@article {pmid41402624,
year = {2025},
author = {Sinkunas, T and Tamulaitiene, G},
title = {A DNA mimic jams the Cas9 scissors.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70374},
pmid = {41402624},
issn = {1742-4658},
support = {S-MIP-20-39//Lietuvos Mokslo Taryba/ ; },
abstract = {Anti-CRISPR (Acr) proteins are small protein inhibitors that block the RNA-guided nucleic acid (DNA or RNA) targeting activity of CRISPR-Cas enzymes. Despite their shared function, Acr proteins display minimal sequence or structural similarity and employ diverse mechanisms to block nuclease activity. Lee and Park characterized the previously undescribed AcrIIA13b protein, which inhibits Cas9 protein. Structural, biochemical, and mutational analyses revealed that AcrIIA13b acts as a DNA mimic, thereby disabling the Cas9 complex from binding to the DNA target.},
}

@article {pmid41402279,
year = {2025},
author = {Wang, D and Ritz, C and Luo, Y and Suresh, A and Pierce, A and Veo, B and Brunt, B and Dahl, N and Serkova, N and Venkataraman, S and Danis, E and Kus, K and Mazan, M and Rzymski, T and Vibhakar, R},
title = {Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11152},
pmid = {41402279},
issn = {2041-1723},
mesh = {*Medulloblastoma/genetics/metabolism/pathology/drug therapy ; Humans ; *Proto-Oncogene Proteins c-myc/metabolism/genetics ; *Cyclin-Dependent Kinase 8/metabolism/genetics/antagonists & inhibitors ; *Protein Biosynthesis/genetics ; Animals ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Mice ; *Cerebellar Neoplasms/genetics/metabolism/pathology/drug therapy ; Transcription, Genetic ; TOR Serine-Threonine Kinases/metabolism/antagonists & inhibitors ; RNA Polymerase II/metabolism ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {MYC-driven medulloblastoma (MB) is a highly aggressive brain tumor with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening, we identify the Mediator-associated kinase CDK8 as a critical regulator of MYC-driven MB. Both genetic loss and pharmacological inhibition of CDK8 impair MB tumor growth. Moreover, we find that CDK8 cooperates with MYC to sustain the MYC-mediated translational program, as CDK8 depletion induces pronounced transcriptional changes in translation-associated gene sets, reduces ribosome biogenesis, and impairs protein synthesis. Mechanistically, CDK8 regulates the occupancy of RNA polymerase II at specific chromatin loci, facilitating epigenetic alterations that promote the transcription of ribosomal genes. Furthermore, combined inhibition of CDK8 and mTOR synergistically enhances therapeutic efficacy in vivo, leading to more pronounced tumor growth suppression. Overall, our findings establish a functional link between CDK8-mediated transcriptional regulation and mRNA translation, suggesting a promising therapeutic approach targeting protein synthesis for MYC-driven MB.},
}

*Medulloblastoma/genetics/metabolism/pathology/drug therapy
Humans
*Proto-Oncogene Proteins c-myc/metabolism/genetics
*Cyclin-Dependent Kinase 8/metabolism/genetics/antagonists & inhibitors
*Protein Biosynthesis/genetics
Animals
Cell Line, Tumor
*Gene Expression Regulation, Neoplastic
Mice
*Cerebellar Neoplasms/genetics/metabolism/pathology/drug therapy
Transcription, Genetic
TOR Serine-Threonine Kinases/metabolism/antagonists & inhibitors
RNA Polymerase II/metabolism
Xenograft Model Antitumor Assays
CRISPR-Cas Systems

@article {pmid41399500,
year = {2025},
author = {Swartjes, T and Bouzetos, E and Adiego-Pérez, B and Pool, VD and Staals, RHJ and van der Oost, J and Wu, WY},
title = {Base editing both DNA strands in distinct editing windows with small CRISPR-associated effector Cas12f1.},
journal = {iScience},
volume = {28},
number = {12},
pages = {114033},
pmid = {41399500},
issn = {2589-0042},
abstract = {CRISPR-associated base editors have been established as genome editing tools that enable base conversions in targeted DNA sequences, without generating double-strand breaks. Here, we describe the development of new base editors based on CRISPR-Cas12f1, a miniature Cas protein of only 422 amino acids. Chimeric constructs have been generated by fusing a catalytically inactive dCas12f1, to either a cytosine deaminase or an adenine deaminase. Using these synthetic fusion proteins, systematic analyses have been performed on base editing of a target sequence on a plasmid in Escherichia coli. Interestingly, apart from the previously described base editing of the displaced non-target DNA strand, we also observed efficient editing of the target DNA strand. This effect was not observed for Un1Cas12f1 BEs. In addition to the small size of AsCas12f1 base editors, its unique editing profile makes it a valuable addition to the CRISPR-Cas toolbox.},
}

@article {pmid41399197,
year = {2026},
author = {Singh, K and Sharma, S and Kalia, A and Manchanda, P},
title = {Advancement in Mushroom Transformation: From Conventional Techniques to Modern Genetic Engineering.},
journal = {Journal of basic microbiology},
volume = {66},
number = {1},
pages = {e70132},
doi = {10.1002/jobm.70132},
pmid = {41399197},
issn = {1521-4028},
mesh = {*Genetic Engineering/methods ; *Agaricales/genetics/growth & development ; *Transformation, Genetic ; CRISPR-Cas Systems ; Gene Editing ; Agrobacterium/genetics ; },
abstract = {Mushrooms have long been valued for their nutritional, pharmaceutical, and culinary benefits. Recent studies showcased mushrooms as bio-factories for protein production, and as a source of value-added products by employing genetic manipulation and molecular transformation techniques. Advancements in molecular tools and transformation methods have enhanced the efficiency of genetic improvements in mushrooms by both conventional and modern genetic engineering techniques, paving the way for their use in various industrial applications. Genetic transformation in mushrooms involves transferring genes within and across species to understand gene functions and improve mushroom qualities. The techniques involved in transformation includes Agrobacterium-mediated transformation, hybridization, mutation breeding, particle bombardment, protoplast fusion, and CRISPR/Cas9. This review outlines the life cycle of mushrooms, major difficulties in mushroom transformation, various transformation techniques, their history, efficiency, and success rate. It also highlights the potential of genetic engineering to revolutionize mushroom cultivation and their applications.},
}

*Genetic Engineering/methods
*Agaricales/genetics/growth & development
*Transformation, Genetic
CRISPR-Cas Systems
Gene Editing
Agrobacterium/genetics

@article {pmid41398311,
year = {2025},
author = {Kang, YJ and Ha, HJ and Jin, HB and Lee, SY and Park, HH},
title = {Structural basis of dimerization and cascade formation by Cas5.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-025-32766-5},
pmid = {41398311},
issn = {2045-2322},
support = {2025//Chung-Ang University/ ; RS-2025-02316334//National Research Foundation of Korea/ ; },
abstract = {CRISPR-Cas systems are essential for prokaryotic adaptive immune mechanisms; however, the structural details of many subtype-specific components remain unclear. Herein, we report the crystal structure and biophysical characterization of Cas5 from Moraxella bovoculi (MboCas5), a component of the type I-C CRISPR-Cas system. We found that M. bovoculi encodes both type I-C and type III-B systems, and that MboCas5 forms a dimer that is stabilized by key interactions, including a salt bridge between R72 and D167. Structural comparisons with other Cas5 homologs and AlphaFold 3 predictions further validated the unique dimer configuration, suggesting that it is conserved across species. Additionally, structural comparison revealed a highly flexible loop region, which likely undergoes conformational changes upon Cascade assembly and might mediate interactions with Cas8 and crRNA. Overall, the findings provided structural and mechanistic insights into Cas5 function and could potentially contribute to our understanding of the assembly of type I-C Cascade complexes.},
}

@article {pmid41397585,
year = {2025},
author = {Hsu, CY and Polatova, D and Hamad, RH and Patel, PN and Akram, M and Singh, G and Arora, V and Nayak, PP and Kadhem, M and Hamzah, HF},
title = {Phage Therapy in Cancer Treatment: Mechanisms, Emerging Innovations, and Translational Progress.},
journal = {Critical reviews in oncology/hematology},
volume = {},
number = {},
pages = {105085},
doi = {10.1016/j.critrevonc.2025.105085},
pmid = {41397585},
issn = {1879-0461},
abstract = {Bacteriophage therapy has re-emerged as a rapidly advancing field in oncology, bridging antimicrobial precision with tumor-targeted biotherapy. Beyond infection control, phages are now recognized as programmable biological systems capable of eradicating multidrug-resistant (MDR) pathogens, modulating tumor-associated microbiota, activating immune responses, and delivering therapeutic genes or drugs. Preclinical evidence shows that phages can selectively eliminate Fusobacterium nucleatum in oral squamous cell carcinoma, restore microbial balance in colorectal cancer, and enhance immune infiltration via cytokine or antigen display. Engineered constructs including GM-CSF-expressing and MAGE-A1-displaying phages, λ-phage ASPH vaccines, and PEGylated nanocarriers delivering MEG3 or TRAIL have demonstrated strong anti-tumor efficacy across melanoma, hepatocellular, and colorectal cancer models. Additionally, CRISPR-Cas-armed phages precisely remove resistance genes such as bla-CTX-M and mecA, while AI-driven selection pipelines enable data-guided design of personalized phage cocktails. These advances represent a paradigm shift from empirical antibacterial use toward mechanistically engineered, multifunctional phage platforms that integrate microbiome modulation, immune activation, and nanocarrier-mediated gene delivery. Although challenges such as immune clearance, bacterial resistance, and regulatory complexity remain, the convergence of AI, CRISPR, and synthetic biology is accelerating the evolution of phage therapy into a clinically viable precision-oncology strategy. In this context, bacteriophages emerge not merely as antibacterial agents but as intelligent, patient-specific nanomedicines poised to redefine therapeutic boundaries in cancer treatment.},
}

@article {pmid41396047,
year = {2025},
author = {Matsuoka, T and Kano, S},
title = {Impact of patent-granting differences between Japan and the United States on patent protection for medical methods: insights from genome editing patents.},
journal = {Expert opinion on therapeutic patents},
volume = {},
number = {},
pages = {},
doi = {10.1080/13543776.2025.2605318},
pmid = {41396047},
issn = {1744-7674},
abstract = {INTRODUCTION: Securing patents in multiple countries has become essential for the development of global medical products. However, differences in national patent systems result in varying patentability standards. Although global claim construction strategies have been applied in practice, these approaches have not yet been systematically organized.

AREA COVERED: This study examines how the patent scope for patent families of international applications related to genome editing technologies filed in 2013, differs between Japan, where medical method patents are prohibited, and the United States, where such patents are permitted.

EXPERT OPINION: For CRISPR-Cas system patents, claim structures varied significantly, even among the corresponding family patents. To navigate these differences, the following strategies were proposed for filing patents in countries that prohibit medical method patents such as Japan:- Convert medical method claims in the U.S. into composition claims that include product inventions, as this process ensures that such claims allow for the enforcement of rights against the suppliers of infringing products.- Clearly define the scope of the claimed use-inventions when specifying the characteristics of the product based on its effects.- Explicitly describe cells produced by a specific manufacturing method within the claimed rights.},
}

@article {pmid41395656,
year = {2025},
author = {Lin, Z and Pu, Z and Wu, J and Zeng, J and Dou, Q and Mao, M and Zhang, Y},
title = {A Versatile CRISPR/Cas12a Autocatalytic Cascade System via Structure-Switching V-Type Split Probe for Highly Sensitive DNA Diagnostics.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c06488},
pmid = {41395656},
issn = {1520-6882},
abstract = {The rapid detection of pathogen nucleic acids is critical for controlling infectious disease outbreaks and providing timely treatment. However, current molecular diagnostic applications, including sensitive CRISPR/Cas-based detection systems, rely on target preamplification, which often requires expensive equipment and strict adherence to sometimes complex workflows. Here, we describe a rapid, simple, and amplification-free CRISPR/Cas-based diagnostic system that employs a structure-switching V-shaped DNA probe with a Cas12a recognition sequence split by an ssDNA loop to establish a positive feedback loop and a signal amplification cascade. This approach exhibited an ultralow background signal, rapid production of an exponential signal, and atto-molar sensitivity. It was incorporated into microfluidic and lateral flow assay applications for multiplex detection of distinct papillomavirus strains and point-of-care detection of monkeypox virus infections, respectively. The approach thus has significant potential for rapid and sensitive detection of specific pathogen-derived DNA targets in both clinical laboratory and point-of-care applications.},
}

@article {pmid41395238,
year = {2025},
author = {Zou, Y and Yao, ZW and Xiao, T and Ma, YR and He, J and Chen, LM and Chen, XQ and Chen, N},
title = {Emerging Trichomonad Infections in Companion Animals: Rapid Visual Detection of Pentatrichomonas hominis and Tritrichomonas foetus Using an RPA-CRISPR/Cas12a Assay.},
journal = {Transboundary and emerging diseases},
volume = {2025},
number = {},
pages = {9995679},
pmid = {41395238},
issn = {1865-1682},
mesh = {Animals ; Cats ; *Protozoan Infections, Animal/diagnosis/parasitology ; *Tritrichomonas foetus/isolation & purification/genetics ; Dogs ; *Trichomonadida/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Cat Diseases/parasitology/diagnosis ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Pets ; *Dog Diseases/diagnosis/parasitology ; },
abstract = {Pentatrichomonas hominis (P. hominis) and Tritrichomonas foetus (T. foetus) are prevalent intestinal protozoa. P. hominis is associated with chronic diarrhea in humans and animals, whereas T. foetus causes gastrointestinal disease in companion animals and reproductive-tract infection in cattle. Rapid and accurate identification of these infections at the point-of-care (POC) is crucial for the diagnosis and effective management of zoonotic diseases. In this study, we developed two novel recombinase polymerase amplification (RPA) assays coupled with CRISPR/Cas12a detection. The dual-species assay, using a lateral-flow format, targeted species-specific regions of the 18S rRNA gene of P. hominis and T. foetus, and under ideal conditions, delivered visual results within 40 min for a single sample at 37°C. P. hominis-specific assay: To differentiate P. hominis in mixed infections with T. foetus, a second assay targeted the highly conserved Spo11-1 gene of P. hominis. Optimal crRNA-412 and RPA primers were selected for maximal Cas12a cleavage efficiency. Analytical sensitivity and specificity were compared with conventional nested polymerase chain reaction (PCR) and Sanger sequencing. The results showed that The dual-species assay detected as few as 50 DNA copies/µL of either parasite with no cross-reactivity to Giardia lamblia, Cystoisospora canis, Cryptosporidium spp., Toxoplasma gondii, Toxocara canis, and Toxascaris leonina. Among 70 fecal samples of companion animal (48 dogs and 22 cats), 14 (29.2%) dogs tested positive for P. hominis, and eight cats (36.4%) tested positive for T. foetus by nested PCR. Due to financial and logistical constraints, we selected a smaller subset for subsequent analysis with the RPA-CRISPR/Cas12a lateral-flow strip (LFS) assay, which showed 100% diagnostic concordance with PCR. The Spo11-1 assay achieved a limit of detection of 20 DNA copies/µL and specifically recognized P. hominis among a panel that included seven non-target protozoa and helminths. Validation on 10 additional canine and feline samples (four positives and six negatives) showed complete agreement with nested-PCR results. In conclusion, this CRISPR-based diagnostic approach significantly enhances the efficiency and accuracy of Trichomonads detection, offering a practical, cost-effective solution particularly suitable for veterinary and potentially human healthcare diagnostics in resource-limited settings.},
}

Animals
Cats
*Protozoan Infections, Animal/diagnosis/parasitology
*Tritrichomonas foetus/isolation & purification/genetics
Dogs
*Trichomonadida/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/veterinary/methods
*Cat Diseases/parasitology/diagnosis
CRISPR-Cas Systems
Sensitivity and Specificity
Pets
*Dog Diseases/diagnosis/parasitology

@article {pmid41394966,
year = {2025},
author = {Punde, A and Dey, S and Pandire, R and Bhattacharjee, A and Patra, C},
title = {Expanding the CRISPR/Cas toolkit: applications in proteomics and theranostics.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {13},
number = {},
pages = {1713700},
pmid = {41394966},
issn = {2296-4185},
abstract = {Conventional methods available for genome editing have proven non-specific, labour-intensive, and time-consuming. In this context, CRISPR/Cas technology represents a significant breakthrough. It is derived from a sophisticated microbial defence system consisting of clustered regularly interspaced short palindromic repeats, or CRISPR, and the RNA-guided DNA endonuclease Cas. Beyond its original role in genome editing, CRISPR continues to play a major role in the field of proteomics, functional genomics, and molecular therapy. Animal models, including mice, Drosophila, zebrafish, etc., have substantially benefited from CRISPR in uncovering protein function through reverse genetics approaches, including knock-in, knockout, CRISPRi, and indel mutation strategies. On the clinical front, CRISPR gene therapy has also seen successes, including applications in sickle cell disease, hypercholesterolemia, and cancer immunotherapy. However, notable challenges remain, including in vivo packaging and delivery efficiency, toxicity, and genomic off-target effects. Ongoing efforts to overcome these include the development of novel delivery formulations (e.g., nanoparticles, exosomes), artificial intelligence-guided experimental design, and miniaturization of Cas proteins. This review focuses on CRISPR/Cas gene editing mechanisms and explores its state-of-the-art applications in the field of proteomics and theranostics.},
}

@article {pmid41392542,
year = {2025},
author = {Qiao, Z and Choi, S and Chen, Z and Rodriguez, RM and Wang, Q and Yang, Z and Theuerkauf, SA and Nabhan, JF and Hensch, TK and Buchholz, CJ and Lu, Q},
title = {Targeted Intracellular Delivery via Precision Programming of ARRDC1-Mediated Microvesicles.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {12},
pages = {e70199},
pmid = {41392542},
issn = {2001-3078},
support = {//Vesigen Therapeutics/ ; P42ES030990/NH/NIH HHS/United States ; R01ES029097/NH/NIH HHS/United States ; R01HL139496/NH/NIH HHS/United States ; },
mesh = {Animals ; Mice ; CD8-Positive T-Lymphocytes/metabolism ; Humans ; *Cell-Derived Microparticles/metabolism ; Neurons/metabolism ; *Drug Delivery Systems/methods ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; },
abstract = {Efficient and cell-specific delivery remains a major barrier to realising the full therapeutic potential of modalities such as mRNA and CRISPR-based gene editors. Here, we report a versatile delivery platform based on engineered ARRDC1-mediated microvesicles (ARMMs) capable of delivering cargo to defined cell populations. By decorating ARMMs with engineered Nipah virus (NiV)-derived fusion and attachment proteins conjugated to cell-specific ligands, we enable selective binding and membrane fusion-mediated cargo release. ARMMs functionalized with anti-CD8 single-chain variable fragment (scFv) delivered protein, mRNA, or CRISPR-Cas9 base editor selectively to CD8[+] T cells. Similarly, ARMMs displaying a designed ankyrin repeat protein (DARPin) targeting the GluA4 receptor enabled delivery to parvalbumin-positive (PV[+]) neurons. In vivo, administration of targeted ARMMs resulted in functional delivery to CD8[+] splenocytes and PV[+] cortical neurons in mice. These findings establish surface-engineered ARMMs as a programmable and modular system for precision delivery of therapeutic macromolecules, with broad applicability in gene and RNA-based medicine.},
}

Animals
Mice
CD8-Positive T-Lymphocytes/metabolism
Humans
*Cell-Derived Microparticles/metabolism
Neurons/metabolism
*Drug Delivery Systems/methods
Mice, Inbred C57BL
CRISPR-Cas Systems

@article {pmid41381532,
year = {2025},
author = {Zaada, DSY and Toren, O and Krsticevic, F and Haber, DA and Gildman, D and Galpaz, N and Häcker, I and Schetelig, MF and Marois, E and Arien, Y and Papathanos, PA},
title = {Mosquito sex separation using complementation of selectable traits and engineered neo-sex chromosomes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11175},
pmid = {41381532},
issn = {2041-1723},
support = {3-1679//Ministry of Science, Technology and Space/ ; 1833/7-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 101059523//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; ANR-23-CE35-0003//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {Animals ; Female ; Male ; *Aedes/genetics/physiology ; *Sex Chromosomes/genetics ; CRISPR-Cas Systems ; Genetic Engineering ; *Sex Determination Processes/genetics ; Animals, Genetically Modified ; Pigmentation/genetics ; Phenotype ; Insect Proteins/genetics ; },
abstract = {Effective sex separation remains a critical challenge for mosquito genetic control. Genetic sexing strains (GSS) address this by linking maleness with selectable traits, enabling efficient female removal. Here, we present a versatile platform for GSS development in the invasive Aedes albopictus mosquito that integrates CRISPR-engineered selectable phenotypes with sex conversion via nix, the male-determining factor. As a proof-of-concept, we disrupt the yellow pigmentation gene and restore its function in males using nix-containing transgenes, producing a stable strain with yellow females and dark males. Beyond serving as a vivid marker, yellow confers added advantages: GSS females pupate later than wild females, enhancing protandry-based sorting, and lay desiccation-sensitive eggs, lowering accidental female release risk. The strain is compatible with size-based separation, improving sexing accuracy through the integration of natural and engineered dimorphisms. To our knowledge, this represents the first engineered sex-linked selectable trait in mosquitoes based on endogenous genes, establishing a foundation for scalable GSS development.},
}

Animals
Female
Male
*Aedes/genetics/physiology
*Sex Chromosomes/genetics
CRISPR-Cas Systems
Genetic Engineering
*Sex Determination Processes/genetics
Animals, Genetically Modified
Pigmentation/genetics
Phenotype
Insect Proteins/genetics

@article {pmid41380779,
year = {2026},
author = {Hill, AC and Schank, MB and Zhang, Y and Sun, N and Wang, L and Zhao, J and Banik, P and Pyburn, JS and Orfield, H and Lightner, JW and Leshaodo, TO and Wu, XY and Ning, S and El Gazzar, M and Moorman, JP and Guo, H and Yao, ZQ},
title = {Suppression of HBV replication and expression by CRISPR/Cas9 ribonucleoproteins.},
journal = {Antiviral research},
volume = {245},
number = {},
pages = {106326},
doi = {10.1016/j.antiviral.2025.106326},
pmid = {41380779},
issn = {1872-9096},
mesh = {*Hepatitis B virus/genetics/physiology/drug effects ; *Virus Replication/drug effects ; Humans ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/pharmacology/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; DNA, Viral/genetics ; Antiviral Agents/pharmacology ; Hep G2 Cells ; Genetic Therapy/methods ; Hepatitis B/therapy/virology ; Cell Line ; CRISPR-Associated Protein 9 ; },
abstract = {HBV infection is a global public health problem. The current treatment using nucleotide analogues (NA) can suppress viral replication but cannot eliminate HBV infection due to the persistence of covalently closed circular DNA (cccDNA), which sustains HBV replication and integration into the host cell genome and is refractory to NA treatment. CRISPR/Cas9 has been used to disrupt integrated HBV DNA and minichromosomal cccDNA for HBV suppression, but its expression and delivery require viral or non-viral vectors, which pose safety concerns for human application. We have previously reported the use of synthetic guide RNA (gRNA)/Cas9 ribonucleoprotein (RNP) as a non-viral formulation for HBV gene editing and viral suppression. To formulate highly effective CRISPR/Cas9 modalities for HBV gene therapy, here we designed additional gRNA/Cas9 RNPs and compared their antiviral efficacy in HBV-transfected as well as -infected cells. We found that two selected gRNA/Cas9 RNPs (gRNA5/Cas9, gRNA9/Cas9, and particularly their combinations) elicited the most potent antiviral efficacy, as evidenced by the significant inhibition of HBV DNA, RNA, and protein productions. DNA sequencing of the treated cells revealed moderate to high rates of insertion and deletion (indel) or knock-out (KO) mutations at the HBV target genes. Gene alignment analysis showed a high level of conservation for both gRNA5 and gRNA9 target sequences across major HBV genotypes, indicating that these CRISPR-based gene editing therapeutics have the potential to target different HBV strains worldwide. Thus, these synthetic gRNA/Cas9 RNPs represent promising novel therapeutics that can be developed and utilized for HBV gene disruption and viral eradication.},
}

*Hepatitis B virus/genetics/physiology/drug effects
*Virus Replication/drug effects
Humans
*CRISPR-Cas Systems
*Ribonucleoproteins/genetics/pharmacology/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing
DNA, Viral/genetics
Antiviral Agents/pharmacology
Hep G2 Cells
Genetic Therapy/methods
Hepatitis B/therapy/virology
Cell Line
CRISPR-Associated Protein 9

@article {pmid41365143,
year = {2025},
author = {Queffeulou, M and Fakhfakh, R and Fani, F and Dos Santos, A and Reis Ferreira, G and Bigot, S and Godin, C and Leprohon, P and Papadopoulou, B and Ouellette, M},
title = {CRISPR-Cas13b mediated gene knockdowns in Leishmania infantum.},
journal = {International journal for parasitology. Drugs and drug resistance},
volume = {29},
number = {},
pages = {100629},
doi = {10.1016/j.ijpddr.2025.100629},
pmid = {41365143},
issn = {2211-3207},
mesh = {*Leishmania infantum/genetics/drug effects ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; Antiprotozoal Agents/pharmacology ; Phosphorylcholine/analogs & derivatives/pharmacology ; Protozoan Proteins/genetics ; Antimony/pharmacology ; Luciferases, Firefly/genetics ; RNA, Messenger/genetics ; },
abstract = {Chemotherapy is an effective means to control infections caused by the protozoan parasite Leishmania. However, available treatments are limited, expensive, and associated with considerable toxicity. Genomic strategies have contributed to a better understanding of Leishmania's response to drugs and in the characterization of drug targets. Nonetheless, there is no knockdown system operational for Leishmania. In this study, we show that the CRISPR-Cas13 system can be an effective strategy to knockdown expression levels of both exogenous and endogenous transcripts. We succeeded in effectively knocking down the expression of the firefly luciferase gene integrated in the genome of L. infantum. This Cas13-mediated decrease in mRNA was paralleled with a significant reduction in both the luciferase protein level and its activity. Furthermore, we tested the effectiveness of the Cas13 system to target the endogenous miltefosine transporter (MT) and the aquaglyceroporin 1 (AQP1) genes. Knockdown was effective and parasites with less MT or AQP1 mRNA levels exhibited reduced susceptibility to miltefosine or antimonials, respectively. While further optimization is warranted, this knockdown system has the potential to facilitate numerous studies related to various aspects of Leishmania biology.},
}

*Leishmania infantum/genetics/drug effects
*CRISPR-Cas Systems/genetics
*Gene Knockdown Techniques/methods
Antiprotozoal Agents/pharmacology
Phosphorylcholine/analogs & derivatives/pharmacology
Protozoan Proteins/genetics
Antimony/pharmacology
Luciferases, Firefly/genetics
RNA, Messenger/genetics

@article {pmid41363172,
year = {2025},
author = {Wen, TT and Xu, L and Jin, R and Liu, Z and Liu, MQ and Dong, CH and Sun, L and Wang, HY and Sun, WJ and Cui, FJ},
title = {Functional Characterization of a β-1,3-Glucanosyltransferase CmGel4 in Cordyceps militaris Using a Precise CRISPR-Cas9 Genome-Editing System.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {50},
pages = {32078-32093},
doi = {10.1021/acs.jafc.5c08653},
pmid = {41363172},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; *Cordyceps/genetics/enzymology/chemistry ; Gene Editing ; *Fungal Proteins/genetics/metabolism/chemistry ; beta-Glucans/metabolism ; Amino Acid Sequence ; Glucan Endo-1,3-beta-D-Glucosidase ; },
abstract = {Cordyceps militaris polysaccharides, especially β-glucans, have presented significant antitumor, hypoglycemic, and immunomodulatory activities. However, the enzymes involved in the branching formation of C. militaris β-glucans remain to be elucidated. In the present study, a 1.69-kb β-1,3-glucanosyltransferase CmGel4 gene putatively involved in β-glucan branching was cloned from C. militaris mycelia and bioinformatically analyzed. The encoded 54.12 kDa CmGel4p consisted of 515 amino acid residues and contained a typical GH72[+] structural characteristic of a signal peptide (1-19aa), a GH72 conserved domain (20-334aa), a GPI-anchor site (485aa), and a CBM43/X8 domain (382-458aa). Using the established CRISPR-Cas9 genome-editing system, the full length of 1.69-kb CmGel4 was precisely inserted at a genomic safe-harbor site CmSh1, and the GH72 conserved domain of CmGel4 was successfully deleted in C. militaris genome for the first time. By comparing the mycelial growth and fermentation performance of WT, control, and CmGel4-overexpressed/knockout mutants, β-1,3-glucanosyltransferase gene CmGel4 was shown to play key roles in cell growth and branching of exo-polysaccharides of C. militaris, accompanied by the transcriptional changes of genes such as CmGel4, CmUgp, and CmPgm. These findings provided the proof of β-1,3-glucanosyltransferases vital for formatting cell walls and maintaining cellular integrity, and a fine regulation strategy for precisely remodeling the β-1,3-glucan with high-branched structures in edible fungi.},
}

CRISPR-Cas Systems
*Cordyceps/genetics/enzymology/chemistry
Gene Editing
*Fungal Proteins/genetics/metabolism/chemistry
beta-Glucans/metabolism
Amino Acid Sequence
Glucan Endo-1,3-beta-D-Glucosidase

@article {pmid41361999,
year = {2025},
author = {Cheng, Y and Zhang, X and Zhao, P and Zhu, D and Meng, YH and Fu, X and Wang, X},
title = {Enhanced Mannan Production of Saccharomyces cerevisiae by CRISPR/Cas9 and Mannoproteins Characteristics on Wine Astringency Modulation.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {50},
pages = {32195-32208},
doi = {10.1021/acs.jafc.5c10790},
pmid = {41361999},
issn = {1520-5118},
mesh = {*Saccharomyces cerevisiae/metabolism/genetics ; *Wine/analysis/microbiology ; *Mannans/metabolism/chemistry ; *Membrane Glycoproteins/metabolism/genetics/chemistry ; CRISPR-Cas Systems ; Taste ; Humans ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; Mannose/metabolism/analysis ; },
abstract = {Mannoproteins are critical in modulating wine astringency, yet the specific impacts of their monosaccharide ratio and side-chain structure remain insufficiently explored. This study employed CRISPR/Cas9 to engineer yeast strains producing mannoproteins with either a high mannose-to-glucose ratio (high-yield-mannan strain BSFA12) or a nonbranched N-glycan structure (BY4741-ΔMNN2). The resulting mannoproteins (MPBSFA, MPBY2) were compared against controls (MPBY extracted from Saccharomyces cerevisiae BY4741, a commercial product MP60) using physicochemical analyses (fluorescence quenching, dynamic light scattering, and isothermal titration calorimetry) and sensory evaluation. Both engineered variants demonstrated superior astringency reduction in model and red wines. Our results establish that an increased mannose-to-glucose ratio and reduced N-glycosylation significantly enhance the astringency-mitigating effect, providing a foundation for the industrial-scale production and application of mannoprotein additives.},
}

*Saccharomyces cerevisiae/metabolism/genetics
*Wine/analysis/microbiology
*Mannans/metabolism/chemistry
*Membrane Glycoproteins/metabolism/genetics/chemistry
CRISPR-Cas Systems
Taste
Humans
*Saccharomyces cerevisiae Proteins/genetics/metabolism
Mannose/metabolism/analysis

@article {pmid41346237,
year = {2025},
author = {Pandit, B and Hanson, E and Dagci, H and Yang, Q and Yigit, MV and Royzen, M},
title = {Effects of N[6]-Methyladenosine (m[6]A) and 5-Methylcytosine (m[5]C) Modifications in the Guide Region of CRISPR RNA on Cas12a Nuclease Activity.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {12},
pages = {2551-2556},
doi = {10.1021/acs.bioconjchem.5c00498},
pmid = {41346237},
issn = {1520-4812},
mesh = {*5-Methylcytosine/chemistry/metabolism ; *Adenosine/analogs & derivatives/chemistry/metabolism ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism ; },
abstract = {CRISPR-Cas12a is a versatile biosensing platform that detects sequence-specific DNA or RNA targets via a CRISPR RNA (crRNA) guide. While Cas12a's specificity is dictated by its crRNA, chemical modifications within the crRNA can influence nuclease performance. Here, we examined the effects of two well-known RNA modifications, N[6]-methyladenosine (m[6]A) and 5-methylcytosine (m[5]C), introduced into the different positions of the guide region of a crRNA. Melting temperature (Tm) analysis showed that m[6]A had a minimal impact on RNA-DNA duplex stability. In contrast, the incorporation of m[5]C residues stabilized the duplex. Using a fluorescence recovery assay, we found that both modifications preserved Cas12a's nuclease activity, indicating that small thermodynamic shifts in duplex formation are insufficient to disrupt its catalytic function. Despite the greater Tm increase with m[5]C, m[6]A incorporation led to a faster fluorescence recovery rate than that with m[5]C.},
}

*5-Methylcytosine/chemistry/metabolism
*Adenosine/analogs & derivatives/chemistry/metabolism
*RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/chemistry
*CRISPR-Cas Systems
*Endodeoxyribonucleases/metabolism
*Bacterial Proteins/metabolism

@article {pmid41266327,
year = {2025},
author = {Corda, L and Volpe, E and Dallali, H and Di Tommaso, E and Colantoni, A and Guarracino, A and Chittoor, SS and Capulli, M and Tassone, E and Giunta, S},
title = {Cell line-matched reference enables high-precision functional genomics.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11194},
pmid = {41266327},
issn = {2041-1723},
support = {#101078838//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; #25189//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; },
mesh = {Humans ; *Genomics/methods/standards ; *Genome, Human/genetics ; Cell Line ; Haplotypes ; Centromere/genetics ; Gene Editing ; CRISPR-Cas Systems ; Kinetochores/metabolism ; },
abstract = {Comparative analyses of newly available human genome assemblies highlight extensive variation that peaks at centromeres. Reliance on a single generic reference genome can thus hinder whole-genome analysis of sequencing data derived from laboratory cell lines and limit their accurate genomic manipulation. Here, we demonstrate that using an "isogenomic" diploid reference genome - specific for the experimental cell line - substantially improves the accuracy of genomic, epigenomic, transcriptomic analyses and genome editing compared to a non-matched reference. Using our recently generated reference genome of the widely used diploid human cell line RPE-1, we uncover haplotype-specific genetic and epigenetic divergence across all centromeres. Mapping quality of RPE-1 data - DNA- and RNA-seq reads, improves both genome-wide and at highly divergent loci when using the matched RPE1v1.1 reference, resolving haplotype-specific enrichment. For genome engineering experiments, centromeric CRISPR guide RNA efficiency and chromosome specificity are best achieved using the RPE-1 reference. Leveraging high-confidence CUT&RUN read mapping using the matched reference, we define the site of the human kinetochore and identify a wide variation in the position, size and structural organization between haplotypes and chromosomes. This work establishes matched-reference genomics as a powerful framework for high-precision cell biology, calling for the systematic assembly of experimentally relevant cell line genomes.},
}

Humans
*Genomics/methods/standards
*Genome, Human/genetics
Cell Line
Haplotypes
Centromere/genetics
Gene Editing
CRISPR-Cas Systems
Kinetochores/metabolism

@article {pmid41250994,
year = {2025},
author = {Bereiter, R and Manjunath, A and Beal, PA},
title = {Clickable RNA via 4'-C-Ethynyl Cytidine─A Novel Design for Metabolically Stable Guide RNAs in RNA Editing.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {12},
pages = {2637-2644},
doi = {10.1021/acs.bioconjchem.5c00480},
pmid = {41250994},
issn = {1520-4812},
mesh = {*Cytidine/chemistry/analogs & derivatives ; Click Chemistry ; Humans ; *RNA Editing ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; Alkynes/chemistry ; Cycloaddition Reaction ; Triazoles/chemistry ; },
abstract = {Chemical modifications in RNA therapeutics have addressed major challenges by enhancing metabolic stability, cellular uptake, and biological activity─regardless of their mechanism of action. Here, we report on the synthesis of 4'-C-ethynyl cytidine (4'-C-EthC) and its 2'-O-methylated derivative (4'-C-EthC-2'-OMe) as phosphoramidite building blocks and their subsequent incorporation into oligonucleotides. These ribose C4-terminal alkyne modifications provide a click handle directly within oligonucleotides. The novel modification is accessible via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and serves as a universal 4'-C-ribose modifier on the oligonucleotide level. We identified both aromatic and aliphatic triazole residues that increase the thermodynamic stability in A-form RNA duplexes. Furthermore, 4'-C-triazole-modified oligonucleotides exhibit high resistance to nuclease-mediated degradation in metabolic stability assays. Finally, we introduced the novel modification and its substituted triazoles into guide RNAs (gRNAs) for site-directed A-to-I editing in mammalian cells and compared their performance with phosphorothioate-modified gRNAs.},
}

*Cytidine/chemistry/analogs & derivatives
Click Chemistry
Humans
*RNA Editing
*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism
Alkynes/chemistry
Cycloaddition Reaction
Triazoles/chemistry

@article {pmid41391580,
year = {2025},
author = {Zarei, S and Hosseiniara, SM and Zijoud, SSH and Hosseiniara, R},
title = {Electrochemical MicroRNA biosensors for kidney Cancer: From biomarker discovery to point-of-care diagnostics.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {120786},
doi = {10.1016/j.cca.2025.120786},
pmid = {41391580},
issn = {1873-3492},
abstract = {Kidney cancer, particularly clear cell renal cell carcinoma (ccRCC), presents a significant clinical burden due to late-stage detection and limited effectiveness of current diagnostic modalities. Minimally invasive strategies, such as liquid biopsy, have emerged as promising alternatives, with microRNAs (miRNAs) gaining attention as stable, disease-specific biomarkers detectable in biofluids. miRNAs function as oncogenes or tumor suppressors, offering advantages over conventional protein biomarkers in early cancer detection and prognostic assessment. Electrochemical biosensors provide a highly sensitive, rapid, and cost-effective platform for miRNA detection, enabling potential point-of-care applications. Recent advances include the integration of nanomaterials, enzymatic and isothermal amplification methods, and CRISPR-Cas systems to enhance specificity and signal sensitivity. Prototype sensors targeting RCC-relevant miRNAs, multiplexed detection for biomarker panels, and smartphone-compatible platforms demonstrate the feasibility of translating these technologies into clinical practice. Despite challenges in assay standardization, pre-analytical variability, and regulatory pathways, electrochemical miRNA biosensors hold transformative potential for non-invasive RCC diagnostics, treatment monitoring, and precision oncology. Continued innovation and clinical validation may establish these platforms as integral tools for personalized patient management.},
}

@article {pmid41390996,
year = {2026},
author = {Wang, AJ and Du, C and Liu, H and Wang, HL},
title = {Effect of the sdc4 gene knockdown on muscle development in zebrafish.},
journal = {Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology},
volume = {281},
number = {},
pages = {111171},
doi = {10.1016/j.cbpb.2025.111171},
pmid = {41390996},
issn = {1879-1107},
mesh = {Animals ; *Zebrafish/genetics/growth & development/metabolism ; *Syndecan-4/genetics/metabolism ; *Muscle Development/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; MicroRNAs/genetics/metabolism ; *Muscle, Skeletal/metabolism/growth & development ; Wnt Signaling Pathway ; CRISPR-Cas Systems ; },
abstract = {The growth and regeneration of skeletal muscle are closely related to syndecan-4 (Sdc4), which is a type I transmembrane heparan sulfate proteoglycan belonging to the syndecan family. However, it remains unclear how the sdc4 gene affects fish muscle development. Therefore, an sdc4 knockdown zebrafish line (sdc4[-/-]) was generated by CRISPR/Cas9 technology in this study, and its phenotypes were analyzed. The results revealed that sdc4[-/-] zebrafish exhibited reduced body length and weight compared to the wild-type (WT) at 90 days post fertilization (dpf). Furthemore, sdc4[-/-] zebrafish also showed a significantly larger number of muscle fibers, and significantly reduced individual muscle fiber cross-sectional area. The mRNA expression levels of genes associated with myogenic regulatory factors (MRFs) and the wnt/β-catenin pathway were all significantly downregulated. Based on dual luciferase reporter assays, sdc4 gene expression was regulated by the transcription factor myocyte enhancer factor 2aa (Mef2aa) and miR-141-3p, which bind to its promoter and 3' untranslated region (UTR), respectively. Additionally, the reduced average swimming speed and distance observed in sdc4[-/-] zebrafish at 90 dpf were concomitant with a significant downregulation of mitochondrial respiratory chain complex-related genes and a reduction in ATP concentration. This research aids understanding of sdc4 function in fish and may provide a new perspective for studying the molecular mechanisms of muscle growth and development.},
}

Animals
*Zebrafish/genetics/growth & development/metabolism
*Syndecan-4/genetics/metabolism
*Muscle Development/genetics
*Zebrafish Proteins/genetics/metabolism
*Gene Expression Regulation, Developmental
Gene Knockdown Techniques
MicroRNAs/genetics/metabolism
*Muscle, Skeletal/metabolism/growth & development
Wnt Signaling Pathway
CRISPR-Cas Systems

@article {pmid41389662,
year = {2025},
author = {Ma, J and Zhao, CF and Liu, X},
title = {Advances in targeted therapeutics and smart delivery systems based on precision nano-oncology.},
journal = {International immunopharmacology},
volume = {169},
number = {},
pages = {115946},
doi = {10.1016/j.intimp.2025.115946},
pmid = {41389662},
issn = {1878-1705},
abstract = {The convergence of nanotechnology and precision oncology is revolutionizing cancer treatment by enabling highly specific, minimally invasive, and personalized therapeutic strategies. This review explores recent breakthroughs in nano-therapeutics and their pivotal role in overcoming the limitations of conventional cancer therapies. Emphasis is placed on the design and function of nanocarriers that facilitate targeted drug delivery via both passive (EPR effect) and active ligand-mediated mechanisms. Special attention is given to stimuli-responsive systems that release therapeutic agents in response to pH, enzymes, temperature, or redox environments, enhancing spatiotemporal control. The article further discusses the integration of nanotechnology with emerging modalities including immunotherapy, photothermal and photodynamic therapies, gene editing tools (e.g., CRISPR/Cas systems), and multifunctional theranostic platforms. While these innovations offer transformative potential, the review also addresses persistent challenges such as tumor heterogeneity, immune clearance, off-target effects, large-scale manufacturing, and regulatory complexity. By highlighting both promise and hurdles, this article provides a comprehensive lens into the future of precision cancer nanomedicine.},
}

@article {pmid41387770,
year = {2025},
author = {Ge, L and Li, W and Dou, Y and Ma, Y and Sun, M and Chen, X and Feng, X and Li, Y and Yu, Q},
title = {Callus and endosperm green fluorescence reporter-assisted selection system in maize CRISPR/Cas9 gene editing.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-025-07429-2},
pmid = {41387770},
issn = {1471-2229},
support = {2022SZX13//Science&Technology Specific Projects in Agricultural High-tech Industrial Demonstration Area of the Yellow River Delta/ ; ZR2024MC067//Natural Science Foundation of Shandong Province/ ; },
abstract = {BACKGROUND: Genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has emerged as a promising approach for functional gene analysis and genetic improvement. Since stable transformation remains the primary method for implementing this system, the ultimate goal in crop breeding programs would require the selection of transgene-free plants with the CRISPR/Cas expression cassette removed.

RESULTS: In this study, we developed an endosperm-specific fluorescence reporter-assisted selection system for CRISPR/Cas9 gene editing (pAZS22-eGFP/CRISPR/Cas9) in maize (Zea mays L.), utilizing enhanced green fluorescent protein (eGFP) expressed specifically in the endosperm to facilitate the easy identification of transgenic and transgene-free plants from the T1 generation on. In addition, the 22 kDa alpha zein (z1C1_10) promoter from maize, employed in this system, has been shown to be active in both callus and endosperm, thereby being able to enhance the accuracy of transformant identification during the tissue culture process by reducing false positives compared to the traditional selective media methods. Our studies targeting the ZmSnRK2.1 or Dwarf1 (D1) genes demonstrated a reasonable editing efficiency, with rates ranging from 56.3% for T0 plants targeting ZmSnRK2.1, to 87.5% and 100% for T1 plants targeting D1 and ZmSnRK2.1, respectively. In addition, we successfully identified 1 transgene-free homozygous d1 mutant in the T1 generation and 7 transgene-free homozygous snrk2.1 mutants in the T2 generation.

CONCLUSIONS: The pAZS22-eGFP/CRISPR/Cas9 system provides an efficient tool for gene editing, transformant selection and transgene status identification in maize breeding.},
}

@article {pmid41385994,
year = {2025},
author = {Luo, Y and Wang, X and Yang, F and Zhao, Y and Hu, S and Liu, S and Li, S and Luo, G and Sun, Q},
title = {Construction and validation of a rapid semen identification system based on SHERLOCK technology.},
journal = {Forensic science international. Genetics},
volume = {82},
number = {},
pages = {103410},
doi = {10.1016/j.fsigen.2025.103410},
pmid = {41385994},
issn = {1878-0326},
abstract = {This study developed a rapid detection system for semen-specific mRNA based on CRISPR/Cas13a system to meet the timeliness requirements of forensic on-site body fluid identification. Specific primers and CRISPR RNA (crRNA) short fragments on semen specific mRNA genes were designed and screened, to establish a SHERLOCK detection method based on technology principles of CRISPR/Cas. Furthermore, nucleic acid rapid release agents for treating samples were screend to construct a new detection method in combination with SHERLOCK, and the specificity and sensitivity of the method were tested. The method can rapidly detect the presence of semen from unknown body fluid samples, and the relative fluorescence unit (RFU) value of the semen sample is significantly higher than those of non-semen samples (P < 0.0001), with a sample detection sensitivity of down to 0.25 μL. The construction of the rapid semen detection method using rapid extraction and SHERLOCK reduces operation time, significantly reduces instrument dependence, and provides an innovative solution for forensic on-site rapid body fluid identification.},
}

@article {pmid41284810,
year = {2025},
author = {Park, S and Kim, MM},
title = {Mutation of the Collagen Type 1α Gene in Human Fibrosarcoma Cells Using the CRISPR/Cas9 System.},
journal = {Biochemistry},
volume = {64},
number = {24},
pages = {4794-4804},
doi = {10.1021/acs.biochem.5c00394},
pmid = {41284810},
issn = {1520-4995},
mesh = {Humans ; Collagen Type I, alpha 1 Chain ; *Fibrosarcoma/genetics/metabolism/pathology ; *Collagen Type I/genetics/metabolism ; *CRISPR-Cas Systems ; *Mutation ; Cell Line, Tumor ; Signal Transduction ; Gene Editing ; },
abstract = {Collagen in the connective tissue plays a key role in the expression the aging phenotypes. While collagen production decreases with aging, collagenase expression increases, resulting in collagen breakdown. The purpose of this study is to investigate the change in the expression of proteins and genes related to the collagen signaling pathway, cell cycle, and aging phenotypes of cells with the collagen type 1 α (COL1A1) gene edited by the CRISPR/Cas9 system. The mutation of the COL1A1 gene was induced by the CRISPR/Cas9 system. Sanger DNA sequencing and Indel analyses, Sanger DNA sequencing analysis and Swiss protein modeling analysis were used to verify the induction of mutation. Aging phenotypes in the mutated cells were evaluated by collagen staining assay, SA-β-galactosidase staining assay, RT-PCR assay, Western blot analysis, gelatin zymography, and immunofluorescent staining assay. Sanger DNA sequencing analysis demonstrated that human fibrosarcoma cells with COL1A1 gene mutations were successfully established in this study. Swiss protein modeling analysis displayed the altered structure of COL1A1 in the edited cells. In addition, while collagen production was decreased, the SA-β-galactosidase staining level was increased in the edited cells. It was also found that the expression levels of CDC2, CDk2, and cyclin D were increased by down-regulating p53 and p21 levels through the increased expression of MDM2 in the edited cells. Moreover, the expression levels of MMP-1, MMP-2, MMP-9, AKT, and p-mTOR were reduced in the edited cells. These findings could provide a crucial clue in elucidating the close relationship between collagen production and senescence.},
}

Humans
Collagen Type I, alpha 1 Chain
*Fibrosarcoma/genetics/metabolism/pathology
*Collagen Type I/genetics/metabolism
*CRISPR-Cas Systems
*Mutation
Cell Line, Tumor
Signal Transduction
Gene Editing

@article {pmid41284414,
year = {2025},
author = {Iyer, KA and Tenchov, R and Lotti Diaz, LM and Jain, P and Thite, T and Deng, Y and Zhou, QA},
title = {CRISPR Technology: Transforming the Future of Medicine and Diagnostics.},
journal = {Biochemistry},
volume = {64},
number = {24},
pages = {4628-4660},
doi = {10.1021/acs.biochem.5c00480},
pmid = {41284414},
issn = {1520-4995},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Genetic Therapy/methods/trends ; Neoplasms/therapy/genetics/diagnosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; },
abstract = {In this report, we examine the extensive research landscape of CRISPR with an emphasis on CRISPR therapeutics and showcase our results from an in-depth analysis of the most up-to-date scientific information consisting of more than 53,000 publications encompassing academic journal articles and patents, spanning nearly three decades, extracted from the CAS Content Collection. Our analysis indicates that cancer and infectious diseases are the most explored in the context of CRISPR. Identified gene targets associated with CRISPR-related publications are led by TP53, c-myc, and hemoglobin beta subunit (HBB). Among the many delivery methods, adeno-associated vectors (AAVs) appear to be highly explored. With >140 CRISPR-based therapeutics in the clinical development pipeline and billions of dollars in investment, the field of CRISPR continues to evolve rapidly. We also briefly discuss the ethical implications of CRISPR technology. While some fundamental challenges persist, the future of CRISPR is undoubtedly bright.},
}

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

@article {pmid41264498,
year = {2025},
author = {Guo, Y and Guo, W and Wu, Z and Xu, H and Zhang, X and Zou, X and Sun, Z},
title = {A Microfluidic Chip-Based Electrochemical Biosensor Coupled with CRISPR/Cas12a for Simultaneous Detection of Foodborne Pathogens.},
journal = {Analytical chemistry},
volume = {97},
number = {49},
pages = {27266-27275},
doi = {10.1021/acs.analchem.5c05232},
pmid = {41264498},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/instrumentation ; *Staphylococcus aureus/isolation & purification/genetics ; Gold/chemistry ; *Salmonella/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Food Microbiology ; Metal Nanoparticles/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; *Lab-On-A-Chip Devices ; Limit of Detection ; Food Contamination/analysis ; Pyrroles/chemistry ; *Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases ; Polymers ; },
abstract = {Staphylococcus aureus (S. aureus) and Salmonella frequently cocontaminate food products, posing significant health threats. We developed a microfluidic electrochemical biosensor that enables simultaneous detection of both pathogens within 65 min. The chip integrates sample loading, recombinase polymerase amplification (RPA), and CRISPR/Cas12a-based recognition into the upper layer. Reaction products enter a detection chamber with a single-walled carbon nanohorns-polypyrrole-gold nanoparticles (SWCNHs-ppy-AuNPs)-modified three-electrode system, where ssDNA probes act as signal reporters. Upon target recognition, Cas12a cleaves the probes, releasing electroactive molecules and reducing the current. The sensor exhibits linear responses for S. aureus (1.06 × 10[1]-1.06 × 10[7] CFU/mL) and Salmonella (1.04 × 10[1]-1.04 × 10[7] CFU/mL), with detection limits of 3 CFU/mL. This platform offers a rapid, sensitive, and accurate tool for on-site detection of foodborne pathogens in food products.},
}

*Biosensing Techniques/methods
*Electrochemical Techniques/instrumentation
*Staphylococcus aureus/isolation & purification/genetics
Gold/chemistry
*Salmonella/isolation & purification/genetics
*CRISPR-Cas Systems
*Food Microbiology
Metal Nanoparticles/chemistry
*CRISPR-Associated Proteins/metabolism/genetics
*Lab-On-A-Chip Devices
Limit of Detection
Food Contamination/analysis
Pyrroles/chemistry
*Bacterial Proteins/genetics/metabolism
Endodeoxyribonucleases
Polymers

@article {pmid41218606,
year = {2025},
author = {Shevade, K and Yang, YA and Feng, K and Mader, K and Sevim, V and Parsons, J and Arora, G and Elfawy, H and Mace, R and Federman, S and Esanov, R and Shafer, S and Chow, ED and Przybyla, L},
title = {Simultaneous capture of single cell RNA-seq, ATAC-seq, and CRISPR perturbation enables multiomic screens to identify gene regulatory relationships.},
journal = {Cell reports methods},
volume = {5},
number = {12},
pages = {101222},
doi = {10.1016/j.crmeth.2025.101222},
pmid = {41218606},
issn = {2667-2375},
mesh = {Humans ; *Single-Cell Analysis/methods ; *Gene Regulatory Networks/genetics ; *RNA-Seq/methods ; Induced Pluripotent Stem Cells/metabolism ; *Chromatin Immunoprecipitation Sequencing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Dasatinib/pharmacology ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chromatin/metabolism/genetics ; Transposases/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Here, we introduce CRISPR and transcriptomics-assay for transposase-accessible chromatin (CAT-ATAC), a technique that adds CRISPR guide RNA (gRNA) capture to the existing 10× Genomics Multiome assay, generating linked transcriptome, chromatin accessibility, and perturbation identity data from the same individual cells. We demonstrate up to 77% capture rate for both arrayed and pooled delivery of lentiviral gRNAs in induced pluripotent stem cells (iPSCs) and cancer cell lines. This capability allows us to construct gene regulatory networks (GRNs) in cells under drug and genetic perturbations. By applying CAT-ATAC, we identified a GRN associated with dasatinib resistance, indirectly activated by the HIC2 gene. Using loss-of-function experiments, we further validated that ZFPM2, a component of the predicted GRN, also contributes to dasatinib resistance. CAT-ATAC can thus be used to generate high-content multidimensional genotype-phenotype maps to reveal gene and cellular interactions and functions.},
}

Humans
*Single-Cell Analysis/methods
*Gene Regulatory Networks/genetics
*RNA-Seq/methods
Induced Pluripotent Stem Cells/metabolism
*Chromatin Immunoprecipitation Sequencing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Dasatinib/pharmacology
*CRISPR-Cas Systems/genetics
Cell Line, Tumor
Chromatin/metabolism/genetics
Transposases/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41202713,
year = {2026},
author = {Wang, Y and Xiang, L and Su, Z},
title = {CRISPR/Cas9 gene editing in muscle-related genetic disorders: Restoring function and exercise capacity.},
journal = {Tissue & cell},
volume = {98},
number = {},
pages = {103217},
doi = {10.1016/j.tice.2025.103217},
pmid = {41202713},
issn = {1532-3072},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Genetic Therapy ; *Muscular Diseases/genetics/therapy ; *Muscular Dystrophies/genetics/therapy ; },
abstract = {Due to their progressive and debilitating character and lack of curative therapies, muscle-related hereditary illnesses such as muscular dystrophies (MDs), myopathies, and motor neuron diseases need immediate care. A pressing need exists for novel therapeutics that can accurately and persistently correct the underlying mutations in muscle-related genetic disorders, as existing treatments are mostly palliative rather than addressing the underlying genetic cause. Traditional therapies for genetic diseases, such as hereditary myopathies and MDs, are often insufficient; however, the advent of CRISPR/Cas9 technology has altered this trajectory. While other evaluations have compiled the potential of gene editing, this one compiles the latest findings on CRISPR/Cas9 applications for musculoskeletal disorders. With an emphasis on the repair of recessive and dominant-negative mutations, we provide a critical evaluation of the development of new therapeutic vectors and in vivo editing techniques that have gone from conceptual models to preclinical triumphs. More specifically, we address the issues of efficient delivery, off-target effects, and immunological responses specific to muscle tissue, all of which are ongoing challenges. By facilitating targeted correction of pathogenic mutations, CRISPR/Cas9 holds promise for restoring muscle integrity and functional performance, potentially translating into improved exercise capacity. This study offers a prospective view on the near-term therapeutic promise of CRISPR/Cas9 for treating crippling muscle diseases by combining recent advances with an evaluation of the translational route.},
}

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

@article {pmid41101973,
year = {2025},
author = {Ishizuka, T and Tsuboyama, K and Tomari, Y},
title = {Comprehensive identification and functional analysis of fully disordered proteins essential for cell survival.},
journal = {RNA (New York, N.Y.)},
volume = {32},
number = {1},
pages = {61-70},
doi = {10.1261/rna.080626.125},
pmid = {41101973},
issn = {1469-9001},
mesh = {*Intrinsically Disordered Proteins/genetics/metabolism/chemistry ; Cell Survival/genetics ; Humans ; Genes, Essential ; Introns ; CRISPR-Cas Systems ; },
abstract = {Proteins have traditionally been understood through their tertiary structures, with well-defined conformations considered essential for biological function. This classical structure-function paradigm implies that proteins with high intrinsic disorder would be less critical for cellular survival. Recent discoveries have suggested that some intrinsically disordered proteins or even fully disordered proteins without any apparent tertiary structures are essential. However, the biological significance of such disordered proteins is not comprehensively understood. Here, using genome-wide CRISPR screening, we demonstrated that highly or fully disordered proteins show comparable essentiality to well-folded proteins. We found that the proportion of essential proteins is comparable across proteins of varying disorder levels, although structured proteins are more prevalent among essential genes. Focusing on FAM32A, one of the essential, fully disordered proteins identified in our screen, we show that its depletion leads to increased intron retention and downregulation of many other essential genes. These findings reshape our understanding of the structure-function paradigm, highlighting that fully disordered proteins can be essential for cellular viability.},
}

*Intrinsically Disordered Proteins/genetics/metabolism/chemistry
Cell Survival/genetics
Humans
Genes, Essential
Introns
CRISPR-Cas Systems

@article {pmid40865518,
year = {2025},
author = {Hong, D and Lyu, Y and Nayak, R and Becker, JS and Booker, MA and Masuzawa, K and Devos, Z and Wang, T and Saito, S and Liu, Q and Li, Y and Li, Z and Knelson, EH and Thai, T and Duplaquet, L and Laimon, YN and Roberti De Oliveira, G and Signoretti, S and Doench, JG and Barbie, DA and Tolystorukov, MY and Qi, J and Bernstein, BE and Ge, Y and Oser, MG},
title = {Loss of NOTCH2 creates a TRIM28-dependent vulnerability in small cell lung cancer.},
journal = {Developmental cell},
volume = {60},
number = {24},
pages = {3462-3479.e13},
doi = {10.1016/j.devcel.2025.07.023},
pmid = {40865518},
issn = {1878-1551},
mesh = {*Receptor, Notch2/metabolism/genetics ; *Tripartite Motif-Containing Protein 28/metabolism/genetics ; *Small Cell Lung Carcinoma/genetics/metabolism/pathology ; Animals ; Humans ; *Lung Neoplasms/metabolism/genetics/pathology ; Mice ; Cell Line, Tumor ; CRISPR-Cas Systems ; Signal Transduction ; },
abstract = {Small cell lung cancer (SCLC) is a highly aggressive malignancy that lacks effective targeted therapies, in part due to frequent loss-of-function mutations in tumor suppressors and the absence of recurrent oncogenic drivers. Approximately 15% of SCLCs harbor inactivating mutations in NOTCH1 or NOTCH2, and most neuroendocrine-high SCLCs exhibit low NOTCH activity. Using CRISPR-Cas9 screening in primary cell lines derived from NOTCH1/2-isogenic SCLC genetically engineered mouse models, we identified TRIM28 as a synthetic lethal dependency in NOTCH2-inactivated SCLCs. Loss of TRIM28 in this context robustly induced expression of endogenous retroviruses (ERVs), activated viral sensing pathways, and triggered a type I interferon response. Mechanistically, NOTCH2 inactivation increased reliance on TRIM28-mediated ERV silencing, creating a hyperdependence on TRIM28 via the STING-MAVS-TBK1 axis. Notably, TRIM28 was essential for tumor growth only in the setting of NOTCH2 loss. These findings identify TRIM28 as a potential therapeutic target in NOTCH2-deficient or low-NOTCH2-expressing SCLC.},
}

*Receptor, Notch2/metabolism/genetics
*Tripartite Motif-Containing Protein 28/metabolism/genetics
*Small Cell Lung Carcinoma/genetics/metabolism/pathology
Animals
Humans
*Lung Neoplasms/metabolism/genetics/pathology
Mice
Cell Line, Tumor
CRISPR-Cas Systems
Signal Transduction

@article {pmid40681864,
year = {2025},
author = {Ronda, C and Perdue, T and Schwanz, L and Rivera Gelsinger, D and Brockmann, L and Kaufman, A and Huang, Y and Sternberg, SH and Wang, HH},
title = {Precise virulence inactivation using a CRISPR-associated transposase for combating Enterobacteriaceae gut pathogens.},
journal = {Nature biomedical engineering},
volume = {9},
number = {12},
pages = {2017-2027},
pmid = {40681864},
issn = {2157-846X},
support = {1R01EB031935//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 2R01AI132403//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 1R01DK118044//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 1R21AI146817//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DP2HG011650//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R21AI68976//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01EB031935//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; S-168-4X5-001//U.S. Department of Defense (United States Department of Defense)/ ; 1016691//Burroughs Wellcome Fund (BWF)/ ; 527896//Simons Foundation/ ; },
mesh = {Animals ; *Transposases/genetics/metabolism ; Mice ; *Enterobacteriaceae/pathogenicity/genetics ; Virulence/genetics ; *CRISPR-Cas Systems/genetics ; *Gastrointestinal Microbiome/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Shiga Toxin/genetics ; *Enterobacteriaceae Infections/microbiology ; Humans ; },
abstract = {Targeted gene manipulation in a complex microbial community is an enabling technology for precise microbiome editing. Here we introduce BACTRINS, an in situ microbiome engineering platform designed for efficient and precise genomic insertion of a desired payload and simultaneous knockout of target genes. This system leverages conjugation-mediated delivery of CRISPR-associated transposases to achieve RNA-guided genomic integration, allowing precise insertion of a therapeutic payload while neutralizing pathogen virulence without causing cell death. When applied against an Enterobacteriaceae Shiga toxin-producing pathogen in the gut, this system delivers a CRISPR-associated transposase by bacterial conjugation for site-specific inactivation of the Shiga toxin gene and integration of a nanobody therapeutic payload to disrupt pathogen attachment. A single dose of this therapy results in high-efficiency Shiga gene inactivation and improved survival in a murine infection model of Shiga-producing pathogen. This work establishes a new type of live bacterial therapeutic capable of reducing gut infections by transforming toxigenic pathogens into commensal protectors.},
}

Animals
*Transposases/genetics/metabolism
Mice
*Enterobacteriaceae/pathogenicity/genetics
Virulence/genetics
*CRISPR-Cas Systems/genetics
*Gastrointestinal Microbiome/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Editing/methods
Shiga Toxin/genetics
*Enterobacteriaceae Infections/microbiology
Humans

@article {pmid41385544,
year = {2025},
author = {Fu, YZ and Luo, FF and Yang, L and Zhang, YX and Li, JY and Wang, SY and Zhang, Y and Wang, YY},
title = {SPNS1 is an essential cellular factor for EV-A71 by acting as a transporter of viral pocket factor.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {50},
pages = {e2510020122},
doi = {10.1073/pnas.2510020122},
pmid = {41385544},
issn = {1091-6490},
support = {2023YFC2306100//The National Key R&D Program China/ ; 82372230//The National Natural Science Foundation of China/ ; U23A20168//The National Natural Science Foundation of China/ ; No.2022338//The Youth Innovation Promotion Association CAS/ ; },
mesh = {Humans ; Animals ; *Enterovirus A, Human/physiology/metabolism/genetics ; Virus Replication ; *Enterovirus Infections/virology/metabolism/genetics ; Lysosomes/metabolism ; Mice ; Capsid Proteins/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; Endosomes/metabolism ; Receptors, Scavenger ; Lysosomal Membrane Proteins ; },
abstract = {Human enterovirus A71 (EV-A71) is a major cause of hand, foot and mouth disease. Cellular factors critical for EV-A71 infection remain enigmatic. Here, we performed CRISPR/Cas9 screens and identified sphingolipid transporter 1 (SPNS1) as an essential factor for EV-A71. SPNS1 deficiency inhibits infection of EV-A71 and 9 of 11 examined enteroviruses. Mechanistically, the endo/lysosomal localization of SPNS1 and the acidification of the endo/lysosomes are essential for SPNS1 to support EV-A71 infection. SPNS1 deficiency inhibits EV-A71 genomic RNA replication, but barely affects replication of EV-A71 RNA directly transfected into the cytoplasm. SPNS1 interacts with the EV-A71 capsid protein VP1 and entry receptor SCARB2 in the endo/lysosomes, where it acts as a transporter to release the viral pocket factor into the cytosol, leading to uncoating. Animal experiments show that SPNS1 deficiency results in reduced viral loads, pathological effects, and lethality following EV-A71 infection. Our findings collectively identified SPNS1 as a transporter of the EV-A71 viral pocket factor.},
}

Humans
Animals
*Enterovirus A, Human/physiology/metabolism/genetics
Virus Replication
*Enterovirus Infections/virology/metabolism/genetics
Lysosomes/metabolism
Mice
Capsid Proteins/metabolism
CRISPR-Cas Systems
HEK293 Cells
Endosomes/metabolism
Receptors, Scavenger
Lysosomal Membrane Proteins

@article {pmid41385323,
year = {2025},
author = {Zahm, AM and Cranney, CW and Gormick, AN and Rondem, KE and Schmitz, B and Himes, SR and English, JG},
title = {ConSeqUMI, an error-free nanopore sequencing pipeline to identify and extract individual nucleic acid molecules from heterogeneous samples.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41385323},
issn = {1362-4962},
support = {1DP2GM146247-01/GM/NIGMS NIH HHS/United States ; //NIH/ ; },
mesh = {*Nanopore Sequencing/methods ; Humans ; SARS-CoV-2/genetics ; Spike Glycoprotein, Coronavirus/genetics ; COVID-19/virology ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems ; *Sequence Analysis, DNA/methods ; Dependovirus/genetics ; *Nucleic Acids/genetics/isolation & purification ; Genome, Viral ; },
abstract = {Nanopore sequencing has revolutionized genetic analysis by offering linkage information across megabase-scale genomes. However, the high intrinsic error rate of nanopore sequencing impedes the analysis of complex heterogeneous samples, such as viruses, bacteria, complex libraries, and edited cell lines. Achieving high accuracy in single-molecule sequence identification would significantly advance the study of diverse genomic populations, where clonal isolation is traditionally employed for complete genomic frequency analysis. Here, we introduce ConSeqUMI, an innovative experimental and analytical pipeline designed to address long-read sequencing error rates using unique molecular indices for precise consensus sequence determination. ConSeqUMI processes nanopore sequencing data without the need for reference sequences, enabling accurate assembly of individual molecular sequences from complex mixtures. We establish robust benchmarking criteria for this platform's performance and demonstrate its utility across diverse experimental contexts, including mixed plasmid pools, recombinant adeno-associated virus genome integrity, and CRISPR/Cas9-induced genomic alterations. Furthermore, ConSeqUMI enables detailed profiling of human pathogenic infections, as shown by our analysis of severe acute respiratory syndrome coronavirus 2 spike protein variants, revealing substantial intra-patient genetic heterogeneity. Lastly, we demonstrate how individual clonal isolates can be extracted directly from sequencing libraries at low cost, allowing for post-sequencing identification and validation of observed variants. Our findings highlight the robustness of ConSeqUMI in processing sequencing data from UMI-labeled molecules, offering a critical tool for advancing genomic research.},
}

*Nanopore Sequencing/methods
Humans
SARS-CoV-2/genetics
Spike Glycoprotein, Coronavirus/genetics
COVID-19/virology
High-Throughput Nucleotide Sequencing/methods
CRISPR-Cas Systems
*Sequence Analysis, DNA/methods
Dependovirus/genetics
*Nucleic Acids/genetics/isolation & purification
Genome, Viral

@article {pmid41384994,
year = {2025},
author = {Zhang, J and Liu, J and Bayani, A},
title = {Phage therapy and the microbiome in hematologic malignancies: opportunities, mechanisms, and early evidence.},
journal = {Journal of cancer research and clinical oncology},
volume = {152},
number = {1},
pages = {8},
pmid = {41384994},
issn = {1432-1335},
mesh = {Humans ; *Hematologic Neoplasms/therapy/microbiology/immunology ; *Phage Therapy/methods ; Animals ; *Bacteriophages ; *Gastrointestinal Microbiome ; *Microbiota ; },
abstract = {Hematologic malignancies remain among the most difficult cancers to treat, challenged by profound heterogeneity, treatment-induced immune dysfunction, and the frequent emergence of drug resistance. Beyond tumor-intrinsic mechanisms, dysbiosis of the gut microbiome is increasingly recognized as a critical determinant of therapeutic outcomes, shaping hematopoiesis, immune responses, and drug metabolism. Bacteriophage (phage) therapy has re-emerged as a precision tool capable of selectively eradicating pathogenic taxa while preserving commensal short-chain fatty acid-producing communities. Preclinical and early human studies demonstrate that phages can recalibrate microbial ecosystems, disrupt antibiotic-tolerant biofilms, and enrich metabolites such as butyrate that support mucosal integrity and immune balance. Mechanistically, phage DNA enriched with CpG motifs engages Toll-like receptor 9, activating dendritic cells and enhancing cytotoxic T lymphocyte responses, suggesting dual benefits in infection control and anti-tumor immunity. Emerging applications extend further, with engineered phages serving as vectors for CRISPR-Cas gene editing, targeted cytokine delivery, and nanocarrier platforms for leukemia therapy. Despite translational promise, major hurdles persist, including immunogenicity, horizontal gene transfer, resistance evolution, and regulatory uncertainty. Addressing these challenges through GMP-compliant manufacturing, metagenomics-guided personalization, and AI-optimized cocktail design could establish phage therapy as a microbiome-informed adjunct to overcome drug resistance in blood cancers. However, direct clinical evidence of phage therapy efficacy in hematologic malignancies remains limited, and current data are largely derived from preclinical and compassionate-use contexts.},
}

Humans
*Hematologic Neoplasms/therapy/microbiology/immunology
*Phage Therapy/methods
Animals
*Bacteriophages
*Gastrointestinal Microbiome
*Microbiota

@article {pmid41381927,
year = {2025},
author = {Yousuf, F and Solanki, M and Singh, SS and Ch, SR and Neeraja, CN and Sundaram, RM and Mangrauthia, SK},
title = {Tissue culture optimization and genome editing for yield improvement of an Indian rice landrace Chittimuthyalu.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {54},
pmid = {41381927},
issn = {1573-9368},
support = {ICAR-EFC Sub-Scheme 10: Enhancing climate resilience and ensuring food security with genome editing tools//Indian Council of Agricultural Research/ ; },
mesh = {*Oryza/genetics/growth & development/drug effects ; *Gene Editing/methods ; *Tissue Culture Techniques/methods ; Plant Breeding ; India ; *Plants, Genetically Modified/genetics/growth & development ; Purines/pharmacology ; CRISPR-Cas Systems ; Phenylurea Compounds ; Thiadiazoles ; },
abstract = {Chittimuthyalu, a rice landrace from Southern India, is known for its pleasant aroma, rich nutritive value, and excellent cooking qualities. However, it has a poor plant type (tall and weak stem prone to lodging) and is low yielding. The efforts to improve such valuable rice accessions with existing cross-breeding or random mutagenesis often result in undesirable traits due to linkage drag or untargeted mutations in large numbers. Genome editing, the most precise breeding tool, offers a viable solution to address such issues. In this study, we developed an efficient tissue culture protocol for callus induction, transformation, and regeneration of Chittimuthyalu. The highest callus induction frequency was achieved on L3 basal media enriched with 2.5 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 600 mg/l of both proline and glutamine. For regeneration, a combination of Thidiazuron (TDZ), 6-Benzylaminopurine (BAP), and kinetin yielded an optimal regeneration frequency. The optimized tissue culture protocol was utilized to transform a multiplex gene editing construct developed by combining the four guide RNAs designed from yield and disease resistance-associated genes OsDEP1, OsTB1, OsCKX2, and OsSWEET14. The OsDEP1genome-edited rice plants exhibit thicker culm, enhanced grain size, ~ 100% increase in the thousand-grain weight, and ~ 50% increase in total grain yield per plant. The optimized tissue culture protocol and development of further edits in the remaining genes will pave the way for improving the agronomic traits of Chittimuthyalu. This study also highlights much-needed efforts to develop efficient tissue culture and genome editing methods for wild rice species and landraces, which will help bring these hardy, climate-resilient, and nutrient-rich accessions into mainstream cultivation.},
}

*Oryza/genetics/growth & development/drug effects
*Gene Editing/methods
*Tissue Culture Techniques/methods
Plant Breeding
India
*Plants, Genetically Modified/genetics/growth & development
Purines/pharmacology
CRISPR-Cas Systems
Phenylurea Compounds
Thiadiazoles

@article {pmid41381092,
year = {2025},
author = {Lane, KR and Jones, SE and Osborne, TH and Geller-McGrath, D and Nwaobi, BC and Chen, L and Thomas, BC and Hudson-Edwards, KA and Banfield, JF and Santini, JM},
title = {Bioleaching Microbial Community Metabolism and Composition Driven by Copper Sulphide Mineral Type.},
journal = {Environmental microbiology reports},
volume = {17},
number = {6},
pages = {e70261},
pmid = {41381092},
issn = {1758-2229},
support = {NE/L002485/1//Natural Environment Research Council/ ; BB/N012674/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //Hellenic Coppers Mines Ltd/ ; },
mesh = {*Copper/metabolism/chemistry ; *Bacteria/metabolism/genetics/classification/isolation & purification ; *Archaea/metabolism/genetics/classification/isolation & purification ; *Sulfides/metabolism/chemistry ; *Microbial Consortia ; Metagenomics ; *Minerals/metabolism/chemistry ; *Microbiota ; Plasmids/genetics ; },
abstract = {Copper bioleaching is a green technology for the recovery of copper from chalcopyrite (CuFeS2) and chalcocite (Cu2S) ores. Much remains to be learned about how mineral type and surface chemistry influence microbial community composition. Here, we established a microbial consortium from a copper bioleaching column in Cyprus on chalcopyrite and then sub-cultured it to chalcocite to investigate how the community composition shifts due to changes in mineral structure and the absence of mineral-derived Fe. The solution chemistry was determined and microbial communities characterised by genome-resolved metagenomics after 4 and 8 weeks of cultivation. Acidithiobacillus species and strains, a Rhodospirilales, Leptospirillum ferrodiazotrophum and Thermoplasmatales archaea dominated all enrichments, and trends in abundance patterns were observed with mineralogy and surface-attached versus planktonic conditions. Many bacteria had associated plasmids, some of which encoded metal resistance pathways, sulphur metabolic capacities and CRISPR-Cas loci. CRISPR spacers on an Acidithiobacillus plasmid targeted plasmid-borne conjugal transfer genes found in the same genus, likely belonging to another plasmid, evidence of intra-plasmid competition. We conclude that the structure and composition of metal sulphide minerals select for distinct consortia and associated mobile elements, some of which have the potential to impact microbial activity during sulphide ore dissolution.},
}

*Copper/metabolism/chemistry
*Bacteria/metabolism/genetics/classification/isolation & purification
*Archaea/metabolism/genetics/classification/isolation & purification
*Sulfides/metabolism/chemistry
*Microbial Consortia
Metagenomics
*Minerals/metabolism/chemistry
*Microbiota
Plasmids/genetics

@article {pmid41380995,
year = {2025},
author = {Roy, S and Nandy, S and Morita, D and Nandy, RK and Veeraraghavan, B and Walia, K and Das, S and Basu, S},
title = {Genomic analysis of a novel high-risk ST5217/ExoU+/O11 clone of carbapenem-resistant OXA-181- and VIM-2-producing Pseudomonas aeruginosa in India.},
journal = {Journal of global antimicrobial resistance},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgar.2025.12.002},
pmid = {41380995},
issn = {2213-7173},
abstract = {OBJECTIVES: Studies of carbapenem-resistant Pseudomonas aeruginosa (CRPA)-harbouring OXA-48-like carbapenemases are rare. The study aimed to report the emergence and characterization of a novel high-risk clone of CRPA-harbouring OXA-48-like from India.

METHODS: Identification, AST, phenotypic detection of carbapenemases and WGS using Ion-Torrent-S5 platform were carried out. Analyses included ResFinder, VFDB, MLST, PAst, Phastest and CRISPR/Cas. SNP-based phylogenetic analysis with global OXA-48-like-harbouring CRPA genomes was carried out by CSI Phylogeny and iTOL for visualization.

RESULTS: The clinical strain of CRPA AMRIR00655 belonged to a novel sequence type ST5217 and serotype O11. The strain was MDR. Phenotypic tests followed by WGS revealed the presence of dual carbapenemases, OXA-181 (serine-carbapenemase) and VIM-2 (zinc-carbapenemase), both located on chromosome. 4,261 bp of blaOXA-181-bearing contig-DNA showed 100% homology to K. pneumoniae plasmid pKP3-A. ISEcp1 was present on upstream and on downstream, △lysR, △ereA and repA genes were detected. blaVIM-2 was located within class 1 integron along with aacC6-II, dfrB5, aac(3)-Id, tniC in surrounding regions. Presence of other ARGs (blaPAO, blaOXA-488,aph(3'')-Ib, aph(6)-Id, crpP, catB7, fosA, sul2) and efflux-pump genes might explain its MDR phenotype. Virulence factors including T3SS (PscF, PopB, PopD, PcrV) and its effectors (ExoT, ExoU, ExoY) indicated the pathogenic potential of ST5217. Core genome analysis showed that ST5217 was closest with other high-risk clones ST1339 and ST773-harbouring blaOXA-48-like.

CONCLUSIONS: To the best of our knowledge, this is the first report of blaOXA-181-harbouring novel high-risk clone of CRPA ST5217/ExoU+/O11 in India which emphasises the spread of OXA-181 among bacteria other than Enterobacteriaceae-family and warrant close monitoring.},
}

@article {pmid41377346,
year = {2025},
author = {Abedin, ZU and Waggan, AI and Khan, E and Suleman, MU and Tabassum, SN},
title = {Letter to the Editor: CRISPR-based gene editing for cardiac protection in Barth syndrome.},
journal = {Annals of medicine and surgery (2012)},
volume = {87},
number = {12},
pages = {9163-9164},
pmid = {41377346},
issn = {2049-0801},
abstract = {Barth syndrome is a rare X-linked mitochondrial disorder caused by mutations in the Tafazzin (TAZ) gene. These mutations make it hard for cardiolipin to remodel and mitochondria to work properly. This condition is characterized by growth retardation, neutropenia, skeletal myopathy, and dilated cardiomyopathy, frequently leading to significant morbidity and mortality, with numerous patients necessitating heart transplants. There are no treatments available at this time to fix the genetic problem. Recent progress in gene editing, especially CRISPR-based methods, holds great promise for fixing TAZ mutations. Research utilizing patient-derived cardiomyocytes has demonstrated that the rectification of TAZ mutations reinstates mitochondrial efficiency and enhances cellular functionality. Animal models, including TAZ-knockout mice, have exhibited substantial enhancements in cardiac function, survival rates, and diminished fibrosis subsequent to gene replacement therapy.},
}

@article {pmid41285673,
year = {2025},
author = {Munusamy, S and Jahani, R and Zheng, H and Chen, J and Kong, J and Zhao, Y and Guan, L and Zhou, S and Guan, X},
title = {Enhanced CRISPR-Cas12a Fluorescent Assay for Detecting Trace Levels of Procalcitonin.},
journal = {ACS applied bio materials},
volume = {8},
number = {12},
pages = {10818-10826},
doi = {10.1021/acsabm.5c01535},
pmid = {41285673},
issn = {2576-6422},
mesh = {*Procalcitonin/analysis/blood ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Biocompatible Materials/chemistry/chemical synthesis ; Gold/chemistry ; Materials Testing ; Metal Nanoparticles/chemistry ; Particle Size ; Fluorescence ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and accurate detection of procalcitonin (PCT), a major biomarker for bacterial infections and sepsis, remains a pressing need in clinical diagnostics because sepsis progresses rapidly and may initially present with nonspecific or even subtle symptoms. Herein, we report a CRISPR-Cas12a-based fluorescence biosensing platform for ultrasensitive detection of PCT. The platform employs antibody-functionalized magnetic beads (MBs) for specific protein enrichment and antibody- and oligonucleotide- dual-functionalized gold nanoparticles (AuNPs) for high-density DNA payload. After sandwich complex formation with the target PCT, a programmed ssDNA strand is released by thermal denaturation, which then activates Cas12a collateral cleavage, thereby generating a fluorescence signal. Thorough physicochemical characterizations, including zeta potential, dynamic light scattering, UV-vis spectroscopy, and TEM, were carried out to confirm the successful functionalization of MBs and AuNPs. The developed PCT sensor was highly sensitive with a limit of detection (LOD) reaching 3 pg/mL. Moreover, the biosensor exhibited an excellent specificity toward PCT against clinically relevant interferents such as C-reactive protein (CRP), interleukin-2β (IL-2β), interleukin-6 (IL-6), human serum albumin (HSA), and bovine serum albumin (BSA), and simulated serum sample analysis was successfully carried out with the recoveries ranging from 108 to 122%. The PCT sensing technique developed in this work offers the potential to be expanded to construct a multiplexing platform for simultaneous detection of multiple biomarker species for early and accurate disease diagnosis.},
}

*Procalcitonin/analysis/blood
Humans
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Biocompatible Materials/chemistry/chemical synthesis
Gold/chemistry
Materials Testing
Metal Nanoparticles/chemistry
Particle Size
Fluorescence
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41272938,
year = {2025},
author = {Salvador, PJ and Lin, S and Chinn, MM and Jauregui-Matos, V and Manjunath, A and Yang, I and Jacobsen, CS and Beal, PA},
title = {Discovery and Tuning of RNA Editing Guides via High-Throughput Screening and Chemical Modification.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {26},
number = {24},
pages = {e202500735},
doi = {10.1002/cbic.202500735},
pmid = {41272938},
issn = {1439-7633},
mesh = {Humans ; *RNA Editing ; *High-Throughput Screening Assays ; HEK293 Cells ; Adenosine Deaminase/metabolism/genetics ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Structure-Activity Relationship ; RNA-Binding Proteins/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; },
abstract = {Adenosine deaminases acting on RNA (ADAR) catalyze the deamination of adenosine to inosine in double-stranded RNA. Because inosine is read as guanosine during translation, this process enables programmable A-to-G recoding at the transcript level. ADARs can be harnessed for therapeutic correction of pathogenic mutations through site-directed RNA editing with guide RNAs. To expand the design space of editing-enabling guides, we applied EMERGe, a high-throughput screening platform, to identify motifs targeting a premature termination codon in the MeCP2 transcript associated with Rett syndrome. This uncovered a guide RNA motif that supported efficient ADAR2-mediated editing in vitro, featuring a 5'-GUG-3' sequence predicted to form an asymmetric loop. To enable therapeutic application, structure-activity relationship studies and chemical optimization were performed, yielding a fully modified guide RNA with 2'-O-methyl, 2'-fluoro, and phosphorothioate linkages. This stabilized guide retained the activity of unmodified RNA and showed enhanced nuclease resistance. The optimized guide induces dose-dependent editing at two MECP2 loci in reporter assays in HEK293T cells, demonstrating that EMERGe-selected motifs can be rendered viable in cells through targeted chemical modification. These findings highlight the utility of EMERGe as a discovery platform and establish a pipeline for identifying and optimizing editing-enabling guide RNA features beyond traditional design rules.},
}

Humans
*RNA Editing
*High-Throughput Screening Assays
HEK293 Cells
Adenosine Deaminase/metabolism/genetics
Methyl-CpG-Binding Protein 2/genetics/metabolism
Structure-Activity Relationship
RNA-Binding Proteins/metabolism
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry

@article {pmid41266370,
year = {2025},
author = {Giddins, M and Kratz, AF and De Los Santos, MB and Forget, A and Tiwari, R and Jang, G and Blazejewski, T and Qin, C and Huang, Y and Lao, YH and Falconer, T and Leong, KW and Krogan, N and Staller, M and Wang, H and Wei, L and Chavez, A},
title = {Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11114},
pmid = {41266370},
issn = {2041-1723},
support = {HR0011-19-2-0009//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; DP2NS131566-01//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 1R01EB031935//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; U54 CA274502/CA/NCI NIH HHS/United States ; U54 CA274502/CA/NCI NIH HHS/United States ; },
mesh = {*Protein Engineering/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; HEK293 Cells ; Protein Domains ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {Current protein engineering methods are inadequate to explore the combinatorial potential offered by nature's vast repertoire of protein domains-limiting our ability to create optimal synthetic tools. To overcome this barrier, we develop an approach to create and test thousands of chimeric proteins and employ it to probe an expansive combinatorial landscape of over 15,000 multi-domain CRISPR activators. Our findings indicate that many activators produce substantial cellular toxicity, often unrelated to their capacity to regulate gene expression. We also explore the biochemical features of activation domains and determine how their combinatorial interactions shape activator behavior. Finally, we identify two potent CRISPR activators, MHV and MMH, and demonstrate their enhanced activity across diverse targets and cell types compared to the gold-standard MCP activator, synergistic activation mediator (SAM).},
}

*Protein Engineering/methods
Humans
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Animals
HEK293 Cells
Protein Domains
Recombinant Fusion Proteins/genetics/metabolism

@article {pmid41102841,
year = {2025},
author = {Li, X and Lv, H and Wu, C and Li, H and Yi, W and Niu, X and Peng, Q and Meng, C and Yuan, Y and Tian, S and Wang, Z and Fang, R and Zhou, Z},
title = {Corynebacterium pseudotuberculosis phospholipase D targets mitochondrial sphingomyelin and induces NLRP3-GSDMD axis-mediated pyroptosis in macrophages to promote infection.},
journal = {Veterinary research},
volume = {56},
number = {1},
pages = {198},
pmid = {41102841},
issn = {1297-9716},
support = {2021YFD1800800//National Key Research and Development Program of China/ ; CSTC2021JCYJ-MSXMX0884//Natural Science Foundation of Chongqing/ ; CSTB2024NSCQ-MSX1262//Natural Science Foundation of Chongqing/ ; XDJK2018C054//Fundamental Research Funds for the Central Universities/ ; XDJK2020B016//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Phospholipase D/metabolism ; *Sphingomyelins/metabolism ; *Corynebacterium Infections/enzymology/pathology/veterinary ; Mitochondria/physiology ; Pyroptosis/physiology ; *Macrophages/microbiology/physiology ; Bacterial Zoonoses/enzymology/pathology ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism ; Gasdermins/metabolism ; Phosphate-Binding Proteins/metabolism ; Mice, Inbred C57BL ; Animals ; Mice/microbiology ; CRISPR-Cas Systems ; *Corynebacterium pseudotuberculosis/enzymology/physiology ; Animals, Outbred Strains ; },
abstract = {Infection by Corynebacterium pseudotuberculosis (Cp), a facultative intracellular bacterium, causes mainly purulent inflammation and chronic granulomas in animals and humans. Pyroptosis is a proinflammatory form of programmed cell death that is important in response to pathogen infection. The role of pyroptosis in Cp infection and the related mechanisms are still unclear. Here, we reveal that Cp infection induces pyroptosis in macrophages. The inhibition of pyroptosis by disulfiram (DSF) or dimethyl fumarate (DMF) decreases the pathogenicity of Cp in mice and prevents the escape of this pathogen from infected macrophages. In contrast, LPS treatment achieves the opposite results in mice and macrophages infected with Cp. In addition, we revealed that phospholipase D (PLD) is a key virulence factor that induces pyroptosis and subsequently promotes Cp escape and spread in macrophages and confirmed that mutation of the enzyme active site at D66S, G80I, K114N, and G242P weakens PLD-induced pyroptosis. Furthermore, the indispensable role of the NLRP3-GSDMD axis in PLD-induced pyroptosis was confirmed using Nlrp3 knockout (Nlrp3[-/-]) and Gsdmd knockout (Gsdmd[-/-]) macrophages. Mechanistically, the PLD of Cp targets mitochondrial sphingomyelins within macrophages, induces cardiolipin externalization to the outer mitochondrial membrane, and releases mtROS, leading to pyroptosis. In conclusion, our data indicate that Cp infection induces mitochondrial dysfunction in macrophages through the secretion of PLD, which targets mitochondrial sphingomyelins and induces NLRP3-GSDMD axis-dependent pyroptosis. Inducing pyroptosis is one of the mechanisms underlying the pathogenesis and transmission of Cp, and inhibiting pyroptosis may be an important strategy for controlling this pathogen.},
}

*Phospholipase D/metabolism
*Sphingomyelins/metabolism
*Corynebacterium Infections/enzymology/pathology/veterinary
Mitochondria/physiology
Pyroptosis/physiology
*Macrophages/microbiology/physiology
Bacterial Zoonoses/enzymology/pathology
NLR Family, Pyrin Domain-Containing 3 Protein/metabolism
Gasdermins/metabolism
Phosphate-Binding Proteins/metabolism
Mice, Inbred C57BL
Animals
Mice/microbiology
CRISPR-Cas Systems
*Corynebacterium pseudotuberculosis/enzymology/physiology
Animals, Outbred Strains

@article {pmid40991373,
year = {2025},
author = {Karimzadeh, A and Kim, R and Garcia, V and Florea, M and Peacker, BL and Kobayashi, S and Watkins, D and Messemer, K and Zeng, J and Bauer, DE and Serwold, T and Wagers, AJ},
title = {In situ gene editing of hematopoietic stem cells via AAV-delivered CRISPR guide RNAs.},
journal = {Blood advances},
volume = {9},
number = {24},
pages = {6563-6574},
doi = {10.1182/bloodadvances.2025016775},
pmid = {40991373},
issn = {2473-9537},
mesh = {*Gene Editing/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; *Dependovirus/genetics ; Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; *CRISPR-Cas Systems ; Mice, Transgenic ; Genetic Vectors/genetics ; Humans ; },
abstract = {Hematopoietic stem cells (HSCs) are self-renewing, multipotent, and engraftable precursors of all blood cells. Efficient delivery of therapeutic gene products and gene editing machinery to correct disease-causing gene variants in endogenous HSCs while they remain in the body holds exciting potential to leverage HSC potency for the treatment of monogenic blood disorders. Toward this goal, we used adeno-associated virus (AAV) to deliver CRISPR guide RNAs (gRNAs) to edit HSC genomes in situ in Ai9;SpCas9-EGFP transgenic mice carrying a Cas9-activatable Lox-STOP-Lox-tdTomato reporter cassette together with a constitutive SpCas9-2A-EGFP. Using a variety of conditions and vector designs, we tested whether systemic administration to these mice of AAVs carrying SpCas9-compatible gRNAs designed to cut DNA upstream and downstream of the STOP cassette would induce tdTomato expression in HSCs. Our findings identify self-complementary AAVs (scAAVs) and increased ratio of guide to Cas9 as parameters facilitating higher editing efficiency. Of note, we find preserved multilineage output and engraftability of HSCs upon scAAV-gRNA editing. In an example application of this technology, we explore the potential for in situ HSC gene editing by dual AAV-CRISPR delivery and demonstrate robust gene modification, concurrent with induction of therapeutic fetal hemoglobin, in a sickle cell disease mouse model modified to express SpCas9. In summary, this work offers a sensitive and adaptable platform that allows robust modification of HSC genomes in situ.},
}

*Gene Editing/methods
*Hematopoietic Stem Cells/metabolism/cytology
*Dependovirus/genetics
Animals
*RNA, Guide, CRISPR-Cas Systems/genetics
Mice
*CRISPR-Cas Systems
Mice, Transgenic
Genetic Vectors/genetics
Humans

@article {pmid40542106,
year = {2025},
author = {Wu, X and Luo, S and Guo, C and Zhao, Y and Zhong, J and Hu, R and Yang, X and Liu, C and Zhang, Q and Zhuang, S and Chen, Y and Liu, Y and Zhang, X},
title = {LbuCas13a directly targets DNA and elicits strong trans-cleavage activity.},
journal = {Nature biomedical engineering},
volume = {9},
number = {12},
pages = {2141-2154},
pmid = {40542106},
issn = {2157-846X},
support = {2024A1515011877//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; 22104048//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *DNA/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Leptotrichia/enzymology/genetics ; Cytochrome P-450 CYP2C19/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {Traditionally perceived as an RNA-specific nuclease, Cas13a has been used primarily for RNA detection. We discover the ability of Leptotrichia buccalis Cas13a (LbuCas13a) to directly target DNA without the restrictions of protospacer flanking sequence and protospacer adjacent motif sequences, coupled with robust trans-cleavage activity. Contrary to conventional understanding, LbuCas13a does not degrade DNA targets. Our study reveals an enhancement in the single-nucleotide specificity of LbuCas13a against DNA compared to RNA. This heightened specificity is attributed to the lower affinity of CRISPR RNA (crRNA) towards DNA, raising the crRNA-DNA binding energy barrier. We introduce a molecular diagnostic platform called superior universal rapid enhanced specificity test with LbuCas13a (SUREST) for high-resolution genotyping. SUREST is capable of detecting DNA concentrations of CYP2C19 (rs4986893) as minute as 0.3 aM (0.18 cps µl[-1]). We also apply SUREST to human genotyping scenarios, indicating that SUREST performs well across a broad range of mutations and sequence contexts. SUREST represents an advancement in real-time nucleic acid detection, making it a useful tool for pathogen identification and mutation analysis in clinical diagnostics.},
}

Humans
*DNA/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Leptotrichia/enzymology/genetics
Cytochrome P-450 CYP2C19/genetics
*CRISPR-Associated Proteins/metabolism/genetics

@article {pmid41375334,
year = {2025},
author = {Lai, CM and Xiao, XS and Liu, LW and Lin, XD and Dou, DL and Cai, HY and Mei, ZF and Yang, F and Cheng, Y and Qin, Y},
title = {Nanotechnology Strategies in Plant Genetic Engineering: Intelligent Delivery and Precision Editing.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {23},
pages = {},
pmid = {41375334},
issn = {2223-7747},
support = {2024NZ029029//Major Science and Technology Project of Fujian Province/ ; },
abstract = {Plant genetic engineering is crucial for enhancing crop yield, quality, and resilience to both abiotic and biotic stresses, thereby promoting sustainable agriculture. Agrobacterium-mediated, biolistic bombardment, electroporation, and poly (ethylene glycol) (PEG)-mediated genetic transformation systems are widely applied in plant genetic engineering. However, these systems have limitations, including species dependency, destruction of plant tissues, low transformation efficiency, and high cost. Recently, gene-delivery methods based on nanotechnology have been developed for plant genetic transformation. This nanostrategy demonstrates remarkable transformation efficiency, excellent biocompatibility, effective protection of exogenous nucleic acids, and the potential for plant regeneration. However, the application of nanomaterial-mediated gene-delivery systems in plants is still in its early stages and faces numerous challenges for widespread adoption. Herein, the conventional genetic transformation techniques utilized in plants are succinctly examined. Subsequently, the advancements in nanomaterial-based gene-delivery systems are reviewed. The applications of CRISPR-Cas-mediated genome editing and its integration with plant nanotechnology are also examined. The innovations, methods, and practical applications of nanomaterial-mediated genetic transformation summarized herein are expected to facilitate the progress of plant genetic engineering in modern agriculture.},
}

@article {pmid41375204,
year = {2025},
author = {Li, W and Shi, Y and Li, D and Wang, Y and Sun, Y and Li, H and Han, Y},
title = {A CRISPR Powered Immobilization-Free, Amplification-Free Carbon-Nanotube Field-Effect Transistor (FET) Sensor for Influenza A Virus (IAV).},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {23},
pages = {},
pmid = {41375204},
issn = {1420-3049},
support = {2023YFC2605101//National Key Research and Development Program of China/ ; },
mesh = {*Influenza A virus/genetics/isolation & purification ; *Biosensing Techniques/methods ; Transistors, Electronic ; *Nanotubes, Carbon/chemistry ; Humans ; *RNA, Viral/genetics/analysis ; CRISPR-Cas Systems ; Limit of Detection ; *Influenza, Human/diagnosis/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The epidemic of infectious diseases, such as influenza A, has imposed a severe health burden on the population. Early detection, diagnosis, reporting, isolation, and treatment are crucial for the prevention, control, and management of infectious diseases. Nucleic acid testing represents a vital approach for the rapid diagnosis of pathogenic microorganism types. However, current nucleic acid detection methods face notable bottlenecks: traditional CRISPR fluorescence assays require time-consuming pre-amplification of target nucleic acids, while existing carbon-nanotube field-effect transistor (FET)-based platforms, though amplification-free, often necessitate complex chip surface modification and probe immobilization, and suffer from non-reusable chips, all limiting their utility in point-of-care testing (POCT) and large-scale screening. This study reports a CRISPR-based amplification-free RNA detection platform (CRISPR-FET) for the rapid identification of influenza A virus. The CRISPR-FET platform described herein enables the detection of viral RNA without amplification within 20 min, with a limit of detection as low as 1 copy/μL. Secondly, a reporter RNA conjugated with gold particles is used to achieve signal amplification in FET detection; meanwhile, the method eliminates probe immobilization, thereby omitting this step and simplifying chip modification to reduce complex work-flows and pre-treatment costs. The chip's reusability further enhances cost-effectiveness. Additionally, streptavidin-modified magnetic bead adsorption minimizes background errors from excessive reporter RNA and non-target nucleic acids. Finally, validation with 24 clinical samples confirmed the platform's efficacy. By integrating rapidity, simplicity, and high sensitivity, alongside cost advantages from reusable chips, this CRISPR-FET platform meets the critical need for early influenza A diagnosis and holds promise for advancing POCT and large-scale epidemiological screening.},
}

*Influenza A virus/genetics/isolation & purification
*Biosensing Techniques/methods
Transistors, Electronic
*Nanotubes, Carbon/chemistry
Humans
*RNA, Viral/genetics/analysis
CRISPR-Cas Systems
Limit of Detection
*Influenza, Human/diagnosis/virology
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40968311,
year = {2025},
author = {Park, H and Yu, S and Koo, T},
title = {Gene editing in cancer therapy: overcoming drug resistance and enhancing precision medicine.},
journal = {Cancer gene therapy},
volume = {32},
number = {12},
pages = {1293-1302},
pmid = {40968311},
issn = {1476-5500},
mesh = {Humans ; *Gene Editing/methods ; *Precision Medicine/methods ; *Neoplasms/therapy/genetics ; *Drug Resistance, Neoplasm/genetics ; CRISPR-Cas Systems ; *Genetic Therapy/methods ; Animals ; },
abstract = {The CRISPR system has revolutionized cancer gene therapy, offering unparalleled precision in genetic manipulation for targeted oncogene disruption, mutation correction, and immune system modulation. This breakthrough tool has demonstrated remarkable potential in overcoming drug resistance, enhancing chemotherapy sensitivity, and improving immunotherapy strategies such as CRISPR-engineered CAR-T cells. Additionally, oncolytic virus-mediated CRISPR delivery has emerged as a novel approach for tumor-specific gene editing, minimizing off-target effects. The rapid transition of CRISPR-based cancer therapeutics from preclinical research to clinical trials underscores its therapeutic potential. This review explores the latest advancements in CRISPR applications for cancer therapy, including gene knockout, base editing for mutation correction, and integration with immune and viral therapies. Despite significant progress, challenges such as off-target effects, immune responses, and delivery limitations remain key hurdles. We discuss current strategies to enhance CRISPR safety and efficacy, emphasizing its potential for personalized cancer treatment.},
}

Humans
*Gene Editing/methods
*Precision Medicine/methods
*Neoplasms/therapy/genetics
*Drug Resistance, Neoplasm/genetics
CRISPR-Cas Systems
*Genetic Therapy/methods
Animals

@article {pmid40753800,
year = {2025},
author = {Behrouzian Fard, G and Ahmadi, MH and Gholamin, M and Hosseini Bafghi, M},
title = {CRISPR-Cas9: a prominent genome editing tool in the management of inherited blood disorders and hematological malignancies.},
journal = {Current research in translational medicine},
volume = {73},
number = {4},
pages = {103531},
doi = {10.1016/j.retram.2025.103531},
pmid = {40753800},
issn = {2452-3186},
mesh = {Humans ; *Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; *Hematologic Neoplasms/therapy/genetics ; Genetic Therapy/methods ; *Hematologic Diseases/therapy/genetics ; },
abstract = {Several hematologic diseases with genetic defects, like sickle cell disease and β-thalassemia can be treated with allogeneic hematopoietic stem cell transplantation (HSCT) from healthy donors. However, suitable tissue-matched donors are often unavailable, and HSCT involves risks such as graft-versus-host disease and potential disease relapse. Due to the genetic heterogeneity of blood disorders and the complexity of the hematopoietic system, identifying effective genes for managing and treating both benign and malignant conditions remains a significant challenge. The genome editing field is rapidly expanding and is essential for identifying genetic factors in pathological processes. These developments highlight the importance of using ex vivo gene therapy approaches for autologous hematopoietic stem cells. Also, gene editing technologies are gaining significant interest in engineered cell therapies for hematological malignancies . Today, various programmable nucleases are available for genome editing, with the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system standing out due to its high efficiency, low cytotoxicity, cost-effectiveness, and precision. This system can serve as a genomic modification tool for treating blood disorders, including hereditary diseases and immunotherapy for cancer using chimeric antigen receptor T cells (CAR-T cells). Advancements in CRISPR-Cas9 are expected to significantly impact medical research and clinical applications. However, challenges such as off-target effects and immunogenicity must be addressed. This review summarizes the mechanism and delivery strategies of CRISPR-Cas9, discusses its applications in treating inherited blood disorders such as sickle cell disease, β-thalassemia, and fanconi anemia, as well as hematological malignancies, and highlights the associated challenges.},
}

Humans
*Gene Editing/methods/trends
*CRISPR-Cas Systems/genetics
*Hematologic Neoplasms/therapy/genetics
Genetic Therapy/methods
*Hematologic Diseases/therapy/genetics

@article {pmid41374373,
year = {2025},
author = {Sambo, CN and Skepu, A and Nxumalo, NP and Polori, KL},
title = {Diagnostic Advances and Public Health Challenges for Monkeypox Virus: Clade-Specific Insight and the Urgent Need for Rapid Testing in Africa.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {15},
number = {23},
pages = {},
pmid = {41374373},
issn = {2075-4418},
support = {B1B0741B-9691-4333-B77E-3B8D42A7B5FF//Technology Innovation Agency/ ; },
abstract = {Background: Monkeypox (MPX), caused by the Monkeypox virus (MPOX) of the Orthopoxvirus genus, has re-emerged as a significant global health threat. Once confined to Central and West Africa, the 2022-2025 multi-country outbreaks, predominantly caused by Clade IIb, demonstrated sustained human-to-human transmission and global spread. Objective: This review summarizes current knowledge on MPX virology, epidemiology, clinical presentation, and diagnostic technologies, with a focus on innovations supporting rapid and field-deployable detection in resource-limited settings. Methods: The recent literature (2019-2025), including peer-reviewed studies, WHO and Africa CDC reports, and clinical guidelines, was critically reviewed. Data were synthesized to outline key developments in diagnostic methodologies and surveillance approaches. Results: MPX comprises two genetic clades: Clade I (Congo Basin) and Clade II (West African), which differ in virulence and transmission. Clade IIb is associated with sexual and close-contact transmission during recent outbreaks. Clinical manifestations have shifted from classic disseminated rash to localized anogenital lesions and atypical or subclinical infections. RT-PCR remains the diagnostic gold standard, while emerging assays such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and CRISPR/Cas-based platforms show promise for rapid point-of-care (POC) testing. Complementary serological tools, including ELISA and lateral flow assays, enhance surveillance and immune profiling. Conclusions: The resurgence of MPX highlights the urgent need for accessible, sensitive, and specific diagnostic platforms to strengthen surveillance and outbreak control, especially in endemic and resource-constrained regions.},
}

@article {pmid41053217,
year = {2025},
author = {Schwämmle, T and Noviello, G and Kanata, E and Froehlich, JJ and Bothe, M and Martitz, A and Altay, A and Scouarnec, J and Feng, VY and Mallie, H and Vingron, M and Schulz, EG},
title = {Reporter CRISPR screens decipher cis-regulatory and trans-regulatory principles at the Xist locus.},
journal = {Nature structural & molecular biology},
volume = {32},
number = {12},
pages = {2465-2475},
pmid = {41053217},
issn = {1545-9985},
mesh = {Animals ; *RNA, Long Noncoding/genetics ; Mice ; Female ; X Chromosome Inactivation ; Transcription Factors/metabolism/genetics ; *CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Octamer Transcription Factor-3/metabolism/genetics ; Otx Transcription Factors/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genes, Reporter ; },
abstract = {Developmental genes are controlled by an ensemble of cis-acting regulatory elements (REs), which in turn respond to multiple trans-acting transcription factors (TFs). Understanding how a cis-regulatory landscape integrates information from many dynamically expressed TFs has remained a challenge. Here we develop a combined CRISPR screening approach using endogenous RNA and RE reporters as readouts. Applied to the murine Xist locus, which is crucial for X-chromosome inactivation in females, this method allows us to comprehensively identify Xist-controlling TFs and map their TF-RE wiring. We find a group of transiently upregulated TFs, including ZIC3, that regulate proximal REs, driving the binary activation of Xist expression. These basal activators are more highly expressed in cells with two X chromosomes, potentially governing female-specific Xist upregulation. A second set of developmental TFs that include OTX2 is upregulated later during differentiation and targets distal REs. This regulatory axis is crucial to achieve high levels of Xist RNA, which is necessary for X-chromosome inactivation. OCT4 emerges as the strongest activator overall, regulating both proximal and distal elements. Our findings support a model for developmental gene regulation, in which factors targeting proximal REs drive binary on-off decisions, whereas factors interacting with distal REs control the transcription output.},
}

Animals
*RNA, Long Noncoding/genetics
Mice
Female
X Chromosome Inactivation
Transcription Factors/metabolism/genetics
*CRISPR-Cas Systems
Gene Expression Regulation, Developmental
Octamer Transcription Factor-3/metabolism/genetics
Otx Transcription Factors/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Genes, Reporter

@article {pmid40907203,
year = {2025},
author = {Tabibian, M and Motevaseli, E and Ghafouri-Fard, S},
title = {CRISPR-mediated modulation of EGFR signaling in lung cancer.},
journal = {Cancer treatment and research communications},
volume = {45},
number = {},
pages = {100992},
doi = {10.1016/j.ctarc.2025.100992},
pmid = {40907203},
issn = {2468-2942},
mesh = {Humans ; *Lung Neoplasms/genetics/therapy/drug therapy/pathology/metabolism ; ErbB Receptors/genetics/metabolism/antagonists & inhibitors ; Signal Transduction/genetics/drug effects ; *CRISPR-Cas Systems ; Gene Editing/methods ; Protein Kinase Inhibitors/pharmacology/therapeutic use ; Mutation ; },
abstract = {Lung cancer is among the most common cancers and the leading source of cancer death. Inhibition of EGFR signaling by small-molecule tyrosine kinase inhibitors and monoclonal antibodies has provided new opportunities for treatment of this type of cancer. However, prognosis remained unfavorable due to the incidence of intrinsic or attained resistance. The advent of CRISPR/Cas9 technology has offered additional chances for cancer genome editing. This technology has been applied in the context of lung cancer research in order to minimize the effects of activating EGFR mutations. In the current manuscript, we address the application of CRISPR/Cas9 method in the modulation of EGFR signaling and its consequence in the treatment of lung cancer.},
}

Humans
*Lung Neoplasms/genetics/therapy/drug therapy/pathology/metabolism
ErbB Receptors/genetics/metabolism/antagonists & inhibitors
Signal Transduction/genetics/drug effects
*CRISPR-Cas Systems
Gene Editing/methods
Protein Kinase Inhibitors/pharmacology/therapeutic use
Mutation

@article {pmid40840103,
year = {2025},
author = {Watts, A and Raipuria, RK and Chauhan, M and Mehta, K and Annamalai, M and Abbas, AZ and Bhattacharya, R and Watts, A and Singh, N},
title = {CRISPR/Cas9-mediated knockout of TRANSPARENT TESTA 8 downregulates flavonoid biosynthetic pathway in seeds of Brassica juncea.},
journal = {Plant physiology and biochemistry : PPB},
volume = {229},
number = {Pt A},
pages = {110330},
doi = {10.1016/j.plaphy.2025.110330},
pmid = {40840103},
issn = {1873-2690},
mesh = {*CRISPR-Cas Systems/genetics ; *Seeds/metabolism/genetics ; *Flavonoids/biosynthesis/genetics ; *Mustard Plant/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Knockout Techniques ; Gene Expression Regulation, Plant ; *Biosynthetic Pathways/genetics ; Down-Regulation ; Gene Editing ; Proanthocyanidins ; },
abstract = {Accumulation of proanthocyanidin (PA) in the seed coat determines black to brown seed color in Brassicaceae members. Consequently, yellow-seed coat results from mutation in the regulatory or biosynthetic genes of the flavonoid pathway which perturb PA accumulation. We identified two homeologs of Transparent Testa 8 (BjTT8) gene in the allotetraploid genome of Brassica juncea with BjTT8A homeolog exhibiting higher expression than BjTT8B. To investigate whether targeted knockout of BjTT8 can result into yellow seed coat color, a CRISPR/Cas9-based genome editing vector was constructed using the binary vector pORE O4 backbone. A single-guide RNA (sgRNA) was designed to precisely target the second exon of both the BjTT8 homeologs. Successful knockout of both BjTT8A and BjTT8B genes deploying pORE O4-CRISPR/Cas9 vector resulted in yellow seed coat color. The mutations were stably inherited over the successive generations, giving consistent yellow seed color including in the lines segregated free from T-DNA bound CRISPR/Cas9 cassette. Comparative transcriptome and gene expression analysis revealed that, a subset of flavonoid pathway genes was downregulated in the Bjtt8 edited lines. Flavonoid profiling demonstrated that, the flavan-3-ol monomer (viz., catechin/epicatechin), the precursor of PA biosynthesis was completely absent in the Bjtt8 edited lines. More significantly, the seeds of Bjtt8 edited lines exhibited higher oil content as compared to their parental Varuna line. In this study, we developed a new yellow-seeded line through CRISPR/Cas9-mediated knockout of BjTT8 genes, providing molecular insights of seed coat color regulation in B. juncea.},
}

*CRISPR-Cas Systems/genetics
*Seeds/metabolism/genetics
*Flavonoids/biosynthesis/genetics
*Mustard Plant/genetics/metabolism
*Plant Proteins/genetics/metabolism
Gene Knockout Techniques
Gene Expression Regulation, Plant
*Biosynthetic Pathways/genetics
Down-Regulation
Gene Editing
Proanthocyanidins

@article {pmid39966655,
year = {2025},
author = {Xia, C and Colognori, D and Jiang, XS and Xu, K and Doudna, JA},
title = {Single-molecule live-cell RNA imaging with CRISPR-Csm.},
journal = {Nature biotechnology},
volume = {43},
number = {12},
pages = {2023-2030},
pmid = {39966655},
issn = {1546-1696},
support = {X-0001//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; D-0001//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35 GM149349/GM/NIGMS NIH HHS/United States ; D-0001//Howard Hughes Medical Institute (HHMI)/ ; K99 GM151484/GM/NIGMS NIH HHS/United States ; },
mesh = {*In Situ Hybridization, Fluorescence/methods ; Humans ; *Single Molecule Imaging/methods ; *CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics/metabolism ; *RNA/genetics/metabolism ; Animals ; Single-Cell Analysis/methods ; Receptor, Notch2/genetics/metabolism ; Mice ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Understanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution in real time. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved in a generalizable manner. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for direct and efficient visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we track individual native NOTCH2 and MAP1B transcripts in living cells and identify two distinct localization mechanisms including the cotranslational translocation of NOTCH2 mRNA at the endoplasmic reticulum and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.},
}

*In Situ Hybridization, Fluorescence/methods
Humans
*Single Molecule Imaging/methods
*CRISPR-Cas Systems/genetics
RNA, Messenger/genetics/metabolism
*RNA/genetics/metabolism
Animals
Single-Cell Analysis/methods
Receptor, Notch2/genetics/metabolism
Mice
RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39820813,
year = {2025},
author = {Zhang, F and Chow, RD and He, E and Dong, C and Xin, S and Mirza, D and Feng, Y and Tian, X and Verma, N and Majety, M and Zhang, Y and Wang, G and Chen, S},
title = {Multiplexed inhibition of immunosuppressive genes with Cas13d for combinatorial cancer immunotherapy.},
journal = {Nature biotechnology},
volume = {43},
number = {12},
pages = {2054-2067},
pmid = {39820813},
issn = {1546-1696},
support = {R33 CA281702/CA/NCI NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; F30 CA250249/CA/NCI NIH HHS/United States ; R33 CA225498/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Immunotherapy/methods ; Mice ; Tumor Microenvironment/genetics/immunology ; *Neoplasms/therapy/immunology/genetics ; Humans ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Gene Silencing ; Dependovirus/genetics ; Mice, Inbred C57BL ; Female ; },
abstract = {The complex nature of the immunosuppressive tumor microenvironment (TME) requires multi-agent combinations for optimal immunotherapy. Here we describe multiplex universal combinatorial immunotherapy via gene silencing (MUCIG), which uses CRISPR-Cas13d to silence multiple endogenous immunosuppressive genes in the TME, promoting TME remodeling and enhancing antitumor immunity. MUCIG vectors targeting four genes delivered by adeno-associated virus (AAV) (Cd274/Pdl1, Lgals9/Galectin9, Lgals3/Galectin3 and Cd47; AAV-Cas13d-PGGC) demonstrate significant antitumor efficacy across multiple syngeneic tumor models, remodeling the TME by increasing CD8[+] T-cell infiltration while reducing neutrophils. Whole transcriptome profiling validates the on-target knockdown of the four target genes and shows limited potential off-target or downstream gene alterations. AAV-Cas13d-PGGC outperforms corresponding shRNA treatments and individual gene knockdown. We further optimize MUCIG by employing high-fidelity Cas13d (hfCas13d), which similarly showed potent gene silencing and in vivo antitumor efficacy, without weight loss or liver toxicity. MUCIG represents a universal method to silence multiple immune genes in vivo in a programmable manner, offering broad efficacy across multiple tumor types.},
}

Animals
*Immunotherapy/methods
Mice
Tumor Microenvironment/genetics/immunology
*Neoplasms/therapy/immunology/genetics
Humans
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Gene Silencing
Dependovirus/genetics
Mice, Inbred C57BL
Female

@article {pmid41373623,
year = {2025},
author = {Petrova, IO and Smirnikhina, SA},
title = {Ex Vivo Gene and Cell Therapy in Hematopoietic Stem Cells.},
journal = {International journal of molecular sciences},
volume = {26},
number = {23},
pages = {},
pmid = {41373623},
issn = {1422-0067},
support = {not applicable//Ministry of Education and Science of Russia/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; *Cell- and Tissue-Based Therapy/methods ; Animals ; *Hematopoietic Stem Cell Transplantation/methods ; Genetic Vectors/genetics ; Lentivirus/genetics ; },
abstract = {Ex vivo cell and gene therapy is a prospective approach to treatment of genetic diseases. To date, one of the most prevalent examples of genetically engineered cell therapies is hematopoietic stem/progenitor cells (HSPCs). This mini review is focused on HSPC therapy methods that have been approved for medical use. Most gene therapy methods rely on the lentiviral integration of the gene into the target cell genome, as lentiviruses are extremely effective, particularly in transduction of non-dividing cells. In this constantly evolving field, it is important to find the balance between safety concerns and efficiency. Analyzing cases of several diseases, for which ex vivo gene therapy was developed, we strive to understand which factors are crucial to success and what the potential drawbacks are. Although in general, viral gene integration demonstrates a considerable therapeutic effect, it has oncogenic potential. Development of self-inactivating vectors was a breakthrough in regard to safety, but the possibility of oncogenesis remains, and strict analysis of integration sites is required.},
}

Humans
*Genetic Therapy/methods
*Hematopoietic Stem Cells/metabolism/cytology
*Cell- and Tissue-Based Therapy/methods
Animals
*Hematopoietic Stem Cell Transplantation/methods
Genetic Vectors/genetics
Lentivirus/genetics

@article {pmid41373550,
year = {2025},
author = {Koller, F},
title = {The Potential of NGTs to Overcome Constraints in Plant Breeding and Their Regulatory Implications.},
journal = {International journal of molecular sciences},
volume = {26},
number = {23},
pages = {},
pmid = {41373550},
issn = {1422-0067},
support = {3522840500//Federal Agency for Nature Conservation/ ; },
mesh = {*Plant Breeding/methods ; CRISPR-Cas Systems ; Genome, Plant ; *Plants/genetics ; *Genomics/methods ; Gene Editing/methods ; },
abstract = {Conventional plant breeding relies on the occurrence of chromosomal crossover and spontaneous or non-targeted mutations in the genome induced by physical or chemical stressors. However, constraints exist concerning the number and variation of genotypes that can be achieved in this way, as the occurrence and combination of mutations are not equally distributed across the genome. The underlying mechanisms and causes of reproductive constraints can be considered the result of evolution to maintain the genomic stability of a species while at the same time allowing necessary adaptations. A continuous horizon scan was carried out to identify plants derived from new genomic techniques (NGTs), which show that CRISPR/Cas is able to circumvent at least some of these mechanisms and constraints. The reason for this is the specific mode of action: While physico-chemical mutagens such as radiation or chemicals merely cause a break in DNA, recombinant enzymatic mutagens (REMs), such as CRISPR/Cas, additionally interfere with cellular repair mechanisms. More recently developed REMs even expand the capabilities of NGTs to introduce new genetic variations within the target sequences. Thus, NGTs introduce genetic changes and combinations that are unknown in the current breeding pool and that are also unlikely to occur as a result of any previously used breeding methods. The resulting genotypes may need to be considered as 'new to the environment'. The technical potential of NGTs should also be taken into account in regulatory provisions. Previously unknown genotypes and phenotypes may negatively impact plant health, ecosystems, biodiversity, and plant breeding. It must further be acknowledged that the different outcomes of NGTs and conventional breeding are not always evident at first sight. As a starting point, within a process-oriented approval process, molecular characterization can inform the following steps in risk assessment and guide requests for further data.},
}

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

@article {pmid41373007,
year = {2025},
author = {Song, J and Yang, D and Kong, L and Tsai, LK and Zhang, J and Chen, YE and Tsai, RY and Xu, J},
title = {Development of a high-yield Rabbit line for enhanced animal pharming.},
journal = {Biological research},
volume = {58},
number = {1},
pages = {73},
pmid = {41373007},
issn = {0717-6287},
support = {R41GM110822/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Rabbits/genetics ; *Animals, Genetically Modified/genetics ; *Recombinant Proteins/biosynthesis/genetics ; *Milk/chemistry ; Gene Knock-In Techniques ; CRISPR-Cas Systems/genetics ; *Caseins/genetics ; Female ; Promoter Regions, Genetic/genetics ; },
abstract = {Animal pharming involves producing recombinant protein drugs using transgenic animals. The United States Food and Drug Administration (FDA) has approved certain drugs produced in the milk of transgenic Rabbits. Traditionally, these pharming Rabbits have been developed using conventional transgenic technology, which often results in an unpredictable success rate, uncontrollable transgene insertion sites, varying copy numbers, and generally low recombinant protein yields, typically 1-2 g/L or lower. We hypothesized that utilizing the promoter of a native major milk protein gene to drive transgene expression could significantly enhance yield. To test this, we developed a rabbit line that expresses tdTomato under the control of the CSN2 gene promoter, responsible for encoding β-casein, the most abundant protein in Rabbit milk. We successfully generated knock-in founder Rabbits using CRISPR/Cas9-mediated knock-in technology, augmented by the homology-directed repair (HDR)-promoting small molecule RS-1. These founder Rabbits were able to transmit the knock-in allele to their offspring, producing both heterozygous and homozygous tdTomato knock-in Rabbits. Remarkably, the recombinant protein yield reached 15-20 g/L in the milk of homozygous animals. Our work demonstrates a promising strategy to enhance recombinant protein production in Rabbit pharming.},
}

Animals
Rabbits/genetics
*Animals, Genetically Modified/genetics
*Recombinant Proteins/biosynthesis/genetics
*Milk/chemistry
Gene Knock-In Techniques
CRISPR-Cas Systems/genetics
*Caseins/genetics
Female
Promoter Regions, Genetic/genetics

@article {pmid41372233,
year = {2025},
author = {Macak, D and Kanis, P and Riesenberg, S},
title = {Repurposing clinically safe drugs for DNA repair pathway choice in CRISPR genome editing and synthetic lethality.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11077},
pmid = {41372233},
issn = {2041-1723},
mesh = {Humans ; *Gene Editing/methods ; *Synthetic Lethal Mutations/drug effects ; *Drug Repositioning/methods ; DNA End-Joining Repair/drug effects ; Induced Pluripotent Stem Cells/drug effects/metabolism ; *DNA Repair/drug effects ; Recombinational DNA Repair/drug effects ; *CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded/drug effects ; Tumor Suppressor p53-Binding Protein 1/metabolism/genetics ; },
abstract = {We evaluate the effect of most FDA-approved drugs (>7,000 conditions) on double-strand DNA break repair pathways by analyzing mutational outcomes in human induced pluripotent stem cells. We identify drugs that can be repurposed as inhibitors and enhancers of repair outcomes attributed to non-homologous and microhomology-mediated end joining (NHEJ, MMEJ), and homology-directed repair (HDR). We also identify functions of the proteins estrogen receptor 2 (ESR2) and aldehyde oxidase 1 (AOX1), affecting several key DNA repair proteins, such as ATM and 53BP1. Silencing of ESR2 can have a synergistic effect on increasing HDR when combined with NHEJ inhibition (mean 4.6-fold increase). We further identify drugs that induce synthetic lethality when NHEJ or HDR is blocked and may therefore be candidates for precision medicine. We anticipate that the ability to modulate the DNA repair outcomes with clinically safe drugs will help disease modeling, gene therapy, chimeric antigen receptor immunotherapy, and cancer treatment.},
}

Humans
*Gene Editing/methods
*Synthetic Lethal Mutations/drug effects
*Drug Repositioning/methods
DNA End-Joining Repair/drug effects
Induced Pluripotent Stem Cells/drug effects/metabolism
*DNA Repair/drug effects
Recombinational DNA Repair/drug effects
*CRISPR-Cas Systems/genetics
DNA Breaks, Double-Stranded/drug effects
Tumor Suppressor p53-Binding Protein 1/metabolism/genetics

@article {pmid41344296,
year = {2025},
author = {Landi, E and Zondag, R and Dehnen, JA and Albert, S and Dickman, MM and LaPointe, VLS and van Bokhoven, H},
title = {Biallelic excision of the CTG18.1 expansion in two Fuchs endothelial corneal dystrophy-derived iPSC lines and one control (SCTCi046-A-1, SCTCi047-A-1 and SCTCi041-A-1) using an episomal vector-based CRISPR/Cas9 approach.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103881},
doi = {10.1016/j.scr.2025.103881},
pmid = {41344296},
issn = {1876-7753},
mesh = {Humans ; *Fuchs' Endothelial Dystrophy/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *CRISPR-Cas Systems/genetics ; *Trinucleotide Repeat Expansion/genetics ; Cell Line ; Transcription Factor 4/genetics ; Alleles ; Plasmids/genetics ; },
abstract = {An expanded CTG repeat in intron 2 of the transcription factor 4 (TCF4) gene is the main cause of Fuchs endothelial corneal dystrophy (FECD), a complex corneal disease. The prevailing paradigm is that the expanded repeat exerts toxic effects, resulting in corneal endothelium degeneration. Here we explored the use of CRISPR/Cas9-mediated, non-homologous end-joining (NHEJ) for disease-modeling purposes, by performing a biallelic excision of the CTG18.1 expansion in two FECD- and one control-derived induced pluripotent stem cell lines (iPSCs). The three Δ/Δ CTG18.1 lines generated by this study provide a platform to investigate the CTG18.1 contribution to FECD pathogenesis.},
}

Humans
*Fuchs' Endothelial Dystrophy/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/pathology
*CRISPR-Cas Systems/genetics
*Trinucleotide Repeat Expansion/genetics
Cell Line
Transcription Factor 4/genetics
Alleles
Plasmids/genetics

@article {pmid41330274,
year = {2025},
author = {Macklin, BL and Runyon, WV and Feliciano, CM and Dierks, PH and Kelly, KR and Watry, HL and Judge, LM and Conklin, BR},
title = {Generation of WTD, a control human iPSC line for genetic research.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103872},
doi = {10.1016/j.scr.2025.103872},
pmid = {41330274},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; Mutation ; },
abstract = {The establishment of well characterized control iPSC lines is essential for robust, reproducible research across laboratories. We used CRISPR/Cas9 to derive an isogeneic control line from a patient-derived iPSC line carrying a mutation in the NEFL gene (E396K). After correction of the E396K mutation, UCSFi003-A (WTD) exhibits multi-lineage differentiation potential, a normal karyotype, no large genomic abnormalities, and has consents for public distribution of cells and genomic data.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
Cell Line
CRISPR-Cas Systems
Cell Differentiation
Mutation

@article {pmid41308567,
year = {2025},
author = {Downton, P and Bates, N and Woods, S and Adamson, A and Sergouniotis, PI},
title = {Genome editing of a low-penetrance albinism-associated variant in TYR in patient-derived pluripotent stem cells.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103855},
doi = {10.1016/j.scr.2025.103855},
pmid = {41308567},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Monophenol Monooxygenase/genetics/metabolism ; *Gene Editing/methods ; *Albinism/genetics/pathology ; CRISPR-Cas Systems ; Cell Differentiation ; Male ; },
abstract = {TYR encodes tyrosinase, the enzyme catalysing the initial steps of melanin biosynthesis in melanocytes and retinal pigment epithelia (RPE). TYR c.1205G>A (p.Arg402Gln) is a common genetic variant associated with several pigmentation traits. Notably, when this variant is encountered in specific haplotypic backgrounds in the homozygous state, it predisposes to albinism. We generated an induced pluripotent stem cell (iPSC) line from an affected individual carrying such a homozygous genotype (UMANi255-A), and then used CRISPR-Cas9 to correct the TYR c.1205G>A variant (UMANi255-A-1). The resulting iPSC lines demonstrate capacity for multi-lineage differentiation, providing a useful in vitro model for studying pigmentation biology.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Monophenol Monooxygenase/genetics/metabolism
*Gene Editing/methods
*Albinism/genetics/pathology
CRISPR-Cas Systems
Cell Differentiation
Male

@article {pmid41275813,
year = {2026},
author = {Zhang, Z and Zhang, T and Li, Z and Zeng, Z},
title = {CRISPR/Cas13a-based colorimetric biosensing platform for point-of-care detection of viral nucleic acids.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {348},
number = {Pt 1},
pages = {127221},
doi = {10.1016/j.saa.2025.127221},
pmid = {41275813},
issn = {1873-3557},
mesh = {*Colorimetry/methods ; *SARS-CoV-2/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Dengue Virus/genetics/isolation & purification ; *COVID-19/diagnosis/virology ; *Point-of-Care Systems ; *RNA, Viral/analysis/genetics ; G-Quadruplexes ; Limit of Detection ; Point-of-Care Testing ; DNA, Catalytic/chemistry ; Sensitivity and Specificity ; },
abstract = {Rapid and accurate diagnosis is important in preventing and effectively combating infectious disease outbreaks. The CRISPR/Cas13a-based Specific High-sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) platform possesses the advantages of high efficiency, good specificity and sensitivity, and it has been widely adopted in molecular diagnostics. However, the traditional SHERLOCK platform requires dual-labeled RNA probes for fluorescence detection or lateral flow assay, which entail tedious modification procedures and sophisticated optical instruments, limiting its broad applications. Herein, we developed a rapid, sensitive, and label-free point-of-care (POC) platform for colorimetric assays of dengue virus (DV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the SHERLOCK method. The adoption of the SHERLOCK-mediated guanine-quadruplex (G4)/hemin DNAzyme-based colorimetric strategy produced cascade signal amplification detection with improved analytical performance. Moreover, it exhibited high sensitivity and specificity for detection in cell-cultured DV samples, and DV and SARS-CoV-2 clinical samples, as well as accurate identification of the four DV serotypes. Hence, the proposed colorimetric biosensing platform has great potential for rapid, accurate, and specific POC detection of viral infections in field-deployable assay.},
}

*Colorimetry/methods
*SARS-CoV-2/genetics/isolation & purification
Humans
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*Dengue Virus/genetics/isolation & purification
*COVID-19/diagnosis/virology
*Point-of-Care Systems
*RNA, Viral/analysis/genetics
G-Quadruplexes
Limit of Detection
Point-of-Care Testing
DNA, Catalytic/chemistry
Sensitivity and Specificity

@article {pmid41265249,
year = {2025},
author = {Papadopoulou, M and Ramachandran, H and Binder, S and Hildebrandt, B and Rossi, A and Krutmann, J},
title = {CRISPR/Cas9-mediated editing of XPA in induced pluripotent stem cells: A model for investigating Xeroderma Pigmentosum and NER dysfunction.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103869},
doi = {10.1016/j.scr.2025.103869},
pmid = {41265249},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *CRISPR-Cas Systems/genetics ; *Xeroderma Pigmentosum/genetics/pathology/metabolism ; *Xeroderma Pigmentosum Group A Protein/genetics/metabolism ; *DNA Repair/genetics ; *Gene Editing ; Cell Differentiation ; Cell Line ; },
abstract = {Xeroderma pigmentosum group A (XPA) is caused by defects in the nucleotide excision repair (NER) pathway, which is essential for repairing UV-induced DNA damage. Mutations in XPA impair lesion recognition and repair, resulting in mutation accumulation, genomic instability, and a high risk of skin cancers. In this study, we generated a CRISPR/Cas9-engineered human induced pluripotent stem cell (iPSC) line, WTSIi018-B-30, carrying a homozygous single nucleotide variant in exon 3 of XPA. The edited iPSCs retained normal morphology, expressed pluripotency markers, and differentiated into all three germ layers. This mutant iPSC line provides a robust isogenic model to dissect the molecular consequences of XPA deficiency and to explore therapeutic strategies for XPA-associated diseases.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*CRISPR-Cas Systems/genetics
*Xeroderma Pigmentosum/genetics/pathology/metabolism
*Xeroderma Pigmentosum Group A Protein/genetics/metabolism
*DNA Repair/genetics
*Gene Editing
Cell Differentiation
Cell Line

@article {pmid41240694,
year = {2026},
author = {Gao, Y and Dong, P and Lin, H and Tian, J},
title = {A circular crRNA-triggered CRISPR/Cas12a fluorescent platform for detection of myeloperoxidase activity.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {348},
number = {Pt 1},
pages = {127149},
doi = {10.1016/j.saa.2025.127149},
pmid = {41240694},
issn = {1873-3557},
mesh = {*Peroxidase/metabolism/analysis ; *CRISPR-Cas Systems/genetics ; Humans ; *RNA, Circular/chemistry ; Limit of Detection ; Spectrometry, Fluorescence/methods ; *CRISPR-Associated Proteins/metabolism ; *Biosensing Techniques/methods ; Animals ; Hypochlorous Acid/metabolism ; *Enzyme Assays/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Myeloperoxidase (MPO) is a key enzymatic biomarker for the diagnosis and therapeutic monitoring of acute leukemia. Here, we developed a CRISPR/Cas12a-based fluorescent sensing platform using a circular phosphorothioate-modified crRNA (crRNA-PS) for selective MPO activity detection. In this system, MPO catalyzes hypochlorous acid (HClO) generation, which oxidizes and linearizes the circular crRNA-PS, activating Cas12a-mediated trans-cleavage of a fluorescent reporter. The assay exhibited high sensitivity with detection limits of 0.79 ng/mL for MPO and 0.21 μM for HClO, along with excellent selectivity against reactive oxygen/nitrogen species and biomolecular interferents. Reliable performance was achieved in 1 % fetal bovine serum with recoveries of 92.87-112.54 %. The sensor also maintained stable responses over 8 weeks, indicating strong structural integrity of the circular crRNA-PS. Furthermore, the system was applied for inhibitor screening, yielding IC50 values of 127.2 μM for SHA and 0.81 μM for 4-ABAH. This strategy provides a rapid, sensitive, and robust platform for MPO detection with promising clinical potential.},
}

*Peroxidase/metabolism/analysis
*CRISPR-Cas Systems/genetics
Humans
*RNA, Circular/chemistry
Limit of Detection
Spectrometry, Fluorescence/methods
*CRISPR-Associated Proteins/metabolism
*Biosensing Techniques/methods
Animals
Hypochlorous Acid/metabolism
*Enzyme Assays/methods
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41238027,
year = {2026},
author = {Zhang, Z and Tang, D and Dong, L and Hu, Y and Li, S and Wang, D and Wang, M and Wang, Q},
title = {Functional divergence of Msp porins in substrate uptake and stress resistance in Mycobacterium smegmatis.},
journal = {Microbial pathogenesis},
volume = {210},
number = {},
pages = {108185},
doi = {10.1016/j.micpath.2025.108185},
pmid = {41238027},
issn = {1096-1208},
mesh = {*Mycobacterium smegmatis/genetics/metabolism/growth & development/drug effects ; *Porins/genetics/metabolism ; *Stress, Physiological ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Anti-Bacterial Agents/pharmacology ; Mutation ; CRISPR-Cas Systems ; Amino Acids/metabolism ; Drug Resistance, Bacterial ; Muramidase/pharmacology ; Hydrogen-Ion Concentration ; *Bacterial Outer Membrane Proteins/genetics/metabolism ; Carbon/metabolism ; },
abstract = {Mycobacterium smegmatis acquires external nutrients primarily through its outer membrane porins, notably the Msp proteins. Although the bacterium encodes four Msp porins (MspA, MspB, MspC, and MspD), previous work has identified MspA as the most abundant and functionally dominant porin, while regarding others as backup porins sharing considerable functional redundancy with MspA; however, their specific physiological roles and transcriptional dynamics remain poorly characterized. To dissect the individual function of each porin, we constructed a precise M. smegmatis Msp quadruple mutant using CRISPR-Cas9, and subsequently generated a panel of isogenic strains expressing individual porins at controlled levels. These strains were assessed in growth assays for their capacity to utilize various nutrients, including carbon sources, amino acids, and ions, as well as for their resistance to antibiotics, low pH, and lysozyme. We also employed Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) to examine the transcriptional responses of each msp gene in the wild-type strain under the tested conditions. Our results demonstrated distinct substrate preferences among the four Msp porins and revealed their differential contributions to resistance against environmental stressors; the transcriptional changes of each Msp porin in the wild-type strain correlated well with their expected roles. Furthermore, the engineered M. smegmatis quadruple mutant provides a robust platform for investigating the functions of other channel proteins in mycobacteria.},
}

*Mycobacterium smegmatis/genetics/metabolism/growth & development/drug effects
*Porins/genetics/metabolism
*Stress, Physiological
Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
Anti-Bacterial Agents/pharmacology
Mutation
CRISPR-Cas Systems
Amino Acids/metabolism
Drug Resistance, Bacterial
Muramidase/pharmacology
Hydrogen-Ion Concentration
*Bacterial Outer Membrane Proteins/genetics/metabolism
Carbon/metabolism

@article {pmid41207276,
year = {2026},
author = {Luo, N and Li, X and Tian, S and Li, X and Zou, L},
title = {Label-free fluorescent aptasensor for sensitive detection of oxytetracycline based on CRISPR/Cas12a and G-quadruplex DNAzyme with rhodamine B reporting.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {348},
number = {Pt 2},
pages = {127141},
doi = {10.1016/j.saa.2025.127141},
pmid = {41207276},
issn = {1873-3557},
mesh = {*Rhodamines/chemistry ; *Oxytetracycline/analysis ; *G-Quadruplexes ; *Aptamers, Nucleotide/chemistry ; Limit of Detection ; *Biosensing Techniques/methods ; *DNA, Catalytic/chemistry/metabolism ; Milk/chemistry ; *CRISPR-Cas Systems ; Spectrometry, Fluorescence/methods ; Animals ; Fluorescent Dyes/chemistry ; *Anti-Bacterial Agents/analysis ; *Endodeoxyribonucleases/metabolism ; Rivers/chemistry ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; },
abstract = {Oxytetracycline (OTC), a widely used broad-spectrum antibiotic, frequently persists in food and the environment due to overuse, posing risks of resistance, allergy, and toxicity. The World Health Organization has set strict limits for OTC residues, highlighting the need for rapid and reliable detection. Conventional assays, though accurate, remain costly and technically demanding, underscoring demand for simpler alternatives. We report a label-free fluorescent aptasensor integrating CRISPR/Cas12a with G-quadruplex (G4) DNAzyme catalysis, using rhodamine B (RhB) as a reporter. In the absence of OTC, the aptamer binds the activator strand, allowing G4 DNAzyme formation that quenches RhB fluorescence. OTC binding liberates the activator, triggering Cas12a cleavage of G4 DNA, thereby preventing DNAzyme formation and preserving fluorescence. This switch provides efficient signal amplification with low background. The assay achieved a detection limit of 0.3 nM and enabled accurate quantification of OTC in river water and milk samples. This platform offers a cost-effective, label-free strategy for antibiotic residue monitoring, eliminating dependence on nanomaterials or external labeling. Its validated sensitivity and robustness in real samples underscore its potential for practical applications in food safety and environmental surveillance.},
}

*Rhodamines/chemistry
*Oxytetracycline/analysis
*G-Quadruplexes
*Aptamers, Nucleotide/chemistry
Limit of Detection
*Biosensing Techniques/methods
*DNA, Catalytic/chemistry/metabolism
Milk/chemistry
*CRISPR-Cas Systems
Spectrometry, Fluorescence/methods
Animals
Fluorescent Dyes/chemistry
*Anti-Bacterial Agents/analysis
*Endodeoxyribonucleases/metabolism
Rivers/chemistry
*CRISPR-Associated Proteins/metabolism
Bacterial Proteins

@article {pmid41192264,
year = {2025},
author = {Raabe, J and Fuchs, S and Augustin, C and Hammerschmidt, A and Krämer, E and Orthey, E and Ehler, E and Cuello, F},
title = {Generation of a NRAP-overexpressing mutant from a human iPSC line.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103863},
doi = {10.1016/j.scr.2025.103863},
pmid = {41192264},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Myocytes, Cardiac/metabolism/cytology ; Cell Differentiation ; Cell Line ; *Mutation ; CRISPR-Cas Systems ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; },
abstract = {Cardiomyopathies are a major contributor to cardiovascular mortality and are frequently linked to abnormalities in intercalated discs, which coordinate mechanical and electrical signaling between cardiomyocytes. The Nebulin-Related Anchoring Protein (NRAP), a key component of these structures, is essential for myofibril formation and force transmission. In various cardiac diseases such as cardiomyopathies with differing genetic mutations, NRAP protein abundance is increased, yet the functional consequences of this expression change remain insufficiently characterized. To investigate the outcome of NRAP-overexpression (NRAP-OE) on cardiac development and disease, we established a human induced pluripotent stem cell (hiPSC) line with stable and specific NRAP-OE in cardiomyocytes using CRISPR-Cas9-based genome editing. The resulting line was rigorously validated for chromosomal integrity, pluripotency markers, absence of off-target effects and mycoplasma contamination, as well as its capacity for trilineage differentiation. This NRAP-OE model offers a novel platform for investigating how increased NRAP levels influence cardiomyocyte structure and function, and may provide insight into its role in the pathogenesis of cardiomyopathy.},
}

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

@article {pmid41187652,
year = {2025},
author = {Masser-Mitchell, BE and McLoughlin, HS},
title = {Generation of an ITPR1[+/-] and isogenic control induced pluripotent stem cell line for SCA15/16 model development.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103858},
doi = {10.1016/j.scr.2025.103858},
pmid = {41187652},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Spinocerebellar Ataxias/pathology/genetics/metabolism ; *Inositol 1,4,5-Trisphosphate Receptors/genetics/metabolism ; Cell Line ; Cell Differentiation ; CRISPR-Cas Systems ; },
abstract = {Spinocerebellar ataxia type 15/16 (SCA15/16) is a rare neurodegenerative disorder caused by heterozygous deletions of ITPR1, leading to haploinsufficiency of the encoded endoplasmic reticulum membrane calcium channel. Patients present with progressive gait disturbances, abnormal eye movements, difficulties with speech and swallowing, and tremors associated with atrophy of the cerebellum. Using CRISPR/Cas9 technology, we generated ITPR1[+/-] and isogenic control induced pluripotent stem cell (iPSC) lines from PGP1 iPSCs for SCA15/16 model development. The clones were genotyped, karyotyped, and assessed for pluripotency and differentiation potential.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*Spinocerebellar Ataxias/pathology/genetics/metabolism
*Inositol 1,4,5-Trisphosphate Receptors/genetics/metabolism
Cell Line
Cell Differentiation
CRISPR-Cas Systems

@article {pmid41167048,
year = {2025},
author = {Wei, S and Ma, R and Zhang, T and Wen, L},
title = {Targeted large-fragment genomic deletion in human pluripotent stem cells (hPSCs) via CRISPR/Cas9.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103859},
doi = {10.1016/j.scr.2025.103859},
pmid = {41167048},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Large genomic deletions are increasingly recognized as contributors to human disease. Here, we describe a CRISPR/Cas9-based protocol to induce targeted large-fragment deletions in human pluripotent stem cells (hPSCs). Two sgRNAs flanking the target region were designed, synthesized, and co-delivered with Cas9 protein into single-cell hPSCs via electroporation. Deletions were identified using junction PCR. Successfully edited cell pools were expanded as single-cell colonies and assessed for genomic deletion, stem cell identity, karyotype integrity, and tri-lineage differentiation capacity. This approach provides a robust method for modeling genomic deletions in hPSCs for disease research and functional genomics.},
}

Humans
*CRISPR-Cas Systems/genetics
*Pluripotent Stem Cells/metabolism/cytology
Cell Differentiation
Gene Editing
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41138562,
year = {2025},
author = {Marteau, S and Duboscq-Bidot, L and Aizawa, T and Hamlin, M and Villard, E and Guicheney, P and Fontaine, V},
title = {Generation of a FAM189A2/ENTREP1 knockout human induced pluripotent stem cell line using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103857},
doi = {10.1016/j.scr.2025.103857},
pmid = {41138562},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Membrane Proteins/genetics/metabolism ; *Gene Knockout Techniques ; },
abstract = {FAM189A2/ENTREP1 encodes a transmembrane protein, EREP1, ubiquitously expressed, with higher levels in thyroid gland, skeletal muscle, heart and glial cells, suggesting tissue-specific roles. It is regulated in various cancers and genetic studies suggested that it could be associated with cardiac and cardiometabolic diseases. EREP1 was reported to activate ITCH, an E3 ubiquitin ligase, involved in endosomal dynamics and protein trafficking. We generated by CRISPR/Cas9 technology a human induced pluripotent stem cells (hiPSCs) line carrying two compound heterozygous deletions leading to gene invalidation, with the aim of further exploring the poorly understood function of this protein.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
Cell Line
*Membrane Proteins/genetics/metabolism
*Gene Knockout Techniques

@article {pmid41067151,
year = {2025},
author = {Li, C and Meng, C and Wang, X and Yuan, C and Chen, Q and Yang, Y and Zhang, Z and Duan, Y and Dong, Q and Sun, X},
title = {CRISPR/Cas9-targeted mutagenesis of the PpTCP4 gene increased tiller number in Poa pratensis.},
journal = {Plant physiology and biochemistry : PPB},
volume = {229},
number = {Pt C},
pages = {110588},
doi = {10.1016/j.plaphy.2025.110588},
pmid = {41067151},
issn = {1873-2690},
mesh = {*CRISPR-Cas Systems/genetics ; *Poa/genetics/growth & development ; *Mutagenesis ; Gene Editing ; Plants, Genetically Modified/genetics ; *Plant Proteins/genetics/metabolism ; *Genes, Plant ; },
abstract = {Poa pratensis, commonly known as Kentucky bluegrass, is a widely used cool-season grass species for turf in lawns and recreational areas worldwide. However, the lack of genome editing platforms has impeded research into enhancing forage crops suitable for land unsuitable for other crops. In this study, we successfully established an Agrobacterium-mediated genetic transformation system for P. pratensis. Furthermore, we successfully produced PpTCP4-edited plants using the CRISPR/Cas9 system. This research represents a significant advancement in the genetic transformation and gene editing of P. pratensis, providing a foundational technology for future investigations into gene function and breeding in P. pratensis.},
}

*CRISPR-Cas Systems/genetics
*Poa/genetics/growth & development
*Mutagenesis
Gene Editing
Plants, Genetically Modified/genetics
*Plant Proteins/genetics/metabolism
*Genes, Plant

@article {pmid41045726,
year = {2025},
author = {Rhode, J and Hagenau, L and Edwards, S and Buettner, FFR and Tzvetkova, A and Jensen, LR and Kuss, AW},
title = {Three modified human IPSC lines containing mutations in the distal DEHMBA associated locus of the SRCAP gene.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103847},
doi = {10.1016/j.scr.2025.103847},
pmid = {41045726},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; *Mutation/genetics ; CRISPR-Cas Systems ; Cell Differentiation ; Frameshift Mutation ; },
abstract = {We modified an existing human iPSC line (MHHi001-A), using CRISPR/Cas9, to introduce heterozygous frameshift mutations in a locus of the SRCAP gene that is associated with the DEHMBA disease (OMIM 619595). The modified iPSCs express several stem cell markers and are able to differentiate into cells originating from all three embryonic germ layers. No additional modifications or chromosomal defects were detected. The modified cells can serve as a model for the investigation of the involvement of SRCAP in DEHMBA (Developmental delay, Hypotonia, Musculoskeletal defects, and Behavioral Abnormalities) disease and/or its molecular functions in different cell types.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
Cell Line
*Mutation/genetics
CRISPR-Cas Systems
Cell Differentiation
Frameshift Mutation

@article {pmid41016181,
year = {2025},
author = {Shibuya, K and Nomizu, T and Morimoto, H and Satou, K},
title = {CRISPR/Cas9-mediated LhNAP mutagenesis extends flower longevity in lily.},
journal = {Plant physiology and biochemistry : PPB},
volume = {229},
number = {Pt C},
pages = {110551},
doi = {10.1016/j.plaphy.2025.110551},
pmid = {41016181},
issn = {1873-2690},
mesh = {*Flowers/genetics/physiology ; *CRISPR-Cas Systems/genetics ; *Lilium/genetics/physiology ; *Plant Proteins/genetics/metabolism ; *Mutagenesis ; Plants, Genetically Modified ; *Transcription Factors/genetics/metabolism ; Gene Editing ; Oryza/genetics ; },
abstract = {Flower longevity is an important trait determining the ornamental plant quality. NAM/ATAF1,2/CUC2 (NAC) transcription factors regulate leaf and petal senescence in several plants. To extend the longevity of lily flowers, in this study we used the CRISPR/Cas9 technology for the targeted mutagenesis of LhNAP encoding the NAC transcription factor in the Oriental hybrid lily (Lilium spp.) 'Acapulco'. Filament-derived calli were transformed with binary vectors containing guide RNA expression cassettes targeting one or two regions of LhNAP and the codon-optimized Cas9 for Oryza sativa driven by Zea mays ubiquitin promoter. Mutant lines harbouring biallelic mutations at the LhNAP locus exhibited clearly delayed tepal senescence and abscission compared to the wild-type plants. Overall, our results highlight the potential of LhNAP-targeted genome editing to produce lilies with extended flower longevity and reveal the crucial role of LhNAP in regulating tepal senescence and abscission in lilies.},
}

*Flowers/genetics/physiology
*CRISPR-Cas Systems/genetics
*Lilium/genetics/physiology
*Plant Proteins/genetics/metabolism
*Mutagenesis
Plants, Genetically Modified
*Transcription Factors/genetics/metabolism
Gene Editing
Oryza/genetics

@article {pmid40976167,
year = {2025},
author = {Park, S and Lee, H and Song, J and Kim, EH and Lim, CJ and Oh, J and Lee, SB and Kim, JA and Kim, BG},
title = {Redirecting flavonoid flux in purple Chinese cabbage via Cas9-mediated BrDFR knockout.},
journal = {Plant physiology and biochemistry : PPB},
volume = {229},
number = {Pt B},
pages = {110534},
doi = {10.1016/j.plaphy.2025.110534},
pmid = {40976167},
issn = {1873-2690},
mesh = {*Flavonoids/metabolism/biosynthesis ; *Alcohol Oxidoreductases/genetics/metabolism ; Gene Knockout Techniques ; *CRISPR-Cas Systems ; Anthocyanins/metabolism/biosynthesis ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; *Brassica/metabolism/genetics ; *Brassica rapa/genetics/metabolism ; },
abstract = {Purple varieties of Chinese cabbage (Brassica rapa subsp. pekinensis) predominantly accumulate cyanidin-based anthocyanins. Although dihydroflavonol 4-reductase (DFR) is a key enzyme in anthocyanin biosynthesis, the function of the putative B. rapa DFR gene (Bra027457) remained unverified. We isolated and sequenced the coding region of Bra027457 from four B. rapa inbred lines with either green or purple phenotypes and detected no sequence variation. Bra027457 expression correlated with anthocyanin accumulation, and in vitro assays confirmed its ability to reduce all three dihydroflavonol substrates. Using CRISPR/Cas9, we knocked out Bra027457 in the purple line 8267 and obtained transgene-free, homozygous BrDFR-KO plants. These exhibited a green phenotype due to complete anthocyanin loss, verifying Bra027457 as the authentic BrDFR gene. Metabolite profiling of BrDFR-KO heads revealed significant increases in quercetin (Q), isorhamnetin (IR), and dihydroquercetin (DHQ). LC/MS analysis further identified five flavonol glycosides and one DHQ glycoside, of whch Q 3,7-di-O-glucoside and IR 3-O-(2‴-O-feruloyl)sophoroside-7-O-glucoside were predominant. These findings advance our understanding of flavonoid biosynthesis in Brassica species and provide valuable genetic resources for Chinese cabbage improvement.},
}

*Flavonoids/metabolism/biosynthesis
*Alcohol Oxidoreductases/genetics/metabolism
Gene Knockout Techniques
*CRISPR-Cas Systems
Anthocyanins/metabolism/biosynthesis
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Gene Expression Regulation, Plant
*Brassica/metabolism/genetics
*Brassica rapa/genetics/metabolism

@article {pmid40857358,
year = {2025},
author = {Sharma, A and Locatelli, F and Bhatia, M and Molinari, L and Mapara, MY and Liem, RI and Dedeken, L and Wall, D and Eckrich, MJ and Kuo, KHM and Smith, W and Imren, S and Kohli, P and Li, N and Liu, T and Rubin, J and Hobbs, W and Grupp, SA and Frangoul, H},
title = {Improvements in health-related quality of life in patients with severe sickle cell disease after exagamglogene autotemcel.},
journal = {Blood advances},
volume = {9},
number = {24},
pages = {6481-6490},
doi = {10.1182/bloodadvances.2025016701},
pmid = {40857358},
issn = {2473-9537},
mesh = {Humans ; *Anemia, Sickle Cell/therapy ; *Quality of Life ; Adolescent ; Male ; Female ; Adult ; Young Adult ; Patient Reported Outcome Measures ; Severity of Illness Index ; Gene Editing ; Treatment Outcome ; CRISPR-Cas Systems ; *Genetic Therapy/methods ; Hematopoietic Stem Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Severe sickle cell disease (SCD), an inherited blood disorder characterized by recurrent vaso-occlusive crises (VOCs), significantly impairs health-related quality of life (HRQoL). Exagamglogene autotemcel (exa-cel), a one-time, ex vivo CRISPR/Cas9 gene-edited cell therapy, eliminated VOCs in 97% of participants with severe SCD in the phase 3 CLIMB SCD-121 trial. Here, we describe changes in patient-reported outcomes (PROs) in 30 adults and 12 adolescents with ≥16 months follow-up. Adult PROs include EuroQol Quality of Life Scale-5 dimensions-5 levels of severity (EQ-5D-5L), Functional Assessment of Cancer Therapy Bone Marrow Transplant (FACT-BMT), Adult Sickle Cell Quality of Life Measurement Information System (ASCQ-Me), and 11-point pain Numerical Rating Scale (NRS). In adolescents, EuroQol Quality of Life Scale-5 dimensions-youth (EQ-5D-Y), Pediatric Quality of Life Inventory (PedsQL), and pain NRS were used. Adult EQ-5D-5L US and UK healthy utility index and EQ visual analog scale (VAS) scores, which were lower than general population norm at baseline, substantially improved by month 6 after exa-cel infusion and were sustained through month 36, with similar improvements in FACT-General (FACT-G) total and bone marrow transplant subscale. All FACT-G (physical, social/family, emotional, functional well-being) and ASCQ-Me (emotional, social functioning, stiffness, sleep impact) subscales showed clinically meaningful improvement, including ASCQ-Me pain subscales (impact, episode frequency, severity), with substantial decreases in pain episode frequency by month 6. Mean pain NRS score decreased by month 6 and was sustained. Consistent with adults, adolescents had improvements in mean EQ-5D-Y VAS score, PedsQL score, and pain NRS. Exa-cel led to broad and clinically meaningful HRQoL benefits in adults and adolescents with SCD. The CLIMB SCD-121 and CLIMB-131 trials were registered at www.clinicaltrials.gov as #NCT03745287 and #NCT04208529, respectively.},
}

Humans
*Anemia, Sickle Cell/therapy
*Quality of Life
Adolescent
Male
Female
Adult
Young Adult
Patient Reported Outcome Measures
Severity of Illness Index
Gene Editing
Treatment Outcome
CRISPR-Cas Systems
*Genetic Therapy/methods
Hematopoietic Stem Cells
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41372121,
year = {2025},
author = {Cheng, KW and Bhave, M and Markhard, AL and Peng, D and Bhatt, KD and Travisano, KA and Medicielo, JV and Anaya, A and Lembirik, S and Njoya, L and Anantpadma, M and Kuhn, JH and Puschnik, AS and Kistler, AL},
title = {Replicon-based genome-wide CRISPR knockout screening for the identification of host factors involved in viral replication.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11028},
pmid = {41372121},
issn = {2041-1723},
mesh = {*Virus Replication/genetics ; Humans ; *Replicon/genetics ; *Dengue Virus/genetics/physiology ; Chikungunya virus/genetics/physiology ; *CRISPR-Cas Systems ; Gene Knockout Techniques/methods ; Ebolavirus/genetics/physiology ; Cell Line ; Animals ; *Host-Pathogen Interactions/genetics ; Membrane Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; HEK293 Cells ; Hexosyltransferases/genetics/metabolism ; },
abstract = {We describe a viral replicon-based CRISPR knockout (KO) screening approach to specifically identify host factors essential for viral replication which are often missed in live virus screens. We benchmark the replicon screening using a stable fluorescent dengue virus type 2 (DENV-2) replicon cell line and successfully identify host genes known to be required for viral DENV-2 replication (e.g., endoplasmic reticulum membrane complex and oligosaccharyltransferase complex components), along with additional genes that have not been reported in prior CRISPR KO screens with DENV-2. We extend this replicon screening approach to chikungunya virus (CHIKV), a positive-sense RNA virus, and Ebola virus (EBOV), a negative-sense RNA virus, and identify distinct sets of genes required for replication of each virus. Our findings indicate that viral replicon-based CRISPR screens are a useful approach to identify host factors essential for replication of diverse viruses and to elucidate potential novel targets for host-directed medical countermeasures.},
}

*Virus Replication/genetics
Humans
*Replicon/genetics
*Dengue Virus/genetics/physiology
Chikungunya virus/genetics/physiology
*CRISPR-Cas Systems
Gene Knockout Techniques/methods
Ebolavirus/genetics/physiology
Cell Line
Animals
*Host-Pathogen Interactions/genetics
Membrane Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
HEK293 Cells
Hexosyltransferases/genetics/metabolism

@article {pmid41371329,
year = {2025},
author = {Chen, B and Gao, J and Sun, H and Zhao, Y and Liu, Y and Qiu, X and Li, Y},
title = {Integrating CRISPR with SERS: Toward intelligent point-of-care diagnostics of the future.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {581},
number = {},
pages = {120782},
doi = {10.1016/j.cca.2025.120782},
pmid = {41371329},
issn = {1873-3492},
abstract = {In recent years, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas) system has emerged as a transformative genome-editing platform. Beyond its editing applications, the CRISPR/Cas system has attracted growing interest in molecular diagnostics particularly for nucleic acid detection due to its exceptional sensitivity and target specificity. Meanwhile, surface-enhanced Raman spectroscopy (SERS), which relies on plasmonic nanoparticles or nanostructures, has become a powerful biosensing technology known for its high sensitivity and distinct spectral fingerprinting capability. The integration of CRISPR/Cas-mediated molecular recognition with the ultrasensitive detection of SERS offers a rapid, low-volume, and direct strategy for identifying diverse nucleic acid targets. This synergistic combination has inspired the development of innovative biosensing platforms designed for ultrasensitive and precise molecular diagnostics. In this review, we first outline the fundamental principles of CRISPR/Cas and SERS, then summarize their hybrid applications in nucleic acid detection. Finally, we discuss the current progress, challenges, and future perspectives of CRISPR/Cas-integrated SERS biosensing.},
}

@article {pmid41371153,
year = {2025},
author = {Longhi Cervantes, DS and Leal, GM and Fortirer, JDS and de Oliveira, LF and Navarro, BV and Buckeridge, MS},
title = {microRNAs and stress adaptation in grasses: A systematic review.},
journal = {Plant physiology and biochemistry : PPB},
volume = {230},
number = {},
pages = {110783},
doi = {10.1016/j.plaphy.2025.110783},
pmid = {41371153},
issn = {1873-2690},
abstract = {MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression after transcription, playing crucial roles in plant development and stress adaptation. In grasses, this regulation is vital under isolated biotic and abiotic stress conditions and combined stress scenarios, although many regulatory modules remain unexplored. This systematic review examined 60 studies out of 1823 publications indexed in Scopus, focusing on grass miRNAs with validated targets through Degradome-Seq and/or RACE approaches. Results indicate that miRNA-target modules were validated more often under abiotic stress than biotic or combined stress conditions. The most frequently studied miRNA families include miR156, miR159, miR164, miR169, and miR396, which are commonly linked to various types of stress, whether isolated or combined. Most research has concentrated on major crops such as rice and maize, with limited studies on other agriculturally important grasses. This review highlights advances in miRNA-phytohormone interactions, systemic signaling, and target validation strategies. It also underscores the potential of biotechnological tools such as RNAi, artificial miRNAs, target mimicry, and CRISPR/Cas for engineering more resilient grasses. Integrating multi-omics approaches and an increasing focus on combined stress responses suggest promising strategies for sustainable agriculture, food security, and bioenergy production amidst climate challenges. Together, these advances strengthen the potential of microRNA-based regulation as a key tool for enhancing crop resilience and adaptation.},
}

@article {pmid41370200,
year = {2025},
author = {Shi, X and Hu, C and Jia, L and Lei, Z and Guo, B and Zhou, J and Wang, F},
title = {An SpC editor targeting pre-mRNA splicing for precise CRISPR control and enhanced antitumor efficacy.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
doi = {10.1093/nar/gkaf1328},
pmid = {41370200},
issn = {1362-4962},
support = {32271512//National Natural Science Foundation of China/ ; 82572281//National Natural Science Foundation of China/ ; 2022JC-56//Natural Science Basic Research Program of Shaanxi/ ; 2023-JC-ZD-43//Natural Science Basic Research Program of Shaanxi/ ; 2024JC-YBQN-0168//Natural Science Basic Research Program of Shaanxi/ ; 2023A1515110886//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA Precursors/genetics/metabolism ; *RNA Splicing/drug effects ; Animals ; Mice ; Cell Line, Tumor ; Spliceosomes/genetics/metabolism ; Macrolides/pharmacology ; Apoptosis/genetics ; Neoplasms/genetics/therapy ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epoxy Compounds ; },
abstract = {The CRISPR/Cas9 system is a powerful genome editing tool that has the potential to be applied to a variety of biomedical applications. Despite the considerable potential of this gene editing technology, there are numerous safety concerns including the possibility of unpredictable off-target effects. The splicing process, which involves the removal of introns from pre-mRNA and the alignment of exons to produce mature transcripts, is a critical step in gene expression in most eukaryotes. In this study, we present a spliceosome-responsive CRISPR/Cas9 (SpC) editor that utilizes the splicing inhibitor pladienolide B (PB) to regulate pre-mRNA splicing and control the expression of the anti-CRISPR protein AcrIIA4, thereby modulating the activity of the Cas9 nuclease. This approach allows for precise regulation of the gene editing process, thereby effectively mitigating off-target effects. The reliability and robustness of the SpC editor were demonstrated through in vitro and in vivo bioluminescence imaging. Furthermore, a dual-target sgRNA was designed to target the diphtheria toxin A gene, resulting in apoptosis induction and growth inhibition of tumor cells across various types of cancer cells. Our results indicate that this SpC editor has the capacity to precisely regulate tumor cell growth, thus providing new insights and significant implications for cancer gene therapy.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*RNA Precursors/genetics/metabolism
*RNA Splicing/drug effects
Animals
Mice
Cell Line, Tumor
Spliceosomes/genetics/metabolism
Macrolides/pharmacology
Apoptosis/genetics
Neoplasms/genetics/therapy
RNA, Guide, CRISPR-Cas Systems/genetics
Epoxy Compounds

@article {pmid41369371,
year = {2025},
author = {Gardner-Kay, A and Le, L and Filla, M and Kibiryeva, N and O'Brien, JE and Bittel, DC},
title = {CRISPR Disruption of scaRNA1 Reduces Pseudouridylation in Spliceosomal RNA U2 at U89 and Perturbs the Transcriptome in HEK293T Cells.},
journal = {Cells},
volume = {14},
number = {23},
pages = {},
pmid = {41369371},
issn = {2073-4409},
support = {Intramural Faculty grant//Kansas City University/ ; },
mesh = {Humans ; HEK293 Cells ; *Spliceosomes/metabolism/genetics ; *Transcriptome/genetics ; *Pseudouridine/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Small Nuclear/metabolism/genetics ; RNA Splicing/genetics ; },
abstract = {Small Cajal body-associated RNAs (scaRNAs) are essential for biochemical modification of spliceosomal RNAs and spliceosome function. Changes in scaRNA expression level have been associated with developmental issues, including cancer and congenital heart defects (CHDs), although the mechanism remains unclear. Small Cajal body-associated RNA 1 (scaRNA1) guides pseudouridylation at uridine 89 (Ψ89) of the spliceosomal RNA U2, a highly conserved modification that may be critical for spliceosome function. To investigate the role of scaRNA1 in splicing regulation, CRISPR-Cas9 genome editing was used to introduce targeted deletions in the scaRNA1 locus in HEK293T cells. Edited clones were identified by T7 endonuclease I assay and confirmed by Sanger sequencing. Pseudouridylation at Ψ89 was quantified using CMC-based reverse transcription followed by quantitative PCR, and global mRNA splicing alterations were assessed by RNA sequencing. Clones harboring scaRNA1 disruptions exhibited a significant reduction in Ψ89 pseudouridylation, consistent with impaired scaRNA1 function. Transcriptome analysis (of mRNA from two clones) revealed >300 protein coding genes with significant changes in transcript isoform level, including >100 genes related to RNA-binding activity. These results indicate that scaRNA1 disruption alters spliceosomal function and leads to substantial changes in mRNA splicing. The dysregulated splicing of RNA-binding proteins may impair RNA processing and gene expression programs required for normal development, providing new insight into how noncoding RNA dysfunction may contribute to developmental pathogenesis.},
}

Humans
HEK293 Cells
*Spliceosomes/metabolism/genetics
*Transcriptome/genetics
*Pseudouridine/metabolism/genetics
*CRISPR-Cas Systems/genetics
*RNA, Small Nuclear/metabolism/genetics
RNA Splicing/genetics

@article {pmid41369349,
year = {2025},
author = {Cheng, X and Wang, D and Zhang, X and Li, L and Liu, Y and Cao, G and Zhang, Y},
title = {Regulation of the Homeostasis of Early Embryo Development in Dairy Cows by Targeted Editing of the PRLR Gene-Mediated Activation of the Anti-Heat Stress Pathway.},
journal = {Cells},
volume = {14},
number = {23},
pages = {},
pmid = {41369349},
issn = {2073-4409},
support = {No. 2023ZD04050//Guifang Cao/ ; },
mesh = {Animals ; Cattle ; *Gene Editing/methods ; *Heat-Shock Response/genetics ; *Homeostasis/genetics ; *Embryonic Development/genetics ; *Receptors, Prolactin/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Female ; Reactive Oxygen Species/metabolism ; Fibroblasts/metabolism ; Nuclear Transfer Techniques ; Oocytes/metabolism ; },
abstract = {The intensification of global climate warming exacerbates the issue of heat stress in dairy cows, making the SLICK mutation in the prolactin receptor (PRLR) gene a critical target for enhancing heat tolerance in these animals. This study aims to investigate the effects of CRISPR/Cas9-mediated editing of the PRLR gene on the biological characteristics of bovine fibroblasts and early embryonic development following somatic cell nuclear transfer (SCNT). Using the CRISPR/Cas9 system, we targeted and edited a 20 bp-150 bp region within exon nine of the PRLR gene. After conducting off-target predictions and activity screenings, we identified optimal guide RNA (sgRNA) sequences and established stable transgenic cell lines. Transcriptome sequencing was performed on edited cells to identify key genes and validate their expression profiles. Edited cells were utilized as donor cells for SCNT, during which we assessed oocyte levels of reactive oxygen species (ROS), glutathione (GSH), and mitochondrial function to analyze embryonic developmental performance. We constructed a cellular stress resistance network aimed at mitigating damage transmission while maintaining embryonic developmental homeostasis. This research provides technical support and theoretical reference for genetic editing breeding programs aimed at improving heat tolerance in dairy cattle.},
}

Animals
Cattle
*Gene Editing/methods
*Heat-Shock Response/genetics
*Homeostasis/genetics
*Embryonic Development/genetics
*Receptors, Prolactin/genetics/metabolism
CRISPR-Cas Systems/genetics
Female
Reactive Oxygen Species/metabolism
Fibroblasts/metabolism
Nuclear Transfer Techniques
Oocytes/metabolism

@article {pmid41367214,
year = {2025},
author = {Selokar, NL and Singh, P and Jose, B and Gautam, D and Patel, K and Verma, R and De, S and Singh, MK and Singh, D},
title = {A Myostatin (MSTN[-/-]) Knockout Buffalo Produced by CRISPR-Cas9 Mediated Genome Editing and Somatic Cell Nuclear Transfer.},
journal = {The CRISPR journal},
volume = {8},
number = {6},
pages = {436-442},
doi = {10.1177/25731599251401528},
pmid = {41367214},
issn = {2573-1602},
mesh = {Animals ; *Myostatin/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Buffaloes/genetics ; *Nuclear Transfer Techniques ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Fibroblasts/metabolism ; Female ; Animals, Genetically Modified/genetics ; },
abstract = {CRISPR-Cas9 genome editing offers significant opportunities to improve livestock traits; however, its application in buffalo has been very limited, with no prior reports of live gene-edited animals. Here, we report the successful birth of a buffalo edited in the myostatin (MSTN) gene. To achieve this, five single-guide RNAs (sgRNAs) targeting the buffalo MSTN gene were designed and tested in skin-derived fibroblasts. Among these, sgRNA5 exhibited the highest editing efficiency, approaching ∼50%, as confirmed by T7 Endonuclease I assay, Tracking of Indels by Decomposition, and Inference of CRISPR Edits analyses. Single-cell cloning identified six edited fibroblast clonal populations, including one with a bi-allelic frameshift mutation predicted to severely truncate the MSTN protein. These bi-allelic clonal cells were subsequently used as nuclear donors to produce somatic cell nuclear transfer (SCNT) embryos, which were transferred into recipient buffaloes (n = 15). This effort established three pregnancies and resulted in the birth of one live MSTN knockout buffalo calf. Phenotypically, the calf displayed accelerated growth and increased muscle fiber number and size while maintaining normal meat composition. In conclusion, this study reports the world's first gene-edited buffalo generated through CRISPR-Cas9-mediated genome editing combined with SCNT. These findings provide a proof-of-concept for genome editing in buffalo and demonstrate that MSTN disruption can effectively enhance muscle growth and meat production traits.},
}

Animals
*Myostatin/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Buffaloes/genetics
*Nuclear Transfer Techniques
*Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Fibroblasts/metabolism
Female
Animals, Genetically Modified/genetics

@article {pmid41367203,
year = {2025},
author = {Patel, J and Patel, D and Raval, A},
title = {Artificial Intelligence for Predictive Modeling in CRISPR/Cas9 Gene Editing: a Survey of Methods and Design Strategies.},
journal = {The journal of gene medicine},
volume = {27},
number = {12},
pages = {e70061},
doi = {10.1002/jgm.70061},
pmid = {41367203},
issn = {1521-2254},
mesh = {*CRISPR-Cas Systems ; *Artificial Intelligence ; *Gene Editing/methods ; Humans ; Machine Learning ; Algorithms ; },
abstract = {Ongoing developments in genome editing most notably the continued evolution of CRISPR-Cas systems and their orthogonal or modified counterparts have substantively altered both experimental and applied practices in biomedicine, agriculture, and therapeutic design. More recently, the systematic incorporation of artificial intelligence and machine learning methodologies has augmented the specificity, throughput, and explanatory capacity of genome-editing workflows, thereby refining the prediction of on-target efficiencies, the appraisal of off-target liabilities, and the tailoring of molecular therapeutic configurations. The present contribution offers an integrative survey of these computational developments, emphasizing (i) predictive algorithms, (ii) machine-learning and deep-learning frameworks, (iii) data-centric procedural strategies, and (iv) dedicated applications in oncology, neurology, rare-disease research, and precision-medicine contexts. Throughout, we evaluate architectural choices, sequence-encoding representations, and lingering dataset-related biases, while additionally addressing current constraints concerning model interpretability, ethical viability, and the procedural prerequisites for clinical translation. Moreover, we advance a structured taxonomy that organizes AI-mediated genome-editing approaches according to methodological lineage and functional scope, and we delineate extant research lacunae. By combining these elements, we supply a prospective assessment of the means by which artificial intelligence might be further leveraged to support secure, efficacious, and equitably accessible genome engineering outcomes.},
}

*CRISPR-Cas Systems
*Artificial Intelligence
*Gene Editing/methods
Humans
Machine Learning
Algorithms

@article {pmid41366257,
year = {2025},
author = {Launspach, M and Macos, J and Afzal, S and Hohmann, J and Appis, ML and Pilgram, M and Beez, S and Ohlendorf, E and van der Ven, CFT and Lachiheb, C and Töws, K and Andersch, L and Jens, M and Zirngibl, F and Kath, J and Stecklum, M and Rodriguez-Fos, E and Anders, K and Wagner, DL and Henssen, AG and Kühn, R and Eggert, A and Künkele, A},
title = {Personalized CRISPR knock-in cytokine gene therapy to remodel the tumor microenvironment and enhance CAR T cell therapy in solid tumors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10987},
pmid = {41366257},
issn = {2041-1723},
mesh = {*Tumor Microenvironment/genetics/immunology ; Humans ; Animals ; Gene Knock-In Techniques ; Mice ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *Genetic Therapy/methods ; Chemokine CXCL10/genetics ; *Cytokines/genetics ; Cell Line, Tumor ; *Neuroblastoma/therapy/immunology/genetics ; Receptors, Chimeric Antigen/genetics ; T-Lymphocytes/immunology ; Chemokine CXCL11/genetics ; *Neoplasms/therapy/immunology/genetics ; Interferon-gamma/genetics ; Precision Medicine ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; },
abstract = {The immunosuppressive tumour microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumours. We present a gene-therapeutic strategy that enables localized remodelling of the TME via tumour-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumour-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma as a model, CancerPAM analysis of tumour sequencing data identifies optimal knock-in sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG), and CancerPAM rankings correlate strongly with target-site specificity and knock-in efficiency, validating its predictive performance. CRISPR-mediated CXCL10 knock-in enhances CAR T cell infiltration and antitumour efficacy in vitro and in vivo, including humanized CD34[+] HuNOG mice, where CXCL10-expressing tumours show stronger immune infiltration and prolonged tumour control within a reconstituted human immune microenvironment. Our findings establish a framework for safe and effective CRISPR-based cytokine delivery, integrating localized TME remodelling with cellular immunotherapies to enhance CAR T cells and other treatments in immune-refractory solid tumours.},
}

*Tumor Microenvironment/genetics/immunology
Humans
Animals
Gene Knock-In Techniques
Mice
*CRISPR-Cas Systems
*Immunotherapy, Adoptive/methods
*Genetic Therapy/methods
Chemokine CXCL10/genetics
*Cytokines/genetics
Cell Line, Tumor
*Neuroblastoma/therapy/immunology/genetics
Receptors, Chimeric Antigen/genetics
T-Lymphocytes/immunology
Chemokine CXCL11/genetics
*Neoplasms/therapy/immunology/genetics
Interferon-gamma/genetics
Precision Medicine
Clustered Regularly Interspaced Short Palindromic Repeats
Female

@article {pmid41366133,
year = {2025},
author = {Bian, W and Mcquarrie, DWJ and Haussmann, IU and Arnold, R and Soller, M},
title = {Genetic evaluation of CRISPR-Cas9 off-target effects from deleterious mutations on Drosophila male single X chromosome.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {270},
pmid = {41366133},
issn = {1438-7948},
mesh = {Animals ; Male ; *CRISPR-Cas Systems ; *X Chromosome/genetics ; *Mutation ; Gene Editing/methods ; *Drosophila/genetics ; *Drosophila melanogaster/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease protein 9 (Cas9) is a powerful tool used for genome engineering, but concerns remain about off-target effects. Here we evaluate potential deleterious effects of CRISPR-Cas9 by combining sequence analysis and the genetics of the male X chromosome in a Drosophila model. Since males have only one X chromosome deleterious mutations on the X chromosome will manifest in reducing viability or result in visible phenotypes and thus provide sensitive readouts of off-target activity. Our data do not support large scale off-target effects in Drosophila. To optimize sgRNA selection, we incorporated off-target evaluation into the PlatinumCRISPr sgRNA selection tool for a broad range of organisms.},
}

Animals
Male
*CRISPR-Cas Systems
*X Chromosome/genetics
*Mutation
Gene Editing/methods
*Drosophila/genetics
*Drosophila melanogaster/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41365538,
year = {2025},
author = {Jung, SC and Oh, H and Eom, W and Jin, YS and Park, SH and Park, K and Koh, HG},
title = {Scarless Genetic Engineering of Saccharomyces cerevisiae for Enhanced Guanosine Monophosphate Production as a Natural Flavor Enhancer.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2508034},
doi = {10.4014/jmb.2508.08034},
pmid = {41365538},
issn = {1738-8872},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Metabolic Engineering/methods ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Fermentation ; *Guanosine Monophosphate/biosynthesis/metabolism ; *Flavoring Agents/metabolism ; *Genetic Engineering/methods ; Promoter Regions, Genetic ; Inosine Monophosphate/metabolism ; Pentose Phosphate Pathway/genetics ; },
abstract = {Saccharomyces cerevisiae and Cyberlindnera jadinii are widely utilized in the natural food seasoning industry as sources of flavor enhancing nucleotides such as inosine monophosphate (IMP) and guanosine monophosphate (GMP), which contribute to umami taste and support sodium reduction in food. However, wild type yeast strains produce GMP at levels that are inadequate for industrial scale applications, necessitating metabolic engineering strategies to increase production efficiency. This study employed a CRISPR-Cas9-based scarless genome engineering approach to enhance GMP biosynthesis in S. cerevisiae via promoter replacement. The key genes IMD3 and GUA1, responsible for converting IMP to GMP, were overexpressed to redirect purine flux toward GMP production. To address precursor limitations, ZWF1 and RKI1, involved in the pentose phosphate pathway, were also overexpressed. In parallel, the expression of STB5 and RAP1 was increased to enhance NADPH regeneration and relieve transcriptional bottlenecks. As a result, the final engineered strain SCJ-7 demonstrated a 1.77-fold increase in GMP titer and a 1.40-fold increase in GMP content during flask fermentation compared to the wild-type. In fed-batch fermentation, GMP titer was further improved by 27.6%. These findings demonstrate that combining metabolic flux enhancement with transcriptional regulation provides an effective and scalable strategy for boosting GMP production in S. cerevisiae, offering strong potential for industrial application in the food industry.},
}

*Saccharomyces cerevisiae/genetics/metabolism
*Metabolic Engineering/methods
Saccharomyces cerevisiae Proteins/genetics/metabolism
CRISPR-Cas Systems
Fermentation
*Guanosine Monophosphate/biosynthesis/metabolism
*Flavoring Agents/metabolism
*Genetic Engineering/methods
Promoter Regions, Genetic
Inosine Monophosphate/metabolism
Pentose Phosphate Pathway/genetics

@article {pmid41365534,
year = {2025},
author = {Skeate, JG and Lee, CJ and Stewart, C and Fischbach, MJ and Kar, B and Tsai, AK and Kenderian, SS and Stromnes, IM and Largaespada, DA and Moriarity, BS and Rogers, LM},
title = {Functional genomics for improving adoptive T-cell transfer therapies.},
journal = {Journal for immunotherapy of cancer},
volume = {13},
number = {12},
pages = {},
doi = {10.1136/jitc-2025-013286},
pmid = {41365534},
issn = {2051-1426},
mesh = {Humans ; *Genomics/methods ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/transplantation ; Gene Editing/methods ; Animals ; *Neoplasms/therapy/immunology/genetics ; CRISPR-Cas Systems ; },
abstract = {Adoptive cell therapy (ACT) has shown remarkable success in the treatment of some malignancies, particularly leukemia. However, there are multiple factors that limit the durability of ACT in solid tumors, including dose-limiting toxicities, the immunosuppressive tumor microenvironment, and T-cell exhaustion. As the manufacture and preparation of adoptive T-cell therapies allows time and adequate conditions for ex vivo T-cell engineering, forward genetic screens can identify novel genetic targets that could improve their effectiveness. CRISPR is a commonly used functional genomics tool that has been successfully used to both enhance our understanding of mechanisms of resistance and to discover potential genetic edits to improve ACT. A complementary approach, Sleeping Beauty transposon mutagenesis provides additional opportunities to identify novel genetic edits without being constrained by the annotated human genome. Here, we summarize forward genetic screens and their tools to uncover strategies to enhance ACT. Complementary approaches can be combined and improved on to identify translatable genetic editing strategies through studies that accurately recapitulate disease-specific challenges.},
}

Humans
*Genomics/methods
*Immunotherapy, Adoptive/methods
*T-Lymphocytes/immunology/transplantation
Gene Editing/methods
Animals
*Neoplasms/therapy/immunology/genetics
CRISPR-Cas Systems

@article {pmid41242662,
year = {2026},
author = {Zhang, N and Zhou, X and Jiao, X and Liang, Z and Jiang, W and Liu, S and Wu, P},
title = {Field-deployable CRISPR-Dx for BmNPV and Nosema bombycis: DNA-extraction-free one-pot RPA-Cas12a and Cas12a/Cas13a dual-gene assays with handheld devices.},
journal = {Insect biochemistry and molecular biology},
volume = {186},
number = {},
pages = {104449},
doi = {10.1016/j.ibmb.2025.104449},
pmid = {41242662},
issn = {1879-0240},
mesh = {Animals ; *Nosema/isolation & purification/genetics ; *Nucleopolyhedroviruses/isolation & purification/genetics ; *Bombyx/virology/microbiology ; *CRISPR-Cas Systems ; },
abstract = {Simple, accurate, sensitive, and rapid pathogen diagnosis is crucial for effective control of silkworm diseases. Although CRISPR-based nucleic acid detection systems show great potential for on-site detection of silkworm pathogens, their practicality is hindered by complex workflows and reagent-storage constraints. To address these limitations and enhance field suitability, we developed a DNA extraction-free one-pot RPA-CRISPR/Cas12a (DEORC) system and a dual-gene assay for detecting Bombyx mori nucleopolyhedrovirus (BmNPV) and Nosema bombycis using a handheld device. The DEORC assay detects BmNPV in hemolymph samples as early as 6 h post-infection (hpi) and N. bombycis at 10[3] spores/mL in spore suspensions. The entire process from sampling to visual readout is completed in approximately 70 min without requiring sophisticated equipment. To further enable off-grid deployment, we lyophilized the Cas12a detection reagents using 1 M betaine as a lyoprotectant, which retained measurable activity for at least one month at 4 °C under our test conditions, facilitating short-term refrigerated transport and field storage. Additionally, the dual-gene assay detects 10[3] copies/μL of a double-reference plasmid and simultaneously detects both BmNPV and N. bombycis in a single tube from midgut samples at 48 hpi; when combined with extraction-free techniques, it enables simultaneous detection of both pathogens in hemolymph samples at 72 hpi. Collectively, these advancements provide sensitive and portable tools for on-site sericulture disease management, offering faster and more practical workflows than two-step single-gene and traditional approaches.},
}

Animals
*Nosema/isolation & purification/genetics
*Nucleopolyhedroviruses/isolation & purification/genetics
*Bombyx/virology/microbiology
*CRISPR-Cas Systems

@article {pmid41241162,
year = {2026},
author = {Huang, G and Tang, Y and Zhang, S and Ning, H and Xu, J and Li, F and Zhang, X},
title = {Multifunctional nano-polymer-based targeted delivery system for CRISPR/Cas9-Mediated hepatocellular carcinoma therapy.},
journal = {International journal of pharmaceutics},
volume = {687},
number = {},
pages = {126392},
doi = {10.1016/j.ijpharm.2025.126392},
pmid = {41241162},
issn = {1873-3476},
mesh = {*Carcinoma, Hepatocellular/therapy/genetics ; *Liver Neoplasms/therapy/genetics ; Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Animals ; *Methyltransferases/genetics ; Cell Line, Tumor ; Plasmids/administration & dosage ; Polyethylene Glycols/chemistry ; *Polymers/chemistry ; Mice, Inbred BALB C ; Deoxycholic Acid/chemistry/administration & dosage ; *Nanoparticles/chemistry/administration & dosage ; Mice, Nude ; Hep G2 Cells ; Apoptosis ; Gene Transfer Techniques ; Mice ; Genetic Therapy/methods ; Polyethyleneimine/chemistry ; Disaccharides ; },
abstract = {CRISPR/Cas9 gene-editing technology exhibits substantial therapeutic potential for hepatocellular carcinoma (HCC); however, the targeted delivery of the CRISPR/Cas9 system into tumor cells remains a critical challenge requiring urgent exploration. Methyltransferase-Like 3 (METTL3), a key methyltransferase, drives HCC proliferation via multiple mechanisms. To address this challenge, a multifunctional delivery system was developed to efficiently deliver CRISPR/Cas9 plasmids targeting METTL3 (pMETTL3) into HCC cells. The cationic PEI, which facilitated the adsorption of pMETTL3 and protected it from lysosomal degradation, served as the polymeric backbone and was modified with deoxycholic acid (DOCA) to enhance its hydrophobicity. Meanwhile, lactobionic acid (LA) was grafted onto the structure to actively target HCC cells. The resulting pMETTL3/LPD was further functionalized with pH-sensitive and cleavable polyethylene glycol (PEG), aiming to reduce toxicity and enhance prolonged circulation. Results demonstrated that the delivery system maintains stability in physiological pH environments while achieving significantly enhanced accumulation in tumor tissues. Furthermore, the efficient cellular uptake of CRISPR/Cas9 plasmids enables precise gene editing, thereby effectively disrupting METTL3 expression, inducing apoptosis, and ultimately inhibiting HCC growth. This study presents a promising therapeutic strategy targeting METTL3 for HCC treatment and further expands the application of CRISPR/Cas9 gene-editing technology in cancer therapy.},
}

*Carcinoma, Hepatocellular/therapy/genetics
*Liver Neoplasms/therapy/genetics
Humans
*CRISPR-Cas Systems
Gene Editing/methods
Animals
*Methyltransferases/genetics
Cell Line, Tumor
Plasmids/administration & dosage
Polyethylene Glycols/chemistry
*Polymers/chemistry
Mice, Inbred BALB C
Deoxycholic Acid/chemistry/administration & dosage
*Nanoparticles/chemistry/administration & dosage
Mice, Nude
Hep G2 Cells
Apoptosis
Gene Transfer Techniques
Mice
Genetic Therapy/methods
Polyethyleneimine/chemistry
Disaccharides

@article {pmid41193657,
year = {2025},
author = {Li, H and Melnyk, JE and Fu, BXH and Shrestha, R and Zhang, M and Sjöström, M and Feng, S and Anderson, JA and Han, W and Chesner, LN and Shin, HJ and Farsh, T and Suarez, HJ and Nath, S and Chou, J and Das, R and Egusa, EA and Calvert, M and Kishishita, A and Barpanda, A and Zhu, J and Maheshwari, A and Chen, WS and Alshalalfa, M and Winters, A and Hua, JT and Liu, T and Davicioni, E and Wiita, AP and Stohr, BA and Siddiqui, J and Huang, B and Small, EJ and Shokat, KM and Nelson, PS and Quigley, DA and Wasmuth, EV and Gilbert, LA and Feng, FY},
title = {Genome-scale CRISPR screens identify PTGES3 as a direct modulator of androgen receptor function in advanced prostate cancer.},
journal = {Nature genetics},
volume = {57},
number = {12},
pages = {3027-3038},
pmid = {41193657},
issn = {1546-1718},
support = {21YOUN12//Prostate Cancer Foundation (PCF)/ ; Young Investigator Award//Prostate Cancer Foundation (PCF)/ ; YI//Prostate Cancer Foundation (PCF)/ ; PC230420//Prostate Cancer Foundation (PCF)/ ; young investigator//Prostate Cancer Foundation (PCF)/ ; 17CHAL06//Prostate Cancer Foundation (PCF)/ ; CA204602//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; P50CA097186 PNW Prostate Cancer SPORE Career Enhancement Program//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 1F32CA236347-01//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; CA230516-02S1//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 1R01CA221969-01//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 1R01CA244550//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; P30 CA015704/CA/NCI NIH HHS/United States ; R01 CA234715/CA/NCI NIH HHS/United States ; R01 CA266452/CA/NCI NIH HHS/United States ; P50 CA097186/CA/NCI NIH HHS/United States ; 1R01CA230516-01//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 1R01CA227025//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; P50CA186786//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; DP2 CA239597/CA/NCI NIH HHS/United States ; Prostate Cancer Program 2021 Pilot Research Awards//UC | UC San Francisco | Department of Medicine, University of California, San Francisco (UCSF Department of Medicine)/ ; 2018-00382//Vetenskapsrådet (Swedish Research Council)/ ; HT9425-23-1-0462//U.S. Department of Defense (United States Department of Defense)/ ; W81XWH-20-1-0136//U.S. Department of Defense (United States Department of Defense)/ ; R01GM124334//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01GM131641//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R00 GM140264/GM/NIGMS NIH HHS/United States ; V2024-016//V Foundation for Cancer Research (V Foundation)/ ; P30 CA015704/CA/NCI NIH HHS/United States ; R01 CA234715/CA/NCI NIH HHS/United States ; R01 CA266452/CA/NCI NIH HHS/United States ; P50 CA097186/CA/NCI NIH HHS/United States ; DP2 CA239597/CA/NCI NIH HHS/United States ; R00 GM140264/GM/NIGMS NIH HHS/United States ; },
mesh = {Male ; *Receptors, Androgen/genetics/metabolism ; Humans ; *Prostatic Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems/genetics ; Homeodomain Proteins/genetics/metabolism ; GATA2 Transcription Factor/genetics/metabolism ; Drug Resistance, Neoplasm/genetics ; },
abstract = {The androgen receptor (AR) is a critical driver of prostate cancer (PCa). Here, to study regulators of AR protein levels and oncogenic activity, we developed a live-cell quantitative endogenous AR fluorescent reporter. Leveraging this AR reporter, we performed genome-scale CRISPRi flow cytometry sorting screens to systematically identify genes that modulate AR protein levels. We identified and validated known AR protein regulators, including HOXB13 and GATA2, and also unexpected top hits including PTGES3-a poorly characterized gene in PCa. PTGES3 repression resulted in loss of AR protein, cell-cycle arrest and cell death in AR-driven PCa models. Clinically, analysis of PCa data demonstrates that PTGES3 expression is associated with AR-directed therapy resistance. Mechanistically, we show PTGES3 binds directly to AR, regulates AR protein stability and is necessary for AR function in the nucleus at AR target genes. PTGES3 represents a potential therapeutic target for overcoming known mechanisms of resistance to existing AR-directed therapies in PCa.},
}

Male
*Receptors, Androgen/genetics/metabolism
Humans
*Prostatic Neoplasms/genetics/pathology/metabolism
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems/genetics
Homeodomain Proteins/genetics/metabolism
GATA2 Transcription Factor/genetics/metabolism
Drug Resistance, Neoplasm/genetics

@article {pmid41170598,
year = {2025},
author = {Yoo, D and Park, H and Lim, H and Kim, G and Kim, D and Seo, Y and An, J and Kang, S and Park, C and Lee, MH and Lee, T},
title = {Versatile Biosensing Tool: CRISPR-Cas12a System-Integrated Electrochemical Biosensor for Severe Fever with Thrombocytopenia Syndrome Virus Detection in Clinical and Environmental Conditions.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {49},
pages = {e06860},
doi = {10.1002/smll.202506860},
pmid = {41170598},
issn = {1613-6829},
support = {2021R1C1C1005583//National Research Foundation of Korea (NRF)/ ; RS-2024-00416117//National Research Foundation of Korea (NRF)/ ; No.2020R1A5A1018052//National Research Foundation of Korea (NRF)/ ; RS-2024-00507931//Materials & Components Technology Development Program/ ; },
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Phlebovirus/isolation & purification/genetics ; *Severe Fever with Thrombocytopenia Syndrome/virology/diagnosis ; *Electrochemical Techniques/methods ; },
abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly lethal zoonotic virus with a maximum mortality rate of 30%. Despite its risks and potential for human-to-human transmission, the standard diagnostic method has been absent for SFTSV detection. Therefore, this study introduces a versatile SFTSV biosensing technology using the electrochemical-clustered regularly interspaced short palindromic repeat (EC-CRISPR) system. The sensing membrane is functionalized with the 2WJ DNA@Au-MoS2 signal probe as a strategy to amplify the EC response resulting from target detection events of the CRISPR system. The sensor exhibits selective, sensitive, and reproducible detection capabilities in phosphate-buffered saline, human serum, and Haemaphysalis longicornis genomic DNA diluted conditions with detection limits of 210.7, 189.0, and 285.1 fM, respectively. This verifies the versatility of the fabricated system, which significantly contributes to the early SFTSV detection in various fields. In the meantime, the sufficient sensing performance is demonstrated in identifying of SFTSV from infectious agent DNA. Furthermore, the proposed EC-CRISPR biosensing platform can be considered as a flexible foundational technique for the diagnosis of zoonotic infectious diseases, as it demonstrates practical applicability for the detection of Orientia tsutsugamushi by utilizing a customized CRISPR system programming strategy.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*Phlebovirus/isolation & purification/genetics
*Severe Fever with Thrombocytopenia Syndrome/virology/diagnosis
*Electrochemical Techniques/methods

@article {pmid41123840,
year = {2025},
author = {Remiszewski, P and Siedlecki, E and Wełniak-Kamińska, M and Mikula, M and Czarnecka, AM},
title = {Genetically Modified Mouse Models for Sarcoma Research: A Comprehensive Review.},
journal = {Current oncology reports},
volume = {27},
number = {11},
pages = {1221-1248},
pmid = {41123840},
issn = {1534-6269},
support = {2019/35/O/NZ2/03761 (AMC)//Narodowe Centrum Nauki/ ; },
mesh = {*Sarcoma/genetics/pathology ; Animals ; Humans ; *Disease Models, Animal ; Mice ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Gene Editing ; },
abstract = {PURPOSE OF REVIEW: Sarcomas are a heterogeneous group of over 170 malignant tumours of mesenchymal origin. The poor prognosis highlights the need for novel therapeutic strategies. Preclinical modelling is essential, yet challenging, given that sarcomas differ substantially from carcinomas and resources are very limited.

RECENT FINDINGS: GEMMs allow for the precise modelling of recurrent sarcoma genetics. The Cre-loxP system offer spatial and temporal control over the activation of oncogenes or the loss of tumour suppressors, while the CRISPR-Cas9 system enables the rapid, simultaneous editing of key drivers such as Trp53, Nf1, Kras and Pten. These models reproduce key features of human sarcomas, including their histopathology, the initiation of tumours in specific lineages and sites, and tumour-immune interactions within immune-competent hosts. GEMMs have been used to investigate hypotheses about the cells of origin, to test radiotherapy and immunotherapy, and to compare fusion-driven sarcomas with those with a complex karyotype. Despite variability, GEMMs remain essential tools for investigating the mechanisms of initiation, progression, and response to therapy. GEMMs offer mechanistic fidelity, but their use is limited by factors such as breeding burden, variability in recombination, off-target effects of CRISPR, underrepresentation of genomic complexity and inconsistent metastasis. These weaknesses reduce their predictive value, particularly with regard to advanced disease and immunotherapy. Progress will require the integration of Cre-loxP with CRISPR-Cas9, the standardisation of induction and reporting, and a closer alignment with distinct sarcoma subtypes, in order to enhance translational relevance.},
}

*Sarcoma/genetics/pathology
Animals
Humans
*Disease Models, Animal
Mice
CRISPR-Cas Systems
Animals, Genetically Modified
Gene Editing

@article {pmid41110509,
year = {2026},
author = {Faber, NR and Ashok, K and Venkatesan, T and Wertheim, B and Bulgarella, M},
title = {Leveraging advances in RNAi and CRISPR for improved biological pest control.},
journal = {Current opinion in insect science},
volume = {73},
number = {},
pages = {101453},
doi = {10.1016/j.cois.2025.101453},
pmid = {41110509},
issn = {2214-5753},
mesh = {*RNA Interference ; *Pest Control, Biological/methods ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Insecta/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The limitations of chemical pesticides and their associated risks highlight the need for more sustainable pest management strategies. Biological control using natural enemies offers an eco-friendly alternative but is sometimes constrained by efficiency and scalability. Emerging molecular tools-RNA interference (RNAi) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based gene editing-present novel opportunities to enhance existing biological control or to control pests directly. RNAi induces targeted gene knockdown via a non-heritable, transient response. CRISPR enables precise genetic modifications and could improve traits in beneficial insects or disrupt essential genes in pests, optionally including a gene drive for increased power. Although limitations remain for several species, these technologies could be valuable tools for integrated pest management. Their future implementation raises biosafety and regulatory considerations, particularly for self-propagating systems like gene drives. This review showcases developments in RNAi and CRISPR-based pest control, and calls for risk-based, adaptive governance to enable their responsible use in sustainable agriculture.},
}

*RNA Interference
*Pest Control, Biological/methods
Animals
*CRISPR-Cas Systems
*Gene Editing
*Insecta/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40997504,
year = {2025},
author = {Berger, P and Wilming, L and Jürgens, R and Minchuk, Y and Leußink, S and Gandhi, S and Walter, C and Wind, SM and Heider, D and Lamparter, L and Galic, M and Stoll, M and Jorch, SK and Roth, J and Austermann, J and Fehler, O},
title = {PSTPIP1 and pyrin, two key regulators of macrophage differentiation.},
journal = {European journal of cell biology},
volume = {104},
number = {4},
pages = {151518},
doi = {10.1016/j.ejcb.2025.151518},
pmid = {40997504},
issn = {1618-1298},
mesh = {*Macrophages/metabolism/cytology ; *Cell Differentiation ; Humans ; *Pyrin/metabolism/genetics ; *Adaptor Proteins, Signal Transducing/metabolism/genetics ; CRISPR-Cas Systems ; *Cytoskeletal Proteins/metabolism/genetics ; Animals ; },
abstract = {BACKGROUND: Monocytes develop from hematopoietic stem cells; migrate into the tissue, where they undergo a stimulation-dependent and tissue specific differentiation into macrophages imprinting specific inflammatory functions. The development of inflammatory functions during differentiation of progenitor cells into macrophages remained incompletely understood.

OBJECTIVE: We intended to identify regulatory factors driving monocyte/macrophage differentiation.

METHODS: A Genome-wide CRISPR/Cas9 knockout screen (GeCKO) in ER-HoxB8 macrophages was used to identify key drivers of macrophage differentiation which were verified in independent knock-out and knock-in cells. Immunophenotyping was studied by FACS, morphology and migration by fluorescence microscopy, the inflammatory response by ELISA. Transcriptomic data were obtained by next generation mRNA sequencing and validated by quantitative polymerase chain reaction and immunoblotting.

RESULTS: Genome-wide CRISPR/Cas9 knockout screen identified the cytosolic cytoskeleton-associated adaptor molecule PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1) as a regulatory factor of macrophage differentiation. Interestingly, mutations in PSTPIP1 cause autoinflammatory disorders (PAPA syndrome). Deletion of PSTPIP1 resulted in hampered differentiation, decreased inflammatory response, changed morphology, altered cell adhesion and migration properties. PSTPIP1 is a regulator of Pyrin inflammasome activity which drives autoinflammation in familial Mediterranean fever (FMF). Deletion of Pyrin also resulted in a strong alteration of cellular dynamics in macrophages.

CONCLUSION: PSTPIP1 and Pyrin are crucial factors in macrophage differentiation. Their deletion or mutation resulted in a hampered differentiation of macrophages resulting in strong morphological alterations and impacting phagocyte key functions as adhesion and migration. Impaired differentiation of macrophages may represent a significant factor in the pathophysiology of autoinflammatory diseases like FMF and PAPA.},
}

*Macrophages/metabolism/cytology
*Cell Differentiation
Humans
*Pyrin/metabolism/genetics
*Adaptor Proteins, Signal Transducing/metabolism/genetics
CRISPR-Cas Systems
*Cytoskeletal Proteins/metabolism/genetics
Animals

@article {pmid40997217,
year = {2025},
author = {Cosgrove, BD and Bounds, LR and Taylor, CK and Su, AL and Rizzo, AJ and Barrera, A and Sun, T and Safi, A and Song, L and Whitlow, T and Tata, A and Iglesias, N and Diao, Y and Tata, PR and Hoffman, BD and Crawford, GE and Gersbach, CA},
title = {Mechanosensitive genomic enhancers potentiate the cellular response to matrix stiffness.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6778},
pages = {eadl1988},
doi = {10.1126/science.adl1988},
pmid = {40997217},
issn = {1095-9203},
mesh = {*Extracellular Matrix/physiology ; Humans ; Fibroblasts/physiology/metabolism ; *Enhancer Elements, Genetic ; *Epigenesis, Genetic ; *Mechanotransduction, Cellular/genetics ; Lung/cytology ; Single-Cell Analysis ; Cell Movement/genetics ; Cell Proliferation/genetics ; Apoptosis/genetics ; Cell Adhesion/genetics ; Gene Editing ; Epigenome ; CRISPR-Cas Systems ; },
abstract = {Epigenetic control of gene expression and cellular phenotype is influenced by changes in the local microenvironment, yet how mechanical cues precisely influence epigenetic state to regulate transcription remains largely unmapped. In this study, we combined genome-wide epigenome profiling, epigenome editing, and phenotypic and single-cell RNA sequencing CRISPR screening to identify a class of genomic enhancers that responds to the mechanical microenvironment. These "mechanoenhancers" can be preferentially activated on either soft or stiff extracellular matrix contexts and regulate transcription to influence critical cell functions including apoptosis, adhesion, proliferation, and migration. Epigenetic editing of mechanoenhancers reprograms the cellular response to the mechanical microenvironment and modulates the activation of disease-related genes in lung fibroblasts from healthy and fibrotic donors. Epigenetic editing of mechanoenhancers holds potential for precise targeting of mechanically driven diseases.},
}

*Extracellular Matrix/physiology
Humans
Fibroblasts/physiology/metabolism
*Enhancer Elements, Genetic
*Epigenesis, Genetic
*Mechanotransduction, Cellular/genetics
Lung/cytology
Single-Cell Analysis
Cell Movement/genetics
Cell Proliferation/genetics
Apoptosis/genetics
Cell Adhesion/genetics
Gene Editing
Epigenome
CRISPR-Cas Systems

@article {pmid41364162,
year = {2025},
author = {Murtaza, M and Gupta, P and Choudhary, P and Manzoor, M and Sharma, S and Jaglan, S},
title = {Strategies to decipher silent biosynthetic gene clusters in actinomycetes.},
journal = {Archives of microbiology},
volume = {208},
number = {1},
pages = {53},
pmid = {41364162},
issn = {1432-072X},
mesh = {*Actinobacteria/genetics/metabolism ; *Multigene Family/genetics ; *Biosynthetic Pathways/genetics ; Anti-Bacterial Agents/biosynthesis ; CRISPR-Cas Systems ; },
abstract = {Actinobacteria have a huge, mainly untapped potential for the production of secondary metabolites. These metabolites are an important source of bioactive compounds. However, a majority of biosynthetic gene clusters (BGCs) are either under-expressed or fully silent under standard laboratory conditions, limiting their potential. The present review article aims to explore the biosynthetic gene clusters (BGCs) of actinobacteria using strategies that aid in unlocking these silent BGCs. The strategies discussed are PCR-Targeted Gene Replacement (PCR-TR); Cre-LoxP recombination system; Transcription factor decoys, Ribosome engineering, and CRISPR/Cas technologies. Besides, elicitors also helped with the identification of these cryptic or silent BGCs and advanced our ability to explore these natural products. Combining experimental and computational platforms provides an opportunity to unlock the hidden chemical diversity in nature, thereby accelerating the identification of new bioactive substances. The new antibiotics explored by all the strategies could help in the fight against antimicrobial resistance (AMR).},
}

*Actinobacteria/genetics/metabolism
*Multigene Family/genetics
*Biosynthetic Pathways/genetics
Anti-Bacterial Agents/biosynthesis
CRISPR-Cas Systems

@article {pmid41362674,
year = {2026},
author = {Toofan, P and Singh, M and Brooks, A and McLuckie, K},
title = {Non-clinical safety considerations on genome editing using the CRISPR/Cas system.},
journal = {Genes & diseases},
volume = {13},
number = {2},
pages = {101785},
pmid = {41362674},
issn = {2352-3042},
abstract = {Recent advances in gene editing using the CRISPR/Cas system have revolutionized genome editing, opening new horizons for human cellular and gene therapy products. Genome editing technologies are rapidly being adopted in clinical trials. However, critical non-clinical safety considerations are required to address challenges in translating research to the clinic. Here, we review current ex vivo and in vivo genome editing approaches using the CRISPR/Cas system and discuss the practical use of these methods in pre-clinical studies and in the clinic. We also discuss known limitations of genome editing in humans and the mitigation of risk factors associated with it from a non-clinical safety perspective. This review aims to aid researchers in acquiring a perspective that is essential for the safe translation of genome editing to the clinic.},
}

@article {pmid41361988,
year = {2025},
author = {Min, YH and Lee, DG and Lee, HY and Yoo, JH and Lee, KH and Shin, YB and Byun, JY},
title = {CRISPR/Cas12a with Antisense Oligonucleotide-Regulated Translational Amplification for Ultrasensitive Nucleic Acid Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c03081},
pmid = {41361988},
issn = {2379-3694},
abstract = {Highly sensitive nucleic acid testing-assisted early disease detection is crucial for effective disease prevention and management, particularly when targeting low-abundance genetic materials in molecular diagnostics. This study describes CRATE (CRISPR/Cas controlled antisense oligonucleotide (ASO)-mediated translational signal enhancement), a novel ultrasensitive approach for nucleic acid detection by integrating Cas12a trans-cleavage, ASO-controlled gene expression, and cell-free signal protein amplification. This assay leverages the target-induced trans-cleavage of ASO-controlled gene expression for the amplification of signal proteins, with luminescent signal allowing for attomolar-level target DNA detection, as well as antigenic protein application enabling visual detection by lateral flow assay. The CRATE assay improves sensitivity using ASO-modified locked nucleic acid, achieving a 10-aM-level DNA detection. The proof of concept demonstrates 0.1 copies/μL detection of HPV genomic DNA from HPV-positive cancer cells as well as colorimetric lateral flow tests with ∼10 copies/μL sensitivity. The CRATE assay can detect the HBV target in plasma from HBV-positive patients with 100% sensitivity and specificity. With high specificity and accuracy, the CRATE assay retains the potential for detecting any nucleic acid of interest. By integration of precise CRISPR-based cleavage, ASO regulation, and efficient protein signal amplification, this approach provides a promising solution for highly selective and sensitive nucleic acid detection and potential applications in clinical diagnostics and point-of-care testing.},
}

@article {pmid41361700,
year = {2025},
author = {Ain, QU and McCarthy, A and Nadeem, A and Javed, M and Niakan, K and Nashta, AF},
title = {CRISPR/Cas9-mediated generation of GATA3 knockout in Bovine Fibroblast and MDBK cell lines to assess sgRNAs targeting efficiency.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {269},
pmid = {41361700},
issn = {1438-7948},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems ; *GATA3 Transcription Factor/genetics/metabolism ; Fibroblasts/metabolism/cytology ; Cell Line ; *Gene Knockout Techniques ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {GATA3 is expressed in the outer cells of the morula stage during embryonic development and is considered a key driver of the regulation of early lineage development in bovines. This research presents an optimised somatic cell validation resource, successfully generating GATA3 knockout (KO) Bovine Fetal Fibroblasts and MDBK cells using CRISPR/Cas9-mediated genome editing for their future implications in vivo studies designed to definitively understand the role of GATA3 in cell lineage specification and bovine embryo development. This involved designing single-guide RNAs (sgRNAs) targeting different regions of the GATA3 gene, cloning them into the px459 plasmid, delivering the CRISPR clone into bovine fibroblast cells and the MDBK cell line, screening for successful targeting and knockouts, and MiSeq analysis to verify successful disruption of the GATA3 gene. A total of eleven guides were designed targeting the functional domains in Exons 4 and 5 and the transcription initiation site in Exon 2. Designed guides were first optimized and screened using an in vitro cleavage assay. The guides with the best cutting efficiencies were then tested in vivo by targeting bovine fetal fibroblast (BFFs) and MDBK cell line followed by MiSeq analysis to verify the successful knockouts. A total of two effective guides were identified targeting the zinc-finger (ZnF) functional domains of the GATA3 gene (sgRNA#5 and sgRNA#8 in Exon 4 and Exon 5, respectively) and one in Exon 2 (sgRNA#1) targeting the transcription initiation site of the GATA3 gene. MiSeq data from targeted bovine cells showed indel frequency of 47.40%, 55.5%, and 42.4% in bovine fetal fibroblasts, 11.03%, 28.9% and 7.3% for MDBK cells for top three sgRNAs. Overall, MiSeq data for 3 selected sgRNAs showed successful disruption of the GATA3 gene, inserting a base pair 2-3 bp upstream of the PAM site, ultimately resulting in a premature stop codon TAA in the downstream region. This study established and validated highly efficient sgRNAs targeting the GATA3 gene, forming a molecular basis for forthcoming functional investigations in bovine embryos to explore gene function and protein-level effects.},
}

Animals
Cattle
*CRISPR-Cas Systems
*GATA3 Transcription Factor/genetics/metabolism
Fibroblasts/metabolism/cytology
Cell Line
*Gene Knockout Techniques
*RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods