmid39831708,
year = {2025},
author = {Cochrane, RW and Robino, RA and Ferreira, LMR},
title = {Generation of Human Chimeric Antigen Receptor Regulatory T Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {215},
pages = {},
doi = {10.3791/67200},
pmid = {39831708},
issn = {1940-087X},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology ; *Receptors, Chimeric Antigen/immunology/genetics ; *CRISPR-Cas Systems ; Lentivirus/genetics ; Dependovirus/genetics/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4[+] T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs' immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.},
}

Humans
*T-Lymphocytes, Regulatory/immunology
*Receptors, Chimeric Antigen/immunology/genetics
*CRISPR-Cas Systems
Lentivirus/genetics
Dependovirus/genetics/immunology

@article {pmid39831695,
year = {2025},
author = {Yan, C and Meng, H and Pei, Y and Sun, W and Zhang, J},
title = {Breeding by Design for Functional Rice with Genome Editing Technologies.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {215},
pages = {},
doi = {10.3791/67336},
pmid = {39831695},
issn = {1940-087X},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Plant Breeding/methods ; *CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; },
abstract = {The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.},
}

*Oryza/genetics
*Gene Editing/methods
*Plant Breeding/methods
*CRISPR-Cas Systems/genetics
Genome, Plant/genetics

@article {pmid39831307,
year = {2025},
author = {Fei, X and Lei, C and Ren, W and Liu, C},
title = {'Splice-at-will' Cas12a crRNA engineering enabled direct quantification of ultrashort RNAs.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkaf002},
pmid = {39831307},
issn = {1362-4962},
support = {22074088//National Natural Science Foundation of China/ ; IRT_15R43//Program for Changjiang Scholars and Innovative Research Team in University/ ; GK202305001//Fundamental Research Funds for the Central Universities/ ; //Shaanxi Normal University/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; RNA/genetics/chemistry/metabolism ; RNA Splicing ; Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases/metabolism/genetics/chemistry ; DNA/genetics/chemistry/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; },
abstract = {We present a robust 'splice-at-will' CRISPR RNA (crRNA) engineering mechanism that overcomes the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in directly detecting ultrashort RNAs. In this strategy, an intact Cas12a crRNA can be split from almost any site of the spacer region to obtain a truncated crRNA (tcrRNA) that cannot activate Cas12a even after binding an auxiliary DNA activator. While splicing tcrRNAs with a moiety of ultrashort RNA, the formed combination can work together to activate Cas12a efficiently, enabling 'splice-at-will' crRNA engineering. Importantly, the 'splice-at-will' crRNA exhibits almost the same trans-cleavage activation efficiency as that of a conventional intact crRNA. Therefore, by rationally designing a DNA auxiliary activator with a conserved tcrRNA-complementary sequence and an arbitrary short RNA-of-interest recognition domain, a general sensing system is established that directly utilizes traditional DNA-activated Cas12a to detect ultrashort RNAs. This 'splice-at-will' crRNA engineering strategy could faithfully detect ultrashort RNA sequences as short as 6-8 nt, which cannot be achieved by conventional Cas12a and Cas13a systems. Additionally, through flexible splicing site design, our method can precisely distinguish single-base differences in microRNA and other short RNA sequences. This work has significantly expanded the Cas12a-based diagnostic toolbox and opened new avenues for ultrashort RNA detection.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
RNA/genetics/chemistry/metabolism
RNA Splicing
Bacterial Proteins/genetics/metabolism
Endodeoxyribonucleases/metabolism/genetics/chemistry
DNA/genetics/chemistry/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Engineering/methods

@article {pmid39829748,
year = {2025},
author = {Nayfach, S and Bhatnagar, A and Novichkov, A and Estevam, GO and Kim, N and Hill, E and Ruffolo, JA and Silverstein, R and Gallagher, J and Kleinstiver, B and Meeske, AJ and Cameron, P and Madani, A},
title = {Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.01.06.631536},
pmid = {39829748},
issn = {2692-8205},
abstract = {CRISPR-Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, significantly limiting the range of targetable sequences in a genome. Machine learning-based protein engineering provides a powerful solution to efficiently generate Cas protein variants tailored to recognize specific PAMs. Here, we present Protein2PAM, an evolution-informed deep learning model trained on a dataset of over 45,000 CRISPR-Cas PAMs. Protein2PAM rapidly and accurately predicts PAM specificity directly from Cas proteins across Type I, II, and V CRISPR-Cas systems. Using in silico deep mutational scanning, we demonstrate that the model can identify residues critical for PAM recognition in Cas9 without utilizing structural information. As a proof of concept for protein engineering, we employ Protein2PAM to computationally evolve Nme1Cas9, generating variants with broadened PAM recognition and up to a 50-fold increase in PAM cleavage rates compared to the wild-type under in vitro conditions. This work represents the first successful application of machine learning to achieve customization of Cas enzymes for alternate PAM recognition, paving the way for personalized genome editing.},
}

@article {pmid39829431,
year = {2025},
author = {Chen, Y and Xue, Y and Jiang, Q and Jin, Y and Chen, W and Hua, M},
title = {Disruption of the FOXM1 Regulatory Region Inhibits Tumor Progression in Ovarian Cancer by CRISPR-Cas9.},
journal = {Drug development research},
volume = {86},
number = {1},
pages = {e70049},
doi = {10.1002/ddr.70049},
pmid = {39829431},
issn = {1098-2299},
support = {//This work was supported by the Nantong Social Science and Technology Plan for People's Livelihood Project (grant No. JCZ2023003) from the Science and Technology Bureau of Nantong, China, and the Research Physician Development Fund (grant No. YJXYY202204-YSC03) from the Affiliated Hospital of Nantong University, China./ ; },
mesh = {Female ; *Forkhead Box Protein M1/genetics ; Humans ; *Ovarian Neoplasms/genetics/drug therapy/pathology ; Animals ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Mice ; *Cell Proliferation/drug effects ; Azepines/pharmacology ; Mice, Nude ; Gene Expression Regulation, Neoplastic/drug effects ; Cell Movement/drug effects ; Triazoles/pharmacology ; Xenograft Model Antitumor Assays ; Disease Progression ; },
abstract = {Ovarian cancer is the seventh most common lethal tumor among women in the world. FOXM1 is a transcription factor implicated in the initiation and progression of ovarian cancer by regulating key oncogenic genes. The role of regulatory regions in regulating the expression of FOXM1 in ovarian cancer is not completely clarified. Treatment with bromodomain and extraterminal (BET) inhibitors JQ-1 and I-BET were explored in ovarian cancer cell lines (OVCAR3, A2780, or SKOV3) to evaluate FOXM1 expression and biological behavior by qPCR, CCK8 assay, colony formation assay, wound-healing, and transwell assays. The regulatory regions (enhancer sequence spanning promoter or exon 1) of FOXM1 were deleted using CRISPR-Cas9 in the OVCAR3 cell line. FOXM1 expression and tumor biological behavior were further assessed in FOXM1 regulatory regions deleted OVCAR3 cell line. The mouse xenograft model was assessed at the indicated time points following subcutaneous injection of enhancer-deleted cells. Treatment with the JQ-1 and I-BET reduced the expression of FOXM1, decreasing cell proliferation, migration, and invasion in a panel of ovarian cancer cell lines including OVCAR3, A2780, and SKOV3 cells. By mining the published ChIP-sequencing data (H3K27Ac) from 12 ovarian cancer cell lines, we identified a potential enhancer and promoter region. Deletion of the spanning enhancer and promoter region of FOXM1 reduced mRNA and protein expression. Similarly, cell proliferation, migration, invasion, and tumorigenesis in both cells and mouse xenograft models were significantly attenuated. Our study demonstrates that JQ-1 and I-BET can regulate the expression of the FOXM1 gene-relating network. These data also indicate that disruption of the span enhancer and promoter region activity of FOXM1 has a vital role in the anti-ovarian cancer effect, hiding a potential opportunity for the evaluation of this non-coding DNA deletion disrupts the FOXM1 transcriptional network in ovarian cancer development.},
}

Female
*Forkhead Box Protein M1/genetics
Humans
*Ovarian Neoplasms/genetics/drug therapy/pathology
Animals
Cell Line, Tumor
*CRISPR-Cas Systems
Mice
*Cell Proliferation/drug effects
Azepines/pharmacology
Mice, Nude
Gene Expression Regulation, Neoplastic/drug effects
Cell Movement/drug effects
Triazoles/pharmacology
Xenograft Model Antitumor Assays
Disease Progression

@article {pmid39828694,
year = {2025},
author = {Matthews, MC and van der Linden, J and Robène, I and Rozsasi, S and Coetzee, B and Campa, M and Burger, J and Akwuruoha, UN and Madufor, NJ and Perold, W and Opara, UL and Viljoen, A and Mostert, D},
title = {A combined recombinase polymerase amplification CRISPR/Cas12a assay for detection of Fusarium oxysporum f. sp. cubense tropical race 4.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {2436},
pmid = {39828694},
issn = {2045-2322},
support = {138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 890856//Horizon 2020 Framework Programme/ ; 890856//Horizon 2020 Framework Programme/ ; 890856//Horizon 2020 Framework Programme/ ; 890856//Horizon 2020 Framework Programme/ ; },
mesh = {*CRISPR-Cas Systems ; *Fusarium/genetics/isolation & purification ; *Musa/microbiology ; Nucleic Acid Amplification Techniques/methods ; Plant Diseases/microbiology ; Recombinases/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The soilborne pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is currently devastating banana production worldwide. Once introduced, it is not possible to eradicate the pathogen from soils where it can survive for decades. The only management option available then is to replace Foc TR4-susceptible with -resistant varieties. Timely detection of the pathogen, however, is an important strategy to prevent the introduction of Foc TR4 into new areas and prevent its spread from infested sites. In this study, a single-tube detection technique was developed by combining recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology (RPA-Cas12a) for detection of Foc TR4. The RPA-Cas12a assay was conducted isothermally, had a sensitivity of up to 10 fg target DNA and did not cross react with any of the 76 non-target isolates included in the specificity testing. The RPA-Cas12a assay detected Foc TR4 from naturally infected banana samples collected in the field and visualization was possible with the naked eye under LED blue light transillumination. The method can be integrated with inexpensive fluorescent or electronic detection devices to accelerate Foc TR4 in-field detection and, thereby, fast-track disease containment strategies.},
}

*CRISPR-Cas Systems
*Fusarium/genetics/isolation & purification
*Musa/microbiology
Nucleic Acid Amplification Techniques/methods
Plant Diseases/microbiology
Recombinases/metabolism/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39827180,
year = {2025},
author = {Van Goethem, MW and Bezuidt, OKI and Pierneef, R and Vikram, S and Hopkins, DW and Aspray, T and Hall, G and Woodborne, S and Hogg, ID and Northen, TR and Kong, W and Daffonchio, D and Cowan, DA and Van de Peer, Y and Delgado-Baquerizo, M and Makhalanyane, TP},
title = {Novel adaptive immune systems in pristine Antarctic soils.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {2368},
pmid = {39827180},
issn = {2045-2322},
support = {118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; },
mesh = {Antarctic Regions ; *Soil Microbiology ; *Phylogeny ; *CRISPR-Cas Systems ; Microbiota/genetics ; Metagenomics/methods ; Adaptive Immunity/genetics ; Genome, Bacterial ; Bacteria/genetics ; Prophages/genetics ; Metagenome ; },
abstract = {Antarctic environments are dominated by microorganisms, which are vulnerable to viral infection. Although several studies have investigated the phylogenetic repertoire of bacteria and viruses in these poly-extreme environments with freezing temperatures, high ultra violet irradiation levels, low moisture availability and hyper-oligotrophy, the evolutionary mechanisms governing microbial immunity remain poorly understood. Using genome-resolved metagenomics, we test the hypothesis that Antarctic poly-extreme high-latitude microbiomes harbour diverse adaptive immune systems. Our analysis reveals the prevalence of prophages in bacterial genomes (Bacteroidota and Verrucomicrobiota), suggesting the significance of lysogenic infection strategies in Antarctic soils. Furthermore, we demonstrate the presence of diverse CRISPR-Cas arrays, including Class 1 arrays (Types I-B, I-C, and I-E), alongside systems exhibiting novel gene architecture among their effector cas genes. Notably, a Class 2 system featuring type V variants lacks CRISPR arrays, encodes Cas1 and Cas2 adaptation module genes. Phylogenetic analysis of Cas12 effector proteins hints at divergent evolutionary histories compared to classified type V effectors and indicates that TnpB is likely the ancestor of Cas12 nucleases. Our findings suggest substantial novelty in Antarctic cas sequences, likely driven by strong selective pressures. These results underscore the role of viral infection as a key evolutionary driver shaping polar microbiomes.},
}

Antarctic Regions
*Soil Microbiology
*Phylogeny
*CRISPR-Cas Systems
Microbiota/genetics
Metagenomics/methods
Adaptive Immunity/genetics
Genome, Bacterial
Bacteria/genetics
Prophages/genetics
Metagenome

@article {pmid39826761,
year = {2025},
author = {Zhou, Z and Liu, S and Saleem, M and Liu, F and Hu, R and Su, H and Dong, D and Luo, Z and Wu, Y and Zhang, Y and He, Z and Wang, C},
title = {Unraveling phase-dependent variations of viral community, virus-host linkage, and functional potential during manure composting process.},
journal = {Bioresource technology},
volume = {},
number = {},
pages = {132081},
doi = {10.1016/j.biortech.2025.132081},
pmid = {39826761},
issn = {1873-2976},
abstract = {The temporal dynamics of bacterial and fungal communities significantly impact the manure composting process, yet viral communities are often underexplored. Bulk metagenomes, viromes, metatranscriptomes, and metabolomes were integrated to investigate dynamics of double-stranded DNA (dsDNA) virus and virus-host interactions throughout a 63-day composting process. A total of 473 viral operational taxonomic units (vOTUs), predominantly Caudoviricetes, showed distinct phase-dependent differentiation. In phase I (initial-mesophilic), viruses targeted Gammaproteobacteria and Firmicutes, utilizing restriction-modification (RM) systems. In phase II (thermophilic-maturing), viruses infected Alphaproteobacteria, Chloroflexi, and Planctomycetes, employing CRISPR-Cas systems. Lysogenic and lytic viruses exerting differential effects on bacterial pathogens across phases. Additionally, six types of auxiliary metabolic genes (AMGs) related to galactose and cysteine metabolisms were identified. The homologous lineages of AMGs with bacterial genes, along with the significant temporal correlation observed between virus-host-metabolite interactions, underscore the critical yet often overlooked role of viral communities in modulating microbial metabolisms and pathogenesis within composting ecosystems.},
}

@article {pmid39826547,
year = {2025},
author = {Hong, SH and An, SY and Park, C and Kim, Y and Kim, EH and Kim, NK and Suh, JY},
title = {Structural variants of AcrIIC5 inhibit Cas9 via divergent binding interfaces.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2024.12.014},
pmid = {39826547},
issn = {1878-4186},
abstract = {CRISPR-Cas is a bacterial defense system that employs RNA-guided endonucleases to destroy invading foreign nucleic acids. Bacteriophages produce anti-CRISPR (Acr) proteins to evade CRISPR-Cas defense during the infection. AcrIIC5, a type II-C Cas9 inhibitor, exhibits unusual variations in the local backbone fold between its orthologs. Here we investigated how the folding variations affect the inhibition of target Cas9 using AcrIIC5 orthologs. Structural comparison of free AcrIIC5Smu and AcrIIC5Nch confirmed that the folding variation correlated with characteristic indels in the helical region. Mutagenesis and biochemical assays combined with AlphaFold2 predictions identified key residues of AcrIIC5 orthologs important for Cas9 inhibition. Remarkably, AcrIIC5 orthologs employed divergent binding interfaces via folding variations to inhibit the Cas9 targets. Our study suggests that Acr proteins have evolved structural variants to diversify key interfaces for target Cas9, which could be beneficial for the adaptation of phages to evasive mutations on the Cas9 surface.},
}

@article {pmid39700063,
year = {2025},
author = {Munusamy, S and Zheng, H and Jahani, R and Zhou, S and Chen, J and Kong, J and Guan, X},
title = {DNA-Assisted CRISPR-Cas12a Enhanced Fluorescent Assay for Protein Detection in Complicated Matrices.},
journal = {ACS applied bio materials},
volume = {8},
number = {1},
pages = {754-762},
doi = {10.1021/acsabm.4c01600},
pmid = {39700063},
issn = {2576-6422},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA/chemistry ; Materials Testing ; Biocompatible Materials/chemistry ; Particle Size ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases/metabolism/genetics ; Proteins/analysis/metabolism ; Bacterial Proteins ; },
abstract = {Proteins are important biological macromolecules that perform a wide variety of functions in the cell and human body, and can serve as important biomarkers for early diagnosis and prognosis of human diseases as well as monitoring the effectiveness of disease treatment. Hence, sensitive and accurate detection of proteins in human biospecimens is imperative. However, at present, there is no ideal method available for the detection of proteins in clinical samples, many of which are present at ultralow (less than 1 pM) concentrations and in complicated matrices. Herein, we report an ultrasensitive and selective DNA-assisted CRISPR-Cas12a enhanced fluorescent assay (DACEA) for protein detection with detection limits reaching as low as attomolar concentrations. The high assay sensitivity was accomplished through the combined DNA barcode amplification (by using dual-functionalized AuNPs) and CRISPR analysis, while the high selectivity and high resistance to the matrix effects of our method were accomplished via the formation of protein-antibody sandwich structure and the specific recognition of Cas12a (under the guidance of crRNA) toward the designed target ssDNA. Given its ability to accurately and sensitively detect trace amounts of proteins in complicated matrices, the DACEA protein assay platform pioneered in this work has a potential application in routine protein biomarker testing.},
}

*CRISPR-Cas Systems/genetics
Humans
*DNA/chemistry
Materials Testing
Biocompatible Materials/chemistry
Particle Size
CRISPR-Associated Proteins/genetics/metabolism
Endodeoxyribonucleases/metabolism/genetics
Proteins/analysis/metabolism
Bacterial Proteins

@article {pmid39825389,
year = {2025},
author = {Abraham, IC and Aboje, JE and Ukoaka, BM and Tom-Ayegunle, K and Amjad, M and Abdulkader, A and Agbo, CE and Akinruli, OA and Akisanmi, TR and Oyetola, EO and Olatunji, G and Kokori, E and Aderinto, N},
title = {Integrating malaria vaccine and CRISPR/Cas9 gene drive: a comprehensive strategy for accelerated malaria eradication.},
journal = {Malaria journal},
volume = {24},
number = {1},
pages = {17},
pmid = {39825389},
issn = {1475-2875},
mesh = {*Malaria Vaccines/immunology/genetics/administration & dosage ; *CRISPR-Cas Systems ; *Malaria/prevention & control ; *Gene Drive Technology/methods ; Humans ; *Disease Eradication/methods ; Animals ; Anopheles/genetics/parasitology ; Mosquito Vectors/genetics ; Mosquito Control/methods ; },
abstract = {Malaria remains a significant public health challenge, particularly in low- and middle-income countries, despite ongoing efforts to eradicate the disease. Recent advancements, including the rollout of malaria vaccines, such as RTS,S/AS01 and R21/Matrix-M™, offer new avenues for prevention. However, the rise of resistance to anti-malarial medications necessitates innovative strategies. This review explores the potential integration of CRISPR/Cas9 gene drive technology with malaria vaccination efforts to enhance vector control and reduce transmission. By employing gene drive mechanisms for population suppression and replacement of malaria-transmitting Anopheles mosquitoes, combined with the immunogenic properties of vaccines, a synergistic approach can be established. This paper discussed the need for integrated strategies to address the biological complexities of malaria and socio-economic factors influencing its prevalence. Challenges such as regulatory hurdles, community acceptance, ecological impacts, and sustainable funding are examined, alongside strategies for implementation within existing malaria control programmes. This integrated approach could significantly contribute to achieving the World Health Organization's targets for malaria reduction by 2030, ultimately enhancing public health outcomes and supporting broader socio-economic development.},
}

*Malaria Vaccines/immunology/genetics/administration & dosage
*CRISPR-Cas Systems
*Malaria/prevention & control
*Gene Drive Technology/methods
Humans
*Disease Eradication/methods
Animals
Anopheles/genetics/parasitology
Mosquito Vectors/genetics
Mosquito Control/methods

@article {pmid39824586,
year = {2025},
author = {Arivarasan, VK and Diwakar, D and Kamarudheen, N and Loganathan, K},
title = {Current approaches in CRISPR-Cas systems for diabetes.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {95-125},
doi = {10.1016/bs.pmbts.2024.08.002},
pmid = {39824586},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Diabetes Mellitus/therapy/genetics ; Animals ; Gene Editing ; Genetic Therapy/methods ; },
abstract = {In the face of advancements in health care and a shift towards healthy lifestyle, diabetes mellitus (DM) still presents as a global health challenge. This chapter explores recent advancements in the areas of genetic and molecular underpinnings of DM, addressing the revolutionary potential of CRISPR-based genome editing technologies. We delve into the multifaceted relationship between genes and molecular pathways contributing to both type1 and type 2 diabetes. We highlight the importance of how improved genetic screening and the identification of susceptibility genes are aiding in early diagnosis and risk stratification. The spotlight then shifts to CRISPR-Cas9, a robust genome editing tool capable of various applications including correcting mutations in type 1 diabetes, enhancing insulin production in T2D, modulating genes associated with metabolism of glucose and insulin sensitivity. Delivery methods for CRISPR to targeted tissues and cells are explored, including viral and non-viral vectors, alongside the exciting possibilities offered by nanocarriers. We conclude by discussing the challenges and ethical considerations surrounding CRISPR-based therapies for DM. These include potential off-target effects, ensuring long-term efficacy and safety, and navigating the ethical implications of human genome modification. This chapter offers a comprehensive perspective on how genetic and molecular insights, coupled with the transformative power of CRISPR, are paving the way for potential cures and novel therapeutic approaches for DM.},
}

Humans
*CRISPR-Cas Systems/genetics
*Diabetes Mellitus/therapy/genetics
Animals
Gene Editing
Genetic Therapy/methods

@article {pmid39824585,
year = {2025},
author = {Munshi, ID and Acharya, M and Mukherjee, S and Mani, I},
title = {Recent development in CRISPR-Cas systems for cardiac disease.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {47-93},
doi = {10.1016/bs.pmbts.2024.08.004},
pmid = {39824585},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Animals ; *Heart Diseases/genetics/therapy ; *Gene Editing ; },
abstract = {The CRISPR-Cas system has emerged as a revolutionary tool in genetic research, enabling highly precise gene editing and significantly advancing the field of cardiovascular science. This chapter provides a comprehensive overview of the latest developments in utilizing CRISPR-Cas technologies to investigate and treat heart diseases. It delves into the application of CRISPR-Cas9 for creating accurate models of complex cardiac conditions, such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and various arrhythmias, which are essential for understanding disease mechanisms and testing potential therapies. The therapeutic potential of gene editing is also explored, with a focus on genes like PCSK9 and ANGPTL3 that play critical roles in lipid metabolism and cardiovascular health, offering promising avenues for new treatments. Furthermore, the expanding applications of CRISPR in heart tissue regeneration are examined, which could revolutionize the repair of damaged heart tissue. Cutting-edge techniques such as base editing and prime editing are discussed, highlighting their potential to further refine genetic interventions. The discussion concludes by addressing the challenges associated with delivering CRISPR components efficiently and safely, while also exploring recent innovations that may overcome these hurdles, providing insights into the future directions of CRISPR technology in cardiovascular medicine.},
}

Humans
*CRISPR-Cas Systems/genetics
Animals
*Heart Diseases/genetics/therapy
*Gene Editing

@article {pmid39824584,
year = {2025},
author = {Khoshandam, M and Soltaninejad, H and Bhia, I and Goudarzi, MTH and Hosseinkhani, S},
title = {CRISPR challenges in clinical developments.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {263-279},
doi = {10.1016/bs.pmbts.2024.08.001},
pmid = {39824584},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; Genetic Therapy/methods ; Neoplasms/genetics/therapy ; },
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) is a novel genome editing technology with potential applications in treating diseases. Currently, its use in humans is restricted to clinical trials, although its growth rate is significant, and some have received initial FDA approval. It is crucial to examine and address the challenges for this technology to be implemented in clinical settings. This review aims to identify and explore new research ideas to increase of CRISPR's efficiency in treating genetic diseases and cancer, as well as its future prospects. Given that a substantial amount of previous research has focused on CRISPR-Cas delivery strategies and materials, this overview introduces specific conditions and strategies. It also discusses some of the challenges and opportunities in this field, offering a unique perspective.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Animals
Genetic Therapy/methods
Neoplasms/genetics/therapy

@article {pmid39824583,
year = {2025},
author = {Mishra, S and Banerjee, S and Tiwari, BS and Tiwari, AK},
title = {Recent progress in CRISPR-Cas-system for neurological disorders.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {231-261},
doi = {10.1016/bs.pmbts.2024.07.017},
pmid = {39824583},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Nervous System Diseases/genetics/therapy ; Animals ; Gene Editing ; Genetic Therapy/methods ; },
abstract = {Different neurological diseases including, Parkinson's, Alzheimer's, and Huntington's diseases extant momentous global disease burdens, affecting millions of lives for imposing a heavy disease burden on the healthcare systems. Despite various treatment strategies aimed at alleviating symptoms, treatments remain elusive and ineffective due to the disease's complexity. However, recent advancements in gene therapy via the CRISPR-Cas system offer ground-breaking and targeted treatment options. Based on a bacterial immune mechanism, the CRISPR-Cas system enables precise genome editing, allowing for the alteration of different genetic mutations and the possible cure of genetic diseases. In the context of neurological disorders, the CRISPR-Cas system shows a promising avenue by allowing researchers to conduct genome-editing which is implicated in neurodegenerative disease therapeutics. This book chapter provides an updated overview of the application of the CRISPR-Cas system for addressing target-specific therapeutic approaches for neurodegenerative disorders. Furthermore, we discuss the principles of the CRISPR-Cas mechanism, its role in modeling neurological disorders, identifying molecular targets, and developing gene-based therapies. Additionally, the chapter explores the recent clinical trials and CRISPR-Cas-mediated treatments for neurological conditions. By leveraging the accuracy and versatility of the CRISPR-Cas system, scientists can more effectively handle the genetic underpinnings of neurodegenerative diseases. Furthermore, the chapter extends the critical viewpoints on ethical considerations and technical limitations related to the clinical deployment of this revolutionizing technique.},
}

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

@article {pmid39824582,
year = {2025},
author = {Gowda, DAA and Birappa, G and Rajkumar, S and Ajaykumar, CB and Srikanth, B and Kim, SL and Singh, V and Jayachandran, A and Lee, J and Ramakrishna, S},
title = {Recent progress in CRISPR/Cas9 system for eye disorders.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {21-46},
doi = {10.1016/bs.pmbts.2024.07.018},
pmid = {39824582},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Eye Diseases/genetics/therapy ; Gene Editing ; },
abstract = {Ocular disorders encompass a broad spectrum of phenotypic and clinical symptoms resulting from several genetic variants and environmental factors. The unique anatomy and physiology of the eye facilitate validation of cutting-edge gene editing treatments. Genome editing developments have allowed researchers to treat a variety of diseases, including ocular disorders. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system holds considerable promise for therapeutic applications in the field of ophthalmology, including repair of aberrant genes and treatment of retinal illnesses related to the genome or epigenome. Application of CRISPR/Cas9 systems to the study of ocular disease and visual sciences have yielded innovations including correction of harmful mutations in patient-derived cells and gene modifications in several mammalian models of eye development and disease. In this study, we discuss the generation of several ocular disease models in mammalian cell lines and in vivo systems using a CRISPR/Cas9 system. We also provide an overview of current uses of CRISPR/Cas9 technologies for the treatment of ocular pathologies, as well as future challenges.},
}

*CRISPR-Cas Systems/genetics
Humans
Animals
*Eye Diseases/genetics/therapy
Gene Editing

@article {pmid39824581,
year = {2025},
author = {Singh, S and Raj, D and Mathur, A and Mani, N and Kumar, D},
title = {Current approaches in CRISPR-Cas systems for hereditary diseases.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {205-229},
doi = {10.1016/bs.pmbts.2024.07.015},
pmid = {39824581},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Diseases, Inborn/therapy/genetics ; *Gene Editing/methods ; Animals ; },
abstract = {CRISPR-Cas technologies have drastically revolutionized genetic engineering and also dramatically changed the potential for treating inherited disorders. The potential to correct genetic mutations responsible for numerous hereditary disorders from single-gene disorders to complex polygenic diseases through precise DNA editing is feasible. The tactic now employed in CRISPR-Cas systems for treating inherited disorders is the usage of particular guide RNAs to target and edit disease-causing mutations in the patient's genome. Several methods such as CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas13 are being thoroughly researched and optimized to increase effectiveness, accuracy, and safety in gene editing. Additionally, it is predicted that CRISPR-based therapies will be able to treat complex genetic illnesses such as cancer predisposition syndromes, neurological disorders, and cardiovascular conditions in addition to single-gene disorders. The available editing tools and creation of base editing technology facilitate the simultaneous correction of many mutations or accurate nucleotide changes leading to further advances in the development of multiplex editing tools and base editing technology fiction. When combined with other paradigms such as gene therapy using stem cell treatment, CRISPR-Cas promises improved efficacy. Patient treatment and lowering side effects significantly in individual genetic profiles will guide CRISPR-based treatments. These procedures will undoubtedly lead to therapies that are both efficient and curative of a wide range of genetic diseases, ushering in a new era of precision medicine. This chapter discusses about CRISPR Cas9 mechanism and its significance in the treatment of Hereditary disorders.},
}

Humans
*CRISPR-Cas Systems/genetics
*Genetic Diseases, Inborn/therapy/genetics
*Gene Editing/methods
Animals

@article {pmid39824580,
year = {2025},
author = {Israr, J and Kumar, A},
title = {Current progress in CRISPR-Cas systems for rare diseases.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {163-203},
doi = {10.1016/bs.pmbts.2024.07.019},
pmid = {39824580},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Rare Diseases/genetics/therapy ; Animals ; *Gene Editing/methods ; Genetic Therapy/methods ; },
abstract = {The groundbreaking CRISPR-Cas gene editing method permits exact genetic code alteration. The "CRISPR" DNA protects bacteria from viruses. CRISPR-Cas utilizes a guide RNA to steer the Cas enzyme to the genome's gene editing target. After attaching to a sequence, Cas enzymes cleave DNA to insert, delete, or modify genes. The influence of CRISPR-Cas technology on molecular biology and genetics is profound. It allows for gene function research, animal disease models, and patient genetic therapy. Gene editing has transformed biotechnology, agriculture, and customized medicine. CRISPR-Cas could revolutionize genetics and medicine. CRISPR-Cas may accurately correct genetic flaws that underlie rare diseases, improving their therapy. Gene mutations make CRISPR-Cas gene editing a viable cure for uncommon diseases. We can use CRISPR-Cas to correct genetic abnormalities at the molecular level. This strategy offers hope for remedies and disease understanding. CRISPR-Cas genome editing may enable more targeted and effective treatments for rare medical illnesses with few therapy options. By developing base- and prime-editing CRISPR technology, CRISPR-Cas allows for accurate and efficient genome editing and advanced DNA modification. This advanced method provides precise DNA alterations without double-strand breakage. These advances have improved gene editing safety and precision, reducing unfavorable effects. Lipid nanoparticles, which use viral vectors, improve therapeutic cell and tissue targeting. In rare disorders, gene therapy may be possible with CRISPR-Cas clinical trials. CRISPR-Cas research is improving gene editing, delivery, and rare disease treatment.},
}

*CRISPR-Cas Systems/genetics
Humans
*Rare Diseases/genetics/therapy
Animals
*Gene Editing/methods
Genetic Therapy/methods

@article {pmid39824579,
year = {2025},
author = {Puri, B and Kulkarni, YA and Gaikwad, AB},
title = {Advances in CRISPR-Cas systems for kidney diseases.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {149-162},
doi = {10.1016/bs.pmbts.2024.07.020},
pmid = {39824579},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Kidney Diseases/genetics/therapy ; Animals ; Gene Editing ; },
abstract = {Recent advances in CRISPR-Cas systems have revolutionised the study and treatment of kidney diseases, including acute kidney injury (AKI), chronic kidney disease (CKD), diabetic kidney disease (DKD), lupus nephritis (LN), and polycystic kidney disease (PKD). CRISPR-Cas technology offers precise and versatile tools for genetic modification in monogenic kidney disorders such as PKD and Alport syndrome. Recent advances in CRISPR technology have also shown promise in addressing other kidney diseases like AKI, CKD, and DKD. CRISPR-Cas holds promise to edit genetic mutations underlying these conditions, potentially leading to more effective and long-lasting treatments. Furthermore, the adaptability of CRISPR-Cas systems allows for developing tailored therapeutic strategies that specifically target the genetic and molecular mechanisms contributing to different kidney diseases. Beyond DNA modifications, CRISPR-Cas technologies also enable editing noncoding RNA, such as lncRNAs and miRNAs, in kidney diseases. Despite these advancements, significant challenges persist, including delivery efficiency to specific kidney cells and potential off-target effects. However, the rapid progress in CRISPR-Cas technology suggests a transformative impact on the future management of kidney diseases, offering the potential for enhanced patient outcomes through personalised and precise therapeutic approaches. This chapter highlights the recent advancement of CRISPR-Cas systems and their potential applications in various kidney diseases.},
}

Humans
*CRISPR-Cas Systems/genetics
*Kidney Diseases/genetics/therapy
Animals
Gene Editing

@article {pmid39824578,
year = {2025},
author = {Ajaykumar, CB and Rajkumar, S and Suresh, B and Birappa, G and Gowda, DAA and Jayachandran, A and Kim, KS and Hong, SH and Ramakrishna, S},
title = {Advances in applications of the CRISPR/Cas9 system for respiratory diseases.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {127-147},
doi = {10.1016/bs.pmbts.2024.07.014},
pmid = {39824578},
issn = {1878-0814},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Respiratory Tract Diseases/genetics/therapy ; Gene Editing ; Genetic Therapy/methods ; },
abstract = {Genetic and environmental factors can have an impact on lung and respiratory disorders which are associated with severe symptoms and have high mortality rates. Many respiratory diseases are significantly influenced by genetic or epigenetic factors. Gene therapy offers a powerful approach providing therapeutic treatment for lung diseases. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) are promising gene modifying tool that can edit the genome. The utilization of CRISPR/Cas9 systems in the investigation of respiratory disorders has resulted in advancements such as the rectification of deleterious mutations in patient-derived cells and the alteration of genes in multiple mammalian lung disease models. New avenues of treatment for lung disorders have been opened up by advances in CRISPR/Cas9 research. In this chapter, we discuss the known genes and mutations that cause several common respiratory disorders such as COPD, asthma, IPF, and ARDS. We further review the current research using CRISPR/Cas9 in numerous respiratory disorders and possible therapeutic treatments.},
}

Humans
*CRISPR-Cas Systems/genetics
Animals
Respiratory Tract Diseases/genetics/therapy
Gene Editing
Genetic Therapy/methods

@article {pmid39824577,
year = {2025},
author = {Arora, Y and Priya, and Kumar, M and Kumar, D},
title = {Current approaches in CRISPR-Cas system for metabolic disorder.},
journal = {Progress in molecular biology and translational science},
volume = {210},
number = {},
pages = {1-19},
doi = {10.1016/bs.pmbts.2024.07.016},
pmid = {39824577},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Metabolic Diseases/therapy/genetics ; Animals ; Gene Editing ; Genetic Therapy/methods ; },
abstract = {A new era in genomic medicine has been brought by the development of CRISPR-Cas technology, which presents hitherto unheard-of possibilities for the treatment of metabolic illnesses. The treatment approaches used in CRISPR/Cas9-mediated gene therapy, emphasize distribution techniques such as viral vectors and their use in preclinical models of metabolic diseases like hypercholesterolemia, glycogen storage diseases, and phenylketonuria. The relevance of high-throughput CRISPR screens for target identification in discovering new genes and pathways associated with metabolic dysfunctions is an important aspect of the discovery of new approaches. With cutting-edge options for genetic correction and cellular regeneration, the combination of CRISPR-Cas technology with stem cell therapy has opened new avenues for the treatment of metabolic illnesses. The integration of stem cell therapy and CRISPR-Cas technology is an important advance in the treatment of metabolic diseases, which are difficult to treat because of their intricate genetic foundations. This chapter addresses the most recent developments in the application of stem cell therapy and CRISPR-Cas systems to treat a variety of metabolic disorders, providing fresh hope for effective and maybe curative therapies. This chapter examines techniques and developments that have been made recently to address a variety of metabolic disorders using CRISPR-Cas systems. Our chapter focuses on the foundational workings of CRISPR-Cas technology and its potential uses in gene editing, gene knockout, and activation/repression-based gene modification.},
}

*CRISPR-Cas Systems/genetics
Humans
*Metabolic Diseases/therapy/genetics
Animals
Gene Editing
Genetic Therapy/methods

@article {pmid39626660,
year = {2025},
author = {Jensen, CL and Chen, LF and Swigut, T and Crocker, OJ and Yao, D and Bassik, MC and Ferrell, JE and Boettiger, AN and Wysocka, J},
title = {Long-range regulation of transcription scales with genomic distance in a gene-specific manner.},
journal = {Molecular cell},
volume = {85},
number = {2},
pages = {347-361.e7},
doi = {10.1016/j.molcel.2024.10.021},
pmid = {39626660},
issn = {1097-4164},
support = {R35 GM131792/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Promoter Regions, Genetic ; *Mouse Embryonic Stem Cells/metabolism/cytology ; Transcription, Genetic ; Chromatin/genetics/metabolism ; Transcription Factor RelA/genetics/metabolism ; Enhancer Elements, Genetic ; Transcriptional Activation ; Gene Expression Regulation ; Transcription Factors/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Although critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here, we bypass the need for sequence-specific transcription factors (TFs) and recruit activators directly using a chimeric array of gRNA oligos to target dCas9 fused to the activator VP64-p65-Rta (CARGO-VPR). We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mouse embryonic stem cells (mESCs), where neither gene is expressed. Although activator recruitment to any tested region results in the transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence, or the presence of repressive chromatin marks at the locus.},
}

Animals
Mice
*Promoter Regions, Genetic
*Mouse Embryonic Stem Cells/metabolism/cytology
Transcription, Genetic
Chromatin/genetics/metabolism
Transcription Factor RelA/genetics/metabolism
Enhancer Elements, Genetic
Transcriptional Activation
Gene Expression Regulation
Transcription Factors/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid39585363,
year = {2025},
author = {Guo, Y and Zhao, X},
title = {CRISPR-based genetic screens in human pluripotent stem cells derived neurons and brain organoids.},
journal = {Cell and tissue research},
volume = {399},
number = {1},
pages = {1-8},
pmid = {39585363},
issn = {1432-0878},
support = {P50HD105353//National Institute of Child Health and Human Development/ ; R01 MH136152/MH/NIMH NIH HHS/United States ; R01 NS138268/NS/NINDS NIH HHS/United States ; P50 HD105353/HD/NICHD NIH HHS/United States ; R01 MH118827/MH/NIMH NIH HHS/United States ; R01MH118827/MH/NIMH NIH HHS/United States ; R01MH118827/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; *Organoids/cytology/metabolism ; *Pluripotent Stem Cells/cytology/metabolism ; *Neurons/cytology/metabolism ; *Brain/cytology ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Testing/methods ; },
abstract = {Recent large-scale genome-wide association and single-cell RNA sequencing (scRNA-seq) studies have uncovered disease-associated genetic risk factors and cell type-specific genetic alterations. However, our understanding of how these genetic variants cause diseases and the underlying mechanisms remains largely unknown. Functional genomics screens using CRISPR-based technologies offer an effective tool for studying genes relevant to disease phenotypes. Here, we summarize recent CRISPR-based functional genomics screen approaches applied to human pluripotent stem cell (hPSC)-derived neurons and brain organoids. These screens have identified genes crucial for neurogenesis, neuronal survival, morphological development, and migration. Combining CRISPR-based genetic screens with scRNA-seq, researchers have revealed downstream genes and cellular pathways impacted by these genetic variants in human neural cells, providing new insights into the pathogenesis of neurodevelopmental disorders, such as microcephaly and autism spectrum disorders. Finally, we discuss current challenges and future directions for using CRISPR-based screens in furthering our understanding of neurological diseases and developing potential therapeutic strategies. Despite challenges, CRISPR-based screens have enormous potential for advancing the therapeutic development of many diseases.},
}

Humans
*Organoids/cytology/metabolism
*Pluripotent Stem Cells/cytology/metabolism
*Neurons/cytology/metabolism
*Brain/cytology
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genetic Testing/methods

@article {pmid39822055,
year = {2025},
author = {Göritzer, K and Melnik, S and Schwestka, J and Arcalis, E and Drapal, M and Fraser, P and Ma, JK and Stoger, E},
title = {Enhancing quality and yield of recombinant secretory IgA antibodies in Nicotiana benthamiana by endoplasmic reticulum engineering.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14576},
pmid = {39822055},
issn = {1467-7652},
support = {760331//Horizon 2020 Framework Programme/ ; 21EBTA/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; J4583//Austrian Science Fund/ ; W1224//Austrian Science Fund/ ; },
abstract = {The production of complex multimeric secretory immunoglobulins (SIgA) in Nicotiana benthamiana leaves is challenging, with significant reductions in complete protein assembly and consequently yield, being the most important difficulties. Expanding the physical dimensions of the ER to mimic professional antibody-secreting cells can help to increase yields and promote protein folding and assembly. Here, we expanded the ER in N. benthamiana leaves by targeting the enzyme CTP:phosphocholine cytidylyltransferase (CCT), which catalyses the rate-limiting step in the synthesis of the key membrane component phosphatidylcholine (PC). We used CRISPR/Cas to perform site-directed mutagenesis of each of the three endogenous CCT genes in N. benthamiana by introducing frame-shifting indels to remove the auto-inhibitory C-terminal domains. We generated stable homozygous lines of N. benthamiana containing different combinations of the edited genes, including plants where all three isofunctional CCT homologues were modified. Changes in ER morphology in the mutant plants were confirmed by in vivo confocal imaging and substantially increased the yields of two fully assembled SIgAs by prolonging the ER residence time and boosting chaperone accumulation. Through a combination of ER engineering with chaperone overexpression, we increased the yields of fully assembled SIgA by an order of magnitude, reaching almost 1 g/kg fresh leaf weight. This strategy removes a major roadblock to producing SIgA and will likely facilitate the production of other complex multimeric biopharmaceutical proteins in plants.},
}

@article {pmid39821831,
year = {2025},
author = {Xie, Y and Liu, X and Wu, T and Luo, Y},
title = {Harnessing the Streptomyces-originating type I-E CRISPR/Cas system for efficient genome editing in Streptomyces.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {39821831},
issn = {1869-1889},
abstract = {Since their discovery, CRISPR/Cas systems have significantly expanded the genetic toolbox, aiding in the exploration and enhanced production of natural products across various microbes. Among these, class 2 CRISPR/Cas systems are simpler and more broadly used, but they frequently fail to function effectively in many Streptomyces strains. In this study, we present an engineered class 1 type I CRISPR/Cas system derived from Streptomyces avermitilis, which enables efficient gene editing in phylogenetically distant Streptomyces strains. Through a plasmid interference assay, we identified the effective protospacer adjacent motif as 5'-AAN-3'. Utilizing this system, we achieved targeted chromosomal deletions ranging from 8 bp to 100 kb, with efficiencies exceeding 92%. We further utilized this system to insert DNA fragments into different Streptomyces genomes, facilitating the heterologous expression of exogenous genes and the activation of endogenous natural product biosynthetic gene clusters. Overall, we established a type I CRISPR/Cas-based gene-editing methodology that significantly advances the exploration of Streptomyces, known for their rich natural product resources. This is the first report of a gene editing tool developed based on the endogenous class 1 type I CRISPR/Cas system in Streptomyces spp. Our work enriches the Streptomyces gene manipulation toolbox and advances the discovery of valuable natural products within these organisms.},
}

@article {pmid39821024,
year = {2025},
author = {Bu, W and Li, Y},
title = {Rat Models of Breast Cancer.},
journal = {Advances in experimental medicine and biology},
volume = {1464},
number = {},
pages = {123-148},
pmid = {39821024},
issn = {0065-2598},
mesh = {Animals ; Female ; Rats ; Humans ; *CRISPR-Cas Systems ; Breast Neoplasms/genetics/pathology/therapy ; Disease Models, Animal ; Mammary Neoplasms, Experimental/genetics/pathology ; Gene Editing/methods ; Mammary Glands, Animal/pathology/metabolism ; },
abstract = {As the first mammal to be domesticated for research purposes, rats served as the primary animal model for various branches of biomedical research, including breast cancer studies, up until the late 1990s and early 2000s. During this time, genetic engineering of mice, but not rats, became routine, and mice gradually supplanted rats as the preferred rodent model. But recent advances in creating genetically engineered rat models, especially with the assistance of CRISPR/Cas9 technology, have rekindled the significance of rats as a critical model in exploring various facets of breast cancer research. This is particularly pronounced in the study of the formation and progression of the estrogen receptor-positive subtype, which remains challenging to model in mice. In this chapter, we embark on a historical journey through the evolution of rat models in biomedical research and provide an overview of the general and histological characteristics of rat mammary glands. Next, we critically review major rat models for breast cancer research, including those induced by carcinogens, hormones, radiation, germline transgenes, germline knockouts, and intraductal injection of retrovirus/lentivirus to deliver oncogenic drivers into mature mammary glands. We also discuss the advances in building rat models using somatic genome editing powered by CRISPR/Cas9. This chapter concludes with our forward-looking perspective on future applications of advanced rat models in critical areas of breast cancer research that have continued to challenge the mouse model community.},
}

Animals
Female
Rats
Humans
*CRISPR-Cas Systems
Breast Neoplasms/genetics/pathology/therapy
Disease Models, Animal
Mammary Neoplasms, Experimental/genetics/pathology
Gene Editing/methods
Mammary Glands, Animal/pathology/metabolism

@article {pmid39820420,
year = {2025},
author = {Dong, Y and Zhu, J and Pan, N},
title = {Recent advances in rapid detection of Helicobacter pylori by lateral flow assay.},
journal = {Archives of microbiology},
volume = {207},
number = {2},
pages = {35},
pmid = {39820420},
issn = {1432-072X},
mesh = {*Helicobacter pylori/isolation & purification/genetics ; *Helicobacter Infections/diagnosis/microbiology ; Humans ; Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; Point-of-Care Systems ; Enzyme-Linked Immunosorbent Assay/methods ; },
abstract = {Infection with H. pylori (Helicobacter pylori) is the most prevalent human infection worldwide and is strongly associated with many gastrointestinal disorders, including gastric cancer. Endoscopy is mainly used to diagnose H. pylori infection in gastric biopsies. However, this approach is invasive, time-consuming and expensive. On the other hand, serology-based methods can be considered as a non-invasive approach to detecting H. pylori infection. The LFA (lateral flow assay) serves as a rapid point-of-care diagnostic tool. This paper-based platform facilitates the detection and quantification of analytes within human fluids such as blood, serum and urine. Due to ease of production, rapid results, and low costs, LFAs have a wide application in clinical laboratories and hospitals. In this comprehensive review, we examined LFA-based approaches for detection of H. pylori infection from human fluids and compare them with other high-sensitivity methods like ELISA (Enzyme-linked immunosorbent assay). Furthermore, we reviewed methods to elevate LFA sensitivity during H. pylori infection including, CRISPR/Cas system and isothermal amplification approaches. The development and optimization of novel labeling agents such as nanozyme to enhance the performance of LFA devices in detecting H. pylori were reviewed. These innovations aim to improve signal amplification and stability, thereby increasing the diagnostic accuracy of LFA devices. A combination of advances in LFA technology and molecular insight could significantly improve diagnostic accuracy, resulting in a significant improvement in clinical and remote diagnostic accuracy.},
}

*Helicobacter pylori/isolation & purification/genetics
*Helicobacter Infections/diagnosis/microbiology
Humans
Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods
Point-of-Care Systems
Enzyme-Linked Immunosorbent Assay/methods

@article {pmid39819332,
year = {2025},
author = {Wang, C and Zhou, Y and Wang, Y and Jiao, P and Liu, S and Guan, S and Ma, Y},
title = {CRISPR-Cas9-mediated editing of ZmPL1 gene improves tolerance to drought stress in maize.},
journal = {GM crops & food},
volume = {16},
number = {1},
pages = {1-16},
doi = {10.1080/21645698.2024.2448869},
pmid = {39819332},
issn = {2164-5701},
mesh = {*Zea mays/genetics/physiology ; *Plants, Genetically Modified/genetics ; *Droughts ; *Plant Proteins/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Stress, Physiological/genetics ; Gene Expression Regulation, Plant ; Germination/genetics ; },
abstract = {Maize (Zea mays L.) is a widely grown food crop around the world. Drought stress seriously affects the growth and development process of plants and causes serious damage to maize yield. In the early stage, our research group conducted transcriptome sequencing analysis on the drought-resistant maize inbred line H8186 and screened out a gene with significantly down-regulated expression, Phylloplanin-like (ZmPL1). The ZmPL1 gee expression pattern was analyzed under various abiotic stresses, and the results showed that this gene was greatly affected by drought stress. Subcellular localization analysis showed that the protein was localized on the cell membrane. In order to verify the role of ZmPL1 in drought stress, we overexpressed ZmPL1 in yeast and found that the expression of ZmPL1 could significantly increase the drought sensitivity of yeast. Next, ZmPL1 transgenic plants were obtained by infecting maize callus using Agrobacterium-mediated method. Under drought stress, compared with overexpression lines, gene-edited lines had higher germination rate and seedling survival rate, lower accumulation of MDA, relative conductivity and ROS, higher antioxidant enzyme activity, and the expression levels of stress-related genes and ROS scavenging-related genes were significantly increased. Exogenous application of ABA to each lines under drought stress attenuated the damage caused by drought stress on ZmPL overexpressing plants. In summary, ZmPL1 negatively regulates drought tolerance in maize.},
}

*Zea mays/genetics/physiology
*Plants, Genetically Modified/genetics
*Droughts
*Plant Proteins/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
Stress, Physiological/genetics
Gene Expression Regulation, Plant
Germination/genetics

@article {pmid39818894,
year = {2025},
author = {Kaiser, J},
title = {'Safe harbor' gene therapy approach may have first success.},
journal = {Science (New York, N.Y.)},
volume = {387},
number = {6731},
pages = {234-235},
doi = {10.1126/science.adv9833},
pmid = {39818894},
issn = {1095-9203},
mesh = {Humans ; *Genetic Therapy/methods ; *Gene Editing/methods ; Infant ; Liver ; CRISPR-Cas Systems ; Agammaglobulinemia ; Severe Combined Immunodeficiency ; },
abstract = {An obscure gene editor was used to restore a missing liver enzyme in an infant with a devastating metabolic condition.},
}

Humans
*Genetic Therapy/methods
*Gene Editing/methods
Infant
Liver
CRISPR-Cas Systems
Agammaglobulinemia
Severe Combined Immunodeficiency

@article {pmid39746849,
year = {2025},
author = {Paialunga, E and Bagheri, N and Rossetti, M and Fabiani, L and Micheli, L and Chamorro-Garcia, A and Porchetta, A},
title = {Leveraging Synthetic Antibody-DNA Conjugates to Expand the CRISPR-Cas12a Biosensing Toolbox.},
journal = {ACS synthetic biology},
volume = {14},
number = {1},
pages = {171-178},
doi = {10.1021/acssynbio.4c00541},
pmid = {39746849},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *DNA/genetics ; *SARS-CoV-2/genetics/immunology ; Humans ; *Spike Glycoprotein, Coronavirus/genetics/immunology ; Antibodies/immunology ; CRISPR-Associated Proteins/genetics ; COVID-19/diagnosis/virology/genetics ; Saliva ; Endodeoxyribonucleases/genetics/metabolism ; Immunoassay/methods ; Limit of Detection ; Enzyme-Linked Immunosorbent Assay/methods ; Bacterial Proteins/genetics ; },
abstract = {We report here the use of antibody-DNA conjugates (Ab-DNA) to activate the collateral cleavage activity of the CRISPR-Cas12a enzyme. Our findings demonstrate that Ab-DNA conjugates effectively trigger the collateral cleavage activity of CRISPR-Cas12a, enabling the transduction of antibody-mediated recognition events into fluorescence outputs. We developed two different immunoassays using an Ab-DNA as activator of Cas12a: the CRISPR-based immunosensing assay (CIA) for detecting SARS-CoV-2 spike S protein, which shows superior sensitivity compared with the traditional enzyme-linked immunosorbent assay (ELISA), and the CRISPR-based immunomagnetic assay (CIMA). Notably, CIMA successfully detected the SARS-CoV-2 spike S protein in undiluted saliva with a limit of detection (LOD) of 890 pM in a 2 h assay. Our results underscore the benefits of integrating Cas12a-based signal amplification with antibody detection methods. The potential of Ab-DNA conjugates, combined with CRISPR technology, offers a promising alternative to conventional enzymes used in immunoassays and could facilitate the development of versatile CRISPR analytical platforms for the detection of non-nucleic acid targets.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*DNA/genetics
*SARS-CoV-2/genetics/immunology
Humans
*Spike Glycoprotein, Coronavirus/genetics/immunology
Antibodies/immunology
CRISPR-Associated Proteins/genetics
COVID-19/diagnosis/virology/genetics
Saliva
Endodeoxyribonucleases/genetics/metabolism
Immunoassay/methods
Limit of Detection
Enzyme-Linked Immunosorbent Assay/methods
Bacterial Proteins/genetics

@article {pmid39623867,
year = {2025},
author = {Giorgetti, OB and Haas-Assenbaum, A and Boehm, T},
title = {Probing TCR Specificity Using Artificial In Vivo Diversification of CDR3 Regions.},
journal = {European journal of immunology},
volume = {55},
number = {1},
pages = {e202451434},
doi = {10.1002/eji.202451434},
pmid = {39623867},
issn = {1521-4141},
support = {//Max Planck Society/ ; 256073931-SFB1160//Deutsche Forschungsgemeinschaft/ ; },
mesh = {Animals ; *Complementarity Determining Regions/genetics/immunology ; Mice ; *Receptors, Antigen, T-Cell, alpha-beta/genetics/immunology ; CRISPR-Cas Systems ; T-Cell Antigen Receptor Specificity ; Mice, Transgenic ; T-Lymphocytes/immunology ; Receptors, Antigen, T-Cell/immunology/genetics ; },
abstract = {The T-cell receptor sequences expressed on cells recognizing a specific peptide in the context of a given MHC molecule can be explored for common features that might explain their antigen specificity. However, despite the development of numerous experimental and bioinformatic strategies, the specificity problem remains unresolved. To address the need for additional experimental paradigms, we report here on an in vivo experimental strategy designed to artificially diversify a transgenic TCR by CRISPR/Cas9-mediated mutagenesis of Tcra and Tcrb chain genes. In this system, an initially monoclonal repertoire of known specificity is converted into an oligoclonal pool of TCRs of altered antigen reactivity. Tracking the fate of individual clonotypes during the intrathymic differentiation process illuminates the strong selective pressures that shape the repertoire of naïve T cells. Sequence analyses of the artificially diversified repertoires identify key amino acid residues in the CDR3 regions required for antigen recognition, indicating that artificial diversification of well-characterized TCR transgene sequences helps to reduce the complexities of learning the rules of antigen recognition.},
}

Animals
*Complementarity Determining Regions/genetics/immunology
Mice
*Receptors, Antigen, T-Cell, alpha-beta/genetics/immunology
CRISPR-Cas Systems
T-Cell Antigen Receptor Specificity
Mice, Transgenic
T-Lymphocytes/immunology
Receptors, Antigen, T-Cell/immunology/genetics

@article {pmid39818188,
year = {2025},
author = {Liu, Z and Liu, Y and Zhang, S and Wen, Y and Wang, X and Han, J and Yin, H},
title = {Crystal structure of the anti-CRISPR protein AcrIE7.},
journal = {Biochemical and biophysical research communications},
volume = {748},
number = {},
pages = {151315},
doi = {10.1016/j.bbrc.2025.151315},
pmid = {39818188},
issn = {1090-2104},
abstract = {Bacterial adaptive immunity, driven by CRISPR-Cas systems, protects against foreign nucleic acids from mobile genetic elements (MGEs), like bacteriophages. The type I-E CRISPR-Cas system employs the Cascade (CRISPR-associated complex for antiviral defense) complex for target DNA cleavage, guided by crRNA. Anti-CRISPR (Acr) proteins, such as AcrIE7, counteract this defense by inhibiting Cascade activity. In this study, we characterized and determined the structure of AcrIE7, a unique member of the AcrIE family, using X-ray crystallography under two distinct crystallization conditions, achieving resolutions of 2.05 Å and 2.68 Å, respectively. Topological analysis revealed that AcrIE7 consists of seven α-helices with two distinct charge regions, likely mediating its inhibitory interactions. Structural flexibility analysis revealed notable structural stability differences between the two crystallization conditions, indicating varying rigidity of the AcrIE7 protein under different conditions. Homology searches and AlphaFold predictions reinforced the unique nature of AcrIE7, which exhibits a novel fold, underscoring its distinct role within the AcrIE family. These findings enhance our understanding of Acr proteins and provide a theoretical foundation for developing CRISPR-based gene-editing regulatory tools.},
}

@article {pmid39817514,
year = {2025},
author = {Jungfer, K and Moravčík, Š and Garcia-Doval, C and Knörlein, A and Hall, J and Jinek, M},
title = {Mechanistic determinants and dynamics of cA6 synthesis in type III CRISPR-Cas effector complexes.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkae1277},
pmid = {39817514},
issn = {1362-4962},
support = {ERC-CoG-820152/ERC_/European Research Council/International ; 51NF40-182880//SNSF/ ; ETH-24-16-2//ETH Zurich/ ; //University of Zurich/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; Adenine Nucleotides/metabolism/biosynthesis ; Cryoelectron Microscopy ; Bacterial Proteins/metabolism/genetics ; Binding Sites ; Models, Molecular ; Second Messenger Systems ; },
abstract = {Type III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign genetic elements, which triggers the generation of cyclic oligoadenylate (cOA) second messengers by the Cas10 subunit of the type III effector complex. In turn, cOAs bind and activate ancillary effector proteins to reinforce the host immune response. Type III systems utilize distinct cOAs, including cyclic tri- (cA3), tetra- (cA4) and hexa-adenylates (cA6). However, the molecular mechanisms dictating cOA product identity are poorly understood. Here we used cryoelectron microscopy to visualize the mechanism of cA6 biosynthesis by the Csm effector complex from Enterococcus italicus (EiCsm). We show that EiCsm synthesizes oligoadenylate nucleotides in 3'-5' direction using a set of conserved binding sites in the Cas10 Palm domains to determine the size of the nascent oligoadenylate chain. Our data also reveal that conformational dynamics induced by target RNA binding results in allosteric activation of Cas10 to trigger oligoadenylate synthesis. Mutations of a key structural element in Cas10 perturb cOA synthesis to favor cA3 and cA4 formation. Together, these results provide comprehensive insights into the dynamics of cOA synthesis in type III CRISPR-Cas systems and reveal key determinants of second messenger product selectivity, thereby illuminating potential avenues for their engineering.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics
Adenine Nucleotides/metabolism/biosynthesis
Cryoelectron Microscopy
Bacterial Proteins/metabolism/genetics
Binding Sites
Models, Molecular
Second Messenger Systems

@article {pmid39817512,
year = {2025},
author = {Zhang, L and Qiu, X and Zhou, Y and Luo, Z and Zhu, L and Shao, J and Xie, M and Wang, H},
title = {A trigger-inducible split-Csy4 architecture for programmable RNA modulation.},
journal = {Nucleic acids research},
volume = {53},
number = {2},
pages = {},
doi = {10.1093/nar/gkae1319},
pmid = {39817512},
issn = {1362-4962},
support = {2023YFF1205400//Ministry of Science and Technology/ ; 32371498//National Natural Science Foundation of China/ ; 202209009//Westlake Laboratory of Life Sciences and Biomedicine/ ; 2024SSYS0007//'Pioneer' and 'Leading Goose' R&D Program of Zhejiang/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; RNA/metabolism/chemistry/genetics ; Endoribonucleases/metabolism/genetics ; Transcriptome ; },
abstract = {The CRISPR-derived endoribonuclease Csy4 is a popular tool for controlling transgene expression in various therapeutically relevant settings, but adverse effects potentially arising from non-specific RNA cleavage remains largely unexplored. Here, we report a split-Csy4 architecture that was carefully optimized for in vivo usage. First, we separated Csy4 into two independent protein moieties whose full catalytic activity can be restored via various constitutive or conditional protein dimerization systems. Next, we show that introduction of split-Csy4 into human cells caused a substantially reduced extent in perturbation of the endogenous transcriptome when directly compared to full-length Csy4. Inspired by these results, we went on to use such split-Csy4 module to engineer inducible CRISPR- and translation-level gene switches regulated by the FDA-approved drug grazoprevir. This work provides valuable resource for Csy4-related biomedical research and discusses important issues for the development of clinically eligible regulation tools.},
}

Humans
*CRISPR-Cas Systems
HEK293 Cells
RNA/metabolism/chemistry/genetics
Endoribonucleases/metabolism/genetics
Transcriptome

@article {pmid39817421,
year = {2025},
author = {Debaenst, S and Jarayseh, T and De Saffel, H and Bek, JW and Boone, M and Josipovic, I and Kibleur, P and Kwon, RY and Coucke, PJ and Willaert, A},
title = {Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {39817421},
issn = {2050-084X},
support = {FWO.OPR.2020.0023.01//Fonds Wetenschappelijk Onderzoek/ ; },
mesh = {*Zebrafish/genetics ; Animals ; Disease Models, Animal ; Bone Density/genetics ; Osteogenesis Imperfecta/genetics ; CRISPR-Cas Systems ; Phenotype ; Zebrafish Proteins/genetics/metabolism ; High-Throughput Nucleotide Sequencing ; },
abstract = {Heritable fragile bone disorders (FBDs), ranging from multifactorial to rare monogenic conditions, are characterized by an elevated fracture risk. Validating causative genes and understanding their mechanisms remain challenging. We assessed a semi-high throughput zebrafish screening platform for rapid in vivo functional testing of candidate FBD genes. Six genes linked to severe recessive osteogenesis imperfecta (OI) and four associated with bone mineral density (BMD) from genome-wide association studies were analyzed. Using CRISPR/Cas9-based crispant screening in F0 mosaic founder zebrafish, Next-generation sequencing confirmed high indel efficiency (mean 88%), mimicking stable knock-out models. Skeletal phenotyping at 7, 14, and 90 days post-fertilization (dpf) using microscopy, Alizarin Red S staining, and microCT was performed. Larval crispants showed variable osteoblast and mineralization phenotypes, while adult crispants displayed consistent skeletal defects, including malformed neural and haemal arches, vertebral fractures and fusions, and altered bone volume and density. In addition, aldh7a1 and mbtps2 crispants experienced increased mortality due to severe skeletal deformities. RT-qPCR revealed differential expression of osteogenic markers bglap and col1a1a, highlighting their biomarker potential. Our results establish zebrafish crispant screening as a robust tool for FBD gene validation, combining skeletal and molecular analyses across developmental stages to uncover novel insights into gene functions in bone biology.},
}

*Zebrafish/genetics
Animals
Disease Models, Animal
Bone Density/genetics
Osteogenesis Imperfecta/genetics
CRISPR-Cas Systems
Phenotype
Zebrafish Proteins/genetics/metabolism
High-Throughput Nucleotide Sequencing

@article {pmid39817182,
year = {2024},
author = {Xue, M and Gonzalez, DH and Osikpa, E and Gao, X and Lillehoj, PB},
title = {Rapid and automated interpretation of CRISPR-Cas13-based lateral flow assay test results using machine learning.},
journal = {Sensors & diagnostics},
volume = {},
number = {},
pages = {},
pmid = {39817182},
issn = {2635-0998},
abstract = {CRISPR-Cas-based lateral flow assays (LFAs) have emerged as a promising diagnostic tool for ultrasensitive detection of nucleic acids, offering improved speed, simplicity and cost-effectiveness compared to polymerase chain reaction (PCR)-based assays. However, visual interpretation of CRISPR-Cas-based LFA test results is prone to human error, potentially leading to false-positive or false-negative outcomes when analyzing test/control lines. To address this limitation, we have developed two neural network models: one based on a fully convolutional neural network and the other on a lightweight mobile-optimized neural network for automated interpretation of CRISPR-Cas-based LFA test results. To demonstrate proof of concept, these models were applied to interpret results from a CRISPR-Cas13-based LFA for the detection of the SARS-CoV-2 N gene, a key marker for COVID-19 infection. The models were trained, evaluated, and validated using smartphone-captured images of LFA devices in various orientations with different backgrounds, lighting conditions, and image qualities. A total of 3146 images (1569 negative, 1577 positive) captured using an iPhone 13 or Samsung Galaxy A52 Android smartphone were analyzed using the trained models, which classified the LFA results within 0.2 s with 96.5% accuracy compared to the ground truth. These results demonstrate the potential of machine learning to accurately interpret test results of CRISPR-Cas-based LFAs using smartphone-captured images in real-world settings, enabling the practical use of CRISPR-Cas-based diagnostic tools for self- and at-home testing.},
}

@article {pmid39816115,
year = {2024},
author = {Syahrani, RA and Wanandi, SI and Arumsari, S and Nihayah, S and Watanabe, Y and Mizuno, S and Louisa, M and Wuyung, PE},
title = {Dual sgRNA-directed knockout survivin gene expression using CRISPR/Cas9 technology for editing survivin gene in triple-negative breast cancer.},
journal = {Narra J},
volume = {4},
number = {3},
pages = {e1177},
pmid = {39816115},
issn = {2807-2618},
mesh = {*Survivin/genetics/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *Triple Negative Breast Neoplasms/genetics/therapy ; Cell Line, Tumor ; Female ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) offers a robust approach for genome manipulation, particularly in cancer therapy. Given its high expression in triple-negative breast cancer (TNBC), targeting survivin with CRISPR/Cas9 holds promise as a therapeutic strategy. The aim of this study was to design specific single guide ribonucleic acid (sgRNA) for CRISPR/Cas9 to permanently knock out the survivin gene, exploring its potential as a therapeutic approach in breast cancer while addressing potential off-target effects. Survivin gene knockout was conducted in the TNBC cell line BT549. Intron 1, exon 2, and intron 2 of the survivin gene were selected as sgRNA targets. These sgRNAs were designed in silico and then cloned into a CRISPR/Cas9 expression plasmid. The cleavage activity was assessed using an enhanced green fluorescent protein (EGFP) expression plasmid. The sgRNAs with higher cleavage activity were selected for the establishment of knockout cells. After transfecting the plasmid into the cells, the success of the survivin gene knockout was validated at the deoxyribonucleic acid (DNA) level using polymerase chain reaction (PCR) and sequencing analysis, and at the protein expression level using Western blotting. The study found that sgRNAs survin1A (targeting intron 1), survex2A (targeting intron 2), and survin2A (targeting intron 2) demonstrated higher cleavage activities compared to the other sgRNAs. However, using the single sgRNA, survex2A did not generate mutations in the survivin gene. At the protein level, survivin was still expressed, indicating that a single sgRNA was ineffective in knocking out the survivin gene. In contrast, the combination of sgRNA survin1A and sgRNA survin2A was more effective in generating mutations in the survivin gene, resulting in the deletion of the entire exon 2 and leading to a loss of survivin protein expression. In conclusion, our work provides specific sgRNAs and demonstrates the utilization of dual sgRNAs strategy in the CRISPR/Cas9 technology to knock out the survivin gene, showing potential in breast cancer therapy.},
}

*Survivin/genetics/metabolism
Humans
*CRISPR-Cas Systems/genetics
*Triple Negative Breast Neoplasms/genetics/therapy
Cell Line, Tumor
Female
*Gene Editing/methods
*Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39778581,
year = {2025},
author = {Lin, DW and Zhang, L and Zhang, J and Chandrasekaran, S},
title = {Inferring metabolic objectives and trade-offs in single cells during embryogenesis.},
journal = {Cell systems},
volume = {16},
number = {1},
pages = {101164},
pmid = {39778581},
issn = {2405-4720},
mesh = {*Embryonic Development/physiology/genetics ; Animals ; *Single-Cell Analysis/methods ; Mice ; Embryonic Stem Cells/metabolism ; Machine Learning ; CRISPR-Cas Systems ; Cell Proliferation/physiology/genetics ; Humans ; Cell Cycle/physiology ; },
abstract = {While proliferating cells optimize their metabolism to produce biomass, the metabolic objectives of cells that perform non-proliferative tasks are unclear. The opposing requirements for optimizing each objective result in a trade-off that forces single cells to prioritize their metabolic needs and optimally allocate limited resources. Here, we present single-cell optimization objective and trade-off inference (SCOOTI), which infers metabolic objectives and trade-offs in biological systems by integrating bulk and single-cell omics data, using metabolic modeling and machine learning. We validated SCOOTI by identifying essential genes from CRISPR-Cas9 screens in embryonic stem cells, and by inferring the metabolic objectives of quiescent cells, during different cell-cycle phases. Applying this to embryonic cell states, we observed a decrease in metabolic entropy upon development. We further uncovered a trade-off between glutathione and biosynthetic precursors in one-cell zygote, two-cell embryo, and blastocyst cells, potentially representing a trade-off between pluripotency and proliferation. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*Embryonic Development/physiology/genetics
Animals
*Single-Cell Analysis/methods
Mice
Embryonic Stem Cells/metabolism
Machine Learning
CRISPR-Cas Systems
Cell Proliferation/physiology/genetics
Humans
Cell Cycle/physiology

@article {pmid39689711,
year = {2025},
author = {Metzner, E and Southard, KM and Norman, TM},
title = {Multiome Perturb-seq unlocks scalable discovery of integrated perturbation effects on the transcriptome and epigenome.},
journal = {Cell systems},
volume = {16},
number = {1},
pages = {101161},
pmid = {39689711},
issn = {2405-4720},
mesh = {Humans ; *Transcriptome/genetics ; *Epigenome/genetics ; Transcription Factors/genetics/metabolism ; Chromatin/genetics ; Polycomb Repressive Complex 2/genetics/metabolism ; DNA-Binding Proteins/genetics/metabolism ; Single-Cell Analysis/methods ; CRISPR-Cas Systems/genetics ; Cell Line ; Neoplasm Proteins/genetics ; Chromatin Assembly and Disassembly/genetics ; },
abstract = {Single-cell CRISPR screens link genetic perturbations to transcriptional states, but high-throughput methods connecting these induced changes to their regulatory foundations are limited. Here, we introduce Multiome Perturb-seq, extending single-cell CRISPR screens to simultaneously measure perturbation-induced changes in gene expression and chromatin accessibility. We apply Multiome Perturb-seq in a CRISPRi screen of 13 chromatin remodelers in human RPE-1 cells, achieving efficient assignment of sgRNA identities to single nuclei via an improved method for capturing barcode transcripts from nuclear RNA. We organize expression and accessibility measurements into coherent programs describing the integrated effects of perturbations on cell state, finding that ARID1A and SUZ12 knockdowns induce programs enriched for developmental features. Modeling of perturbation-induced heterogeneity connects accessibility changes to changes in gene expression, highlighting the value of multimodal profiling. Overall, our method provides a scalable and simply implemented system to dissect the regulatory logic underpinning cell state. A record of this paper's transparent peer review process is included in the supplemental information.},
}

Humans
*Transcriptome/genetics
*Epigenome/genetics
Transcription Factors/genetics/metabolism
Chromatin/genetics
Polycomb Repressive Complex 2/genetics/metabolism
DNA-Binding Proteins/genetics/metabolism
Single-Cell Analysis/methods
CRISPR-Cas Systems/genetics
Cell Line
Neoplasm Proteins/genetics
Chromatin Assembly and Disassembly/genetics

@article {pmid39528429,
year = {2025},
author = {Ding, S and Zheng, J and Jia, C},
title = {DeepMEns: an ensemble model for predicting sgRNA on-target activity based on multiple features.},
journal = {Briefings in functional genomics},
volume = {24},
number = {},
pages = {},
pmid = {39528429},
issn = {2041-2657},
support = {62071079//National Natural Science Foundation of China/ ; 3132024197//Fundamental Research Funds for the Central Universities/ ; JSKX202203//Hainan Normal University, Ministry of Education/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Neural Networks, Computer ; Deep Learning ; Gene Editing/methods ; Streptococcus pyogenes/genetics ; Humans ; },
abstract = {The CRISPR/Cas9 system developed from Streptococcus pyogenes (SpCas9) has high potential in gene editing. However, its successful application is hindered by the considerable variability in target efficiencies across different single guide RNAs (sgRNAs). Although several deep learning models have been created to predict sgRNA on-target activity, the intrinsic mechanisms of these models are difficult to explain, and there is still scope for improvement in prediction performance. To overcome these issues, we propose an ensemble interpretable model termed DeepMEns based on deep learning to predict sgRNA on-target activity. By using five different training and validation datasets, we constructed five sub-regressors, each comprising three parts. The first part uses one-hot encoding, wherein 0-1 representation of the secondary structure is used as the input to the convolutional neural network (CNN) with Transformer encoder. The second part uses the DNA shape feature matrix as the input to the CNN with Transformer encoder. The third part uses positional encoding feature matrices as the proposed input into a long short-term memory network with an attention mechanism. These three parts are concatenated through the flattened layer, and the final prediction result is the average of the five sub-regressors. Extensive benchmarking experiments indicated that DeepMEns achieved the highest Spearman correlation coefficient for 6 of 10 independent test datasets as compared to previous predictors, this finding confirmed that DeepMEns can accomplish state-of-the-art performance. Moreover, the ablation analysis also indicated that the ensemble strategy may improve the performance of the prediction model.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems/genetics
Neural Networks, Computer
Deep Learning
Gene Editing/methods
Streptococcus pyogenes/genetics
Humans

@article {pmid39814846,
year = {2025},
author = {Shin, SW and Kim, SH and Gasselin, A and Lee, GM and Lee, JS},
title = {Comprehensive genome-scale CRISPR knockout screening of CHO cells.},
journal = {Scientific data},
volume = {12},
number = {1},
pages = {71},
pmid = {39814846},
issn = {2052-4463},
support = {RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; },
mesh = {CHO Cells ; Animals ; *Cricetulus ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome ; High-Throughput Nucleotide Sequencing ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Chinese hamster ovary (CHO) cells play a pivotal role in the production of recombinant therapeutics. In the present study, we conducted a genome-scale pooled CRISPR knockout (KO) screening using a virus-free, recombinase-mediated cassette exchange-based platform in CHO-K1 host and CHO-K1 derived recombinant cells. Genome-wide guide RNA (gRNA) amplicon sequencing data were generated from cell libraries, as well as short- and long-term KO libraries, and validated through phenotypic assessment and gRNA read count distribution. Additionally, we obtained gRNA amplicon sequencing data from the highly productive recombinant cell populations. By analyzing these datasets, essential genes involved in cell fitness as well as functional target genes associated with therapeutic protein production can be identified. Collectively, our next-generation sequencing datasets, derived from a robust and reliable CRISPR screening method, provide valuable insights into CHO genomic functions, advancing the development of next-generation CHO factories.},
}

CHO Cells
Animals
*Cricetulus
*CRISPR-Cas Systems
Gene Knockout Techniques
RNA, Guide, CRISPR-Cas Systems/genetics
Genome
High-Throughput Nucleotide Sequencing
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid39812375,
year = {2025},
author = {Wang, C and Xu, X and Yao, W and Wang, L and Pang, X and Xu, S and Luo, X},
title = {Programmable DNA Nanoswitch-Regulated Plasmonic CRISPR/Cas12a-Gold Nanostars Reporter Platform for Nucleic Acid and Non-Nucleic Acid Biomarker Analysis Assisted by a Spatial Confinement Effect.},
journal = {Nano letters},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.nanolett.4c05829},
pmid = {39812375},
issn = {1530-6992},
abstract = {CRISPR/Cas 12a system based nucleic acid and non-nucleic acid targets detection faces two challenges including (1) multiple crRNAs are needed for multiple biomarkers detection and (2) insufficient sensitivity resulted from photobleaching of fluorescent dyes and the low kinetic cleavage rate for a traditional single-strand (ssDNA) reporter. To address these limitations, we developed a programmable DNA nanoswitch (NS)-regulated plasmonic CRISPR/Cas12a-gold nanostars (Au NSTs) reporter platform for detection of nucleic acid and non-nucleic acid biomarkers with the assistance of the spatial confinement effect. Through simply programming the target recognition sequence in NS, only one crRNA is required to detect both nucleic acid and non-nucleic acid biomarkers. The detection limit decreased by ∼196-fold for miRNA-375 and 122-fold for prostate-specific antigen (PSA), respectively. Moreover, versatile evaluation of miRNA-375 and PSA in clinical urine samples can also be achieved, according to which prostate cancer and healthy groups can be well identified.},
}

@article {pmid39810563,
year = {2025},
author = {Wang, W and Du, H and Dai, C and Ma, H and Luo, S and Wang, X and Guo, M and Kong, D and Wei, D},
title = {Amplification-free detection of Mycobacterium tuberculosis using CRISPR-Cas12a and graphene field-effect transistors.},
journal = {Nanoscale},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4nr03852e},
pmid = {39810563},
issn = {2040-3372},
abstract = {Current molecular tests for tuberculosis (TB), such as whole genome sequencing and Xpert Mycobacterium tuberculosis/rifampicin resistance assay, exhibit limited sensitivity and necessitate the pre-amplification step of target DNA. This limitation greatly increases detection time and poses an increased risk of infection. Here, we present a graphene field-effect transistor (GFET) based on the CRISPR/Cas system for detecting Mycobacterium tuberculosis. The CRISPR/Cas12a system has the ability to specifically recognize and cleave target DNA. By integrating the system onto the FET platform and utilizing its electrical amplification capability, we achieve rapid and sensitive detection without requiring sample pre-amplification, with a limit of detection (LoD) as low as 2.42 × 10[-18] M. Cas12a-GFET devices can differentiate 30 positive cases from 56 serum samples within 5 minutes. These findings highlight its immense potential in future biological analysis and clinical diagnosis.},
}

@article {pmid39774325,
year = {2025},
author = {Funk, JS and Klimovich, M and Drangenstein, D and Pielhoop, O and Hunold, P and Borowek, A and Noeparast, M and Pavlakis, E and Neumann, M and Balourdas, DI and Kochhan, K and Merle, N and Bullwinkel, I and Wanzel, M and Elmshäuser, S and Teply-Szymanski, J and Nist, A and Procida, T and Bartkuhn, M and Humpert, K and Mernberger, M and Savai, R and Soussi, T and Joerger, AC and Stiewe, T},
title = {Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations.},
journal = {Nature genetics},
volume = {57},
number = {1},
pages = {140-153},
pmid = {39774325},
issn = {1546-1718},
support = {STI 182/15-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; STI 182/13-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; GRK2573//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 109546710 Project A10//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SA 1923/7-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC1213 A10N//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; JO 1473/1-3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 031L0063//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; DZL//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; LOEWE iCANx//Hessisches Ministerium für Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; 65-0004//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 66-LV06//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 71-0012//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 09 R/2018//José Carreras Leukämie-Stiftung (Deutsche José Carreras Leukämie-Stiftung)/ ; TACTIC//Deutsche Krebshilfe (German Cancer Aid)/ ; },
mesh = {Humans ; *Tumor Suppressor Protein p53/genetics ; *Gene Editing/methods ; *Mutagenesis ; *CRISPR-Cas Systems ; *Neoplasms/genetics ; Mutation, Missense/genetics ; Cell Line, Tumor ; Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nonsense Mediated mRNA Decay/genetics ; },
abstract = {The mutational landscape of TP53, a tumor suppressor mutated in about half of all cancers, includes over 2,000 known missense mutations. To fully leverage TP53 mutation status for personalized medicine, a thorough understanding of the functional diversity of these mutations is essential. We conducted a deep mutational scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 TP53 variants in cancer cells. This high-resolution approach, covering 94.5% of all cancer-associated TP53 missense mutations, precisely mapped the impact of individual mutations on tumor cell fitness, surpassing previous deep mutational scan studies in distinguishing benign from pathogenic variants. Our results revealed even subtle loss-of-function phenotypes and identified promising mutants for pharmacological reactivation. Moreover, we uncovered the roles of splicing alterations and nonsense-mediated messenger RNA decay in mutation-driven TP53 dysfunction. These findings underscore the power of saturation genome editing in advancing clinical TP53 variant interpretation for genetic counseling and personalized cancer therapy.},
}

Humans
*Tumor Suppressor Protein p53/genetics
*Gene Editing/methods
*Mutagenesis
*CRISPR-Cas Systems
*Neoplasms/genetics
Mutation, Missense/genetics
Cell Line, Tumor
Mutation
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Nonsense Mediated mRNA Decay/genetics

@article {pmid39754554,
year = {2025},
author = {Hu, F and Liu, K and Zhang, Y and Zhao, S and Zhang, T and Yao, C and Lv, X and Wang, J and Liu, X and Peng, N},
title = {Short-Time Preamplification-Assisted One-Pot CRISPR Nucleic Acid Detection Method with Portable Self-Heating Equipment for Point-of-Care Diagnosis.},
journal = {Analytical chemistry},
volume = {97},
number = {1},
pages = {658-666},
doi = {10.1021/acs.analchem.4c05026},
pmid = {39754554},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; Humans ; *Nucleic Acid Amplification Techniques ; *COVID-19/diagnosis/virology ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Point-of-Care Testing ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Point-of-Care Systems ; COVID-19 Nucleic Acid Testing/methods/instrumentation ; Limit of Detection ; },
abstract = {Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve "sample-in-answer-out" detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.},
}

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