@article {pmid40379664,
year = {2025},
author = {Nam, H and Xie, K and Majumdar, I and Wang, J and Yang, S and Starzyk, J and Lee, D and Shan, R and Li, J and Wu, H},
title = {Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4569},
pmid = {40379664},
issn = {2041-1723},
support = {DP2 GM154019/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; Transgenes/genetics ; DNA, Single-Stranded/genetics/metabolism ; HEK293 Cells ; Receptors, Chimeric Antigen/genetics/metabolism ; T-Lymphocytes/metabolism ; DNA, Circular/genetics ; Genetic Engineering/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; Homologous Recombination ; Gene Knock-In Techniques/methods ; },
abstract = {Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination (HR), with efficiency improved by CRISPR/Cas9 technology. Circular single-stranded DNA (cssDNA) has been used as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in. Here, we introduce enGager, an enhanced GATALYST associated genome editor system that increases transgene integration efficiency by tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs. This approach further improves targeted integration and expression of reporter genes at multiple genomic loci in various cell types, showing up to 6-fold higher efficiency compared to unfused Cas9, especially for large transgenes in primary cells. Notably, enGager enables efficient integration of a chimeric antigen receptor (CAR) transgene in 33% of primary human T cells, enhancing anti-tumor functionality. This 'tripartite editor with ssDNA optimized genome engineering (TESOGENASE) offers a safer, more efficient alternative to viral vectors for therapeutic gene modification.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems/genetics
Transgenes/genetics
DNA, Single-Stranded/genetics/metabolism
HEK293 Cells
Receptors, Chimeric Antigen/genetics/metabolism
T-Lymphocytes/metabolism
DNA, Circular/genetics
Genetic Engineering/methods
CRISPR-Associated Protein 9/metabolism/genetics
Homologous Recombination
Gene Knock-In Techniques/methods

@article {pmid40378977,
year = {2025},
author = {Qin, B and Shen, S and Chen, H and Wang, Y and Ding, J and Ding, J},
title = {Inactivation of the key ORFs of HBV for antiviral therapy by non-cleavage base editing.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {107689},
doi = {10.1016/j.micpath.2025.107689},
pmid = {40378977},
issn = {1096-1208},
abstract = {OBJECTIVES: Hepatitis B virus (HBV) infection is the key cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Currently available anti-HBV drugs are more or less defective owing to the unremovable covalently closed circular DNA (cccDNA). Thus, CRISPR/Cas9 is a promising therapeutic strategy for anti-HBV therapy. Double-strand breaks (DSBs) and uncontrolled genomic rearrangements occur inevitably. In this study, we aimed to use base editors to control HBV infection.

METHODS: Base editors precisely instal targeted point mutations without requiring DSBs or donor DNA templates, and without relying on homology-directed repair (HDR) or nonhomologous end joining (NHEJ). Adenine base editors (ABEs) and cytosine base editors (CBEs) catalyse A• T to G •C and C• G to T •A conversions, respectively. In this study, to control HBV replication by modifying and inactivating key HBV genes, recently developed CRISPR/Cas-mediated SpRY-ABE8e and CBE4-max were utilised to falsify and invalidate the ATG initiation codons of the S, Pre-S1, PreS2, C, Pre-C, X, and P genes.

RESULTS: The ATG initiation codons of HBV genes were edited by ABE/CBE. The expected point mutations were successfully introduced, resulting in the simultaneous suppression of HBV antigen expression and replication to varying degrees.

CONCLUSIONS: Our study focused on clearing HBV using base and provided experimental and theoretical evidence for the treatment of chronic HBV infection. Thus, base editing is a potential strategy for curing CHB by permanently inactivating the integrated DNA and cccDNA without using DSBs.},
}

@article {pmid40378975,
year = {2025},
author = {Kumar, V and Nagano, T and Takasuka, TE},
title = {Genome integration and expression of β-glucosidase in Priestia megaterium enhanced poly(3-hydroxybutyrate) production from cellobiose and cellulose.},
journal = {Bioresource technology},
volume = {432},
number = {},
pages = {132681},
doi = {10.1016/j.biortech.2025.132681},
pmid = {40378975},
issn = {1873-2976},
abstract = {Polyhydroxyalkanoates (PHAs) production using cellulosic biomass is a promising way for sustainable manufacturing of bioplastics. Priestia megaterium is an ideal choice as it can use glucose and xylose for PHA production. To further improve the strain for PHA production from cellobiose, we integrate exogenous β-glucosidase (Bgl) from Bacillus sp. GL1 (Bsbgl) in the PHA depolymerase (phaZ1) deletion background (ΔZ1) using CRISPR-Cas. The deletion of phaZ1 in P. megaterium showed a significant improvement in the PHA accumulation whereas BsBgl expression resulted in robust activity and improved growth using cellobiose as a sole carbon source compared to other Bgl targets. To further improve the strain, four native promoters were examined for intracellular BsBgl expression, and the PHA promoter (PphaR) and citrate synthase promoter (Pcitz) showed 2.0- and 4.5-fold higher activities of BsBgl, compared to the xylose promoter (Pxyl). The rate of cellobiose utilization in engineered strains P2 (PphaRBsbgl_ΔZ1) and P4 (PcitzBsbgl_ΔZ1) was improved to 1.6-fold and 2.6-fold, whereas poly(3-hydroxybutyrate) (P3HB) yield for the respective strains was around 3-fold to the wild-type. The strain P2 turned out to be better for cellobiose to PHA production. Further, the strain P2 in a co-culture with cellulolytic Streptomyces sp. SirexAA-E in a consolidated bioprocessing yielded 76 mg of P3HB/ g of carboxymethylcellulose, which is 4.3-times higher than the co-culture with the wild-type. Thus, the present work improved the cellobiose utilization and P3HB accumulation of P. megaterium. The current study paves the way for designing efficient cell factories for cellulosic biomass into bioplastic in the future.},
}

@article {pmid40377871,
year = {2025},
author = {Rahmati, R and Zarimeidani, F and Ghanbari Boroujeni, MR and Sadighbathi, S and Kashaniasl, Z and Saleh, M and Alipourfard, I},
title = {CRISPR-Assisted Probiotic and In Situ Engineering of Gut Microbiota: A Prospect to Modification of Metabolic Disorders.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {40377871},
issn = {1867-1314},
abstract = {The gut microbiota, a substantial group of microorganisms residing in the human body, profoundly impacts various physiological and pathological mechanisms. Recent studies have elucidated the association between gut dysbiosis and multiple organ diseases. Gut microbiota plays a crucial role in maintaining gastrointestinal stability, regulating the immune system and metabolic processes not only within the gastrointestinal tract but also in other organs such as the brain, lungs, and skin. Dysbiosis of the gut microbiota can disrupt biological functioning and contribute to the development of metabolic disorders. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) modules are adaptive immune systems in numerous archaea and bacteria. CRISPR/Cas is a versatile gene-editing tool that enables modification of the genome in live cells, including those within the gut microbiota. This technique has revolutionized gene editing due to its simplicity and effectiveness. It finds extensive applications in diverse scientific arenas, facilitating the functional screening of genomes during various biological processes. Additionally, CRISPR has been instrumental in creating model organisms and cell lines for research purposes and holds great potential for developing personalized medical treatments through precise genetic alterations. This review aims to explore and discuss the possibilities of CRISPR/Cas and the current trends in using this technique for editing gut microbiota genes in various metabolic disorders. By uncovering the valuable potential of CRISPR/Cas in modifying metabolic disorders through the human gut microbiota, we shed light on its promising applications.},
}

@article {pmid40377313,
year = {2025},
author = {Scarrone, M and Turner, D and Dion, M and Tremblay, D and Moineau, S},
title = {In silico and in vitro comparative analysis of 79 Acinetobacter baumannii clinical isolates.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0284924},
doi = {10.1128/spectrum.02849-24},
pmid = {40377313},
issn = {2165-0497},
abstract = {Acinetobacter baumannii is a significant nosocomial bacterial pathogen that poses a substantial infection risk due to its high resistance to antibiotics and ability to survive in hospital environments. In this study, we performed comprehensive in silico and in vitro analyses on 79 A. baumannii clinical isolates from different geographical locations to uncover their genomic and epidemiological characteristics as well as their antibiotic and phage susceptibilities. Our findings revealed considerable genomic diversity among the isolates, as shown by average nucleotide identity (ANI) heat maps, multilocus sequence typing (MLST), and core genome MLST (cgMLST). We identified several international clones known for their high antibiotic resistance and global prevalence. Surprisingly, we also observed that the number of antimicrobial resistance genes (ARGs) was higher in isolates containing CRISPR-Cas systems. Plaque assays with 13 phages indicated that Acinetobacter phages have a narrow host range, with capsule loci (KL) serving as a good indicator of phage-bacteria interactions. The presence of CRISPR-Cas systems and other antiviral defense mechanisms in A. baumannii genomes also appears to play a key role in providing phage resistance, regardless of the phage receptors. We also found that spacers associated with subtypes I-F1 and I-F2 CRISPR-Cas systems predominantly target prophages, suggesting a role in maintaining genomic stability and contributing to phage-bacteria co-evolution. Overall, this study provides a set of highly characterized A. baumannii clinical isolates for future studies on antibiotic-phage-bacteria interactions.IMPORTANCEAcinetobacter baumannii poses a significant challenge to the healthcare system due to its antibiotic resistance and strong survival mechanisms. This study examines a diverse collection of 79 clinical isolates to deepen our understanding of A. baumannii's genetic characteristics and its defense mechanisms against both antibiotics and phages. Genomic analysis revealed globally prevalent, highly resistant clones and uncovered a complex role for CRISPR-Cas systems. Although CRISPR-Cas systems were not widespread among these isolates, they primarily targeted prophages. Additionally, the study emphasizes the importance of capsule types as indicators of phage susceptibility. Together, these findings provide insights into the pathogen's resilience and evolutionary adaptations, potentially guiding future research on infection control strategies and new therapeutic approaches to combat A. baumannii infections.},
}

@article {pmid40377112,
year = {2025},
author = {Kellari, LM and Dalakouras, A and Tsiouri, O and Vletsos, P and Katsaouni, A and Uslu, VV and Papadopoulou, KK},
title = {Cross-kingdom RNAi induced by a beneficial endophytic fungus to its host requires transitivity and amplification of silencing signals.},
journal = {Plant biology (Stuttgart, Germany)},
volume = {27},
number = {4},
pages = {504-514},
doi = {10.1111/plb.70026},
pmid = {40377112},
issn = {1438-8677},
support = {6236//the Hellenic Foundation for Research and Innovation (HFRI)/ ; 7322//Research Committee of UTH/ ; //PRIMA Programme/ ; },
mesh = {*Nicotiana/microbiology/genetics/metabolism ; *Endophytes/physiology ; *Fusarium/physiology/genetics ; *RNA Interference ; Green Fluorescent Proteins/metabolism/genetics ; },
abstract = {Cross-kingdom transfer of small RNA (sRNA) molecules has been identified as a means of communication between plants and interacting microorganisms, but the mechanistic details of this sRNA-based interaction remain elusive. We have previously shown that the beneficial root-colonizing fungus Fusarium solani strain K (FsK) translocates sRNAs to its host, Nicotiana benthamiana (Nb), leading to systemic silencing of a reporter gene. Here, we investigated the mechanistic details of the endophyte-induced systemic silencing using an RNAi sensor system. We inoculated three Nb GFP expressing lines with conidia of an FsK transformant containing a transgene that targets host GFP (FsK-hpGF). The efficiency of silencing mediated by FsK-hpGF was monitored both phenotypically under ultraviolet light as well as quantitatively by RT-qPCR. sRNA sequencing was performed to evaluate the production of sRNAs targeting host GFP. Finally, bisulfite sequencing was used to assess plant GFP methylation levels. We show that the translocated fungal sRNAs induced production of secondary sRNAs, mainly of 22-24-nt in size, with the conspicuous absence of 21-nt sRNAs. Importantly, systemic silencing could not be induced in an RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) CRISPR/Cas knockout background, nor in an intron-containing target gene. Overall, our data show that endophyte-induced silencing in the host requires RDR6-mediated transitivity and amplification of silencing signals. Despite being based on an artificial RNAi sensor system, our observations may reflect a more generalized and so far unexplored facet of cross-kingdom RNAi, with RDR6-based transitivity influencing the way symbionts and pathogens elicit systemic phenotypes in their host plants.},
}

*Nicotiana/microbiology/genetics/metabolism
*Endophytes/physiology
*Fusarium/physiology/genetics
*RNA Interference
Green Fluorescent Proteins/metabolism/genetics

@article {pmid40376763,
year = {2025},
author = {Wang, K and Liu, Z},
title = {Plant synthetic biology-based biofortification, strategies and recent progresses.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13934},
pmid = {40376763},
issn = {1744-7909},
support = {startup fund//Shenzhen University of Advanced Technology/ ; },
abstract = {Hidden hunger, caused by chronic micronutrient deficiencies, affects billions of people worldwide and remains a critical public health issue despite progress in food production. Biofortification offers a promising solution by enhancing nutrient levels within plant tissues through traditional breeding or advanced biotechnologies. Recent advancements in plant synthetic biology have significantly improved biofortification strategies, enabling precise and targeted nutrient enrichment. This mini-review outlines five core strategies in synthetic biology-based biofortification: overexpression of endogenous biosynthetic genes, introduction of heterologous biosynthetic pathways, expression of nutrient-specific transporters, optimization of transcriptional regulation, and protein (directed) evolution. Vitamin B1 biofortification serves as a primary illustrative example due to its historical importance and ongoing relevance. Recent breakthroughs, particularly from Chinese research teams, are also highlighted. Together, these strategies offer transformative potential for addressing global nutritional challenges through precise, sustainable and innovative plant-based approaches.},
}

@article {pmid40376717,
year = {2025},
author = {Marzluf, JP and Daniela, K and Klein, J and Zehe, C and Leroux, AC},
title = {Utilizing Stable Gene-Edited Knockout Pools for Genetic Screening and Engineering in Chinese Hamster Ovary Cells.},
journal = {Biotechnology journal},
volume = {20},
number = {5},
pages = {e70033},
pmid = {40376717},
issn = {1860-7314},
mesh = {CHO Cells ; Animals ; Cricetulus ; *Gene Knockout Techniques/methods ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Cricetinae ; *Genetic Testing/methods ; Fibronectins/genetics ; Genetic Engineering/methods ; },
abstract = {Chinese hamster ovary (CHO) cells are the primary host for biopharmaceutical production. To meet increasing demands for productivity, quality, and complex molecule expression, genetic engineering, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene knockout (KO), is widely used to optimize host cell performance. However, systematic screening of KO targets remains challenging due to the labor-intensive process of generating and evaluating individual clones. In this study, we present a robust, high-throughput CRISPR workflow using stable KO pools in CHO cells. These pools maintain genetic stability for over 6 weeks, including in multiplexed configurations targeting up to seven genes simultaneously. Compared to clonal approaches, KO pools reduce variability caused by clonal heterogeneity and better reflect the host cell population phenotype. We demonstrate the utility of this approach by reproducing the beneficial phenotypic effects of fibronectin 1 (FN1) KO, specifically prolonged culture duration and improved late-stage viability in fed-batch processes. This workflow enables efficient identification and evaluation of promising KO targets without the need to generate and test large numbers of clones. Overall, screening throughput is increased 2.5-fold and timelines are compressed from 9 to 5 weeks. This provides a scalable, efficient alternative to traditional clonal screening, accelerating discovery for CHO cell line engineering for biopharmaceutical development.},
}

CHO Cells
Animals
Cricetulus
*Gene Knockout Techniques/methods
*Gene Editing/methods
CRISPR-Cas Systems/genetics
Cricetinae
*Genetic Testing/methods
Fibronectins/genetics
Genetic Engineering/methods

@article {pmid40376300,
year = {2025},
author = {Feussner, M and Migur, A and Mitrofanov, A and Alkhnbashi, OS and Backofen, R and Beisel, CL and Weinberg, Z},
title = {Disparate mechanisms counteract extraneous CRISPR RNA production in type II-C CRISPR-Cas systems.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf007},
pmid = {40376300},
issn = {2633-6693},
abstract = {CRISPR-Cas adaptive immune systems in bacteria and archaea enable precise targeting and elimination of invading genetic elements. An inherent feature of these systems is the 'extraneous' CRISPR RNA (ecrRNA), which is produced via the extra repeat in a CRISPR array lacking a corresponding spacer. As ecrRNAs would interact with the Cas machinery yet not direct acquired immunity, they pose a potential barrier to defence. Type II-A CRISPR-Cas systems resolve this barrier through the leader sequence upstream of a CRISPR array, which forms a hairpin structure with the extra repeat that inhibits ecrRNA production. However, the fate of ecrRNAs in other CRISPR types and subtypes remains to be explored. Here, we report that II-C systems likely employ disparate strategies to resolve the ecrRNA due to their distinct configuration in comparison to II-A. Applying bioinformatics analyses to over 650 II-C systems followed by experimental validation, we identified three strategies applicable to these systems: formation of an upstream Rho-independent terminator, formation of a hairpin that sequesters the ecrRNA guide, and mutations in the repeat expected to disrupt ecrRNA formation. These findings expand the list of mechanisms in CRISPR-Cas systems that could resolve the ecrRNA to optimize immune response.},
}

@article {pmid40376084,
year = {2024},
author = {Shi, H and Al-Sayyad, N and Wasko, KM and Trinidad, MI and Doherty, EE and Vohra, K and Boger, RS and Colognori, D and Cofsky, JC and Skopintsev, P and Bryant, Z and Doudna, JA},
title = {Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.10.01.616117},
pmid = {40376084},
issn = {2692-8205},
abstract = {RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a 20-base-pair DNA sequence adjacent to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical and cell-based assays on S. pyogenes Cas9 (Spy Cas9) variants with wide-ranging genome editing efficiencies that differ in PAM binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.},
}

@article {pmid40375877,
year = {2025},
author = {Khamwut, A and Nimnual, J and Chomta, N and Nimsamer, P and Mayuramart, O and Kaewsapsak, P and Pasittungkul, S and Poovorawan, Y and Payungporn, S},
title = {Detection of respiratory syncytial virus based on RT-RPA and CRISPR-Cas12a.},
journal = {Experimental biology and medicine (Maywood, N.J.)},
volume = {250},
number = {},
pages = {10387},
pmid = {40375877},
issn = {1535-3699},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Respiratory Syncytial Virus, Human/isolation & purification/genetics ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Human respiratory syncytial virus (hRSV) is one of the most prevalent viruses infecting children globally. In this study, we employed the RT-RPA with CRISPR/Cas12a detection methodology to detect and differentiate RSV-A and RSV-B, particularly in resource-limited settings. The detection limit for RSV-A and RSV-B was approximately 10[2] and 10[3] copies/reaction, respectively. The assay revealed 100% specificity in detecting both RSV-A and RSV-B. Diagnostic accuracy was 90.32 and 93.55% for RSV-A and RSV-B, respectively, compared to RT-qPCR. These data indicate a proficient strategy for RSV screening, demonstrating promise for prospective applications in detecting diverse viral infections.},
}

Humans
*CRISPR-Cas Systems/genetics
*Respiratory Syncytial Virus, Human/isolation & purification/genetics
*Respiratory Syncytial Virus Infections/diagnosis/virology
Sensitivity and Specificity
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40374732,
year = {2025},
author = {Hagman, A and Stenström, O and Carlström, G and Akke, M and Grey, C and Carlquist, M},
title = {Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {16972},
pmid = {40374732},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Amination ; *Saccharomyces cerevisiae/genetics/metabolism ; *Metabolic Engineering/methods ; Biocatalysis ; Transaminases/genetics/metabolism ; Chromobacterium/enzymology/genetics ; Alanine/metabolism ; Alanine Transaminase/genetics/metabolism ; Bioreactors ; },
abstract = {Metabolically engineered baker's yeast can be used to produce chiral amines through whole-cell bioconversion of prochiral ketones. This study investigates the modulation of the alanine-pyruvate metabolic node to enhance reductive amination, using the stereoselective conversion of benzylacetone to (S)-1-methyl-3-phenylpropylamine (MPPA) as a model reaction. Chromosomal integration of multiple copies of the promiscuous omega transaminase from Chromobacterium violaceum (cv-ATA) resulted in an active yeast catalyst. Physiological characterization in bioreactors under aerobic batch cultivation revealed that amine production occurred only under post-diauxic growth on ethanol. To reduce native alanine utilization, the endogenous alanine aminotransferase (ALT1) was knocked out and replaced with cv-ATA. To rapidly employ this strategy in other strains, a simple CRISPR/cas9 method for universal gene replacement was developed. The replacement of ALT1 with cv-ATA improved the reaction by 2.6-fold compared to the control strain with intact ALT1. NMR measurements of metabolites originating from [15]N L-alanine and [13]C glucose indicated that pyruvate formation during growth on glucose inhibited amine production. Under optimal conditions, the biocatalytic bioconversion of benzylacetone to MPPA reached a yield of 58%.},
}

*CRISPR-Cas Systems
Amination
*Saccharomyces cerevisiae/genetics/metabolism
*Metabolic Engineering/methods
Biocatalysis
Transaminases/genetics/metabolism
Chromobacterium/enzymology/genetics
Alanine/metabolism
Alanine Transaminase/genetics/metabolism
Bioreactors

@article {pmid40373829,
year = {2025},
author = {Trujillo, E and Angulo, C},
title = {Perspectives on the use of the CRISPR system in plants to improve recombinant therapeutic protein production.},
journal = {Journal of biotechnology},
volume = {405},
number = {},
pages = {111-123},
doi = {10.1016/j.jbiotec.2025.05.010},
pmid = {40373829},
issn = {1873-4863},
abstract = {The plant-based system is a promising platform for producing biotherapeutics due to its scalability, cost-effectiveness, and lower risk of contamination by human pathogens. However, several challenges remain, including optimizing yield, stability, functionality, and the immunogenic properties of recombinant proteins. In this context, this review explores the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology to improve the production of recombinant therapeutic proteins in plants. Traditional tools and strategies for plant-based recombinant protein production are discussed, highlighting their limitations and the potential of CRISPR to overcome these boundaries. It delves into the components of the CRISPR-Cas system and its application in optimizing therapeutic protein function and yield. Major strategies include modifying glycosylation patterns to humanize plant-produced proteins, metabolic pathway engineering to increase protein accumulation, and the precise integration of transgenes into specific genomic loci to enhance expression stability and productivity. These advancements demonstrate how CRISPR system can overcome bottlenecks in plant molecular farming and enable the production of high-quality therapeutic proteins. Lastly, future trends and perspectives are examined, emphasizing ongoing innovations and challenges in the field. The review underscores the potential of CRISPR to reshape plant biotechnology and support the growing demand for recombinant therapeutics, offering new avenues for sustainable and efficient protein production systems. KEY MESSAGE: CRISPR technology has the potential to improve plant-based therapeutic protein production by optimizing yield, stability, and humanization, overcoming bottlenecks, and enabling sustainable, efficient systems for recombinant biotherapeutics.},
}

@article {pmid40373688,
year = {2025},
author = {Thakur, RK and Aggarwal, K and Sood, N and Kumar, A and Joshi, S and Jindal, P and Maurya, R and Patel, P and Kurmi, BD},
title = {Harnessing advances in mechanisms, detection, and strategies to combat antimicrobial resistance.},
journal = {The Science of the total environment},
volume = {982},
number = {},
pages = {179641},
doi = {10.1016/j.scitotenv.2025.179641},
pmid = {40373688},
issn = {1879-1026},
abstract = {Antimicrobial resistance (AMR) is a growing global health crisis, threatening the effectiveness of antibiotics and other antimicrobial agents, leading to increased morbidity, mortality, and economic burdens. This review article provides a comprehensive analysis of AMR, beginning with a timeline of antibiotics discovery and the year of first observed resistance. Main mechanisms of AMR in bacteria, fungi, viruses, and parasites are summarized, and the main mechanisms of bacteria are given in detail. Additionally, we discussed in detail methods for detecting AMR, including phenotypic, genotypic, and advanced methods, which are crucial for identifying and monitoring AMR. In addressing AMR mitigation, we explore innovative interventions such as CRISPR-Cas systems, nanotechnology, antibody therapy, artificial intelligence (AI), and the One Health approach. Moreover, we discussed both finished and ongoing clinical trials for AMR. This review emphasizes the urgent need for global action and highlights promising technologies that could shape the future of AMR surveillance and treatment. By integrating interdisciplinary research and emerging clinical insights, this study aims to guide individuals toward impactful solutions in the battle against AMR.},
}

@article {pmid40373119,
year = {2025},
author = {Witte, IP and Lampe, GD and Eitzinger, S and Miller, SM and Berríos, KN and McElroy, AN and King, RT and Stringham, OG and Gelsinger, DR and Vo, PLH and Chen, AT and Tolar, J and Osborn, MJ and Sternberg, SH and Liu, DR},
title = {Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase.},
journal = {Science (New York, N.Y.)},
volume = {388},
number = {6748},
pages = {eadt5199},
doi = {10.1126/science.adt5199},
pmid = {40373119},
issn = {1095-9203},
mesh = {Humans ; *Transposases/genetics/metabolism ; *CRISPR-Cas Systems ; *Directed Molecular Evolution/methods ; *Gene Editing/methods ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems ; *Mutagenesis, Insertional ; },
abstract = {Programmable gene integration in human cells has the potential to enable mutation-agnostic treatments for loss-of-function genetic diseases and facilitate many applications in the life sciences. CRISPR-associated transposases (CASTs) catalyze RNA-guided DNA integration but thus far demonstrate minimal activity in human cells. Using phage-assisted continuous evolution (PACE), we generated CAST variants with >200-fold average improved integration activity. The evolved CAST system (evoCAST) achieves ~10 to 30% integration efficiencies of kilobase-size DNA cargoes in human cells across 14 tested genomic target sites, including safe harbor loci, sites used for immunotherapy, and genes implicated in loss-of-function diseases, with undetected indels and low levels of off-target integration. Collectively, our findings establish a platform for the laboratory evolution of CASTs and advance a versatile system for programmable gene integration in living systems.},
}

Humans
*Transposases/genetics/metabolism
*CRISPR-Cas Systems
*Directed Molecular Evolution/methods
*Gene Editing/methods
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems
*Mutagenesis, Insertional

@article {pmid40372912,
year = {2025},
author = {Xiang, W and Lin, X and Yang, Y and Huang, L and Chen, Y and Chen, J and Liu, L},
title = {Cas12h is a crRNA-guided DNA nickase that can be utilized for precise gene editing.},
journal = {Cell reports},
volume = {44},
number = {5},
pages = {115718},
doi = {10.1016/j.celrep.2025.115718},
pmid = {40372912},
issn = {2211-1247},
abstract = {Type V-H CRISPR-Cas system, an important subtype of type V CRISPR-Cas systems, has remained enigmatic in terms of its structure and function despite being discovered several years ago. Here, we comprehensively characterize the type V-H CRISPR-Cas system and elucidate its role as a DNA nicking system. The unique CRISPR RNA (crRNA) employed by Cas12h effector protein enables specific targeting of double-stranded DNA (dsDNA), while its RuvC domain is responsible for cleaving the non-target strand (NTS) of dsDNA. We present the structure of Cas12h bound to crRNA and target DNA. Our structural analysis reveals that the RuvC domain possesses a narrow active pocket that facilitates recognition of NTS but potentially hinders access to the target strand. Furthermore, we demonstrate that Cas12h confers adaptive immunity against invading mobile genetic elements through transcriptional gene inhibition. We have engineered an adenine base editor by fusing Cas12h with an adenine deaminase, achieving effective A-to-G substitution.},
}

@article {pmid40371045,
year = {2025},
author = {Tewari, M and Rana, P and Pande, V},
title = {Nanomaterial-Based Biosensors for the Detection of COVID-19.},
journal = {Indian journal of microbiology},
volume = {65},
number = {1},
pages = {120-136},
pmid = {40371045},
issn = {0046-8991},
abstract = {The COVID-19 outbreak began in December 2019 and has affected people worldwide. It was declared a pandemic in 2020 by the World Health Organization. Developing rapid and reliable diagnostic techniques is crucial for identifying COVID-19 early and preventing the disease from becoming severe. In addition to conventional diagnostic techniques such as RT-PCR, computed tomography, serological assays, and sequencing methods, biosensors have become widely accepted for identifying and screening COVID-19 infection with high accuracy and sensitivity. Their low cost, high sensitivity, specificity, and portability make them ideal for diagnostics. The use of nanomaterials improves the performance of biosensors by increasing their sensitivities and limiting detection by several orders of magnitude. This manuscript briefly reviews the COVID-19 outbreak and its pathogenesis. Furthermore, it comprehensively discusses the currently available biosensors for SARS-CoV-2 detection, with a special emphasis on nanomaterials-based biosensors developed to detect this emerging virus and its variants efficiently.},
}

@article {pmid40369543,
year = {2025},
author = {Zhang, T and Liu, G and Sun, S and Meng, Z and Qiu, Y and Ding, P},
title = {A novel tri-mode detection platform for ampicillin and drug resistance genes by CRISPR-driven luminescent nanozymes.},
journal = {Journal of nanobiotechnology},
volume = {23},
number = {1},
pages = {346},
pmid = {40369543},
issn = {1477-3155},
support = {82373635//the National Natural Science Foundation of China/ ; },
mesh = {*Ampicillin/analysis/pharmacology ; *Biosensing Techniques/methods ; *Anti-Bacterial Agents/analysis/pharmacology ; *CRISPR-Cas Systems ; Colorimetry ; Aptamers, Nucleotide/chemistry ; Smartphone ; Manganese Compounds/chemistry ; Oxides/chemistry ; Polymers/chemistry ; Ferrosoferric Oxide/chemistry ; Limit of Detection ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Drug Resistance, Bacterial/genetics ; beta-Lactamases/genetics ; Indoles ; },
abstract = {The antibiotic residues pose significant risks for bacterial resistance. To address the practical requirements for rapid, accurate, and on-site detection of antibiotic residues and monitoring the abundance of associated resistance genes, we report a smartphone-integrated multi-mode platform. The platform is aimed to simultaneous, accurate, and visual quantitative detection of ampicillin (AMP) and β-lactam antibiotic resistance genes (blaTEM). Specifically, we developed a magnetically controlled fluorescence, colorimetric, and photothermal biosensor based on a magnetic separation unit (aminated modified complementary DNA chain (NH2-cDNA) loading on the surface of Ferrosoferric Oxide@polydopamine (Fe3O4@PDA, FP), FP@cDNA) and a signal unit (the aptamer nucleic acid chain modified by phosphate group linked to Prussian blue@UiO-66@manganese dioxide (PB@UiO-66@MnO2, PUM) through Zr-O-P bond, PUM@Apt), for the integrated detection of AMP and blaTEM. By utilizing complementary base pairing between FP@cDNA and PUM@Apt, along with precise aptamer recognition the AMP, we achieved the fluorescence quantitative detection of AMP by measuring the signal unit in the supernatant. Subsequently, the difference of signal units in colorimetric process leads to a varying conversion rate of oxidized 3,3',5,5'-Tetramethylbenzidine (oxTMB), enabling the output of colorimetric and photothermal signals. The competitive binding of aptamers permitting the determination of AMP in the range of 0-160 pM with a low detection limit (0.34 pM). Additionally, in the presence of blaTEM, the activated CRISPR/Cas12a indiscriminately cleaves the single-stranded portion of the FP@DNA@PUM complex obtained by magnetic separation. A PUM-based three-signal detection scheme was established for the sensitive determination of blaTEM with the limit of detection (LOD) of 1.03 pM. The integration of smartphone-assisted analysis broadens the potential of the platform for visual detection. Notably, the innovative platform, with its excellent stability, exhibits great potential as a simple yet robust approach for the simultaneously visually monitoring antibiotics and drug resistance genes, and holds promise in the field of kit development.},
}

*Ampicillin/analysis/pharmacology
*Biosensing Techniques/methods
*Anti-Bacterial Agents/analysis/pharmacology
*CRISPR-Cas Systems
Colorimetry
Aptamers, Nucleotide/chemistry
Smartphone
Manganese Compounds/chemistry
Oxides/chemistry
Polymers/chemistry
Ferrosoferric Oxide/chemistry
Limit of Detection
Clustered Regularly Interspaced Short Palindromic Repeats
*Drug Resistance, Bacterial/genetics
beta-Lactamases/genetics
Indoles

@article {pmid40369382,
year = {2025},
author = {Ye, Y and Li, L and Chen, Y and Li, B and Xu, Z},
title = {Molecular methods for rapid detection and identification of foodborne pathogenic bacteria.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {5},
pages = {175},
pmid = {40369382},
issn = {1573-0972},
mesh = {*Foodborne Diseases/microbiology/diagnosis ; *Bacteria/genetics/isolation & purification/classification/pathogenicity ; *Molecular Diagnostic Techniques/methods ; *Food Microbiology/methods ; Humans ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction/methods ; Nucleic Acid Hybridization/methods ; Food Safety ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
abstract = {Foodborne pathogenic bacteria are one of the main factors causing food safety issues. The rapid and accurate detection of pathogenic bacteria using molecular techniques is an effective and powerful strategy for preventing and controlling outbreaks of foodborne diseases, thereby ensuring food safety. This article summarizes the rapid and efficient molecular diagnostic techniques for detecting pathogenic bacteria, including polymerase chain reaction and its derivatives, isothermal amplification, DNA hybridization, genomic sequencing, and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated (CRISPR/Cas)-based detection technique. Through a comparative analysis of the technical principles, advantages, and potential limitations of these diagnostic methods, as well as an outlook on the future development directions for molecular biological detection technology, which will provide a valuable reference for developing more accurate, convenient, and sensitive methods for foodborne pathogens detection, and will help better address the challenges posed by foodborne diseases, thereby ensuring public health and safety.},
}

*Foodborne Diseases/microbiology/diagnosis
*Bacteria/genetics/isolation & purification/classification/pathogenicity
*Molecular Diagnostic Techniques/methods
*Food Microbiology/methods
Humans
Nucleic Acid Amplification Techniques/methods
Polymerase Chain Reaction/methods
Nucleic Acid Hybridization/methods
Food Safety
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems

@article {pmid40354613,
year = {2025},
author = {Zhao, S and Zhang, Q and Sun, J and Li, S and Wang, S and Zhou, D and Gong, X},
title = {Simply Engineered crRNA with CRISPR/Cas12a System Enables Wide-Scope Nucleic Acid Biomarker Analysis.},
journal = {Nano letters},
volume = {25},
number = {20},
pages = {8431-8441},
doi = {10.1021/acs.nanolett.5c01939},
pmid = {40354613},
issn = {1530-6992},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *MicroRNAs/genetics/analysis ; Point Mutation ; Animals ; *RNA/genetics/analysis ; Cell Line, Tumor ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a systems have emerged as versatile tools for molecular diagnostics, but directly detecting RNA and identifying specific DNA point mutations remain challenging. Herein, we report a simple engineering approach with a split site in the spacer sequence, enabling activation of CRISPR/Cas12a (LbCas12a) for trans-cleavage with similar efficiency to wild-type crRNA. The engineered crRNA facilitated RNA target recognition by replacing the 3'-end with RNA fragments, enhancing point mutation specificity for ssDNA targets. Based on this, we achieved amplification-free detection of microRNAs and DNA point mutations with high sensitivity and specificity. For clinical sample validation, we constructed reverse fluorescence-enhanced lateral flow test strips (rLFTS), which achieved femtomole-level detection. Moreover, the engineered crRNA-based CRISPR/Cas12a system also effectively recognized tumor cells via intracellular and in vivo imaging of miRNA-21. In conclusion, this engineered crRNA platform enhances CRISPR/Cas12a-based nucleic acid detection, promoting its wide application in molecular diagnostics and bioimaging.},
}

*CRISPR-Cas Systems/genetics
Humans
*MicroRNAs/genetics/analysis
Point Mutation
Animals
*RNA/genetics/analysis
Cell Line, Tumor
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40256973,
year = {2025},
author = {Zhao, J and Wang, X and Zhang, H and Zhang, Q and Bo, D and Zhong, H and Jiao, L and Yuan, H and Lu, G},
title = {Tumor Cell Membrane Biomimetic Mesoporous Silicon Materials in Combination with PD-L1 Knockout Achieved through the CRISPR/Cas9 System for Targeted and Immunotherapeutic Purposes.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {5},
pages = {971-979},
doi = {10.1021/acs.bioconjchem.5c00001},
pmid = {40256973},
issn = {1520-4812},
mesh = {*CRISPR-Cas Systems ; Animals ; *B7-H1 Antigen/genetics ; *Silicon/chemistry ; Mice ; *Cell Membrane/metabolism/chemistry ; Porosity ; *Biomimetic Materials/chemistry ; Cell Line, Tumor ; Immunotherapy/methods ; *Antineoplastic Agents/pharmacology/chemistry ; Gene Knockout Techniques ; Humans ; Mice, Inbred C57BL ; Drug Delivery Systems ; },
abstract = {Nanoparticle-based drug delivery systems, which enable the effective and targeted delivery of chemotherapeutic drugs to tumors, have revolutionized cancer therapy. Mesoporous silicon materials (MSN) have emerged as promising candidates for drug delivery due to their unique properties. The therapeutic efficacy can be significantly enhanced when treatments exhibit both targeting and antiphagocytic properties. In this study, cell membranes extracted from B16-F10 cells were used to encapsulate carboplatin (CBP)-loaded MSN via physical extrusion. Additionally, we intratumorally injected a plasmid containing the CRISPR/Cas9 system to achieve PD-L1 knockout, thereby reactivating the immune system. The cell membrane coating endowed the CBP@MSN with excellent slow-release capability and cytocompatibility. Enhanced tumor cell uptake of CBP@MSN@M was observed due to homologous targeting by cancer cell membranes. Moreover, CBP@MSN@M demonstrated enhanced antitumor efficacy in vivo and promoted the proliferation of immune cells. Finally, the antitumor effect was further improved by the knockout of PD-L1 within the tumor microenvironment. These results suggest that the newly prepared CBP@MSN@M, combined with PD-L1 knockout, holds significant potential as an effective therapeutic approach for treating tumors.},
}

*CRISPR-Cas Systems
Animals
*B7-H1 Antigen/genetics
*Silicon/chemistry
Mice
*Cell Membrane/metabolism/chemistry
Porosity
*Biomimetic Materials/chemistry
Cell Line, Tumor
Immunotherapy/methods
*Antineoplastic Agents/pharmacology/chemistry
Gene Knockout Techniques
Humans
Mice, Inbred C57BL
Drug Delivery Systems

@article {pmid39307577,
year = {2025},
author = {Lau, CH and Huang, S and Zhu, H},
title = {Amplification-free nucleic acids detection with next-generation CRISPR/dx systems.},
journal = {Critical reviews in biotechnology},
volume = {45},
number = {4},
pages = {859-886},
doi = {10.1080/07388551.2024.2399560},
pmid = {39307577},
issn = {1549-7801},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Biosensing Techniques ; *Nucleic Acids/analysis/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR-based diagnostics (CRISPR/Dx) have revolutionized the field of molecular diagnostics. It enables home self-test, field-deployable, and point-of-care testing (POCT). Despite the great potential of CRISPR/Dx in diagnoses of biologically complex diseases, preamplification of the template often is required for the sensitive detection of low-abundance nucleic acids. Various amplification-free CRISPR/Dx systems were recently developed to enhance signal detection at sufficient sensitivity. Broadly, these amplification-free CRISPR/Dx systems are classified into five groups depending on the signal enhancement strategies employed: CRISPR/Cas12a and/or CRISPR/Cas13a are integrated with: (1) other catalytic enzymes (Cas14a, Csm6, Argonaute, duplex-specific nuclease, nanozyme, or T7 exonuclease), (2) rational-designed oligonucleotides (multivalent aptamer, tetrahedral DNA framework, RNA G-quadruplexes, DNA roller machine, switchable-caged guide RNA, hybrid locked RNA/DNA probe, hybridized cascade probe, or "U" rich stem-loop RNA), (3) nanomaterials (nanophotonic structure, gold nanoparticle, micromotor, or microbeads), (4) electrochemical and piezoelectric plate biosensors (SERS nanoprobes, graphene field-effect transistor, redox probe, or primer exchange reaction), or (5) cutting-edge detection technology platforms (digital bioanalysis, droplet microfluidic, smartphone camera, or single nanoparticle counting). Herein, we critically discuss the advances, pitfalls and future perspectives for these amplification-free CRISPR/Dx systems in nucleic acids detection. The continued refinement of these CRISPR/Dx systems will pave the road for rapid, cost-effective, ultrasensitive, and ultraspecific on-site detection without resorting to target amplification, with the ultimate goal of establishing CRISPR/Dx as the paragon of diagnostics.},
}

*CRISPR-Cas Systems/genetics
Humans
Biosensing Techniques
*Nucleic Acids/analysis/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid39084403,
year = {2025},
author = {Ding, L and Wang, X and Chen, X and Xu, X and Wei, W and Yang, L and Ji, Y and Wu, J and Xu, J and Peng, C},
title = {Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops.},
journal = {Journal of advanced research},
volume = {72},
number = {},
pages = {97-106},
doi = {10.1016/j.jare.2024.07.027},
pmid = {39084403},
issn = {2090-1224},
mesh = {*Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; Animals ; *CRISPR-Associated Proteins/genetics ; Nucleic Acid Amplification Techniques/methods ; Amino Acid Oxidoreductases ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {INTRODUCTION: Genetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation.

OBJECTIVE: In this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of CaMV35S and NOS genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection.

METHODS: The RPA reaction used for amplification CaMV35S and NOS targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure CaMV35S through the FAM channel and NOS through the HEX channel. When using lateral flow strips, CaMV35S was detected using rabbit anti-digoxin (blue line), whilst NOS was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically.

RESULTS: Detection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability.

CONCLUSION: The development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments.},
}

*Plants, Genetically Modified/genetics
*CRISPR-Cas Systems/genetics
*Crops, Agricultural/genetics
Animals
*CRISPR-Associated Proteins/genetics
Nucleic Acid Amplification Techniques/methods
Amino Acid Oxidoreductases
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid38996967,
year = {2025},
author = {Huang, X and Wu, W and Qi, H and Yan, X and Dong, L and Yang, Y and Zhang, Q and Ma, G and Zhang, G and Lei, H},
title = {Exploitation of enhanced prime editing for blocking aberrant angiogenesis.},
journal = {Journal of advanced research},
volume = {72},
number = {},
pages = {121-133},
doi = {10.1016/j.jare.2024.07.006},
pmid = {38996967},
issn = {2090-1224},
mesh = {Animals ; *Gene Editing/methods ; Mice ; *Vascular Endothelial Growth Factor Receptor-2/genetics/metabolism ; Humans ; Disease Models, Animal ; Lentivirus/genetics ; Genetic Vectors/genetics ; *Neovascularization, Pathologic/genetics/therapy ; *Retinal Neovascularization/genetics ; Genetic Therapy/methods ; *Diabetic Retinopathy/genetics/therapy ; CRISPR-Cas Systems ; Angiogenesis ; },
abstract = {INTRODUCTION: Aberrant angiogenesis plays an important part in the development of a variety of human diseases including proliferative diabetic retinopathy, with which there are still numerous patients remaining a therapeutically challenging condition. Prime editing (PE) is a versatile gene editing approach, which offers a novel opportunity to genetically correct challenging disorders.

OBJECTIVES: The goal of this study was to create a dominant-negative (DN) vascular endothelial growth factor receptor (VEGFR) 2 by editing genomic DNA with an advanced PE system to block aberrant retinal angiogenesis in a mouse model of oxygen-induced retinopathy.

METHODS: An advanced PE system (referred to as PE6x) was established within two lentiviral vectors, with one carrying an enhanced PE guide RNA and a canonical Cas9 nickase fused with an optimized reversal transcriptase, and the other conveying a nicking guide RNA and a DN-MLH1 to improve PE efficiency. Dual non-integrating lentiviruses (NILVs) produced with the two lentiviral PE6x vectors were then employed to create a mutation of VEGFR2 T17967A by editing the Mus musculus VEGFR2 locus in vitro and in vivo, leading to generation of a premature stop codon (TAG, K796stop) to produce DN-VEGFR2, to interfere with the wild type VEGFR2 which is essential for angiogenesis.

RESULTS: NILVs targeting VEGFR2 delivered into cultured murine vascular endothelial cells led to 51.06 % VEGFR2 T17967A in the genome analyzed by next generation sequencing and the production of DN-VEGFR2, which was found to hamper VEGF-induced VEGFR2 phosphorylation, as demonstrated by Western blot analysis. Intravitreally injection of the dual NILVs into postnatal day 12 mice in a model of oxygen-induced retinopathy, led to production of retinal DN-VEGFR2 in postnatal day 17 mice which blocked retinal VEGFR2 expression and activation as well as abnormal retinal angiogenesis without interfering with retinal structure and function, as assessed by electroretinography, optical coherence tomography, fundus fluorescein angiography and histology.

CONCLUSION: DN-VEGFR2 resulted from editing genomic VEGFR2 using the PE6x system can be harnessed to treat intraocular pathological angiogenesis.},
}

Animals
*Gene Editing/methods
Mice
*Vascular Endothelial Growth Factor Receptor-2/genetics/metabolism
Humans
Disease Models, Animal
Lentivirus/genetics
Genetic Vectors/genetics
*Neovascularization, Pathologic/genetics/therapy
*Retinal Neovascularization/genetics
Genetic Therapy/methods
*Diabetic Retinopathy/genetics/therapy
CRISPR-Cas Systems
Angiogenesis

@article {pmid40369306,
year = {2025},
author = {Nie, Y and Li, X and Yang, W and Fei, S and Wang, Y and Li, Y and Zhang, K and Kang, J and Cheng, Y and Wang, H and Liu, D},
title = {Concanavalin-A-assisted extraction-free one-pot RPA-CRISPR/Cas12a assay for rapid detection of HPV16.},
journal = {Mikrochimica acta},
volume = {192},
number = {6},
pages = {354},
pmid = {40369306},
issn = {1436-5073},
support = {22QNFC098//Youth Independent Innovation Science Foundation of the General Hospital of the Chinese People's Liberation Army/ ; 22QNFC098//Youth Independent Innovation Science Foundation of the General Hospital of the Chinese People's Liberation Army/ ; 24JSZ12//Military Family Planning Program of China/ ; 24JSZ12//Military Family Planning Program of China/ ; },
mesh = {*Human papillomavirus 16/genetics/isolation & purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Concanavalin A/chemistry ; DNA, Viral/genetics/analysis ; Papillomavirus Infections/diagnosis/virology ; Female ; Recombinases/metabolism ; },
abstract = {Human papillomavirus (HPV) infection is a major threat to women's health worldwide. High-risk subtypes, particularly HPV16, require rigorous screening and long-term surveillance to control cervical cancer. However, traditional HPV testing is hampered by the need for nucleic acid extraction, reliance on specialized technicians, and fluorescence detection equipment, limiting its suitability for rapid on-site testing. In this study, we developed a Concanavalin A-assisted extraction-free one-pot recombinase polymerase amplification (RPA) CRISPR/Cas12a assay (ConRCA) for HPV16. Concanavalin A-coated magnetic beads were used for target enrichment and nucleic acid-extraction-free processing. Suboptimal protospacer-adjacent motifs were used to achieve a one-pot RPA-CRISPR/Cas12a assay. The ConRCA assay can be completed in approximately 25 min under isothermal conditions and can detect at least 1.2 copies/μL of HPV16 genomic DNA using a fluorescence reader or test strip, demonstrating comparable sensitivity to qPCR. The feasibility of this detection method was evaluated with 31 unextracted clinical samples. Compared with qPCR, the overall sensitivity was 95% (19/20), and the specificity was 100% (11/11). Our results indicate that the ConRCA assay has great potential utility as a point-of-care testing for the rapid identification of HPV.},
}

*Human papillomavirus 16/genetics/isolation & purification
Humans
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Concanavalin A/chemistry
DNA, Viral/genetics/analysis
Papillomavirus Infections/diagnosis/virology
Female
Recombinases/metabolism

@article {pmid40369106,
year = {2025},
author = {Guo, T and Yang, J and Zhou, N and Sun, X and Huan, C and Lin, T and Bao, G and Hu, J and Li, G},
title = {Cas3 of type I-Fa CRISPR-Cas system upregulates bacterial biofilm formation and virulence in Acinetobacter baumannii.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {750},
pmid = {40369106},
issn = {2399-3642},
support = {82073611//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Biofilms/growth & development ; *Acinetobacter baumannii/pathogenicity/genetics/physiology ; Animals ; Virulence/genetics ; Mice ; *CRISPR-Cas Systems ; *Acinetobacter Infections/microbiology ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Virulence Factors/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Female ; Mice, Inbred BALB C ; },
abstract = {Acinetobacter baumannii (A. baumannii) is an important pathogen causing various nosocomial infections. CRISPR-Cas system is the adaptive immune system of bacteria, which is also closely related to the drug resistance and virulence of bacteria. However, the effect and mechanism of cas3 (type I-Fa) in A. baumannii is still unclear. In this study, we successfully constructed a cas3 deletion mutant (19606Δcas3) and complemented strain (19606Δcas3/pcas3) to study the regulatory mechanism of type I-Fa cas3 on bacterial virulence. Our results showed that deletion of cas3(type I-Fa) significantly reduced the biofilm formation, virulence and pathogenicity to mice. The organ bacterial load of mice infected with cas3 deletion strain was significantly reduced, the lung inflammation was slightly changed, and the serum cytokine level was also decreased. All results demonstrated that cas3 enhanced the virulence and pathogenicity of A. baumannii. Mechanism analysis showed that deletion of cas3 can lead to the down-regulation of virulence factors such as biofilm formation related factors and outer membrane protein A(ompA). In addition, cas3 was also involved in the regulation of carbon metabolism and oxidative phosphorylation pathway of A. baumannii. Altogether, our study may provide cas3 as a therapeutic target in the future because of the close link to the virulence of A. baumannii.},
}

*Biofilms/growth & development
*Acinetobacter baumannii/pathogenicity/genetics/physiology
Animals
Virulence/genetics
Mice
*CRISPR-Cas Systems
*Acinetobacter Infections/microbiology
*Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
Virulence Factors/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
Female
Mice, Inbred BALB C

@article {pmid40349461,
year = {2025},
author = {Li, W and Song, H and Cai, X and Wang, Y and Zhang, Y and Sun, C and Han, B and Zhang, J},
title = {Deletion of a novel upstream promoter of p53 impairs cold tolerance capacity in zebrafish.},
journal = {Biochemical and biophysical research communications},
volume = {769},
number = {},
pages = {151969},
doi = {10.1016/j.bbrc.2025.151969},
pmid = {40349461},
issn = {1090-2104},
mesh = {Animals ; *Zebrafish/genetics/physiology ; *Promoter Regions, Genetic/genetics ; *Tumor Suppressor Protein p53/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Cold-Shock Response/genetics ; Oxidative Stress/genetics ; Cold Temperature ; CRISPR-Cas Systems ; Reactive Oxygen Species/metabolism ; *Sequence Deletion ; },
abstract = {P53 is a tumor suppressor that plays a crucial role in stress responses. We previously identified a novel upstream p53 promoter in cold acclimated zebrafish cells, however the functional significance of this upstream promoter under cold stress in zebrafish remains unclear. We generated novel p53 promoter[-/-] zebrafish using CRISPR/Cas9 and characterized their responses to cold stress. While novel p53 promoter[-/-] zebrafish exhibited normal development, growth, and locomotion at 28 °C, they showed enhanced locomotor capacity at 18 °C and reduced cold tolerance capacity at 8 °C. RNA-seq revealed the expression of genes related to oxidative stress was downregulated at 8 °C in the gill of novel p53 promoter[-/-] zebrafish, compared with WT zebrafish. Further experiments confirmed that the ROS levels increased, and the expression of SOD and GSH-PX reduced under cold stress in novel p53 promoter[-/-] zebrafish, compared with WT zebrafish. The novel p53 promoter plays a critical role in maintaining antioxidant defense and low-temperature tolerance in zebrafish. Our findings provide new theoretical insights into the environmental adaptation mechanisms of fish and offer experimental evidence for the regulatory role of the p53 gene in response to low-temperature stress.},
}

Animals
*Zebrafish/genetics/physiology
*Promoter Regions, Genetic/genetics
*Tumor Suppressor Protein p53/genetics
*Zebrafish Proteins/genetics/metabolism
*Cold-Shock Response/genetics
Oxidative Stress/genetics
Cold Temperature
CRISPR-Cas Systems
Reactive Oxygen Species/metabolism
*Sequence Deletion

@article {pmid40340328,
year = {2025},
author = {Liu, H and Yang, Y and Zhang, N and Hou, Y and Zhang, Z and Yu, X and Huang, Y and Yan, B and Zhou, H and Mu, M and Gao, Y and Guo, K and Yang, M and Sun, X},
title = {Overcoming Photothermal Resistance of Gastric Cancer by Bionic 2D Iron-Based Nanoplatforms with Precise CRISPR/Cas9 Delivery.},
journal = {ACS nano},
volume = {19},
number = {19},
pages = {18188-18202},
doi = {10.1021/acsnano.4c16846},
pmid = {40340328},
issn = {1936-086X},
mesh = {*CRISPR-Cas Systems ; Humans ; Animals ; *Stomach Neoplasms/therapy/pathology/diagnostic imaging ; *Iron/chemistry/pharmacology ; Mice ; *Photothermal Therapy ; Ferroptosis/drug effects ; Cell Line, Tumor ; *Nanoparticles/chemistry ; Mice, Inbred BALB C ; Mice, Nude ; },
abstract = {The development of new CRISPR/Cas9 delivery systems with a synergistic therapeutic mode can provide a new perspective for efficient tumor treatment. In this work, we developed a bionic 2D FeS nanoplatform with high CRISPR/Cas9 loading (FCRM), highlighting the synergy of CRISPR/Cas9 and ferroptosis in regulating heat shock proteins (HSPs) for enhanced tumor photothermal therapy (PTT). Due to the ultrathin structure and pH-response of FeS nanosheets, FCRM can quickly decompose in a tumor microenvironment and effectively release CRISPR/Cas9 and Fe[2+], which can be further enhanced by a photothermal process. CRISPR/Cas9 can accurately downregulate the level of intracellular antiapoptosis protein Survivin. Fe[2+] can induce lipid peroxidation and ferroptosis of tumor cells. Ferroptosis and the regulation of Survivin protein can synergistically downregulate the expression of HSPs, thereby reducing the photothermal resistance of tumor cells in PTT. Additionally, FCRM caused significant tumor magnetic resonance contrast enhancement, which aided in the accurate diagnosis of tumors. Therefore, FCRM has great potential in achieving targeted magnetic resonance imaging and dual regulation of HSPs for ferroptosis-gene enhanced tumor PTT.},
}

*CRISPR-Cas Systems
Humans
Animals
*Stomach Neoplasms/therapy/pathology/diagnostic imaging
*Iron/chemistry/pharmacology
Mice
*Photothermal Therapy
Ferroptosis/drug effects
Cell Line, Tumor
*Nanoparticles/chemistry
Mice, Inbred BALB C
Mice, Nude

@article {pmid40363763,
year = {2025},
author = {Wang, L and Liu, Y and Song, H and Zhang, X and Wang, Y},
title = {Conditional Control of CRISPR/Cas9 Function by Chemically Modified Oligonucleotides.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {9},
pages = {},
pmid = {40363763},
issn = {1420-3049},
support = {22307107//the National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Oligonucleotides/chemistry/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Animals ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats) system has emerged as a revolutionary gene-editing tool with immense potential in gene therapy, functional genomics, and beyond. However, achieving precise spatiotemporal control of gene editing in specific cells and tissues while effectively mitigating potential risks, such as off-target effects, remains a key challenge for its clinical translation. To overcome these limitations, researchers have developed innovative strategies based on chemical modifications of oligonucleotides to enhance the precision, efficiency, and controllability of CRISPR/Cas9-mediated gene editing. By introducing conditional responsive elements, such as photosensitive groups, small-molecule responsive units, and supramolecular structures, they have successfully achieved precise spatiotemporal and dose-dependent regulation of CRISPR/Cas9 function. This review provides a comprehensive overview of recent advancements in gRNA regulation strategies based on chemical modifications of oligonucleotides, discussing their applications in improving the efficiency, specificity, and controllability of CRISPR/Cas9 editing. We also highlight the challenges associated with the conditional control of gRNA and offer insights into future directions for the chemical regulation of gRNA to further advance CRISPR/Cas9 technology.},
}

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

@article {pmid40362657,
year = {2025},
author = {Seijas, A and Cora, D and Novo, M and Al-Soufi, W and Sánchez, L and Arana, ÁJ},
title = {CRISPR/Cas9 Delivery Systems to Enhance Gene Editing Efficiency.},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362657},
issn = {1422-0067},
support = {ND//Campus Terra, University of Santiago de Compostela/ ; ND//Fundación Caixa Rural Galega Tomás Notario Vacas/ ; investment line no.1 of its component number 17//Spain's Recovery and Resilience Plan, Complementary RTDI Plan for Marine Science/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; Animals ; *Gene Transfer Techniques ; Nanoparticles/chemistry ; Lipids/chemistry ; },
abstract = {CRISPR/Cas9 has revolutionized genome editing by enabling precise and efficient genetic modifications across multiple biological systems. Despite its growing therapeutic potential, key challenges remain in mitigating off-target effects, minimizing immunogenicity, and improving the delivery of CRISPR components into target cells. This review provides an integrated analysis of physical, viral, and non-viral delivery systems, highlighting recent advances in the use of lipid nanoparticles, polymeric carriers, and hybrid platforms. We also examine an often overlooked factor: the aggregation behavior of the Cas9 protein, which may interfere with cellular uptake, the encapsulation efficiency, and nuclear localization. By comparing delivery platforms and their reported editing outcomes, we identify critical physicochemical parameters that influence therapeutic success. Finally, we propose standardized methods to assess Cas9 encapsulation and aggregation and discuss translational barriers such as manufacturing scalability and regulatory requirements. These insights aim to guide the development of safer and more effective CRISPR/Cas9-based therapies.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans
Animals
*Gene Transfer Techniques
Nanoparticles/chemistry
Lipids/chemistry

@article {pmid40362595,
year = {2025},
author = {Luo, X and Weidinger, E and Burghardt, T and Höhn, M and Wagner, E},
title = {CRISPR/Cas9 Ribonucleoprotein Delivery Enhanced by Lipo-Xenopeptide Carriers and Homology-Directed Repair Modulators: Insights from Reporter Cell Lines.},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362595},
issn = {1422-0067},
support = {SFB1032 (project-ID 201269156) sub-project B4//Deutsche Forschungsgemeinschaft/ ; No 825825 UPGRADE//European Union/ ; CNATM, number 03ZU1201AA//Federal Ministry of Education and Research/ ; doctoral fellowship to XL//China Scholarship Council/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Recombinational DNA Repair/drug effects ; *Ribonucleoproteins/genetics/metabolism ; HeLa Cells ; DNA End-Joining Repair ; Peptides ; },
abstract = {CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Recombinational DNA Repair/drug effects
*Ribonucleoproteins/genetics/metabolism
HeLa Cells
DNA End-Joining Repair
Peptides

@article {pmid40362588,
year = {2025},
author = {Arana, ÁJ and González-Llera, L and Barreiro-Iglesias, A and Sánchez, L},
title = {Emerging Frontiers in Zebrafish Embryonic and Adult-Derived Cell Lines.},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362588},
issn = {1422-0067},
support = {PID2020-115121GB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; PID2023-147266NB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; ED431C 2022/33//Xunta de Galicia/ ; },
mesh = {Animals ; *Zebrafish/embryology/genetics ; Cell Line ; *Embryo, Nonmammalian/cytology ; CRISPR-Cas Systems ; Cell Culture Techniques/methods ; Humans ; },
abstract = {Zebrafish (Danio rerio) has become a pivotal vertebrate model in biomedical research, renowned for its genetic similarity to humans, optical transparency, rapid embryonic development, and amenability to experimental manipulation. In recent years, the derivation of cell lines from zebrafish embryos has unlocked new possibilities for in vitro studies across developmental biology, toxicology, disease modeling, and genetic engineering. These embryo-derived cultures offer scalable, reproducible, and ethically favorable alternatives to in vivo approaches, enabling high-throughput screening and mechanistic exploration under defined conditions. This review provides a comprehensive overview of protocols for establishing and maintaining zebrafish embryonic cell lines, emphasizing culture conditions, pluripotency features, transfection strategies, and recent innovations such as genotype-defined mutant lines generated via CRISPR/Cas9 and feeder-free systems. We also highlight emerging applications in oncology, regenerative medicine, and functional genomics, positioning zebrafish cell lines as versatile platforms bridging animal models and next-generation in vitro systems. Its continued optimization holds promise for improved reproducibility, reduced animal use, and expanded translational impact in biomedical research.},
}

Animals
*Zebrafish/embryology/genetics
Cell Line
*Embryo, Nonmammalian/cytology
CRISPR-Cas Systems
Cell Culture Techniques/methods
Humans

@article {pmid40362571,
year = {2025},
author = {Herreno-Pachón, AM and Leal, AF and Khan, S and Alméciga-Díaz, CJ and Tomatsu, S},
title = {CRISPR/nCas9-Edited CD34+ Cells Rescue Mucopolysaccharidosis IVA Fibroblasts Phenotype.},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362571},
issn = {1422-0067},
support = {1R01HD102545-01A1/GF/NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Fibroblasts/metabolism/pathology ; *Gene Editing/methods ; *Antigens, CD34/metabolism/genetics ; Genetic Therapy/methods ; *Mucopolysaccharidosis IV/therapy/genetics/metabolism/pathology ; Phenotype ; Animals ; Mice ; Chondroitinsulfatases/genetics/metabolism ; },
abstract = {Mucopolysaccharidosis (MPS) IVA is a bone-affecting lysosomal storage disease (LSD) caused by impaired degradation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) due to deficient N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity. Previously, we successfully developed and validated a CRISPR/nCas9-based gene therapy (GT) to insert an expression cassette at the AAVS1 and ROSA26 loci in human MPS IVA fibroblasts and MPS IVA mice, respectively. In this study, we have extended our approach to evaluate the effectiveness of our CRISPR/nCas9-based GT in editing human CD34+ cells to mediate cross-correction of MPS IVA fibroblasts. CD34+ cells were electroporated with the CRISPR/nCas9 system, targeting the AAVS1 locus. The nCas9-mediated on-target donor template insertion, and the stemness of the CRISPR/nCas-edited CD34+ cells was evaluated. Additionally, MPS IVA fibroblasts were co-cultured with CRISPR/nCas-edited CD34+ cells to assess cross-correction. CRISPR/nCas9-based gene editing did not affect the stemness of CD34+ cells but did lead to supraphysiological levels of the GALNS enzyme. Upon co-culture, MPS IVA fibroblasts displayed a significant increase in the GALNS enzyme activity along with lysosomal mass reduction, pro-oxidant profile amelioration, mitochondrial mass recovery, and pro-apoptotic and pro-inflammatory profile improvement. These results show the potential of our CRISPR/nCas9-based GT to edit CD34+ cells to mediate cross-correction.},
}

Humans
*CRISPR-Cas Systems
*Fibroblasts/metabolism/pathology
*Gene Editing/methods
*Antigens, CD34/metabolism/genetics
Genetic Therapy/methods
*Mucopolysaccharidosis IV/therapy/genetics/metabolism/pathology
Phenotype
Animals
Mice
Chondroitinsulfatases/genetics/metabolism

@article {pmid40362308,
year = {2025},
author = {Haider, S and Mussolino, C},
title = {Fine-Tuning Homology-Directed Repair (HDR) for Precision Genome Editing: Current Strategies and Future Directions.},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362308},
issn = {1422-0067},
support = {CA311-41//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Recombinational DNA Repair ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; Animals ; DNA Breaks, Double-Stranded ; },
abstract = {CRISPR-Cas9 is a powerful genome-editing technology that can precisely target and cleave DNA to induce double-strand breaks (DSBs) at almost any genomic locus. While this versatility holds tremendous therapeutic potential, the predominant cellular pathway for DSB repair-non-homologous end-joining (NHEJ)-often introduces small insertions or deletions that disrupt the target site. In contrast, homology-directed repair (HDR) utilizes exogenous donor templates to enable precise gene modifications, including targeted insertions, deletions, and substitutions. However, HDR remains relatively inefficient compared to NHEJ, especially in postmitotic cells where cell cycle constraints further limit HDR. To address this challenge, numerous methodologies have been explored, ranging from inhibiting key NHEJ factors and optimizing donor templates to synchronizing cells in HDR-permissive phases and engineering HDR-enhancing fusion proteins. These strategies collectively aim to boost HDR efficiency and expand the clinical and research utility of CRISPR-Cas9. In this review, we discuss recent advances in manipulating the balance between NHEJ and HDR, examine the trade-offs and practical considerations of these approaches, and highlight promising directions for achieving high-fidelity genome editing in diverse cell types.},
}

*Gene Editing/methods
Humans
*Recombinational DNA Repair
*CRISPR-Cas Systems
DNA End-Joining Repair
Animals
DNA Breaks, Double-Stranded

@article {pmid40362304,
year = {2025},
author = {Shiryaeva, O and Tolochko, C and Alekseeva, T and Dyachuk, V},
title = {Targets and Gene Therapy of ALS (Part 1).},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362304},
issn = {1422-0067},
mesh = {Humans ; *Amyotrophic Lateral Sclerosis/therapy/genetics/pathology ; *Genetic Therapy/methods ; Animals ; Superoxide Dismutase-1/genetics ; C9orf72 Protein/genetics ; Mutation ; Gene Editing ; RNA-Binding Protein FUS/genetics ; Oligonucleotides, Antisense/therapeutic use ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; RNA Interference ; MicroRNAs/genetics ; Disease Models, Animal ; RNA, Small Interfering/genetics ; Motor Neurons/metabolism/pathology ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motor neurons, which causes muscle atrophy. Genetic forms of ALS are recorded only in 10% of cases. However, over the past decade, studies in genetics have substantially contributed to our understanding of the molecular mechanisms underlying ALS. The identification of key mutations such as SOD1, C9orf72, FUS, and TARDBP has led to the development of targeted therapy that is gradually being introduced into clinical trials, opening up a broad range of opportunities for correcting these mutations. In this review, we aimed to present an extensive overview of the currently known mechanisms of motor neuron degeneration associated with mutations in these genes and also the gene therapy methods for inhibiting the expression of their mutant proteins. Among these, antisense oligonucleotides, RNA interference (siRNA and miRNA), and gene-editing (CRISPR/Cas9) methods are of particular interest. Each has shown its efficacy in animal models when targeting mutant genes, whereas some of them have proven to be efficient in human clinical trials.},
}

Humans
*Amyotrophic Lateral Sclerosis/therapy/genetics/pathology
*Genetic Therapy/methods
Animals
Superoxide Dismutase-1/genetics
C9orf72 Protein/genetics
Mutation
Gene Editing
RNA-Binding Protein FUS/genetics
Oligonucleotides, Antisense/therapeutic use
CRISPR-Cas Systems
DNA-Binding Proteins/genetics
RNA Interference
MicroRNAs/genetics
Disease Models, Animal
RNA, Small Interfering/genetics
Motor Neurons/metabolism/pathology

@article {pmid40361099,
year = {2025},
author = {Xie, H and Bourgade, B and Stensjö, K and Lindblad, P},
title = {dCas12a-mediated CRISPR interference for multiplex gene repression in cyanobacteria for enhanced isobutanol and 3-methyl-1-butanol production.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {104},
pmid = {40361099},
issn = {1475-2859},
support = {2021-01669//Svenska Forskningsrådet Formas/ ; CTS 20:412//Carl Tryggers Stiftelse för Vetenskaplig Forskning/ ; 2024-00443//Vinnova/ ; },
mesh = {*Butanols/metabolism ; *Metabolic Engineering/methods ; *CRISPR-Cas Systems ; *Synechocystis/genetics/metabolism ; *Pentanols/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {BACKGROUND: Cyanobacteria of the genera Synechocystis and Synechococcus have emerged as promising platforms for metabolic engineering endeavors aimed at converting carbon dioxide into valuable fuels and chemicals, thus addressing the pressing energy demand and mitigating global climate change. Notably, Synechocystis sp. strain PCC 6803 (Synechocystis) has been engineered to produce isobutanol (IB) and 3-methyl-1-butanol (3M1B) via heterologous expression of α-ketoisovalerate decarboxylase (Kivd). Despite these advances, the achieved IB/3M1B titers remain low. CRISPR interference (CRISPRi), an emerging tool for targeted gene repression, has demonstrated success in various cellular systems to enhance biochemical productivity.

RESULTS: In this study, we developed a dCas12a-mediated CRISPRi system (CRISPRi-dCas12a) that effectively blocked the transcriptional initiation/elongation of essential gene(s), resulting in up to 60% gene repression in Synechocystis. Subsequently, the CRISPRi-dCas12a system was successfully integrated into an IB/3M1B producer strain, where it exhibited target gene repression under optimal cultivation conditions. To identify gene targets involved in metabolic pathways potentially limiting IB/3M1B biosynthesis, we initially designed a CRISPR RNA (crRNA) library targeting fifteen individual gene(s), where repression of ten genes significantly increased IB/3M1B production per cell. Moreover, a synergetic effect was observed on IB/3M1B production by designing a single crRNA targeting multiple genes for simultaneous repression. A final strain HX106, featuring dual repression of ppc and gltA, both involved in the TCA cycle, resulted in 2.6-fold and 14.8-fold improvement in IB and 3M1B production per cell, respectively.

CONCLUSIONS: Our findings underscore the effectiveness of the CRISPRi-dCas12a system in Synechocystis for identifying competing pathways and redirecting carbon flux to enhance IB/3M1B production. Furthermore, this study established a solid groundwork for utilizing an expanded CRISPRi-crRNA library to undertake genome-wide exploration of potential competing pathways not only for IB/3M1B biosynthesis but also for other diverse biofuels and biochemical production processes.},
}

*Butanols/metabolism
*Metabolic Engineering/methods
*CRISPR-Cas Systems
*Synechocystis/genetics/metabolism
*Pentanols/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40273314,
year = {2025},
author = {Montagud-Martínez, R and Márquez-Costa, R and Ruiz, R and Martínez-Aviñó, A and Ballesteros-Garrido, R and Navarro, D and Campins-Falcó, P and Rodrigo, G},
title = {Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay.},
journal = {ACS applied bio materials},
volume = {8},
number = {5},
pages = {4221-4229},
doi = {10.1021/acsabm.5c00307},
pmid = {40273314},
issn = {2576-6422},
mesh = {*CRISPR-Cas Systems ; Humans ; *SARS-CoV-2/isolation & purification/genetics ; *COVID-19/diagnosis/virology ; Nucleic Acid Amplification Techniques/methods ; Chromatography, Affinity/methods ; },
abstract = {In public health emergencies or in resource-constrained settings, laboratory-based diagnostic methods, such as RT-qPCR, need to be complemented with accurate, rapid, and accessible approaches to increase testing capacity, as this will translate into better outcomes in disease prevention and management. Here, we develop an original nucleic acid detection platform by leveraging CRISPR-Cas9 and lateral flow immunochromatography technologies. In combination with an isothermal amplification that runs with a biotinylated primer, the system exploits the interaction between the CRISPR-Cas9 R-loop formed upon targeting a specific nucleic acid and a fluorescein-labeled probe to generate a visual readout on a lateral flow device. Our method enables rapid, sensitive detection of nucleic acids, achieving a limit of 1-10 copies/μL in 1 h at a low temperature. We validated the efficacy of the method by using clinical samples of patients infected with SARS-CoV-2. Compared with other assays, it operates with more accessible molecular elements and showcases a robust signal-to-noise ratio. Moreover, multiplexed detection was demonstrated using primers labeled with biotin and digoxigenin, achieving the simultaneous identification of target genes on lateral flow devices with two test lines. We successfully detected SARS-CoV-2 and Influenza A (H1N1) in spiked samples, highlighting the potential of the method for multiplexed diagnostics of respiratory viruses. All in all, this represents a versatile and manageable platform for point-of-care testing, thereby supporting better patient outcomes and enhanced pandemic preparedness.},
}

*CRISPR-Cas Systems
Humans
*SARS-CoV-2/isolation & purification/genetics
*COVID-19/diagnosis/virology
Nucleic Acid Amplification Techniques/methods
Chromatography, Affinity/methods

@article {pmid39431534,
year = {2025},
author = {Hu, K and Geng, M and Ma, L and Yao, G and Zhang, M and Zhang, H},
title = {The H2S-responsive transcription factor ERF.D3 regulates tomato abscisic acid metabolism, leaf senescence, and fruit ripening.},
journal = {Plant physiology},
volume = {197},
number = {2},
pages = {},
doi = {10.1093/plphys/kiae560},
pmid = {39431534},
issn = {1532-2548},
support = {32170315//National Natural Science Foundation of China/ ; 202304a05020081//Key Technologies Research and Development Program/ ; JZ2021HGPA0063//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Plant Senescence ; *Abscisic Acid/metabolism ; Plant Leaves/physiology ; *Hydrogen Sulfide/metabolism ; Transcription Factors ; *Solanum lycopersicum/genetics/physiology ; Real-Time Polymerase Chain Reaction ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Genes, Plant/physiology ; Plant Proteins/genetics/metabolism ; },
abstract = {Hydrogen sulfide (H2S) is a signaling molecule that regulates plant senescence. In this study, we found that H2S delays dark-induced senescence in tomato (Solanum lycopersicum) leaves. Transcriptome and reverse transcription quantitative PCR (RT-qPCR) analyses revealed an ethylene response factor ERF.D3 is quickly induced by H2S. H2S also persulfidated ERF.D3 at amino acid residues C115 and C118. CRISPR/Cas9-mediated gene editing, and gene overexpression analyses showed that ERF.D3 negatively regulates leaf senescence and fruit ripening. Abscisic acid (ABA) levels were reduced by ERF.D3 overexpression, suggesting ERF.D3 might regulate ABA metabolism. Additionally, the ABA 8'-hydroxylase-encoding gene CYP707A2, which is required for ABA degradation, was identified as an ERF.D3 target gene through transcriptome data, RT-qPCR, dual-luciferase reporter assays, and electrophoretic mobility shift assays. ERF.D3 persulfidation enhanced its transcriptional activity toward CYP707A2. Moreover, the E3 ligase RNF217 ubiquitinated ERF.D3, which may accelerate fruit ripening during the late stage of fruit development. Overall, our study provides valuable insights into the roles of a H2S-responsive ERF.D3 and its persulfidation state in delaying leaf senescence and fruit ripening and provides a link between H2S and ABA degradation.},
}

*Plant Senescence
*Abscisic Acid/metabolism
Plant Leaves/physiology
*Hydrogen Sulfide/metabolism
Transcription Factors
*Solanum lycopersicum/genetics/physiology
Real-Time Polymerase Chain Reaction
CRISPR-Cas Systems
Gene Expression Regulation, Plant
Genes, Plant/physiology
Plant Proteins/genetics/metabolism

@article {pmid40319546,
year = {2025},
author = {Tavella, S and di Lillo, A and Conti, A and Iannelli, F and Mancheno-Ferris, A and Matti, V and Di Micco, R and Fagagna, FDD},
title = {Weaponizing CRISPR/Cas9 for selective elimination of cells with an aberrant genome.},
journal = {DNA repair},
volume = {149},
number = {},
pages = {103840},
doi = {10.1016/j.dnarep.2025.103840},
pmid = {40319546},
issn = {1568-7856},
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; HeLa Cells ; *Gene Editing/methods ; Green Fluorescent Proteins/genetics/metabolism ; DNA Repair ; Cell Survival ; },
abstract = {The CRISPR/Cas9 technology is a powerful and versatile tool to disrupt genes' functions by introducing sequence-specific DNA double-strand breaks (DSBs). Here, we repurpose this technology to eradicate aberrant cells by specifically targeting silent and non-functional genomic sequences present only in target cells to be eliminated. Indeed, an intrinsic challenge of most current therapies against cancer and viral infections is the non-specific toxicity that they can induce in normal tissues because of their impact on important cellular mechanisms shared, to different extents, between unhealthy and healthy cells. The CRISPR/Cas9 technology has potential to overcome this limitation; however, so far effectiveness of these approaches was made dependent on the targeting and inactivation of a functional gene product. Here, we generate proof-of-principle evidence by engineering HeLa and RKO cells with a promoterless Green Fluorescent Protein (GFP) construct. The integration of this construct simulates either a genomic alteration, as in cancer cells, or a silent proviral genome. Cas9-mediated DSBs in the GFP sequence activate the DNA damage response (DDR), reduce cell viability and increase mortality. This is associated with increased cell size, multinucleation, cGAS-positive micronuclei accumulation and the activation of an inflammatory response. Pharmacological inhibition of the DNA repair factor DNA-PK enhances cell death. These results demonstrate the therapeutic potential of the CRISPR/Cas9 system in eliminating cells with an aberrant genome, regardless of the expression or the function of the target DNA sequence.},
}

Humans
*CRISPR-Cas Systems
*DNA Breaks, Double-Stranded
HeLa Cells
*Gene Editing/methods
Green Fluorescent Proteins/genetics/metabolism
DNA Repair
Cell Survival

@article {pmid40250571,
year = {2025},
author = {Ali, A and Azmat, U and Khatoon, A and Akbar, K and Murtaza, B and Ji, Z and Irshad, U and Su, Z},
title = {From gene editing to tumor eradication: The CRISPR revolution in cancer therapy.},
journal = {Progress in biophysics and molecular biology},
volume = {196},
number = {},
pages = {114-131},
doi = {10.1016/j.pbiomolbio.2025.04.003},
pmid = {40250571},
issn = {1873-1732},
mesh = {*Gene Editing/methods ; Humans ; *Neoplasms/therapy/genetics/immunology ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods ; Immunotherapy/methods ; },
abstract = {Cancer continues to be a significant worldwide health concern, characterized by high rates of occurrence and death. Unfortunately, existing treatments frequently fall short of delivering satisfying therapeutic outcomes. Immunotherapy has ushered in a new era in the treatment of solid tumors, yet its effectiveness is still constrained and comes with unwanted side effects. The advancement of cutting-edge technology, propelled by gene analysis and manipulation at the molecular scale, shows potential for enhancing these therapies. The advent of genome editing technologies, including CRISPR-Cas9, can greatly augment the efficacy of cancer immunotherapy. This review explores the mechanism of CRISPR-Cas9-mediated genome editing and its wide range of tools. The study focuses on analyzing the effects of CRISPR-induced double-strand breaks (DSBs) on cancer immunotherapy, specifically by gene knockdown or knockin. In addition, the study emphasizes the utilization of CRISPR-Cas9-based genome-wide screening to identify targets, the potential of spatial CRISPR genomics, and the extensive applications and difficulties of CRISPR-Cas9 in fundamental research, translational medicine, and clinical environments.},
}

*Gene Editing/methods
Humans
*Neoplasms/therapy/genetics/immunology
*CRISPR-Cas Systems/genetics
Animals
*Genetic Therapy/methods
Immunotherapy/methods

@article {pmid40123001,
year = {2025},
author = {Zhang, T and Zhang, Y and Wang, X and Hu, H and Lin, CG and Xu, Y and Zheng, H},
title = {Genome-wide CRISPR activation screen identifies ARL11 as a sensitivity determinant of PARP inhibitor therapy.},
journal = {Cancer gene therapy},
volume = {32},
number = {5},
pages = {521-537},
pmid = {40123001},
issn = {1476-5500},
support = {81972462//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Female ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Breast Neoplasms/genetics/drug therapy/pathology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Neoplasm/genetics ; },
abstract = {Resistance to poly-(ADP)-ribose polymerase inhibitors (PARPi) remains a significant challenge in clinical practice, leading to treatment failure in many patients. It is crucial to better understand the molecular mechanisms that underlie PARPi resistance. In this study, utilizing a genome-wide CRISPR activation screen with olaparib, we identified ARL11 as a potential modulator of PARPi treatment response in BRCA-wild-type MDA-MB-231 cells. Mechanistically, ARL11 interacts with STING to enhance innate immunity and forms positive feedback with type I interferon (IFN) induction, which induces ARL11 up-regulation and contributes to resistance to PARPi therapy. Additionally, we observed that ARL11 interacts with the RUVBL1 and RUVBL2 (RUVBL1/2) complex, the key DNA double-strand repair proteins, facilitating DNA homologous recombination (HR) repair and significantly reducing PARPi-induced DNA double-strand damages. Clinical sample analysis reveals that the expression levels of ARL11 and RUVBL1/2 are significantly elevated in breast cancer patients compared to healthy controls. Collectively, our findings suggested that ARL11 and RUVBL1/2 may be promising therapeutic targets to sensitize breast cancer cells to PARPi therapy.},
}

Humans
*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use
Female
CRISPR-Cas Systems
Cell Line, Tumor
*Breast Neoplasms/genetics/drug therapy/pathology
Clustered Regularly Interspaced Short Palindromic Repeats
Drug Resistance, Neoplasm/genetics

@article {pmid40361067,
year = {2025},
author = {Avci, FG and Prasun, T and Wendisch, VF},
title = {Metabolic engineering for microbial production of sugar acids.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {36},
pmid = {40361067},
issn = {1472-6750},
support = {031B1737C//German Federal Ministry of Education and Research (BMBF) as part of the collaborative research project ForceYield2/ ; online publication fund//Deutsche Forschungsgemeinschaft/ ; open access publication fund//Universität Bielefeld/ ; },
mesh = {*Metabolic Engineering/methods ; *Sugar Acids/metabolism ; Fermentation ; Synthetic Biology ; Oxidation-Reduction ; *Bacteria/metabolism/genetics ; },
abstract = {Carbohydrates including sugar acids are commonly used as carbon sources in microbial biotechnology. These sugar acids are themselves desirable and often overlooked targets for biobased production since they find applications in a broad range of industries, examples include food, construction, medical, textile, and polymer industries. Different stages of oxidation for natural sugar acids can be distinguished. Oxidation of the aldehyde group yields aldonic acids, oxidation of the primary hydroxy group leads to uronic acids, and both oxidations combined yield aldaric acids. While the chemical oxidation of sugars to their acid forms often is a one-pot reaction under harsh conditions, their biosynthesis is much more delicate. Bio-based production can involve enzymatic conversion, whole-cell biotransformation, and fermentation. Generally, the in vivo approaches are preferred because they are less resource-intensive than enzymatic conversion. Metabolic engineering plays a crucial role in optimizing microbial strains for efficient sugar acid production. Strategies include pathway engineering to overexpress key enzymes involved in sugar oxidation, deletion of competing pathways to enhance the precursor availability and eliminate the product consumption, cofactor balancing for efficient redox reactions, and transporter engineering to facilitate precursor import or sugar acid export. Synthetic biology tools, such as CRISPR-Cas and dynamic regulatory circuits, have further improved strain development by enabling precise genetic modifications and adaptive control of metabolic fluxes. The usage of plant biomass hydrolysates for bio-based production further adds to the environmental friendliness of the in vivo approaches. This review highlights the different approaches for the production of C5 and C6 sugar acids, their applications, and their catabolism in microbes.},
}

*Metabolic Engineering/methods
*Sugar Acids/metabolism
Fermentation
Synthetic Biology
Oxidation-Reduction
*Bacteria/metabolism/genetics

@article {pmid40360740,
year = {2025},
author = {Tei, C and Hata, S and Mabuchi, A and Okuda, S and Ito, KK and Genova, M and Fukuyama, M and Yamamoto, S and Chinen, T and Toyoda, A and Kitagawa, D},
title = {Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {749},
pmid = {40360740},
issn = {2399-3642},
support = {18K06246, 19H05651, 20K15987, 20K22701, 21H02623, 22H02629, 22K19305, 22K19370, 22K20624, 23K14176//Japan Society for the Promotion of Science London (JSPS London)/ ; JPMJPR21EC//MEXT | JST | Precursory Research for Embryonic Science and Technology (PRESTO)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; Humans ; *DNA End-Joining Repair ; *Gene Editing/methods ; *DNA Repair ; Gene Knock-In Techniques ; Recombinational DNA Repair ; HEK293 Cells ; },
abstract = {CRISPR-mediated endogenous tagging is a powerful tool in biological research. Inhibiting the non-homologous end joining (NHEJ) pathway has been shown to improve the low efficiency of accurate knock-in via homology-directed repair (HDR). However, the influence of alternative double-stranded break (DSB) repair pathways on knock-in remains to be fully explored. In this study, our long-read amplicon sequencing analysis reveals various patterns of imprecise repair in CRISPR-mediated knock-in, even with NHEJ inhibition. Further suppressing either microhomology-mediated end joining (MMEJ) or single-strand annealing (SSA) reduces nucleotide deletions around the cut site, thereby elevating knock-in accuracy. Additionally, imprecise donor integration is reduced by inhibiting SSA, but not MMEJ. Particularly, SSA suppression reduced asymmetric HDR, a specific imprecise integration pattern, which we further confirm using a novel reporter system. These findings demonstrate the complex interplay of multiple DSB repair pathways in CRISPR-mediated knock-in and offer novel strategies, including SSA pathway targeting, to improve precise gene editing efficiency.},
}

*DNA Breaks, Double-Stranded
*CRISPR-Cas Systems
Humans
*DNA End-Joining Repair
*Gene Editing/methods
*DNA Repair
Gene Knock-In Techniques
Recombinational DNA Repair
HEK293 Cells

@article {pmid40359574,
year = {2025},
author = {Zhou, J and Pang, R and Han, Y and Guo, Y and Wang, Y and Yang, H and Wang, W and Fu, X and Zhang, R and Zheng, X and Zhang, T and Zhang, Y and Wang, Q},
title = {CRISPR-Cas9-mediated knockout of OsKCS11 in rice reveals potential crosstalk between very-long-chain fatty acids and cytokinin.},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {3},
pages = {e70208},
doi = {10.1111/tpj.70208},
pmid = {40359574},
issn = {1365-313X},
support = {2020B1515120086//Guangdong Basic and Applied Basic Research Foundation/ ; SZKF2103//Open Project Program of Panxi Crops Research and Utilization Key Laboratory of Sichuan Province/ ; 2023ZD04074//the Biological Breeding- National Science and Technology Major Projects/ ; 2021B0707010006//Key-Area Research and Development Program of Guangdong Province/ ; CAAS-CSIAF-202303//Innovation Program of CAAS/ ; 31961143015//National Natural Science Foundation of China/ ; 32070202//National Natural Science Foundation of China/ ; 2021YFYZ0016//Sichuan Science and Technology Program/ ; 2024NSFSC0314//Sichuan Science and Technology Program/ ; },
mesh = {*Oryza/genetics/metabolism ; *Cytokinins/metabolism ; CRISPR-Cas Systems/genetics ; *Fatty Acids/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Knockout Techniques ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; *3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/genetics/metabolism ; },
abstract = {Very-long-chain fatty acids (VLCFAs) play crucial roles in various physiological processes in plants. Through our investigation using a CRISPR-Cas9 knockout mutant library in rice, we identified a semi-dwarf rice mutant named CRISPR-Cas-based dwarf-1 (csd-1). This mutant displayed multiple developmental defects, such as decreased plant height, panicle length, seed size, and seed-setting rate. Whole-genome resequencing analysis revealed that a T-nucleotide insertion in β-ketoacyl-CoA synthase 11 (KCS11), responsible for the initial step in fatty acid elongation, was responsible for the observed defects in csd-1. The identity of csd-1 was confirmed through genetic complementation and CRISPR-Cas9-mediated knockout. Expression analysis indicated that OsKCS11 was present in various tissues, with differential abundance observed through RT-qPCR and promoter GUS staining, and strong localization at the node position by RNA in situ hybridization; furthermore, OsKCS11 protein was confirmed to be in the endoplasmic reticulum. Furthermore, csd-1 exhibited significantly reduced levels of linolenic acid (18:3), C24:0-OH, C28:0-alkanes, C29:0-alkanes, alpha-tocopherol, and C33:0-alkanes, while trans-nonadecenoic acid and behenic acid levels were increased. Cytokinin analysis revealed significant increases in isopentenyladenine (IPA) and cis-zeatin (cZ) levels in csd-1. Molecular investigations indicated upregulation of genes involved in cytokinin biosynthesis or signaling, suggesting a potential link between VLCFAs and cytokinin synthesis through acetyl-CoA. This study not only proposed an alternative gene mapping method based on whole-genome resequencing but also elucidated the mechanism by which VLCFAs influence cytokinin synthesis and signaling.},
}

*Oryza/genetics/metabolism
*Cytokinins/metabolism
CRISPR-Cas Systems/genetics
*Fatty Acids/metabolism
*Plant Proteins/genetics/metabolism
Gene Knockout Techniques
Gene Expression Regulation, Plant
Plants, Genetically Modified
*3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/genetics/metabolism

@article {pmid40358185,
year = {2025},
author = {Boff, MO and Xavier, FAC and Diz, FM and Gonçalves, JB and Ferreira, LM and Zambeli, J and Pazzin, DB and Previato, TTR and Erwig, HS and Gonçalves, JIB and Bruzzo, FTK and Marinowic, D and da Costa, JC and Zanirati, G},
title = {mTORopathies in Epilepsy and Neurodevelopmental Disorders: The Future of Therapeutics and the Role of Gene Editing.},
journal = {Cells},
volume = {14},
number = {9},
pages = {},
pmid = {40358185},
issn = {2073-4409},
support = {Not applicable//Pontifical Catholic University of Rio Grande do Sul (PUCRS) - Brain Institute of Rio Grande do Sul (BraIns)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Neurodevelopmental Disorders/therapy/genetics ; *TOR Serine-Threonine Kinases/metabolism/genetics ; *Epilepsy/therapy/genetics ; Animals ; Genetic Therapy ; CRISPR-Cas Systems ; },
abstract = {mTORopathies represent a group of neurodevelopmental disorders linked to dysregulated mTOR signaling, resulting in conditions such as tuberous sclerosis complex, focal cortical dysplasia, hemimegalencephaly, and Smith-Kingsmore Syndrome. These disorders often manifest with epilepsy, cognitive impairments, and, in some cases, structural brain anomalies. The mTOR pathway, a central regulator of cell growth and metabolism, plays a crucial role in brain development, where its hyperactivation leads to abnormal neuroplasticity, tumor formation, and heightened neuronal excitability. Current treatments primarily rely on mTOR inhibitors, such as rapamycin, which reduce seizure frequency and tumor size but fail to address underlying genetic causes. Advances in gene editing, particularly via CRISPR/Cas9, offer promising avenues for precision therapies targeting the genetic mutations driving mTORopathies. New delivery systems, including viral and non-viral vectors, aim to enhance the specificity and efficacy of these therapies, potentially transforming the management of these disorders. While gene editing holds curative potential, challenges remain concerning delivery, long-term safety, and ethical considerations. Continued research into mTOR mechanisms and innovative gene therapies may pave the way for transformative, personalized treatments for patients affected by these complex neurodevelopmental conditions.},
}

Humans
*Gene Editing/methods
*Neurodevelopmental Disorders/therapy/genetics
*TOR Serine-Threonine Kinases/metabolism/genetics
*Epilepsy/therapy/genetics
Animals
Genetic Therapy
CRISPR-Cas Systems

@article {pmid40356437,
year = {2025},
author = {Kong, G and Li, R and Huang, W and Yang, Y and Guan, T and Liu, J and Li, W and Hsiang, T and Xi, P and Li, M and Jiang, Z},
title = {A RACK1 family protein regulates pathogenicity of Peronophythora litchii by acting as a scaffold for MAPK signal modules.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2503429},
pmid = {40356437},
issn = {2150-5608},
mesh = {*Plant Diseases/microbiology ; *Receptors for Activated C Kinase/metabolism/genetics ; *MAP Kinase Signaling System ; *Litchi/microbiology ; Virulence ; *Mitogen-Activated Protein Kinases/metabolism/genetics ; *Fungal Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; },
abstract = {Litchi downy blight caused by Peronophythora litchii is the most destructive disease of litchi (Litchi chinensis). RACK1 (Receptor for activated C kinase 1) is a group of scaffold proteins, mainly involved in the regulation of various signaling pathways by interacting with signal transduction proteins and affecting the activity of these proteins. In this study, a RACK1 homologue identified in P. litchii, and named PlRACK1. The protein was found to interact with the mitogen-activated protein kinases, PlMAPK1 and PlMAPK2. CRISPR/Cas9-mediated genome editing technology was used to knock out PlRACK1, and we found that it was involved in mycelial growth, cell wall integrity, ROS metabolism, laccase activity, and pathogenicity of P. litchii. PlMAPK1 interacted with RACK1, and they jointly regulated sporangiophore branching of P. litchii. Transcriptome analysis showed that P. litchii MAPK Phosphatase 1 (PlMKP1) and beta-glucoside (PlBglX) were regulated by PlRACK1, both of which were also required for the pathogenicity of P. litchii. As well, PlMKP1 also interacted with PlMAPK1 and PlMAPK2. These results provide insights into the direct interactions between RACK1, MAPKs, and MKP, and their functions in growth, development, and pathogenesis in a plant pathogenic oomycete.},
}

*Plant Diseases/microbiology
*Receptors for Activated C Kinase/metabolism/genetics
*MAP Kinase Signaling System
*Litchi/microbiology
Virulence
*Mitogen-Activated Protein Kinases/metabolism/genetics
*Fungal Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Expression Regulation, Fungal

@article {pmid40356298,
year = {2025},
author = {Zubair, A and Sujan, A and Ali, M and Hussain, SM},
title = {Current Challenges With Highly Active Antiretroviral Therapy and New Hope and Horizon With CRISPR-CAS9 Technology for HIV Treatment.},
journal = {Chemical biology & drug design},
volume = {105},
number = {5},
pages = {e70121},
doi = {10.1111/cbdd.70121},
pmid = {40356298},
issn = {1747-0285},
mesh = {Humans ; *CRISPR-Cas Systems ; *HIV Infections/drug therapy/genetics ; Gene Editing ; *Antiretroviral Therapy, Highly Active ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR/Cas system) is now the predominant approach for genome editing. Compared to conventional genetic editing methods, CRISPR/Cas technology offers several advantages that were previously unavailable. Key benefits include the ability to simultaneously modify multiple locations, reduced costs, enhanced efficiency, and a more user-friendly design. By directing Cas-mediated DNA cleavage to specific genomic targets and utilizing intrinsic DNA repair processes, this system can produce site-specific gene modifications. This goal is achieved through an RNA-guided procedure. As the most effective gene editing method currently available, the CRISPR/Cas system has proven to be highly valuable in genomic research across a wide range of species since its discovery as a component of the adaptive immune system in bacteria. Its applicability extends to various organisms, making it increasingly prevalent in the medical field, where it shows great promise in investigating viral infections, cancer, and genetic disorders. Furthermore, it enhances our understanding of fundamental genetics. This article outlines the current antiretroviral therapy and its adverse effects but also CRISPR/Cas technology. This review article also discusses its mechanism of action and potential applications in the treatment of HIV/AIDS.},
}

Humans
*CRISPR-Cas Systems
*HIV Infections/drug therapy/genetics
Gene Editing
*Antiretroviral Therapy, Highly Active

@article {pmid40355359,
year = {2025},
author = {Gao, YM and Chang, LX and Zhu, XF},
title = {[Advancements in CRISPR-Cas9 for Fanconi anemia].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {46},
number = {3},
pages = {276-280},
pmid = {40355359},
issn = {0253-2727},
mesh = {*Fanconi Anemia/therapy/genetics ; Humans ; *CRISPR-Cas Systems ; *Gene Editing ; *Genetic Therapy ; },
abstract = {Fanconi anemia (FA) is a hereditary bone marrow failure syndrome that is characterized by genomic instability and heightened sensitivity to DNA cross-linking agents. In recent years, the CRISPR-Cas9 technology has exhibited groundbreaking progress in the field of gene therapy for FA. The traditional CRISPR-Cas9 technology has been successfully applied in FA gene editing. Further, single-base editing technology, based on the CRISPR/Cas9 system, performs precise and efficient gene repair for prevalent gene mutations in patients with FA. The prime editing technology provides new possibilities for gene editing; however, its application in FA has not been initiated. Despite significant advancements in FA gene editing technology, several challenges remain, including the collection of sufficient hematopoietic stem cells, the risk of increased tumorigenesis postgene editing, chromosomal instability, and off-target effects. Future research is recommended to focus on optimizing sgRNA and Cas9 nucleases, designing stricter PAM sequences to reduce off-target effects, and devising personalized gene editing strategies. Further, ethical and regulatory issues as well as long-term follow-ups are crucial priorities for future gene editing work. With continuous technological advancements and in-depth clinical trials, we expect more breakthroughs in FA treatment using the CRISPR-Cas9 technology in the future. This article reviews the latest research progress of CRISPR technology in FA treatment and analyzes the advantages and disadvantages of this technology in FA gene therapy.},
}

*Fanconi Anemia/therapy/genetics
Humans
*CRISPR-Cas Systems
*Gene Editing
*Genetic Therapy

@article {pmid40334450,
year = {2025},
author = {Zhang, X and Wang, X and Zhu, L and Zhu, J and Zheng, Q and Yuan, J and Xu, W and Cao, J},
title = {Target responsive-regulated CRISPR/Cas12a electrochemiluminescence sensing of salmonella typhimurium integrating ultrafine Pt NCs-anchored MXenes-boosted luminol/O2 system.},
journal = {Biosensors & bioelectronics},
volume = {283},
number = {},
pages = {117558},
doi = {10.1016/j.bios.2025.117558},
pmid = {40334450},
issn = {1873-4235},
mesh = {*Salmonella typhimurium/isolation & purification/genetics ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Luminol/chemistry ; Luminescent Measurements/methods ; Platinum/chemistry ; Electrochemical Techniques/methods ; Oxygen/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Humans ; Salmonella Infections/microbiology/diagnosis ; },
abstract = {Salmonella typhimurium (S. typhimurium), as common and highly pathogenic foodborne pathogen, poses a significant risk to public safety worldwide. The development of highly sensitive, rapid and on-site method for S. typhimurium analysis is urgently needed to prevent bacterial infections. Herein, we introduced a CRISPR/Cas12a-driven electrochemiluminescence (ECL) sensor based on luminol/O2 binary systems for S. typhimurium detection, employing ultrafine Pt nanoclusters-anchored 2D delaminated-MXenes (Pt NCs/D-MXenes) as the co-reactant accelerator. The ultrathin D-MXenes support regulates the size and dispersibility of Pt NCs and facilitates the full exposure of active sites, and synergistic interactions between D-MXenes and Pt NCs improves electrocatalytic properties toward the reduction of O2, which promotes the generation of ROS for boosting ECL emission. Using target responsive-regulated CRISPR/Cas12a system, the ECL sensor for S. typhimurium showed a broad concentration range from 10[1] to 10[6] CFU/mL and limit of detection of 6 CFU/mL, with satisfactory recoveries in spiked-actual samples.},
}

*Salmonella typhimurium/isolation & purification/genetics
*Biosensing Techniques/methods
CRISPR-Cas Systems/genetics
Luminol/chemistry
Luminescent Measurements/methods
Platinum/chemistry
Electrochemical Techniques/methods
Oxygen/chemistry
Metal Nanoparticles/chemistry
Limit of Detection
Humans
Salmonella Infections/microbiology/diagnosis

@article {pmid40311445,
year = {2025},
author = {Yu, Y and Liu, Y and Xu, N and Li, L and Yang, Y and Liu, X and Zhao, L and Bai, X},
title = {A CRISPR/Cas12a mediated click immunoassay catalyzed by in situ formation of clickase for highly sensitive detection of Trichinella spiralis.},
journal = {Biosensors & bioelectronics},
volume = {283},
number = {},
pages = {117521},
doi = {10.1016/j.bios.2025.117521},
pmid = {40311445},
issn = {1873-4235},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Trichinella spiralis/isolation & purification/genetics ; Immunoassay/methods ; Limit of Detection ; Metal Nanoparticles/chemistry ; Gold/chemistry ; *Trichinellosis/diagnosis/parasitology ; Copper/chemistry ; Click Chemistry ; DNA, Single-Stranded/chemistry ; Meat/parasitology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The accurate and high-throughput detection of trace protein targets remains an ongoing challenge in the field of food safety testing. This research article presents a highly sensitive CRISPR/Cas12a-mediated click immunoassay for the sensitive detection of Trichinella spiralis (T. spiralis) in meat samples. By simultaneously conjugating activator ssDNA and monoclonal antibodies to gold nanoparticles, the CRISPR/Cas12a system was introduced into the immunoassay. To overcome the challenges associated with the preparation, storage, and transportation of FQ probes in CRISPR/Cas12a systems, the Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was employed instead. The designed ssDNA was both the substrate of trans-cleavage activity of Cas12a and the synthetic template of the artificial clicking enzyme copper nanoparticles (CuNPs), which could effectively catalyze the CuAAC reaction to generate the desired signal output. The fluorescence intensity showed a linear relationship with T. spiralis crude protein concentration ranging from 3.125 to 100 ng/mL, and the detection limit was 0.35 ng/mL, which is three orders of magnitude lower than that of ELISA (LOD: 309.75 ng/mL). This method can accurately detect a single T. spiralis larva in 100 g of pork. Collectively, the strategy of combining CRISPR/Cas12a system and CuAAC reaction opens up a novel avenue for developing a highly sensitive, simple and convenient fluorescence assay.},
}

Animals
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Trichinella spiralis/isolation & purification/genetics
Immunoassay/methods
Limit of Detection
Metal Nanoparticles/chemistry
Gold/chemistry
*Trichinellosis/diagnosis/parasitology
Copper/chemistry
Click Chemistry
DNA, Single-Stranded/chemistry
Meat/parasitology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40209705,
year = {2025},
author = {Geilenkeuser, J and Armbrust, N and Steinmaßl, E and Du, SW and Schmidt, S and Binder, EMH and Li, Y and Warsing, NW and Wendel, SV and von der Linde, F and Schiele, EM and Niu, X and Stroppel, L and Berezin, O and Santl, TH and Orschmann, T and Nelson, K and Gruber, C and Palczewska, G and Menezes, CR and Risaliti, E and Engfer, ZJ and Koleci, N and Schmidts, A and Geerlof, A and Palczewski, K and Westmeyer, GG and Truong, DJ},
title = {Engineered nucleocytosolic vehicles for loading of programmable editors.},
journal = {Cell},
volume = {188},
number = {10},
pages = {2637-2655.e31},
doi = {10.1016/j.cell.2025.03.015},
pmid = {40209705},
issn = {1097-4172},
mesh = {*Gene Editing/methods ; Animals ; Mice ; Humans ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Ribonucleoproteins/metabolism ; T-Lymphocytes/metabolism ; HEK293 Cells ; },
abstract = {Advanced gene editing methods have accelerated biomedical discovery and hold great therapeutic promise, but safe and efficient delivery of gene editors remains challenging. In this study, we present a virus-like particle (VLP) system featuring nucleocytosolic shuttling vehicles that retrieve pre-assembled Cas-effectors via aptamer-tagged guide RNAs. This approach ensures preferential loading of fully assembled editor ribonucleoproteins (RNPs) and enhances the efficacy of prime editing, base editing, trans-activators, and nuclease activity coupled to homology-directed repair in multiple immortalized, primary, stem cell, and stem-cell-derived cell types. We also achieve additional protection of inherently unstable prime editing guide RNAs (pegRNAs) by shielding the 3'-exposed end with Csy4/Cas6f, further enhancing editing performance. Furthermore, we identify a minimal set of packaging and budding modules that can serve as a platform for bottom-up engineering of enveloped delivery vehicles. Notably, our system demonstrates superior per-VLP editing efficiency in primary T lymphocytes and two mouse models of inherited retinal disease, highlighting its therapeutic potential.},
}

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

@article {pmid38956324,
year = {2025},
author = {Zhu, M and Xu, R and Yuan, J and Wang, J and Ren, X and Cong, T and You, Y and Ju, A and Xu, L and Wang, H and Zheng, P and Tao, H and Lin, C and Yu, H and Du, J and Lin, X and Xie, W and Li, Y and Lan, X},
title = {Tracking-seq reveals the heterogeneity of off-target effects in CRISPR-Cas9-mediated genome editing.},
journal = {Nature biotechnology},
volume = {43},
number = {5},
pages = {799-810},
pmid = {38956324},
issn = {1546-1696},
support = {81972680//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32171448//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system.},
}

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

@article {pmid38907037,
year = {2025},
author = {Mathis, N and Allam, A and Tálas, A and Kissling, L and Benvenuto, E and Schmidheini, L and Schep, R and Damodharan, T and Balázs, Z and Janjuha, S and Ioannidi, EI and Böck, D and van Steensel, B and Krauthammer, M and Schwank, G},
title = {Machine learning prediction of prime editing efficiency across diverse chromatin contexts.},
journal = {Nature biotechnology},
volume = {43},
number = {5},
pages = {712-719},
pmid = {38907037},
issn = {1546-1696},
support = {185293/SNSF_/Swiss National Science Foundation/Switzerland ; 201184/SNSF_/Swiss National Science Foundation/Switzerland ; 214936/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {*Machine Learning ; *Chromatin/genetics ; *Gene Editing/methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {The success of prime editing depends on the prime editing guide RNA (pegRNA) design and target locus. Here, we developed machine learning models that reliably predict prime editing efficiency. PRIDICT2.0 assesses the performance of pegRNAs for all edit types up to 15 bp in length in mismatch repair-deficient and mismatch repair-proficient cell lines and in vivo in primary cells. With ePRIDICT, we further developed a model that quantifies how local chromatin environments impact prime editing rates.},
}

*Machine Learning
*Chromatin/genetics
*Gene Editing/methods
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
Animals

@article {pmid40354233,
year = {2025},
author = {Zhang, X and Duan, M and Zhao, Y and Zhang, K and Liu, F and Jie, J and Li, C and Chen, D and Li, D and Hua, S and Wang, C and Guan, Q and Wu, J and Liu, B and Song, L},
title = {Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {218},
pages = {},
doi = {10.3791/67542},
pmid = {40354233},
issn = {1940-087X},
mesh = {*Acinetobacter baumannii/isolation & purification/genetics/enzymology ; *Nucleic Acid Amplification Techniques/methods ; *Acinetobacter Infections/diagnosis/microbiology ; *Recombinases/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *Bacterial Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; Humans ; DNA, Bacterial/genetics/analysis ; },
abstract = {Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.},
}

*Acinetobacter baumannii/isolation & purification/genetics/enzymology
*Nucleic Acid Amplification Techniques/methods
*Acinetobacter Infections/diagnosis/microbiology
*Recombinases/chemistry/metabolism/genetics
CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/metabolism/chemistry
*Bacterial Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism/chemistry
Humans
DNA, Bacterial/genetics/analysis

@article {pmid40353913,
year = {2025},
author = {Wu, G and Yin, C and Zheng, J and Wang, M and Abdalmegeed, D and Zhang, F and Sun, S and Sun, S and Shao, Y and Xin, Z},
title = {Dynamic regulation of iturin production via reconstructing the quorum-sensing system ComQXPA in Bacillus subtilis.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {5},
pages = {173},
pmid = {40353913},
issn = {1573-0972},
mesh = {*Bacillus subtilis/genetics/metabolism ; *Quorum Sensing/genetics ; Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Promoter Regions, Genetic ; *Peptides, Cyclic/biosynthesis/genetics/metabolism ; *Lipopeptides/biosynthesis/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Pheromone ComX is a critical element of the quorum-sensing (QS) system in Bacillus subtilis. It activates the surfactin promoter (Psrf) to initiate surfactin production in a cell-density-dependent manner. In this study, the natural promoter Pitu of B. subtilis 1A751 WR-itu, a lipopeptide iturin mono-producing parent strain, was replaced by the constitutive promoter P43, QS Psrf, and the mutated QS PM-srf, generating dynamic regulation systems to improve iturin production. HPLC analysis revealed that the PM-srf promoter-harbouring strain significantly enhanced iturin production to 409.33 ± 16.77 mg·L[- 1], 2.15 times higher than the parent strain. Further identification by UPLC-HRESI-MS/MS and GC-MS indicated that the strain could produce four new C10-C13 iturins. Moreover, the ComX degrading enzymes AprE or NprE were down-regulated by the CRISPR interference (CRISPRi) system, increasing iturin production to 526.46 ± 18.43 mg·L[- 1] and 416.99 ± 17.02 mg·L[- 1], respectively. Interestingly, iturin production was further increased to 579.85 ± 19.83 mg·L[- 1] under simultaneous down-regulation of AprE and NprE, 3.05 times higher than the parent strain. The reconstructed ComQXPA QS system in B. subtilis combines the inhibitory of AprE and NprE to dynamically up and down-regulate the expression of iturin operon, providing an effective pipeline for regulating other bioactive molecules.},
}

*Bacillus subtilis/genetics/metabolism
*Quorum Sensing/genetics
Gene Expression Regulation, Bacterial
*Bacterial Proteins/genetics/metabolism
Promoter Regions, Genetic
*Peptides, Cyclic/biosynthesis/genetics/metabolism
*Lipopeptides/biosynthesis/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40352342,
year = {2025},
author = {Kaushik, A and Singh, J and Fatima, Z and Hameed, S},
title = {Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settings.},
journal = {Drug target insights},
volume = {19},
number = {},
pages = {31-40},
pmid = {40352342},
issn = {1177-3928},
abstract = {INTRODUCTION: The current scenario of tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has presented an almost insurmountable challenge to hospitals with high patient numbers. Delayed diagnosis of TB is a major hurdle in preventing the employment of efficient therapeutics, leading to the development of drug resistance. Hence, an easily accessible diagnostic method, particularly for resource for resource-limited settings, is pertinent for the rapid identification of MTB-infected patients. In pursuit of developing such an assay, the present study offers a CLAP-TB (CRISPR-Cas coupled RT-LAMP Amplification Protocol for Tuberculosis) assay, which will allow us to diagnose TB rapidly and visually.

METHODS AND RESULTS: Herein, the visual MTB detection consists of a method utilizing 232 different samples (sputum, urine, serum) from 82 patients for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of a CRISPR-Cas12-based system using different guide RNAs of IS6110 and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dipsticks with the unaided eye (~134 min). Overall, the limit of detection for CLAP-TB assay was up to 1 ag of RNA, while the clinical sensitivity and specificity were 98.27% and 100%, respectively, on the pilot scale.

CONCLUSION: Together, our CLAP-TB assay offers proof of concept for a rapid, sensitive, and specific method with the minimum technical expertise required for TB diagnosis in developing and resource-limited settings.},
}

@article {pmid40352243,
year = {2025},
author = {Prasetia, IG and Kurniati, NF and Riani, C and Mudhakir, D},
title = {Design of lipid nanoparticle (LNP) containing genetic material CRISPR/Cas9 for familial hypercholesterolemia.},
journal = {Narra J},
volume = {5},
number = {1},
pages = {e2217},
pmid = {40352243},
issn = {2807-2618},
mesh = {*Hyperlipoproteinemia Type II/therapy/genetics ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Humans ; Receptors, LDL/genetics ; *Lipids/chemistry ; *Genetic Therapy/methods ; Particle Size ; Liposomes ; },
abstract = {Familial hypercholesterolemia is a genetic disorder caused by mutations in the low- density lipoprotein receptor gene (LDLR) and the current treatment still focuses on symptom management. The aim of this study was to develop a lipid nanoparticle (LNP)- based delivery system for the CRISPR/Cas9 component in correcting LDLR gene mutations. LNPs were prepared using an ultrasonic-solvent emulsification technique by varying the surfactant: oil ratio (SOR), homogenization speed and time, and sonication time. Next, the LNP surface was modified by adding DSPE-PEG2000-NH2 and polyethyleneimine. The next stage is to design the single guide RNA (sgRNA) and Donor DNA wildtype (Donor DNA wt). This genetic material was complexed with LNP and then transfected into Hepa1-6 LDLR mt cells, an in vitro representation of cells suffering from familial hypercholesterolemia. This optimization process produced LNPs with a particle size of 118.6 ± 0.8 nm and a polydispersity index of 0.34 ± 0.03. The LNP surface modification resulted in a zeta potential of +7.5 mV. A transmission electron microscope (TEM) analysis showed spherical morphology with size distribution following a regular pattern. LNP cell viability tests showed good biocompatibility at concentrations <15 mM with a half-maximal inhibitory concentration (IC50) value of 27.7 mM. The dominant cellular uptake mechanism of LNP was through the clathrin-mediated endocytosis (CME) pathway. The Hepa1-6 LDLR mt cell model was successfully produced with the transfecting agent Lipofectamine 3000 by homology-directed repair (HDR) mechanism. The LNP-genetic material complex with a ratio of sgRNA:Cas9:Donor DNA wt (1:1:0.04) showed an increase in LDLR gene expression of 3.3 ± 0.2 times and LDLR protein levels reached 12.95 ± 0.25 ng/mL on day 4 after transfection. The results of this study indicate that the developed LNP-based delivery system has the potential for gene therapy applications in familial hypercholesterolemia.},
}

*Hyperlipoproteinemia Type II/therapy/genetics
*Nanoparticles/chemistry
*CRISPR-Cas Systems
Humans
Receptors, LDL/genetics
*Lipids/chemistry
*Genetic Therapy/methods
Particle Size
Liposomes

@article {pmid40351170,
year = {2025},
author = {Zang, SS and Zhang, R and Zhang, JR and Zhang, X and Li, J},
title = {Progress, Applications and Prospects of CRISPR-Based Genome Editing Technology in Gene Therapy for Cancer and Sickle Cell Disease.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2024.262},
pmid = {40351170},
issn = {1557-7422},
abstract = {The advent of genome-editing technologies, particularly the RNA-guided the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) 9, which originates from prokaryotic CRISPR/Cas adaptive immune mechanisms, has revolutionized molecular biology. Renowned for its simplicity, cost-effectiveness, and capacity for multiplexed gene editing, CRISPR/Cas9 has emerged as the most versatile and widely adopted genome-editing platform. Its applications span fundamental research, biotechnology, medicine, and therapeutics. This review highlights recent advancements in CRISPR-based technologies, focusing on CRISPR/Cas9, CRISPR/Cas12a, and CRISPR/Cas12f. It emphasizes precision editing methods like base editing and prime editing, which enable targeted nucleotide changes without double-strand breaks. The specificity of these tools, including on-target accuracy and off-target risks, is critically evaluated. Additionally, recent preclinical and clinical efforts to treat diseases such as cancer and sickle cell disease using CRISPR are summarized. Finally, the challenges and future directions of CRISPR-mediated gene therapy are discussed, emphasizing its potential to integrate with other molecular approaches to address unmet medical needs.},
}

@article {pmid40350476,
year = {2025},
author = {Kumari, G and Gupta, P and Goswami, SG and Jain, R and Anand, S and Biswas, S and Garg, S and Thakur, P and Saravanakumar, V and Arvinden, VR and Goswami, B and Bhowmick, IP and Mohandas, N and Burrows, J and Ramalingam, S and Singh, S},
title = {CRISPR/Cas9-engineering of Kell null erythrocytes to unveil host targeted irresistible antimalarial.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {730},
pmid = {40350476},
issn = {2399-3642},
support = {NER/84/2022-ECD-I//Indian Council of Medical Research (ICMR)/ ; },
mesh = {*Erythrocytes/parasitology/metabolism/drug effects ; *Plasmodium falciparum/drug effects/genetics ; *Antimalarials/pharmacology ; *CRISPR-Cas Systems ; Animals ; Humans ; *Malaria, Falciparum/drug therapy/parasitology ; Mice ; },
abstract = {Malaria elimination faces challenges from drug resistance, stemming from mutations within the parasite's genetic makeup. Genetic adaptations in key erythrocyte proteins offer malaria protection in endemic regions. Emulating nature's approach, and implementing methodologies to render indispensable host proteins inactive, holds the potential to reshape antimalarial therapy. This study delves into the functional implication of the single-span membrane protein Kell ectodomain, which shares consensus sequence with the zinc endopeptidase family, possesses extracellular enzyme activity crucial for parasite invasion into host erythrocytes. Through generating Kell-null erythrocytes from an erythroid progenitor, BEL-A, we demonstrate the indispensable nature of Kell activity in P. falciparum invasion. Additionally, thiorphan, a metallo-endopeptidase inhibitor, which specifically inhibits Kell activity, inhibited Plasmodium infection at nanomolar concentrations. Interestingly, individuals in malaria-endemic regions exhibit low Kell expression and activity, indicating a plausible Plasmodium-induced evolutionary pressure. Both thiorphan and its prodrug racecadotril, demonstrated potent antimalarial activity in vivo, highlighting Kell's protease role in invasion and proposing thiorphan as a promising host-oriented antimalarial therapeutic.},
}

*Erythrocytes/parasitology/metabolism/drug effects
*Plasmodium falciparum/drug effects/genetics
*Antimalarials/pharmacology
*CRISPR-Cas Systems
Animals
Humans
*Malaria, Falciparum/drug therapy/parasitology
Mice

@article {pmid40350377,
year = {2025},
author = {Zhang, L and Zhao, D and Wei, Z and Zhu, X and Sha, T and Tang, W and Bi, C and Zhang, X},
title = {The Advancement of Prime Editing Technology.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {},
number = {},
pages = {e202500193},
doi = {10.1002/cbic.202500193},
pmid = {40350377},
issn = {1439-7633},
abstract = {The advent of CRISPR/Cas genome editing has spurred major breakthroughs across life sciences, offering vast potential across numerous research and application fields. Among the expanding toolkit of CRISPR/Cas-derived methods, prime editing (PE) stands out for its versatility and specificity, enabling precise point mutations and small insertions or deletions without requiring double-stranded DNA breaks. Since its introduction, PE has undergone multiple rounds of optimization to improve performance. In this review, we first outline the core components and mechanism of prime editors, followed by four key evolution strategies: protein engineering, pegRNA modifications, accessory protein recruitment, and paired pegRNA approaches. We then discuss persistent challenges and outline possible refinements, highlighting how further innovations can expand prime editing's utility across diverse areas of research, biotechnology, and potential therapeutic interventions.},
}

@article {pmid40348775,
year = {2025},
author = {Jalan, M and Brambati, A and Shah, H and McDermott, N and Patel, J and Zhu, Y and Doymaz, A and Wu, J and Anderson, KS and Gazzo, A and Pareja, F and Yamaguchi, TN and Vougiouklakis, T and Ahmed-Seghir, S and Steinberg, P and Neiman-Golden, A and Azeroglu, B and Gomez-Aguilar, J and da Silva, EM and Hussain, S and Higginson, D and Boutros, PC and Riaz, N and Reis-Filho, JS and Powell, SN and Sfeir, A},
title = {RNA transcripts serve as a template for double-strand break repair in human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4349},
pmid = {40348775},
issn = {2041-1723},
support = {U01CA231019//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; U2C CA271894/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; P30 CA016042/CA/NCI NIH HHS/United States ; R01 CA244729/CA/NCI NIH HHS/United States ; P50 CA247749/CA/NCI NIH HHS/United States ; U01 CA231019/CA/NCI NIH HHS/United States ; R01 CA229161/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *DNA Breaks, Double-Stranded ; *DNA Repair/genetics ; CRISPR-Cas Systems/genetics ; *RNA, Messenger/genetics/metabolism ; *RNA/genetics/metabolism ; DNA-Directed DNA Polymerase/metabolism/genetics ; Templates, Genetic ; Genomic Instability ; },
abstract = {Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, growing evidence suggests that RNA:DNA hybrids and nearby transcripts can influence repair outcomes. However, whether transcript RNA can directly serve as a template for DSB repair in human cells remains unclear. In this study, we develop fluorescence and sequencing-based assays to show that RNA-containing oligonucleotides and messenger RNA can serve as templates during DSB repair. We conduct a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR). Of the candidate polymerases, we identify DNA polymerase zeta (Polζ) as a potential reverse transcriptase that facilitates RT-DSBR. Furthermore, analysis of cancer genome sequencing data reveals whole intron deletions - a distinct genomic signature of RT-DSBR that occurs when spliced mRNA guides repair. Altogether, our findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.},
}

Humans
*DNA Breaks, Double-Stranded
*DNA Repair/genetics
CRISPR-Cas Systems/genetics
*RNA, Messenger/genetics/metabolism
*RNA/genetics/metabolism
DNA-Directed DNA Polymerase/metabolism/genetics
Templates, Genetic
Genomic Instability

@article {pmid40348052,
year = {2025},
author = {Koike, A and Brindley, PJ},
title = {CRISPR/Cas genome editing, functional genomics, and diagnostics for parasitic helminths.},
journal = {International journal for parasitology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ijpara.2025.05.001},
pmid = {40348052},
issn = {1879-0135},
abstract = {Functional genomics using CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated endonuclease)-based approaches has revolutionized biomedical sciences. Gene editing is also widespread in parasitology generally and its use is increasing in studies on helminths including flatworm and roundworm parasites. Here, we survey the progress, specifically with experimental CRISPR-facilitated functional genomics to investigate helminth biology and pathogenesis, and also with the burgeoning use of CRISPR-based methods to assist in diagnosis of helminth infections. We also provide an historical timeline of the introduction and uses of CRISPR in helminth species to date.},
}

@article {pmid40347261,
year = {2025},
author = {Botelho, FBS and Nandy, S and Srivastava, V},
title = {CRISPR/Cas9-based modulation of V-PPase expression in rice improves grain quality and yield under high nighttime temperature.},
journal = {Plant cell reports},
volume = {44},
number = {6},
pages = {119},
pmid = {40347261},
issn = {1432-203X},
support = {1826836//Directorate for Biological Sciences/ ; 2023-11092//National Institute of Food and Agriculture/ ; },
mesh = {*Oryza/genetics/enzymology/growth & development/physiology ; Gene Expression Regulation, Plant ; *Edible Grain/genetics/growth & development ; *CRISPR-Cas Systems/genetics ; *Inorganic Pyrophosphatase/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Hot Temperature ; Plants, Genetically Modified ; Endosperm/genetics ; },
abstract = {Transcriptional modulation of the vacuolar H[+] translocating pyrophosphatase expressed specifically in the endosperm and reproductive tissue of rice improves its spikelet fertility and reduces grain chalkiness under high nighttime temperature.},
}

*Oryza/genetics/enzymology/growth & development/physiology
Gene Expression Regulation, Plant
*Edible Grain/genetics/growth & development
*CRISPR-Cas Systems/genetics
*Inorganic Pyrophosphatase/genetics/metabolism
Plant Proteins/genetics/metabolism
Hot Temperature
Plants, Genetically Modified
Endosperm/genetics

@article {pmid40346697,
year = {2025},
author = {Guo, T and Wang, H and Wu, F and Lu, W and Zhu, M and Ma, S and Zhang, Y and Yan, Y and Zhou, M and Talanaite, D and Liu, S and Qi, M and Lan, F and Liu, X},
title = {Functional analysis of JPH2-knockout cardiomyocytes identifies ECCD as a novel indicator in a human cardiac modelJPH2.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {234},
pmid = {40346697},
issn = {1757-6512},
support = {2021-RC310-12, 2022I2M-2-001 and 2023-I2M-1-003//CAMS Innovation Fund for Medical Sciences/ ; 2021YFC2701703, 2023YFA0915002//National Key Research and Devel opment Program of China/ ; 2022-GSP-GG-7//National High-Level Hospital Clinical Research Funding/ ; 2019PT320026//Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences/ ; ZDSYS20200923172000001//Shenzhen Fundamental Research Program/ ; 2024SKL-TJ001//Open Project Fund of State Key Laboratory of Cardiovascular Diseases，Shanghai East Hospital/ ; 2024T170070//China Postdoctoral Science Foundation/ ; 82400381//National Natural Science Foundation of China/ ; NCRCG-PLAGH-2024008//Open Project Fund of National Clinical Research Center for Geriatric Diseases, Chinese PLA General Hospital/ ; CXPY202304//Chinese PLA General Hospital Sixth medical center Innovation and Culture Grant/ ; },
mesh = {Humans ; *Myocytes, Cardiac/metabolism/cytology ; *Membrane Proteins/genetics/metabolism ; Calcium/metabolism ; Human Embryonic Stem Cells/metabolism/cytology ; *Excitation Contraction Coupling ; Myocardial Contraction ; Animals ; CRISPR-Cas Systems ; Muscle Proteins ; },
abstract = {BACKGROUND: Junctophilin-2 (JPH2) is a vital protein in cardiomyocytes, anchoring T-tubule and sarcoplasmic reticulum membranes to facilitate excitation-contraction coupling, a process essential for cardiac contractile function. Dysfunction of JPH2 is associated with cardiac disorders such as heart failure; however, prior studies using mouse models or primary human cardiomyocytes are limited by interspecies differences or poor cell viability, respectively. This study aimed to investigate JPH2's role in human cardiac function and disease using a novel stem cell-derived model, while introducing a new indicator to evaluate related cardiac impairments.

METHODS: We generated a JPH2-knockout model using human embryonic stem cell-derived cardiomyocytes (hESC-CMs) with CRISPR/Cas9. Cellular morphology, contractile function, calcium dynamics, and electrophysiological properties were assessed via transmission electron microscopy, the CardioExcyte96 system, calcium imaging with Fluo-4 AM, and multi-electrode array recordings, respectively. Wild-type JPH2 was overexpressed through lentiviral transfection to evaluate rescue effects, and two JPH2 variants-one benign (G505S) and one pathogenic (E85K)-were introduced to study mutation-specific effects.

RESULTS: JPH2 knockout disrupted excitation-contraction coupling in hESC-CMs by impairing junctional membrane complex structure, leading to heart failure-like phenotypes with reduced contractility, altered calcium dynamics, and electrophysiological irregularities. Overexpression of wild-type JPH2 restored these functions, affirming its critical role in cardiac physiology. We identified excitation-contraction coupling delay (ECCD) as a novel indicator that precisely quantified coupling impairment severity, with its applicability validated across distinct JPH2 variants (G505S and E85K).

CONCLUSIONS: This study demonstrates JPH2's essential role in sustaining excitation-contraction coupling by stabilizing the junctional membrane complex, with its deficiency driving heart failure-like cardiac dysfunction. ECCD is established as a sensitive, comprehensive indicator for assessing JPH2-related impairment severity. These findings advance our understanding of JPH2 in cardiac pathology and position ECCD as a valuable tool for research and potential clinical evaluation, with JPH2 and calcium regulation emerging as promising therapeutic targets.},
}

Humans
*Myocytes, Cardiac/metabolism/cytology
*Membrane Proteins/genetics/metabolism
Calcium/metabolism
Human Embryonic Stem Cells/metabolism/cytology
*Excitation Contraction Coupling
Myocardial Contraction
Animals
CRISPR-Cas Systems
Muscle Proteins

@article {pmid40346034,
year = {2025},
author = {Villa, S and Jafri, Q and Lazzari-Dean, JR and Sangha, M and Olsson, N and Lefebvre, AEYT and Fitzgerald, ME and Jackson, K and Chen, Z and Feng, BY and Nile, AH and Stokoe, D and Bersuker, K},
title = {BiDAC-dependent degradation of plasma membrane proteins by the endolysosomal system.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4345},
pmid = {40346034},
issn = {2041-1723},
mesh = {*Lysosomes/metabolism/drug effects ; Humans ; *Proteolysis/drug effects ; Ubiquitination/drug effects ; ErbB Receptors/metabolism ; *Membrane Proteins/metabolism ; *Cell Membrane/metabolism/drug effects ; Proteasome Endopeptidase Complex/metabolism ; Receptor, ErbB-2/metabolism/genetics ; HEK293 Cells ; Endocytosis/drug effects ; CRISPR-Cas Systems ; *Endosomes/metabolism ; Animals ; Protein Transport/drug effects ; },
abstract = {The discovery of bifunctional degradation activating compounds (BiDACs) has led to the development of a new class of drugs that promote the clearance of their protein targets. BiDAC-induced ubiquitination is generally believed to direct cytosolic and nuclear proteins to proteolytic destruction by proteasomes. However, pathways that govern the degradation of other classes of BiDAC targets, such as integral membrane and intraorganellar proteins, have not been investigated in depth. In this study we use morphological profiling and CRISPR/Cas9 genetic screens to investigate the mechanisms by which BiDACs induce the degradation of plasma membrane receptor tyrosine kinases (RTKs) EGFR and Her2. We find that BiDAC-dependent ubiquitination triggers the trafficking of RTKs from the plasma membrane to lysosomes for degradation. Notably, functional proteasomes are required for endocytosis of RTKs upstream of the lysosome. Additionally, our screen uncovers a non-canonical function of the lysosome-associated arginine/lysine transporter PQLC2 in EGFR degradation. Our data show that BiDACs can target proteins to proteolytic machinery other than the proteasome and motivate further investigation of mechanisms that govern the degradation of diverse classes of BiDAC targets.},
}

*Lysosomes/metabolism/drug effects
Humans
*Proteolysis/drug effects
Ubiquitination/drug effects
ErbB Receptors/metabolism
*Membrane Proteins/metabolism
*Cell Membrane/metabolism/drug effects
Proteasome Endopeptidase Complex/metabolism
Receptor, ErbB-2/metabolism/genetics
HEK293 Cells
Endocytosis/drug effects
CRISPR-Cas Systems
*Endosomes/metabolism
Animals
Protein Transport/drug effects

@article {pmid40338137,
year = {2025},
author = {Wang, X and Yang, S and Qian, F and Dong, F and Zhou, X and Yin, M and Zhang, Y and Huang, Z and Jiang, Y and Yang, S},
title = {RAGATH-Associated DNA Nuclease Assisted DNA Insertion in Corynebacterium glutamicum.},
journal = {ACS synthetic biology},
volume = {14},
number = {5},
pages = {1861-1867},
doi = {10.1021/acssynbio.5c00022},
pmid = {40338137},
issn = {2161-5063},
mesh = {*Corynebacterium glutamicum/genetics/metabolism ; Plasmids/genetics/metabolism ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Deoxyribonucleases/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; DNA, Bacterial/genetics ; },
abstract = {Corynebacterium glutamicum serves as a key microbial chassis for the industrial production of feed and food ingredients. While long DNA fragment insertion technologies have advanced strain engineering capabilities, previous approaches such as utilizing a chromosome-integrated Cas9-RecET system were constrained by a maximum insertion fragment size of 7.5 kb. Through systematic evaluation of Cas9, gRNA, and recombinase expression driven by five distinct promoters and their implementation on 1 or 2 plasmids with compatible replicons (resulting in a total of 17 combinations), we developed an optimized genome editing vector capable of inserting DNA fragments of up to 8.0 kb in C. glutamicum. Parallel implementation of the Cpf1 system also successfully achieved 8.0 kb of DNA insertions. However, the construction of plasmids carrying insertion sequences larger than 8.0 kb was hindered by the plasmid vector capacity. To address this limitation, we screened six smaller RAGATH-associated DNA nucleases, ultimately identifying two with high cleavage activity in C. glutamicum. These nucleases demonstrated superior editing efficiencies compared to both Cas9 and Cpf1, enabling the integration of DNA fragments up to 11.3 kb─surpassing previously reported size limitations for C. glutamicum. These RAGATH-associated DNA nuclease-based systems effectively overcome the previous size constraints for long fragment insertions, thereby advancing metabolic engineering and fundamental research applications.},
}

*Corynebacterium glutamicum/genetics/metabolism
Plasmids/genetics/metabolism
Gene Editing/methods
CRISPR-Cas Systems/genetics
*Deoxyribonucleases/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
DNA, Bacterial/genetics

@article {pmid40335033,
year = {2025},
author = {Bryson, JW},
title = {Array Assembler Provides Greatly Simplified crRNA Array Design for CRISPR Cas12 and Cas13 Variants.},
journal = {ACS synthetic biology},
volume = {14},
number = {5},
pages = {1868-1872},
doi = {10.1021/acssynbio.5c00100},
pmid = {40335033},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Associated Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {As newer CRISPR variants have emerged and corresponding toolkits have been developed, researchers can now readily target multiple genes simultaneously for knockout, activation, or repression alongside being able to bind or cleave mRNA. However, as larger multitargeting crRNA arrays are required for these experiments, the design process becomes more complicated, taking more time and increasing risks of errors being introduced. The Array Assembler seeks to address the critical bottleneck that emerges during longer crRNA array design by providing a highly user-friendly tool to process input crRNA spacer sequences into the oligos required for efficient assembly of the corresponding crRNA array. By enabling rapid and reliable design of oligos for efficient assembly of crRNA arrays from a user-defined list of crRNA spacer sequences this tool should prove useful for a wide range of laboratories employing genomic perturbations.},
}

*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Associated Proteins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40326732,
year = {2025},
author = {Li, Z and Li, C and Xiao, S and Liang, H},
title = {Efficient and Precise Integration of Large DNA Sequences Using Precise Interstrand Cross-Linking of Long ssDNA and sgRNA.},
journal = {ACS synthetic biology},
volume = {14},
number = {5},
pages = {1451-1463},
doi = {10.1021/acssynbio.4c00715},
pmid = {40326732},
issn = {2161-5063},
mesh = {Humans ; *DNA, Single-Stranded/genetics/chemistry/metabolism ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; CRISPR-Cas Systems/genetics ; Cross-Linking Reagents/chemistry ; Recombinational DNA Repair/genetics ; },
abstract = {Homology-directed repair (HDR) allows the precise introduction of functional constructs into the human genome through nonviral gene-editing reagents. However, its application in large DNA sequence gene editing remains limited due to challenges such as low efficiency and the off-target effect. To address these limitations, a new method named AOLP was developed to synthesize chemically modified long single-stranded DNA (lssDNA) as the template donor for Cas9-based gene editing, which has been proven to be more stable than that prepared using the commercial phosphorylation method. We propose a novel strategy involving precise ligation-based interstrand cross-linking between lssDNA and sgRNA using cyanovinylcarbazole nucleoside ([CNV]K), enhancing the upregulation of the HDR pathway for DSB repair induced by Cas9. The light-activated ligation between Cas9/sgRNA and lssDNA improves the knock-in (KI) efficiency, overcomes the challenges of low KI efficiency, and surpasses the low off-target effect accompanied by the lssDNA donor. Moreover, the interstrand cross-linking of lssDNA and sgRNA can subtly control the ligation sites and the degree of cross-linking of lssDNA and sgRNA to enhance the KI accuracy of HDR. Our approach improves the KI efficiency of lssDNA in K562, HEK293T, and HepG2 cells by 4- to 12-fold relative to conventional lssDNA donors prepared using the phosphorylation method. Furthermore, the KI accuracy of HDR pathway in HEK293T cells is enhanced by >4.7-fold relative to previous commercial lssDNA. Leveraging this approach, we achieved an unprecedented KI rate of approximately 36% for a gene-sized 1.4 kilobase lssDNA insertion in HEK293T cells.},
}

Humans
*DNA, Single-Stranded/genetics/chemistry/metabolism
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
HEK293 Cells
CRISPR-Cas Systems/genetics
Cross-Linking Reagents/chemistry
Recombinational DNA Repair/genetics

@article {pmid40316310,
year = {2025},
author = {Kang, H and Fitch, JC and Varghese, RP and Thorne, CA and Cusanovich, DA},
title = {Optimization of a Cas12a-Driven Synthetic Gene Regulatory Network System.},
journal = {ACS synthetic biology},
volume = {14},
number = {5},
pages = {1732-1744},
doi = {10.1021/acssynbio.5c00084},
pmid = {40316310},
issn = {2161-5063},
mesh = {*Gene Regulatory Networks/genetics ; *CRISPR-Cas Systems/genetics ; *Synthetic Biology/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Gene regulatory networks, which control gene expression patterns in development and in response to stimuli, use regulatory logic modules to coordinate inputs and outputs. One example of a regulatory logic module is the gene regulatory cascade (GRC), where a series of transcription factor genes turn on in order. Synthetic biologists have derived artificial systems that encode regulatory rules, including GRCs. Furthermore, the development of single-cell approaches has enabled the discovery of gene regulatory modules in a variety of experimental settings. However, the tools available for validating these observations remain limited. Based on a synthetic GRC using DNA cutting-defective Cas9 (dCas9), we designed and implemented an alternative synthetic GRC utilizing DNA cutting-defective Cas12a (dCas12a). Comparing the ability of these two systems to express a fluorescent reporter, the dCas9 system was initially more active, while the dCas12a system was more streamlined. Investigating the influence of individual components of the systems identified nuclear localization as a major driver of differences in activity. Improving nuclear localization for the dCas12a system resulted in 1.5-fold more reporter-positive cells and a 15-fold increase in reporter intensity relative to the dCas9 system. We call this optimized system the "Synthetic Gene Regulatory Network" (SGRN, pronounced "sojourn").},
}

*Gene Regulatory Networks/genetics
*CRISPR-Cas Systems/genetics
*Synthetic Biology/methods
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism

@article {pmid40300480,
year = {2025},
author = {Richard, C and Allais-Bonnet, A and André, M and Gelin, V and Deloche, MC and Thépot, D and Canon, E and Laffont, L and Dubois, O and Congar, P and Lasserre, O and Aguirre-Lavin, T and Gomot, G and Moussu, C and Barbey, S and Pailhoux, E and Dardente, H},
title = {CRISPR-Cas9 gene-editing to assess the role of RF-amide-related peptide 3 in ovine seasonal breeding.},
journal = {Animal : an international journal of animal bioscience},
volume = {19},
number = {5},
pages = {101508},
doi = {10.1016/j.animal.2025.101508},
pmid = {40300480},
issn = {1751-732X},
mesh = {Animals ; Seasons ; Female ; *CRISPR-Cas Systems ; *Gene Editing/veterinary ; Sheep/genetics/physiology ; *Neuropeptides/genetics/metabolism ; Male ; Breeding ; Reproduction/genetics ; },
abstract = {Seasonal breeding is an adaptive strategy that ensures the birth of the offspring during the best time of year and allows energy saving in times of food scarcity and adverse environmental conditions. At temperate and polar latitudes, photoperiod is the main synchroniser of seasonal functions, which exerts its action through melatonin. Within the pars tuberalis of the anterior pituitary, melatonin triggers a series of events that lead to enhanced local triiodothyronine (T3) production in the medio-basal hypothalamus specifically under long days and ultimately drive the appropriate GnRH output at the median eminence. How T3 governs GnRH output is mostly unknown but neuronal populations that respectively produce KISS1 and RFRP3 appear to be involved. However, while the role of KISS1 as a major GnRH secretagogue is undisputed, the function of RFRP3 in the control of (seasonal) breeding remains enigmatic, with conflicting results hinting at elusive mechanisms. Therefore, we launched an extensive series of experiments in sheep, aimed at invalidating the NPVF gene, which encodes RFRP3, using CRISPR-Cas9 technology. Here, we report on the generation of six sheep for which the NPVF gene has been successfully edited. Four of these animals bear at least one allele expected to behave as a null and constitute founders for distinct ovine lines. These founder sheep will now enter a standard breeding scheme in order to obtain male and female homozygotes for distinct mutations. These animals are expected to provide a clear delineation of the function of RFRP3 in seasonal breeding.},
}

Animals
Seasons
Female
*CRISPR-Cas Systems
*Gene Editing/veterinary
Sheep/genetics/physiology
*Neuropeptides/genetics/metabolism
Male
Breeding
Reproduction/genetics

@article {pmid40259084,
year = {2025},
author = {Nadig, A and Replogle, JM and Pogson, AN and Murthy, M and McCarroll, SA and Weissman, JS and Robinson, EB and O'Connor, LJ},
title = {Transcriptome-wide analysis of differential expression in perturbation atlases.},
journal = {Nature genetics},
volume = {57},
number = {5},
pages = {1228-1237},
pmid = {40259084},
issn = {1546-1718},
support = {T32GM007753//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; T32GM007618//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; F31NS115380//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {*Transcriptome/genetics ; Humans ; *Gene Expression Profiling/methods ; Single-Cell Analysis/methods ; Gene Expression Regulation ; CRISPR-Cas Systems/genetics ; },
abstract = {Single-cell CRISPR screens such as Perturb-seq enable transcriptomic profiling of genetic perturbations at scale. However, the data produced by these screens are noisy, and many effects may go undetected. Here we introduce transcriptome-wide analysis of differential expression (TRADE)-a statistical model for the distribution of true differential expression effects that accounts for estimation error appropriately. TRADE estimates the 'transcriptome-wide impact', which quantifies the total effect of a perturbation across the transcriptome. Analyzing several large Perturb-seq datasets, we show that many transcriptional effects remain undetected in standard analyses but emerge in aggregate using TRADE. A typical gene perturbation affects an estimated 45 genes, whereas a typical essential gene affects over 500. We find moderate consistency of perturbation effects across cell types, identify perturbations where transcriptional responses vary qualitatively across dosage levels and clarify the relationship between genetic and transcriptomic correlations across neuropsychiatric disorders.},
}

*Transcriptome/genetics
Humans
*Gene Expression Profiling/methods
Single-Cell Analysis/methods
Gene Expression Regulation
CRISPR-Cas Systems/genetics

@article {pmid40210680,
year = {2025},
author = {Koh, GCC and Nanda, AS and Rinaldi, G and Boushaki, S and Degasperi, A and Badja, C and Pregnall, AM and Zhao, SJ and Chmelova, L and Black, D and Heskin, L and Dias, J and Young, J and Memari, Y and Shooter, S and Czarnecki, J and Brown, MA and Davies, HR and Zou, X and Nik-Zainal, S},
title = {A redefined InDel taxonomy provides insights into mutational signatures.},
journal = {Nature genetics},
volume = {57},
number = {5},
pages = {1132-1141},
pmid = {40210680},
issn = {1546-1718},
support = {Award 2019//Dr. Josef Steiner Krebsstiftung (Dr. Josef Steiner Cancer Research Foundation)/ ; C60100/A23433//Cancer Research UK (CRUK)/ ; C60100/A25274//Cancer Research UK (CRUK)/ ; CGCATF-2021/100013//Cancer Research UK (CRUK)/ ; C60100/A27815//Cancer Research UK (CRUK)/ ; NIHR301627//DH | National Institute for Health Research (NIHR)/ ; BRC-1215-20014//DH | National Institute for Health Research (NIHR)/ ; },
mesh = {Humans ; *INDEL Mutation/genetics ; DNA Mismatch Repair/genetics ; *Neoplasms/genetics ; DNA Polymerase II/genetics ; DNA Polymerase III/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Cell Line, Tumor ; },
abstract = {Despite their deleterious effects, small insertions and deletions (InDels) have received far less attention than substitutions. Here we generated isogenic CRISPR-edited human cellular models of postreplicative repair dysfunction (PRRd), including individual and combined gene edits of DNA mismatch repair (MMR) and replicative polymerases (Pol ε and Pol δ). Unique, diverse InDel mutational footprints were revealed. However, the prevailing InDel classification framework was unable to discriminate these InDel signatures from background mutagenesis and from each other. To address this, we developed an alternative InDel classification system that considers flanking sequences and informative motifs (for example, longer homopolymers), enabling unambiguous InDel classification into 89 subtypes. Through focused characterization of seven tumor types from the 100,000 Genomes Project, we uncovered 37 InDel signatures; 27 were new. In addition to unveiling previously hidden biological insights, we also developed PRRDetect-a highly specific classifier of PRRd status in tumors, with potential implications for immunotherapies.},
}

Humans
*INDEL Mutation/genetics
DNA Mismatch Repair/genetics
*Neoplasms/genetics
DNA Polymerase II/genetics
DNA Polymerase III/genetics
CRISPR-Cas Systems/genetics
Gene Editing
Cell Line, Tumor

@article {pmid39803701,
year = {2025},
author = {Deniz, AH and Aydemir, E and Ekimci Gürcan, N and Dabo Jakir, A and Faruk Bayrak, Ö and Şahin, F and Ayaz, F},
title = {Improved Malignancy of Colon Cancer Cells at Gene Expression Level With Constitutive Activation of the Eukaryotic Elongation Factor 2 Under Nutrition-Deficient Conditions.},
journal = {Chemistry & biodiversity},
volume = {22},
number = {5},
pages = {e202402547},
doi = {10.1002/cbdv.202402547},
pmid = {39803701},
issn = {1612-1880},
support = {BİRUNİ-BAP-06-2023-01-01//Biruni University/ ; BAP2023-01-01//Biruni University/ ; },
mesh = {Humans ; *Colonic Neoplasms/pathology/genetics/metabolism ; HCT116 Cells ; *Peptide Elongation Factor 2/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; },
abstract = {Regulation of protein production in response to physiological signals is achieved through precise control of eukaryotic Elongation Factor 2 (eEF2), whose distinct translocase function is crucial for cell survival. Phosphorylation of eEF2 at its Thr56 (T56) residue inactivates this function in translation. Using a genetically modified paralogue of a colon cancer cell line, HCT116 which carries a point mutation at Ser595-to-Alanine in the eEF2 gene we were able to create a constitutively active form of eEF2. Then, we compared these cells with their wild-type counterparts under complete growth and nutrient deprivation conditions. The mutant forms were created by using the CRISPR/Cas9-mediated gene cassette knock-in (KI) strategy. The samples were analyzed by a microarray-based approach. Carrying out a WikiPath analysis, we identified distinct signaling pathways and gene expression patterns that are differentially regulated in the mutant cells (HCT116[KI)], but not in wild-type HCT116 (HCT116[WT]) cells, particularly under nutrient-deprived conditions. Our results suggest that the constitutive activation of eEF2 enables an increase in the malignant gene expression in colon cancer cells compared to the wild-type counterparts under nutrient-deprived conditions.},
}

Humans
*Colonic Neoplasms/pathology/genetics/metabolism
HCT116 Cells
*Peptide Elongation Factor 2/genetics/metabolism
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems

@article {pmid40342201,
year = {2025},
author = {Chulei, Y and Yiyuan, Z and Hong, T and Yanhua, G and Limin, W and Ping, Z},
title = {Analysis of Oestrus and Hormonal Changes in the Progeny of 2 Genotypes of FecB Gene-Edited Sheep.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {60},
number = {5},
pages = {e70038},
doi = {10.1111/rda.70038},
pmid = {40342201},
issn = {1439-0531},
support = {2023TSYCTD0021//the foundation Bingtuan Science and Technology Project/ ; NYHXGG2023AA101//Bingtuan Science and Technology Project/ ; NCG202221//Bingtuan Science and Technology Project/ ; },
mesh = {Animals ; Female ; *Estrus/genetics/physiology ; Genotype ; *Gene Editing/veterinary ; Ovarian Follicle ; Luteinizing Hormone/blood ; *Sheep, Domestic/genetics/physiology ; Follicle Stimulating Hormone/blood ; Estradiol/blood ; Sheep/genetics/physiology ; CRISPR-Cas Systems ; },
abstract = {The FecB gene, the first gene identified for multiple births, can improve reproductive performance in sheep. We used the CRISPR/Cas9 editing system to obtain the FecB gene in Kazakh sheep and mated with unedited Kazakh sheep. After the genotypic screening of the progeny, four B+ and ++ Kazakh ewes, each 2 weeks old and healthy, and close to the same body weight, were selected to study oestrus and hormonal changes in the progeny population of FecB gene-edited Kazakh sheep. Our results showed that the FecB gene was moderately polymorphic (0.25 < PIC < 0.5) in the progeny population, was able to be stably inherited, and showed increased lambing numbers. The number of follicles and tiny follicles differed significantly (p < 0.05) between the 2 genotypes of ewes in the progeny population. Physiologically, FecB gene-edited sheep did not differ from wild-type sheep. Throughout the oestrous cycle, FSH, LH, and E2 in serum hormones of both sheep genotypes showed variability (p < 0.05) between 72-360 h, 360-528 h, and 12-72 h, respectively, compared to other periods. The concentration of FSH and PROG in follicular fluid was much higher than that of LH and E2 (p < 0.05). Overall, it is possible to make Kazakh sheep carry the FecB gene by CRISPR/Cas9 editing and stably inherit it into the progeny population. The enhancement of reproductive hormones by the FecB gene is beneficial to follicular development and ovulation and improves the reproductive efficiency of sheep.},
}

Animals
Female
*Estrus/genetics/physiology
Genotype
*Gene Editing/veterinary
Ovarian Follicle
Luteinizing Hormone/blood
*Sheep, Domestic/genetics/physiology
Follicle Stimulating Hormone/blood
Estradiol/blood
Sheep/genetics/physiology
CRISPR-Cas Systems

@article {pmid40342053,
year = {2025},
author = {Allemailem, KS and Rahmani, AH and Almansour, NM and Aldakheel, FM and Albalawi, GM and Albalawi, GM and Khan, AA},
title = {Current updates on the structural and functional aspects of the CRISPR/Cas13 system for RNA targeting and editing: A next‑generation tool for cancer management (Review).},
journal = {International journal of oncology},
volume = {66},
number = {5},
pages = {},
pmid = {40342053},
issn = {1791-2423},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy ; *Gene Editing/methods ; Animals ; Genetic Therapy/methods ; },
abstract = {For centuries, a competitive evolutionary race between prokaryotes and related phages or other mobile genetic elements has led to the diversification of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR‑associated sequence (Cas) genome‑editing systems. Among the different CRISPR/Cas systems, the CRISPR/Cas9 system has been widely studied for its precise DNA manipulation; however, due to certain limitations of direct DNA targeting, off‑target effects and delivery challenges, researchers are looking to perform transient knockdown of gene expression by targeting RNA. In this context, the more recently discovered type VI CRISPR/Cas13 system, a programmable single‑subunit RNA‑guided endonuclease system that has the capacity to target and edit any RNA sequence of interest, has emerged as a powerful platform to modulate gene expression outcomes. All the Cas13 effectors known so far possess two distinct ribonuclease activities. Pre‑CRISPR RNA processing is performed by one RNase activity, whereas the two higher eukaryotes and prokaryotes nucleotide‑binding domains provide the other RNase activity required for target RNA degradation. Recent innovative applications of the type VI CRISPR/Cas13 system in nucleic acid detection, viral interference, transcriptome engineering and RNA imaging hold great promise for disease management. This genome editing system can also be employed by the Specific High Sensitivity Enzymatic Reporter Unlocking platform to identify any tumor DNA. The discovery of this system has added a new dimension to targeting, tracking and editing circulating microRNA/RNA/DNA/cancer proteins for the management of cancer. However, there is still a lack of thorough understanding of the mechanisms underlying some of their functions. The present review summarizes the recent updates on the type VI CRISPR/Cas system in terms of its structural and mechanistic properties and some novel applications of this genome‑editing tool in cancer management. However, some issues, such as collateral degradation of bystander RNA, impose major limitations on its in vivo application. Furthermore, additional challenges and future prospects for this genome editing system are described in the present review.},
}

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

@article {pmid40341582,
year = {2025},
author = {Dacquay, LC and Antoniou, P and Mentani, A and Selfjord, N and Mårtensson, H and Hsieh, PP and Mustfa, S and Thom, G and Wimberger, S and Firth, M and Akrap, N and Maresca, M and Peterka, M},
title = {Dual inhibition of DNA-PK and Polϴ boosts precision of diverse prime editing systems.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4290},
pmid = {40341582},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; *DNA-Activated Protein Kinase/antagonists & inhibitors/metabolism/genetics ; Humans ; DNA Polymerase theta ; *DNA-Directed DNA Polymerase/metabolism/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Repair ; HEK293 Cells ; Animals ; },
abstract = {Prime editing is a genome engineering tool that allows installation of various small edits with high precision. However, prime editing efficiency and purity can vary widely across different edits, genomic targets, and cell types. Prime editing typically offers more precise editing outcomes compared to other genome editing methods such as homology-directed repair. However, it can still result in significant rates of unintended editing outcomes, such as indels or imprecise prime edits. This issue is particularly notable in systems utilizing a second nicking gRNA, such as PE3 and PE5, as well as in dual pegRNA systems and fully active nuclease systems such as PEn, which increase efficiency but compromise precision. In this work, we show that pharmacological inhibition of DNA-PK and Polϴ, two major mediators of mutagenic DNA repair pathways, improves precision of PEn, PE3, PE5, PE7, and dual pegRNA editing systems, including TwinPE, HOPE, and Bi-PE, across multiple genomic loci and edit types. We show that co-inhibition of DNA-PK and Polϴ mitigates both prime editing-unrelated indels and prime editing by-products such as template duplications. Moreover, in the case of PEn, this strategy also substantially improved the off-target editing profile. Together, our data establish small molecule modulation of DNA repair pathways as a general strategy to maximize the precision of diverse prime editing systems.},
}

*Gene Editing/methods
*DNA-Activated Protein Kinase/antagonists & inhibitors/metabolism/genetics
Humans
DNA Polymerase theta
*DNA-Directed DNA Polymerase/metabolism/genetics
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
DNA Repair
HEK293 Cells
Animals

@article {pmid40341025,
year = {2025},
author = {Wang, M and Krueger, JB and Gilkey, AK and Stelljes, EM and Kluesner, MG and Pomeroy, EJ and Skeate, JG and Slipek, NJ and Lahr, WS and Claudio Vázquez, PN and Zhao, Y and Bell, JB and Clement, K and Eaton, EJ and Laoharawee, K and Chang, JW and Webber, BR and Moriarity, BS},
title = {Precision enhancement of CAR-NK cells through non-viral engineering and highly multiplexed base editing.},
journal = {Journal for immunotherapy of cancer},
volume = {13},
number = {5},
pages = {},
pmid = {40341025},
issn = {2051-1426},
support = {R01 AI161017/AI/NIAID NIH HHS/United States ; P30 CA077598/CA/NCI NIH HHS/United States ; R21 CA237789/CA/NCI NIH HHS/United States ; R21 AI163731/AI/NIAID NIH HHS/United States ; T32 HL007062/HL/NHLBI NIH HHS/United States ; U24 OD026641/OD/NIH HHS/United States ; P01 CA254849/CA/NCI NIH HHS/United States ; R01 AI146009/AI/NIAID NIH HHS/United States ; U54 CA268069/CA/NCI NIH HHS/United States ; P50 CA136393/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *Killer Cells, Natural/immunology/metabolism ; *Receptors, Chimeric Antigen/genetics/metabolism ; Animals ; Mice ; *Immunotherapy, Adoptive/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Natural killer (NK) cells' unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials.

METHODS: Advanced genome engineering may thus be used to unlock their full potential. Multiplex genome editing with CRISPR/Cas9 base editors (BEs) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here, we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations.

RESULTS: We observed highly efficient single and multiplex base editing, resulting in significantly enhanced NK cell function in vitro and in vivo. Next, we combined multiplex BE with non-viral TcBuster transposon-based integration to generate interleukin-15 armored CD19 chimeric antigen receptor (CAR)-NK cells with significantly improved functionality in a highly suppressive model of Burkitt's lymphoma both in vitro and in vivo.

CONCLUSIONS: The use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated.},
}

Humans
*Gene Editing/methods
*Killer Cells, Natural/immunology/metabolism
*Receptors, Chimeric Antigen/genetics/metabolism
Animals
Mice
*Immunotherapy, Adoptive/methods
CRISPR-Cas Systems

@article {pmid40334424,
year = {2025},
author = {Baranova, SV and Zhdanova, PV and Pestryakov, PE and Chernonosov, AA and Koval, VV},
title = {Key thermodynamic characteristics of Cas9 and Cas12a endonucleases' cleavage of a DNA substrate containing a nucleotide mismatch in the region complementary to RNA.},
journal = {Biochemical and biophysical research communications},
volume = {768},
number = {},
pages = {151892},
doi = {10.1016/j.bbrc.2025.151892},
pmid = {40334424},
issn = {1090-2104},
mesh = {Thermodynamics ; *Base Pair Mismatch ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; *DNA/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/chemistry ; Substrate Specificity ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Molecular Dynamics Simulation ; *Bacterial Proteins/metabolism/chemistry ; DNA Cleavage ; Kinetics ; *RNA/metabolism/chemistry/genetics ; },
abstract = {CRISPR-Cas9 and CRISPR-Cas12a are endonuclease systems widely used for genome editing, but their mechanisms of DNA cleavage, particularly in the presence of nucleotide mismatches, remain incompletely understood. This study deals with thermodynamic parameters governing the cleavage of DNA substrates-containing a mismatch in the region complementary to RNA-by Cas9 and Cas12a. Using a series of 55 bp DNA substrates with various mismatches, we investigated the cleavage efficiency, reaction kinetics, and thermodynamic stability of the Cas12a-crRNA complex and compared it with Cas9-sgRNA on the same substrates. Cas12a manifested strict specificity, with a mismatch leading to a significant reduction in cleavage efficiency or to nonspecific trans-cleavage, whereas Cas9 showed higher tolerance to each mismatch, especially in internal and distal regions. Thermodynamic calculations indicated that Cas12a-crRNA complexes are generally stabler with fully complementary DNA but are more destabilized by a mismatch than Cas9-sgRNA complexes are. Molecular dynamics simulations revealed that a mismatch causes greater structural destabilization in Cas12a than in Cas9, correlating with reduced cleavage efficiency. These findings highlight distinct mechanisms of mismatch recognition by Cas9 and Cas12a, provide insights into their enzymatic behavior, and inform the design of more precise genome-editing tools.},
}

Thermodynamics
*Base Pair Mismatch
*CRISPR-Associated Proteins/metabolism/chemistry
*Endodeoxyribonucleases/metabolism/chemistry
*DNA/metabolism/chemistry/genetics
CRISPR-Cas Systems
*CRISPR-Associated Protein 9/metabolism/chemistry
Substrate Specificity
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
Molecular Dynamics Simulation
*Bacterial Proteins/metabolism/chemistry
DNA Cleavage
Kinetics
*RNA/metabolism/chemistry/genetics

@article {pmid40331901,
year = {2025},
author = {Ai, Z and Wang, W and Li, X and Wang, X and Chen, J and Wu, J and Zhou, S},
title = {A CRISPR/Cas12a-coupled multiplexed amplification system for ultrasensitive detection of miRNA-155.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {19},
pages = {4044-4050},
doi = {10.1039/d5ay00415b},
pmid = {40331901},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {miRNA plays an important role in gene regulation and can be an effective biomarker for disease diagnosis. Herein, a new miRNA detection platform based on the CRISPR/Cas12a-coupled multiplexed amplification system is developed. In this strategy, miRNA-155 acts as an intermediary to trigger the recombinase polymerase amplification (RPA). Due to the introduction of endonuclide recognition sites in the amplification template, the resulting double-stranded DNA (dsDNA) can in turn initiate a strand replacement reaction (SDA), generating a great deal of single-stranded DNA (ssDNA). The ssDNA can directly unlock the trans-cleavage activity of CRSIPR/Cas12a, and the process is independent of PAM sites. Subsequently, the activated Cas12a trans-cleaves nearby signaling molecules, outputting a fluorescence/visualization signal. This method achieves miRNA detection as low as 68.69 fM, with a linear range of 200 fM to 1 nM, and shows good selectivity and repeatability. Meanwhile, the target of 10 pM can be distinguished by the naked eye. Moreover, the proposed method can achieve miRNA-155 detection in complicated cell extracts. The excellent detection sensitivity is mainly due to the integration of two amplification techniques, while the CRISPR/Cas12a system enables fast and accurate visual detection. More importantly, the actual detection results are consistent with standard methods (RT-qPCR), indicating that the CRISPR/Cas12a-coupled multiplexed amplification system is reliable and has potential clinical application value.},
}

*MicroRNAs/genetics/analysis
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
*Endodeoxyribonucleases/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40292762,
year = {2025},
author = {Qin, Y and Xie, JL and Mao, K and Li, YF and Huang, CZ and Zou, HY and Zhen, SJ},
title = {A novel CRISPR-Cas12a-based fluorescence anisotropy method with a high signal-to-background ratio for sensitive biosensing.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {41},
pages = {7458-7461},
doi = {10.1039/d5cc01565k},
pmid = {40292762},
issn = {1364-548X},
mesh = {*Biosensing Techniques/methods ; *Fluorescence Polarization/methods ; *CRISPR-Cas Systems ; *DNA, Viral/analysis ; *Hepatitis B virus/genetics ; DNA/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Here, a CRISPR-Cas12a system with high trans-cleavage ability integrating a DNA nanochain formed by DNA tetrahedrons with a large molecular mass was employed to enhance the signal-to-background ratio of the fluorescence anisotropy method for achieving sensitive detection of hepatitis B virus DNA.},
}

*Biosensing Techniques/methods
*Fluorescence Polarization/methods
*CRISPR-Cas Systems
*DNA, Viral/analysis
*Hepatitis B virus/genetics
DNA/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40192002,
year = {2025},
author = {Wang, W and Shan, Y and Liu, R and Li, D and Zhou, J and Lu, Q and Zhao, H},
title = {Coordination of IFT20 With Other IFT Components Is Required for Ciliogenesis.},
journal = {Journal of clinical laboratory analysis},
volume = {39},
number = {9},
pages = {e70000},
doi = {10.1002/jcla.70000},
pmid = {40192002},
issn = {1098-2825},
support = {2022HWYQ-075;ZR2024MC040//Natural Science Foundation of Shandong Province/ ; 32170695//National Natural Science Foundation of China/ ; },
mesh = {*Cilia/metabolism/physiology ; Humans ; *Carrier Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {BACKGROUND: Primary cilia are organelles formed on the cell surface. They can act as cellular antennae to sense signals and play important roles in various biological processes. Abnormalities in primary cilia lead to a variety of diseases collectively known as ciliopathies. Intraflagellar transport protein 20 (IFT20) has been implicated in ciliogenesis.

METHODS: IFT20 knockout cell lines were established using the CRISPR-Cas9 gene editing technology. The GFP-IFT20 plasmid was constructed with the Gateway cloning system. Protein levels were detected via immunoblotting, and the localization of IFT20, acetylated α-tubulin, ARL13B, CP110, MKS3, IFT88, and IFT140 in wild-type and IFT20 knockout cells was examined by immunofluorescence microscopy. The fluorescence intensities were analyzed using ImageJ. Data quantifications and mass spectrometry results were analyzed using GraphPad Prism and Metascape.

RESULTS: The IFT20 deficiency impaired ciliogenesis and reduced cilium length. IFT20 depletion did not affect the removal of centriolar coiled-coil protein 110 (CP110) from the mother centriole or the recruitment of Meckel-Gruber syndrome type 3 (MKS3) to the transition zone. Mass spectrometry analysis revealed that proteins interacting with IFT20 were mainly IFT components. IFT20 knockout decreased the levels of both IFT88 and IFT140, and abrogated IFT88 localization at the basal body and ciliary axoneme. IFT20 knockout also impaired IFT140 localization at the ciliary axoneme but did not affect its localization at the basal body.

CONCLUSIONS: IFT20 is involved in ciliogenesis by regulating the level and localization of other IFT proteins and may have important implications in ciliopathies and related diseases.},
}

*Cilia/metabolism/physiology
Humans
*Carrier Proteins/metabolism/genetics
CRISPR-Cas Systems
Gene Knockout Techniques

@article {pmid40163310,
year = {2025},
author = {Rathje, F and Sykora, MM and Aberger, F and Krenn, PW},
title = {High Efficiency Lentiviral Transduction of Colon Organoids Using Reversible 2D/3D Culture Techniques.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2905},
number = {},
pages = {245-254},
pmid = {40163310},
issn = {1940-6029},
mesh = {*Organoids/cytology/metabolism ; Animals ; *Lentivirus/genetics ; Mice ; *Colon/cytology/metabolism ; *Transduction, Genetic/methods ; *Cell Culture Techniques/methods ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Humans ; Stem Cells/cytology/metabolism ; },
abstract = {Organoids are a promising research tool for studying tissue development and disease in vitro. While organoids are frequently considered a replacement or complementary model for in vivo mouse experiments, exploiting their full potential often requires genetically engineered mice as a source of transgenic stem cells, also because genetic manipulation of organoids is rather inefficient and cumbersome. Here, we describe an alternative and optimized murine colon organoid manipulation protocol that reversibly and temporarily interrupts the 3D organoid structure for short-term 2D monolayer culture. This approach allows highly efficient viral transduction and genetic manipulation of stem cells in a 2D setting, followed by 3D stem cell embedding and restoration of the original organoid architecture. This method greatly improves the efficiency of lentiviral-mediated genetic manipulation of organoids and increases their potential applications in CRISPR/Cas9 and compound screens, immune-competent co-cultures, and disease modeling.},
}

*Organoids/cytology/metabolism
Animals
*Lentivirus/genetics
Mice
*Colon/cytology/metabolism
*Transduction, Genetic/methods
*Cell Culture Techniques/methods
Genetic Vectors/genetics
CRISPR-Cas Systems
Humans
Stem Cells/cytology/metabolism

@article {pmid40140553,
year = {2025},
author = {Xi, JF and Liu, BD and Tang, GR and Ren, ZH and Chen, HX and Lan, YL and Yin, F and Li, Z and Cheng, WS and Wang, J and Chen, L and Yuan, SC and Zhang, Z and Luo, GZ},
title = {m[6]A modification regulates cell proliferation via reprogramming the balance between glycolysis and pentose phosphate pathway.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {496},
pmid = {40140553},
issn = {2399-3642},
mesh = {*Pentose Phosphate Pathway/genetics ; *Cell Proliferation/genetics ; *Glycolysis/genetics ; Humans ; *Adenosine/analogs & derivatives/metabolism/genetics ; Cell Line, Tumor ; Glucosephosphate Dehydrogenase/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Neoplasms/genetics/metabolism/pathology ; CRISPR-Cas Systems ; },
abstract = {N6-methyladenosine (m[6]A) stands as the predominant modification in eukaryotic mRNA and is involved in various biological functions. Aberrant m[6]A has been implicated in abnormal cellular phenotypes, including defects in stem cell differentiation and tumorigenesis. However, the precise effects of m[6]A on cell proliferation and the underlining mechanism of metabolic gene regulation remain incompletely understood. Here, we established a cellular environment with low-m[6]A levels and observed a severe impairment of cell proliferation. Mechanistic studies revealed that the depletion of m[6]A on TIGAR mRNA led to increased expression, subsequently inhibiting glycolysis while promoting the pentose phosphate pathway (PPP). A genome-wide CRISPR-Cas9 screen identified numerous genes involved in cell proliferation that are sensitive to m[6]A modification, with G6PD emerging as a key regulator. Integration of gene expression and survival data from cancer patients suggested that patients with elevated G6PD expression may exhibit enhanced responsiveness to tumor growth inhibition through m[6]A suppression. Our findings elucidate the critical role of m[6]A in cell proliferation, highlighting the therapeutic potential of targeting m[6]A-mediated metabolic pathways in cancer.},
}

*Pentose Phosphate Pathway/genetics
*Cell Proliferation/genetics
*Glycolysis/genetics
Humans
*Adenosine/analogs & derivatives/metabolism/genetics
Cell Line, Tumor
Glucosephosphate Dehydrogenase/genetics/metabolism
Gene Expression Regulation, Neoplastic
Neoplasms/genetics/metabolism/pathology
CRISPR-Cas Systems

@article {pmid39841016,
year = {2025},
author = {Araújo, JL and Wagenblast, E and Voisin, V and McLeod, J and Gan, OI and Bansal, S and Jin, L and Mitchell, A and Gratton, B and Cutting, S and Arruda, A and Doedens, M and Travas, A and Kim, D and Capo-Chichi, JM and Abelson, S and Minden, MD and Wang, JCY and Sobrinho-Simões, MA and Pinto-do-Ó, P and Lechman, E and Dick, JE},
title = {FLT3 is genetically essential for ITD-mutated leukemic stem cells but dispensable for human hematopoietic stem cells.},
journal = {Blood},
volume = {145},
number = {20},
pages = {2361-2373},
doi = {10.1182/blood.2024025886},
pmid = {39841016},
issn = {1528-0020},
mesh = {*fms-Like Tyrosine Kinase 3/genetics/metabolism ; Humans ; *Hematopoietic Stem Cells/metabolism/pathology ; Animals ; *Neoplastic Stem Cells/pathology/metabolism ; Mice ; *Leukemia, Myeloid, Acute/genetics/pathology ; *Mutation ; *Tandem Repeat Sequences ; CRISPR-Cas Systems ; },
abstract = {Leukemic stem cells (LSCs) fuel acute myeloid leukemia (AML) growth and relapse, but therapies tailored toward eradicating LSCs without harming normal hematopoietic stem cells (HSCs) are lacking. FMS-like tyrosine kinase 3 (FLT3) is considered an important therapeutic target due to frequent mutation in AML and association with relapse. However, there has been limited clinical success with FLT3 drug targeting, suggesting either that FLT3 is not a vulnerability in LSC or that more potent inhibition is required, a scenario where HSC toxicity could become limiting. We tested these possibilities by ablating FLT3 using CRISPR/Cas9-mediated FLT3 knockout (FLT3-KO) in human LSCs and HSCs followed by functional xenograft assays. FLT3-KO in LSCs from FLT3-internal tandem duplication (ITD)-mutated but not FLT3-wild-type AMLs resulted in short-term leukemic grafts of FLT3-KO edited cells that disappeared by 12 weeks. By contrast, FLT3-KO in HSCs from the fetal liver, cord blood, and adult bone marrow did not impair multilineage hematopoiesis in primary and secondary xenografts. Our study establishes FLT3 as an ideal therapeutic target where ITD-positive LSCs are eradicated upon FLT3 deletion whereas HSCs are spared. These findings support the development of more potent FLT3-targeting drugs or gene-editing approaches for LSC eradication to improve clinical outcomes.},
}

*fms-Like Tyrosine Kinase 3/genetics/metabolism
Humans
*Hematopoietic Stem Cells/metabolism/pathology
Animals
*Neoplastic Stem Cells/pathology/metabolism
Mice
*Leukemia, Myeloid, Acute/genetics/pathology
*Mutation
*Tandem Repeat Sequences
CRISPR-Cas Systems

@article {pmid40333994,
year = {2025},
author = {Chen, H and LaFlamme, CW and Wang, YD and Blan, AW and Koehler, N and Mendonca Moraes, R and Olszewski, AR and Almanza Fuerte, EP and Bonkowski, ES and Bajpai, R and Lavado, A and Pruett-Miller, SM and Mefford, HC},
title = {Patient-derived models of UBA5-associated encephalopathy identify defects in neurodevelopment and highlight potential therapeutic avenues.},
journal = {Science translational medicine},
volume = {17},
number = {797},
pages = {eadn8417},
doi = {10.1126/scitranslmed.adn8417},
pmid = {40333994},
issn = {1946-6242},
mesh = {Humans ; Organoids/metabolism/pathology ; *Ubiquitin-Activating Enzymes/genetics/metabolism ; *Brain Diseases/genetics/pathology/therapy ; Endoplasmic Reticulum/metabolism ; *Models, Biological ; Male ; Female ; Unfolded Protein Response ; CRISPR-Cas Systems/genetics ; Neurons/metabolism/pathology ; },
abstract = {UBA5 encodes for the E1 enzyme of the UFMylation cascade, which plays an essential role in endoplasmic reticulum (ER) homeostasis. The clinical phenotypes of UBA5-associated encephalopathy include developmental delays, epilepsy, and intellectual disability. To date, there is no humanized neuronal model to study the cellular and molecular consequences of UBA5 pathogenic variants. We developed and characterized patient-derived cortical organoid cultures from two patients with compound heterozygous variants in UBA5. Both shared the same missense variant, which encodes a hypomorphic allele (p.A371T), along with a nonsense variant (p.G267* or p.A123fs*4). Single-cell RNA sequencing of 100-day organoids identified defects in GABAergic interneuron development. We demonstrated aberrant neuronal firing and reduction in size of patient-derived organoids. Mechanistically, we showed that ER homeostasis is perturbed along with an exacerbated unfolded protein response pathway in engineered U87-MG cells and patient-derived organoids expressing UBA5 pathogenic variants. We also assessed two potential therapeutic modalities that augmented UBA5 protein abundance to rescue aberrant molecular and cellular phenotypes. We assessed SINEUP, a long noncoding RNA that augments translation efficiency, and CRISPRa, a modified CRISPR-Cas9 approach to augment transcription efficiency to increase UBA5 protein production. Our study provides a humanized model that allows further investigations of UBA5 variants in the brain and highlights promising approaches to alleviate cellular aberrations for this rare, developmental disorder.},
}

Humans
Organoids/metabolism/pathology
*Ubiquitin-Activating Enzymes/genetics/metabolism
*Brain Diseases/genetics/pathology/therapy
Endoplasmic Reticulum/metabolism
*Models, Biological
Male
Female
Unfolded Protein Response
CRISPR-Cas Systems/genetics
Neurons/metabolism/pathology

@article {pmid40333840,
year = {2025},
author = {Chang, J and Yang, X and Zhang, T and Sun, H and Cheng, H and Jia, Z and Li, Y and Ma, S and Sun, T and Cao, J},
title = {High-Throughput Screening to Identify Novel Compounds Affecting the Genome Editing Efficiency of CRISPR System.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {8},
pages = {},
pmid = {40333840},
issn = {1420-3049},
support = {202203021212372//Applied Basic Research Program of Shanxi Province/ ; 82170523//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/drug effects ; HEK293 Cells ; *High-Throughput Screening Assays/methods ; },
abstract = {Genome editing is a promising therapeutic strategy for genetic disorders by modifying the genome precisely, especially the CRISPR/Cas9 system. However, a major limitation of CRISPR/Cas9 in gene therapy is the biosafety issues caused by off-target effects. Compounds that can modulate the genome editing efficiency of the CRISPR/Cas9 system, especially those reducing the off-target effects, are potentially useful pharmacological tools for improving the effectiveness and safety of genome editing. Here, we performed high-throughput screening in HEK 293FT cells to discover compounds that decrease or increase the genome editing efficiency of the CRISPR/Cas9 system from 9930 compounds. After two rounds of screening, we identified that CP-724714, a ErbB2 (HER2) tyrosine kinase inhibitor, decreased the CRISPR/Cas9 efficiency and reduced the off-target effects by suppressing the efficiency of CRISPR/Cas9, and was thus named a CRISPR decelerator (or inhibitor), while Clofarabine, a DNA synthesis inhibitor, increased the efficiency of CRISPR/Cas9, and was named a CRISPR accelerator. We further identified four compounds (Tranilast, Cerulenin, Rosolic acid and Resveratrol) that affected the efficiency of single-strand annealing (SSA) repair. Among them, Tranilast, Cerulenin and Rosolic acid are potential SSA decelerators, while Resveratrol is a potential SSA accelerator. These identified compounds may be useful in optimizing mammalian genetic manipulation techniques.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/drug effects
HEK293 Cells
*High-Throughput Screening Assays/methods

@article {pmid40333207,
year = {2025},
author = {Ouyang, Y and Xia, Y and Tang, X and Qin, L and Xia, S},
title = {Trans-Kingdom sRNA Silencing in Sclerotinia sclerotiorum for Crop Fungal Disease Management.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {4},
pages = {},
pmid = {40333207},
issn = {2076-0817},
support = {31971836//the National Nature Science Foundation of China/ ; },
mesh = {*Ascomycota/genetics/pathogenicity ; *Plant Diseases/microbiology/prevention & control ; *RNA Interference ; *Crops, Agricultural/microbiology ; Gene Silencing ; Disease Management ; *RNA, Fungal/genetics ; },
abstract = {Sclerotinia sclerotiorum is a globally widespread and vast destructive plant pathogenic fungus that causes significant yield losses in crops. Due to the lack of effective resistant germplasm resources, the control of diseases caused by S. sclerotiorum largely relies on chemical fungicides. However, excessive use of these chemicals not only causes environmental concerns but also leads to the increased development of resistance in S. sclerotiorum. In contrast, trans-kingdom sRNA silencing-based technologies, such as host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), offer novel, effective, and environmentally friendly methods for the management of S. sclerotiorum infection. This review summarizes recent advances in the identification of S. sclerotiorum pathogenic genes, target gene selection, categories, and application of trans-kingdom RNA interference (RNAi) technologies targeting this pathogen. Although some challenges, including off-target effects and the efficiency of external sRNA uptake, exist, recent findings have proposed solutions for further improvement. Combined with the latest developments in CRISPR/Cas gene editing and other technologies, trans-kingdom RNAi has significant potential to become a crucial tool in the control of sclerotinia stem rot (SSR), mitigating the impact of S. sclerotiorum on crop production.},
}

*Ascomycota/genetics/pathogenicity
*Plant Diseases/microbiology/prevention & control
*RNA Interference
*Crops, Agricultural/microbiology
Gene Silencing
Disease Management
*RNA, Fungal/genetics

@article {pmid40333073,
year = {2025},
author = {Saenz-Garcia, JL and Souza-Melo, N and Miranda, JS and Borges, B and Pacheco-Lugo, LA and Quintero-Solano, JM and Moretti, N and Wheeler, R and Soares-Medeiros, LC and DaRocha, WD},
title = {Kharon Is Crucial for Trypanosoma cruzi Morphology but Does Not Impair In Vitro Infection.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {4},
pages = {},
pmid = {40333073},
issn = {2076-0817},
support = {2020221000012//Fundação Araucária/ ; 2018/09948-0//FAPESP/ ; 2020/07870-4//FAPESP/ ; 2022/03075-0//FAPESP/ ; },
mesh = {*Trypanosoma cruzi/genetics/metabolism/growth & development ; *Protozoan Proteins/genetics/metabolism ; *Chagas Disease/parasitology ; Animals ; Cytoskeleton/metabolism ; CRISPR-Cas Systems ; },
abstract = {Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease with few options for treatment and no available vaccine. Deletion mutants for live attenuated vaccines, particularly deletions of proteins related to the cytoskeleton, have been widely tested in related parasites but candidates have not been tested in T. cruzi. Kharon is one such protein, identified as being associated with the cytoskeleton in Leishmania and essential for amastigote replication. Here we investigated the T. cruzi Kharon ortholog (TcKharon) to test if it has orthologous function and thus potential in generating a live attenuated vaccine. In silico analysis predicted TcKharon to be an intrinsically disordered protein, consistent with its ortholog feature, and GFP fusion protein revealed that TcKharon is associated with the cytoskeleton of epimastigotes. CRISPR-Cas9-mediated gene disruption impaired epimastigote proliferation and cytokinesis, resulting in altered nucleus-to-kinetoplast ratios and pronounced morphological defects, particularly in the posterior cell region. Despite these abnormalities, TcKharon[-/-] mutants retained the ability to differentiate into metacyclic trypomastigotes and exhibited in vitro infection rates comparable to wild-type parasites. Our data show that TcKharon is crucial for cell morphology. However, in contrast to close related parasites, TcKharon is not essential for in vitro infectivity.},
}

*Trypanosoma cruzi/genetics/metabolism/growth & development
*Protozoan Proteins/genetics/metabolism
*Chagas Disease/parasitology
Animals
Cytoskeleton/metabolism
CRISPR-Cas Systems

@article {pmid40311322,
year = {2025},
author = {He, Z and Cole, KD and He, HJ},
title = {A novel immortalization method for immortalizing human primary CD8[+] T cells by inserting a single copy of human telomerase reverse transcriptase via CRISPR/Cas9.},
journal = {Tissue & cell},
volume = {95},
number = {},
pages = {102908},
doi = {10.1016/j.tice.2025.102908},
pmid = {40311322},
issn = {1532-3072},
mesh = {Humans ; *Telomerase/genetics/metabolism ; *CD8-Positive T-Lymphocytes/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Existing cell immortalization methods made the cells obtain oncogenesis phenotype and/or caused the cells gain and/or lose chromosomes. Immortalized normal human T cells lines provide critical in vitro models for basic research and therapeutic products development.

METHODS: We developed a novel method utilizing a CRISPR/Cas9 system to replace the exon 2 of the cell cycle inhibitor gene CDKN2A (encoding p16 and p14 proteins) with a single copy of human telomerase reverse transcriptase (hTERT) to immortalize human primary CD8[+] T cells (hCD8[+]T-TERT).

RESULTS: By using Cas9 protein and low donor DNA copies/cell, we successfully immortalized hCD8[+]T cells with a single copy of hTERT transgene, which also avoided uncontrolled insertion of Cas9 gene and guide RNA vector. Human primary CD8[+] cells from independent donors were immortalized and expanded more than 2.6 × 10[7] times. Characterization of one of the immortalized CD8[+] T-TERT cell lines revealed that the cells retained most of the cell surface markers and normal karyotype. The CD8[+] T-TERT cells also retained the dependence of IL-2 and CD3/CD28 activator for survival and expansion.

CONCLUSION: We established a stable immortalized cell lines using the novel immortalization method, and the immortalized CD8[+] T cells had a phenotype consistent with T cells.},
}

Humans
*Telomerase/genetics/metabolism
*CD8-Positive T-Lymphocytes/cytology/metabolism
*CRISPR-Cas Systems/genetics

@article {pmid40214251,
year = {2025},
author = {Mao, S and Yang, X and Wang, Y and Chen, F and Jiang, H and Wang, Y and Kang, Y and Li, S},
title = {Ultrasensitive and highly specific detection of the Brucella genus and B. melitensis by CRISPR/Cas12b-multiple cross displacement amplification technique.},
journal = {Journal of clinical microbiology},
volume = {63},
number = {5},
pages = {e0153224},
doi = {10.1128/jcm.01532-24},
pmid = {40214251},
issn = {1098-660X},
support = {82273758//National Natural Science Foundation of China/ ; gzwkj2024-055//Health Commission of Guizhou Province/ ; Guo Jikong Zong Ren Han [2024]122//National Disease Control and Prevention Administration/ ; QJJ [2022]019//Foundation of Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou/ ; },
mesh = {*Brucellosis/diagnosis/microbiology ; Sensitivity and Specificity ; *Brucella/genetics/isolation & purification/classification ; *Brucella melitensis/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; *Molecular Diagnostic Techniques/methods ; DNA Primers/genetics ; },
abstract = {Brucellosis is caused by members of the Brucella spp. and remains one of the world's major zoonotic diseases. Brucella melitensis (B. melitensis) as the most contagious Brucella species cannot be ignored as an essential source of infection for brucellosis, especially in countries/regions dominated by animal husbandry. Thus, the identification of the Brucella genus and B. melitensis is crucial for rapid diagnosis of brucellosis to control disease transmission and clinical treatment. Here, we developed the CRISPR/Cas12b nuclease combined with a multiple cross displacement amplification (MCDA) assay (CRISPR-MCDA) for highly specific and sensitive detection of Brucella genus and B. melitensis in clinical applications. Two sets of specific primers were designed targeting the specific gene of Brucella genus (Bcsp31) and B. melitensis (BMEII0466), respectively. The CRISPR-MCDA assay showed high specificity and sensitivity in 28 non-Brucella isolates and 64 clinical samples. The detection limit of CRISPR-MCDA assay was 2 copies/μL in the plasmid dilution template, and the whole detection process took within 90 minutes with nanoparticle-based lateral flow biosensor (LFB) to validate experimental results. Taken together, the CRISPR-MCDA-LFB assay is a visual, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Brucella genus and B. melitensis.IMPORTANCEThe prevention and control of Brucellosis urgently require rapid and accurate diagnostic methods. This work validates a new method for the simultaneous detection of Brucella genus and B. melitensis. The method can effectively reduce the chances of contamination and provides a more rapid, sensitive, and specific on-site detection of Brucella. It also offers a solution for the rapid screening of Brucellosis in resource-limited environments, which is crucial for effective disease prevention and control. This technology can also be widely applied to the rapid detection of other pathogens beyond Brucella.},
}

*Brucellosis/diagnosis/microbiology
Sensitivity and Specificity
*Brucella/genetics/isolation & purification/classification
*Brucella melitensis/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
Humans
*CRISPR-Cas Systems
Animals
*Molecular Diagnostic Techniques/methods
DNA Primers/genetics

@article {pmid40211002,
year = {2025},
author = {Cheng, Y and Hu, M and Yang, B and Jensen, TB and Zhang, Y and Yang, T and Yu, R and Ma, Z and Radda, JSD and Jin, S and Zang, C and Wang, S},
title = {Perturb-tracing enables high-content screening of multi-scale 3D genome regulators.},
journal = {Nature methods},
volume = {22},
number = {5},
pages = {950-961},
pmid = {40211002},
issn = {1548-7105},
support = {DP2GM137414//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 5T32GM007205//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 2T32GM007499//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35GM133712//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01HG013503//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *Chromatin/genetics/chemistry/metabolism ; *Genome, Human ; CRISPR-Cas Systems ; In Situ Hybridization, Fluorescence/methods ; },
abstract = {Three-dimensional (3D) genome organization becomes altered during development, aging and disease, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multi-scale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screens, a cellular barcode readout method (BARC-FISH) and chromatin tracing. We performed a loss-of-function screen in human cells, and visualized alterations to their 3D chromatin folding conformations, alongside perturbation-paired barcode readout in the same single cells. We discovered tens of new regulators of chromatin folding at different length scales, ranging from chromatin domains and compartments to chromosome territory. A subset of the regulators exhibited 3D genome effects associated with loop extrusion and A-B compartmentalization mechanisms, while others were largely unrelated to these known 3D genome mechanisms. Finally, we identified new regulators of nuclear architectures and found a functional link between chromatin compaction and nuclear shape. Altogether, our method enables scalable, high-content identification of chromatin and nuclear topology regulators that will stimulate new insights into the 3D genome.},
}

Humans
*Chromatin/genetics/chemistry/metabolism
*Genome, Human
CRISPR-Cas Systems
In Situ Hybridization, Fluorescence/methods

@article {pmid40140468,
year = {2025},
author = {Rodríguez-Sánchez, A and Quijada-Álamo, M and Pérez-Carretero, C and Herrero, AB and Arroyo-Barea, A and Dávila-Valls, J and Rubio, A and García de Coca, A and Benito-Sánchez, R and Rodríguez-Vicente, AE and Hernández-Rivas, JM and Hernández-Sánchez, M},
title = {SAMHD1 dysfunction impairs DNA damage response and increases sensitivity to PARP inhibition in chronic lymphocytic leukemia.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {10446},
pmid = {40140468},
issn = {2045-2322},
support = {JCYL-EDU/1868/2022//Predoctoral research fellowship 2022 from Junta de Castilla y León/ ; },
mesh = {Humans ; *Leukemia, Lymphocytic, Chronic, B-Cell/genetics/drug therapy/metabolism/pathology ; *SAM Domain and HD Domain-Containing Protein 1/genetics/metabolism ; *DNA Damage ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Mutation ; Female ; Male ; BRCA1 Protein/metabolism/genetics ; Middle Aged ; Aged ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {Chronic lymphocytic leukemia (CLL) is a clinically and genetically heterogenous disease. Recent next-generation sequencing (NGS) studies have uncovered numerous low-frequency mutated genes in CLL patients, with SAMHD1 emerging as a candidate driver gene. However, the biological and clinical implications of SAMHD1 mutations remain unclear. Using CRISPR/Cas9, we generated CLL models to investigate the impact of SAMHD1 deficiency on pathogenesis and explore therapeutic strategies. Moreover, we performed NGS in treatment-naïve CLL patients to characterize SAMHD1 mutations and employed RNA-sequencing to evaluate their clinical significance. Our study shows that SAMHD1 inactivation impairs the DNA damage response by reducing homologous recombination efficiency through BRCA1 and RAD51 dysregulation. Importantly, SAMHD1 colocalizes with BRCA1 at DNA damage sites in CLL cells. This research also identifies that SAMHD1-mutated cells are more sensitive to PARP inhibition. Clinically, SAMHD1 dysfunction negatively impacts clinical outcome of CLL cases: SAMHD1 mutations reduce failure-free survival (median 46 vs 57 months, p = 0.033), while low SAMHD1 expression associates with shorter time to first treatment (median 47 vs 77 months; p = 0.00073). Overall, this study elucidates that SAMHD1 dysfunction compromises DNA damage response mechanisms, potentially contributing to unfavorable clinical outcomes in CLL, and proposes PARP-inhibitors as a potential therapeutic approach for SAMHD1-mutated CLL cells.},
}

Humans
*Leukemia, Lymphocytic, Chronic, B-Cell/genetics/drug therapy/metabolism/pathology
*SAM Domain and HD Domain-Containing Protein 1/genetics/metabolism
*DNA Damage
*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use
Mutation
Female
Male
BRCA1 Protein/metabolism/genetics
Middle Aged
Aged
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid40135601,
year = {2025},
author = {Yee, PS and Chai, AWY and Yee, SM and Ooi, S and Tan, YH and Garnett, MJ and Ng, SK and Cheong, SC},
title = {Interferon-Inducible ADAR1 p150 Is Essential for the Survival of Oral Squamous Cell Carcinoma.},
journal = {Molecular carcinogenesis},
volume = {64},
number = {6},
pages = {1066-1077},
doi = {10.1002/mc.23910},
pmid = {40135601},
issn = {1098-2744},
support = {//This study was funded by the Dr. Siti Hasmah Mohd Ali Professorial Chair appointment (PV061-2019) and Fundamental Research Grant Scheme (FRGS/1/2022/SKK10/USM/02/14)./ ; },
mesh = {Humans ; *Adenosine Deaminase/genetics/metabolism ; *RNA-Binding Proteins/genetics/metabolism ; *Mouth Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; *Carcinoma, Squamous Cell/genetics/pathology/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; Apoptosis ; Cell Survival/genetics ; CRISPR-Cas Systems ; eIF-2 Kinase/metabolism/genetics ; Interferon-Induced Helicase, IFIH1/metabolism/genetics ; *Squamous Cell Carcinoma of Head and Neck/genetics/pathology ; },
abstract = {We identified ADAR1 as one of the top essential genes for oral squamous cell carcinoma (OSCC) survival from our genome-wide CRISPR/Cas9 screen in OSCC cell lines. In this study, we confirm that ADAR1-knockout (KO) inhibits cell viability and colony forming ability, and induces apoptosis. We report that IFN-β treatment sensitizes less-dependent cell lines to ADAR1 KO-induced cell lethality. Overexpression of ADAR1-p150, but not ADAR1-p110, rescued cell lethality upon ADAR1 KO, confirming that the IFN-inducible p150 is responsible for OSCC survival. Using a deaminase inactive mutant, we demonstrate that the editing function of ADAR1 is important for OSCC survival. Furthermore, we show that ADAR1 KO-induced cell death is mediated by both PKR and MDA5. We compute gene signatures of ADAR1 dependency in OSCC tumors, and found that those with high ADAR1 dependency score are associated with well or moderate differentiation, likely due to high PKR expression or activation. While a majority of ADAR1-dependent tumors exhibit a low T cell-inflamed gene expression profile, ADAR1 KO upregulates PD-L1, a marker of anti-PD1 response, indicating that ADAR1 inhibition may enhance immunotherapy response in OSCC. Collectively, these findings suggest that targeting ADAR1-p150 not only induces OSCC cell death but could induce a favorable response to anti-PD1.},
}

Humans
*Adenosine Deaminase/genetics/metabolism
*RNA-Binding Proteins/genetics/metabolism
*Mouth Neoplasms/genetics/pathology/metabolism
Cell Line, Tumor
*Carcinoma, Squamous Cell/genetics/pathology/metabolism
Gene Expression Regulation, Neoplastic/drug effects
Apoptosis
Cell Survival/genetics
CRISPR-Cas Systems
eIF-2 Kinase/metabolism/genetics
Interferon-Induced Helicase, IFIH1/metabolism/genetics
*Squamous Cell Carcinoma of Head and Neck/genetics/pathology

@article {pmid40332816,
year = {2025},
author = {Xu, Q and Wang, M and Zeng, J and Sun, H and Wei, X and Jiang, H and Shentu, X and Sun, D},
title = {CRISPR/Cas Technology in Insect Insecticide Resistance.},
journal = {Insects},
volume = {16},
number = {4},
pages = {},
pmid = {40332816},
issn = {2075-4450},
support = {32302354//National Natural Science Foundation of China/ ; LQ24C140002//Zhejiang Provincial Programs for Natural Science/ ; U21A20223//National Natural Science Foundation of China/ ; 2023SNJF034//Zhejiang Provincial Programs for Science and Technology Development/ ; "Pioneer" and "Leading Goose" R&D Program of Zhejiang//"Pioneer" and "Leading Goose" R&D Program of Zhejiang/ ; },
abstract = {Chemicals and biological insecticides play a crucial role as pest management strategies in modern agriculture and forestry. However, their excessive and unreasonable use inevitably leads to varying degrees of resistance among insect populations, which seriously affects the sustainability of insecticide use. One primary reason for this resistance is alterations or mutations in insect gene expression. One class of genes encodes proteins that serve as critical targets for insecticides to exert their toxic effects in insects, while another class of genes encodes proteins involved in the detoxification process of insecticides within insects. Reverse genetics has become a vital research tool for studying the molecular mechanisms underlying changes and mutations in these target genes and their impact on insect resistance. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the CRISPR-associated gene Cas as gene-editing technologies has significantly advanced our understanding of how insects adapt to and resist insecticides. This article aims to provide a comprehensive and objective review of the progress made using the CRISPR/Cas system in various arthropods within the field of pest control.},
}

@article {pmid40332635,
year = {2025},
author = {Mukhtiar, A and Ullah, S and Yang, B and Jiang, YQ},
title = {Unlocking genetic potential: a review of the role of CRISPR/Cas technologies in rapeseed improvement.},
journal = {Stress biology},
volume = {5},
number = {1},
pages = {31},
pmid = {40332635},
issn = {2731-0450},
abstract = {Rapeseed (Brassica napus L.) is a globally important oil crop, providing edible vegetable oil and other valuable sources for humans. Being an allotetraploid, rapeseed has a complex genome that has undergone whole-genome duplication, making molecular breeding rather difficult. Fortunately, clustered regularly interspacedshort palindromic repeat (CRISPR)/CRISPR-associated (Cas) technologies have emerged as a potent tool in plant breeding, providing unprecedented accuracy as well as effectiveness in genome editing. This review focuses on the application and progresses of CRISPR/Cas technologies in rapeseed. We discussed the principles and mechanisms of CRISPR/Cas systems focusing on their use in rapeseed improvement such as targeted gene knockout, gene editing and transcriptional regulation. Furthermore, we summarized the regulatory frameworks governing CRISPR-edited crops as well as the challenges and opportunities for their commercialization and adoption. The potential advantages of CRISPR-mediated traits in rapeseed such as increased yield, disease and stress resistance and oil quality are discussed along with biosafety and environmental implications. The purpose of this review is to provide insights into the transformative role of CRISPR/Cas technologies in rapeseed breeding and its potential to address global agricultural challenges while ensuring sustainable crop production.},
}

@article {pmid40304259,
year = {2025},
author = {Kim, J and Orozaliev, A and Sahloul, S and Van, AD and Dang, VT and Pham, VS and Oh, Y and Chehade, I and Al-Sayegh, M and Song, YA},
title = {Accelerating Cleavage Activity of CRISPR-Cas13 System on a Microfluidic Chip for Rapid Detection of RNA.},
journal = {Analytical chemistry},
volume = {97},
number = {18},
pages = {9858-9865},
doi = {10.1021/acs.analchem.5c00256},
pmid = {40304259},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Humans ; *Lab-On-A-Chip Devices ; *MicroRNAs/analysis/genetics ; *Microfluidic Analytical Techniques ; SARS-CoV-2/genetics ; },
abstract = {It is extremely advantageous to detect nucleic acid levels in the early phases of disease management; such early detection facilitates timely treatment, and it can prevent altogether certain cancers and infectious diseases. A simple, rapid, and versatile detection platform without enzymatic amplification for both short and long sequences would be highly desirable in this regard. Our study addresses this need by introducing IMACC, an ICP-based Microfluidic Accelerator Combined with CRISPR, for amplification-free nucleic acid detection. It exploits electrokinetically induced ion concentration polarization (ICP) to concentrate target nucleic acids and CRISPR reagents near the depletion zone boundary within a microfluidic channel. This localized accumulation accelerates the CRISPR-guided promiscuous cleavage of reporter molecules while enhancing their fluorescence signals simultaneously. Simultaneous accumulation of RNA and ribonucleoproteins (RNP) in confined spaces was validated experimentally and numerically, showing overlapping regions. IMACC enabled detection of miRNA-21 (22 bp) down to 10 pM within 2 min of ICP. IMACC ensured CRISPR specificity (single mismatch (N = 1) sensitivity) during ICP, as shown by off-target and mismatch sequence experiments. IMACC was applied to long RNA samples (i.e., SARS-CoV-2), but it statistically remained challenging at this point due to nonlinear intensity trends with copy numbers and large deviations. IMACC enabled rapid detection of short RNAs such as microRNAs using only basic CRISPR reagents in a single microfluidic channel, eliminating the need for extra enzymes or buffer sets, streamlining workflow and reducing turnaround time. IMACC has the potential to advance CRISPR diagnostics and holds promise for improved detection and future prescreening applications.},
}

*CRISPR-Cas Systems
Humans
*Lab-On-A-Chip Devices
*MicroRNAs/analysis/genetics
*Microfluidic Analytical Techniques
SARS-CoV-2/genetics

@article {pmid40288511,
year = {2025},
author = {Sharma, N and Whinn, KS and Ghodke, H and van Oijen, AM and Lewis, JS and Spenkelink, LM},
title = {nCas9-based method for rolling-circle DNA substrate generation.},
journal = {Analytical biochemistry},
volume = {703},
number = {},
pages = {115883},
doi = {10.1016/j.ab.2025.115883},
pmid = {40288511},
issn = {1096-0309},
mesh = {*DNA, Circular/genetics/chemistry/metabolism ; *CRISPR-Associated Protein 9/metabolism ; *DNA Replication ; RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA ; },
abstract = {Rolling-circle DNA replication is a DNA-duplication mechanism whereby circular DNA templates are continuously copied to produce long DNA products. It is widely used in molecular diagnostics, DNA sequencing, nanotechnology, and in vitro DNA replication studies. The efficiency of rolling-circle replication reaction heavily relies on the quality of the rolling-circle DNA template. Existing methods to create rolling-circle DNA substrates often rely on unique restriction sites and have limited control over replication fork topology and position. To address these limitations, we present a straightforward, customizable, and efficient strategy for producing rolling-circle DNA substrates with control over gap size and fork position. Our method relies on the use of nickase Cas9 (nCas9), which can be programmed to target specific DNA sequences using guide RNAs. In a one-pot reaction, we target nCas9 to four sites on an 18-kb plasmid to create 8-11-bp fragments. These fragments are removed and a flap oligo is ligated, to construct a fork with precisely controlled flap length and gap size. We demonstrate the application of this DNA substrate in an in vitro single-molecule rolling-circle DNA-replication assay. With our method, any plasmid DNA can be converted into a rolling-circle template, permitting generation of more physiologically-relevant DNA templates.},
}

*DNA, Circular/genetics/chemistry/metabolism
*CRISPR-Associated Protein 9/metabolism
*DNA Replication
RNA, Guide, CRISPR-Cas Systems/genetics
*DNA

@article {pmid40203601,
year = {2025},
author = {Li, X and Zhang, Y and He, M and Sun, J and Xiong, B and Wang, G},
title = {An ultrasensitive and specific fluorescence split-aptasensor for D-VP detection based on target-induced self-propelled 3D DNA walkers coupled with CRISPR-Cas12a.},
journal = {Talanta},
volume = {293},
number = {},
pages = {128102},
doi = {10.1016/j.talanta.2025.128102},
pmid = {40203601},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; *Aptamers, Nucleotide/chemistry/genetics/metabolism ; Humans ; *DNA/chemistry/genetics ; Fluorescence ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism ; DNA, Catalytic/chemistry/metabolism ; Spectrometry, Fluorescence ; },
abstract = {In this work, we present an ultrasensitive, specific, and high-signal-to-background ratio fluorescence split-aptasensor for D-vasopressin (D-VP) detection. This sensor is based on target-induced self-propelled 3D DNA walkers in conjunction with CRISPR-Cas12a technology. Two split probes (SDA 1 and SDA 2) were designed to undergo structural recombination and function as a walking chain (SDA) under the induction of D-VP. Simultaneously, an intact Mg[2+]-dependent DNAzyme domain was formed at the tail of SDA and subsequently activated. The activated Mg[2+]-dependent DNAzyme continuously propelled the 3D DNA walker, enabling the generation of signal strand DNA (activator DNA). The activator DNA can subsequently trigger the activation of the Cas12a protein, enabling it to cleave the FAM-ssDNA-BHQ1 substrate. This process leads to signal amplification and the specific detection of D-VP. Under optimal conditions, the designed split-aptasensor exhibits excellent linearity across a concentration range of 5 ng/mL to 1215 ng/mL, with a detection limit (LOD) as low as 0.22 ng/mL. This split-aptasensor were employed to identify D-VP in human serum and urine samples, yielding highly satisfactory results. This unique design acts as a proof of concept and illustrates considerable promise for the detection of a wide range of analytes.},
}

*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
*Aptamers, Nucleotide/chemistry/genetics/metabolism
Humans
*DNA/chemistry/genetics
Fluorescence
*Endodeoxyribonucleases/metabolism/genetics
*CRISPR-Associated Proteins/metabolism
*Bacterial Proteins/metabolism
DNA, Catalytic/chemistry/metabolism
Spectrometry, Fluorescence

@article {pmid40194458,
year = {2025},
author = {Liu, S and Hu, J and Zhang, R and Tian, H and Wang, F and Chou, SH and He, J and Ma, L and Yin, W},
title = {Catalytic hairpin assembly assists CRISPR/Cas12a-mediated high-sensitivity detection of aflatoxin B1.},
journal = {Talanta},
volume = {293},
number = {},
pages = {128043},
doi = {10.1016/j.talanta.2025.128043},
pmid = {40194458},
issn = {1873-3573},
mesh = {*Aflatoxin B1/analysis ; *CRISPR-Cas Systems ; *Food Contamination/analysis ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism/genetics ; Biocatalysis ; *Inverted Repeat Sequences ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Aflatoxin B1 (AFB1) is recognized the most toxic and carcinogenic mycotoxin and is widely present in cereals and various foods. Therefore, its precise detection is crucial to safeguard food quality and human health. In this study, we proposed a highly sensitive detection system for AFB1 by combining the catalytic hairpin assembly (CHA) and CRISPR/Cas12a techniques. The Aptamer of Aptamer-Initiator interacts with AFB1 to release the blocked Antisense. As a result, the Initiator of the Aptamer-Initiator becomes free and can act as a toehold to bind with H1, which can initiate the CHA to generate a large amount of double-stranded DNA, which hybridized with the Cas12a-crRNA duplex to form the Cas12a-crRNA-DNA ternary complex, wherein Cas12a subsequently cleaves the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. After optimization, we observed a linear relationship between fluorescence intensity and the AFB1 concentration in the range of 50 pM to 1 nM, with a limit of detection (LOD) of 10 pM. Also, the system was robust and could operate with excellent reliability and accuracy even in complex samples. The recovery values in food samples ranged from 92.23 % to 111.72 %, with relative standard deviation (RSD) below 5.68 %. The system exhibited remarkable advantages, including high sensitivity, strong specificity, and rapid response, thereby showed great potential in the efficient detection of AFB1 contaminants in food.},
}

*Aflatoxin B1/analysis
*CRISPR-Cas Systems
*Food Contamination/analysis
Limit of Detection
Aptamers, Nucleotide/chemistry
*Biosensing Techniques/methods
*Endodeoxyribonucleases/metabolism/genetics
Biocatalysis
*Inverted Repeat Sequences
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40187288,
year = {2025},
author = {Li, Z and Wang, J and Shen, K and Zhao, X and Lin, Z and Yi, H},
title = {Signal-on electrochemiluminescence resonance energy transfer biosensor for miRNA-543 based on CRISPR/Cas13a and magnetic separation.},
journal = {Talanta},
volume = {293},
number = {},
pages = {128085},
doi = {10.1016/j.talanta.2025.128085},
pmid = {40187288},
issn = {1873-3573},
mesh = {*MicroRNAs/analysis/genetics/blood ; *Biosensing Techniques/methods ; Humans ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Silicon Dioxide/chemistry ; *Luminescent Measurements/methods ; Gold/chemistry ; Energy Transfer ; Female ; Limit of Detection ; Metal Nanoparticles/chemistry ; DNA, Single-Stranded/chemistry ; },
abstract = {In this study, an electrochemiluminescence resonance energy transfer (ECL-RET) biosensor with high sensitivity and strong resistance to interference was constructed based on the CRISPR/Cas13a system and magnetic separation for ovarian cancer biomarker miR-543 detection. Mesoporous silica nanoparticles embedded with Ru(bpy)3[2+] (Ru@SiO2) have high electrochemiluminescence (ECL) response was chosen as energy donor. Single-stranded DNA S1 containing "rUrU" motif was immobilized on AuNRs (AuNRs-S1), which hybridized with single-stranded DNA S2 modified SAMBs (SAMBs-S2) to form AuNRs-S1/S2-SAMBs complex, this has been used as energy acceptor. In the absence of the target, Cas13a remained inactive, preventing the cleavage of S1, thereby maintaining the association of AuNRs with SAMBs. Then they were added in Ru@SiO2 solution after magnetic separation. The electrostatic adsorption between the negatively charged AuNRs and the positively charged Ru@SiO2 cause the occurrence of ECL-RET and low ECL signal had been detected. When the target was added, Cas13a was activated and resulted in the non-specifically cleaving of S1, so AuNRs detached from SAMBs. After magnetic separation, fewer AuNRs participated in ECL-RET, leading to an enhanced ECL signal detected. The change in ECL intensity (ΔECL) exhibited a linear correlation with the logarithm of miR-543 concentration within the range of 10 fM to 10 nM, with a detection limit of 6.91 fM. The biosensor had been applied to detect miR-543 in clinical samples with high accuracy.},
}

*MicroRNAs/analysis/genetics/blood
*Biosensing Techniques/methods
Humans
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
Silicon Dioxide/chemistry
*Luminescent Measurements/methods
Gold/chemistry
Energy Transfer
Female
Limit of Detection
Metal Nanoparticles/chemistry
DNA, Single-Stranded/chemistry