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Bibliography on: CRISPR-Cas

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Robert J. Robbins is a biologist, an educator, a science administrator, a publisher, an information technologist, and an IT leader and manager who specializes in advancing biomedical knowledge and supporting education through the application of information technology. More About:  RJR | OUR TEAM | OUR SERVICES | THIS WEBSITE

RJR: Recommended Bibliography 18 Oct 2025 at 01:46 Created: 

CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: ( "CRISPR.CAS" OR "crispr/cas" ) NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

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RevDate: 2025-10-17
CmpDate: 2025-10-17

Chen L, Yun M, Chen B, et al (2025)

Loss of CsCLV2 function causes dwarfism and determinates growth in cucumber.

The Plant journal : for cell and molecular biology, 124(1):e70525.

Cucumber (Cucumis sativus L.) is a globally important vegetable crop. Ideal plant architecture optimizes spatial utilization, enhances economic coefficient, and facilitates mechanized cultivation. In this study, we identified a dwarf mutant, csdw3, exhibiting reduced plant height, shortened internodes, and fewer internodes. Genetic analysis showed that this dwarf phenotype is controlled by a single recessive gene. Fine-mapping localized the causal locus to an 80 kb region on chromosome 1, where we discovered a 102 bp deletion in CsCLV2, a gene encoding a leucine-rich repeat receptor-like protein homologous to Arabidopsis CLAVATA2. CRISPR-Cas9-generated loss-of-function mutants recapitulated the dwarf phenotype, confirming the role of CsCLV2 in plant height regulation. Histological examination revealed that CsCLV2 disruption causes premature termination of shoot apical meristem (SAM) development, reducing both internode number and length. Protein interaction assays further demonstrated that CsCLV2 associates with receptor-like kinase CsCIK1 (CLAVATA3 INSENSITIVE RECEPTOR KINASES 1), indicating their cooperative function in the CLV-WUS signaling pathway to maintain meristem activity. Our findings uncover a regulator of plant height in cucumber and provide valuable genetic resources for breeding ideotypes optimized for yield and cultivation efficiency.

RevDate: 2025-10-17
CmpDate: 2025-10-17

Marchenko N, Nesbitt NM, Alexandrova E, et al (2025)

Biliverdin reductase B as a new target in breast cancer.

Breast cancer research : BCR, 27(1):179.

BACKGROUND: Enhanced metabolic and mitochondrial activity inherent in actively proliferating cancer cells is associated with intracellular redox imbalance that impacts cellular viability. To restore redox homeostasis cancer cells evolve to activate redox protective mechanisms. This differential activation of redox defense pathways compared to normal cells provides a therapeutic window for novel targeted therapies in cancer. Although heme metabolism emerges as a crucial regulator of redox homeostasis and iron metabolism in cancer cells with frequent alteration in breast cancer, it remains largely unexplored, and no targeted translational approaches have been developed. Heme-regulated redox homeostasis is coordinately maintained through biosynthetic and degradation pathways. As a byproduct of TCA cycle, cytotoxic heme is initially derivatized by heme oxygenases and progressively metabolized to the potent antioxidant bilirubin by two non-redundant biliverdin reductases, BLVRA and BLVRB. BLVRB overexpression has been observed in breast cancers, although its function in breast cancer pathogenesis remains unknown.

METHODS: CRISPR/Cas9 deletion of BLVRB in multiple breast cancer cell lines demonstrated its profound effect on intracellular redox state and cell proliferation in vitro and in xenograft models. Integrated proteomic, metabolomic, and lipidomic studies identified and validated BLVRB-mediated adaptive metabolic responses required for breast cancer cell cytoprotection.

RESULTS: We have established BLVRB as a requisite component of the pro-survival redox defense mechanism in breast cancer cells. Targeted deletion of BLVRB induces reductive stress, leading to alterations in endoplasmic reticulum proteostasis and lipid composition. These defects impact plasma membrane functionality and endosomal recycling of multiple oncogenic receptors, such as HER2 and transferrin receptors.

CONCLUSIONS: These data collectively identify BLVRB as a novel metabolic target in breast cancer, distinct from other redox-regulating pathways. This study, along with our recent progress in developing novel specific BLVRB inhibitors, offers a unique translational opportunity for targeted therapies in personalized breast cancer medicine.

RevDate: 2025-10-16
CmpDate: 2025-10-17

Savage N, Danis E, Chokshi CR, et al (2025)

CRISPR screen reveals SOX2 as a critical regulator of CD133 and cellular stress response in glioblastoma.

Scientific reports, 15(1):36228.

Glioblastoma (GBM) remains a formidable challenge in clinical settings due to limited treatments available. The surface protein CD133 marks glioblastoma stem cells (GSCs), cells capable of overcoming therapeutic pressures and correlate with more aggressiveness tumor phenotypes. In this study, we employed a CRISPR-Cas9 functional screen to deconvolute CD133 dynamics in tumors. This led us to establish that SOX2 is a key player in controlling the PROM1 gene, which in turn influences how cells react to stress factors, including those induced by chemoradiation treatment. The discoveries in this study shed light on the complex web of mechanisms that control the survival and resistance of GSCs, offering promising new avenues for targeting and potentially overcoming therapy resistance.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Lim SL, Chin CH, Chiou YJ, et al (2025)

Unveiling Unusual Ecofunctional Traits of Endozoicomonas Species Through Comprehensive Comparative Genomics.

Environmental microbiology, 27(10):e70191.

Endozoicomonas is an omnipresent marine bacterial genus, associated with various marine organisms, that contributes to host health, nutrient cycling and disease resistance. Nonetheless, its genomic features remain poorly characterised due to a paucity of high-quality genomes. In this study, we sequenced 5 novel Endozoicomonas strains and re-sequenced 1 known strain to improve genomic resolution. By integrating these 6 high-quality genomes with 31 qualified published genomes, our pan-genomic analysis revealed variation in genetic traits among clades. Notably, Endozoicomonas lacks quorum-sensing capabilities, suggesting resistance to quorum quenching mechanisms. It also lacks the capacity to synthesise and transport vitamin B12, indicating that it does not supply this nutrient to holobionts. Remarkably, Endozoicomonas genomes encode 92 identified giant proteins (15-65 kbp). These proteins cluster into three major groups associated with antimicrobial peptide synthesis, exotoxin production and cell adhesion. Additionally, we found that Endozoicomonas has acquired prophages from diverse sources via infection or other types of gene transfer. Notably, CRISPR-Cas sequences suggest evolutionary trajectories independent of both prophage acquisition and phylogenetic lineage, implying potential geographic influences or environmental pressures. This study provides new insights into the genomic diversity of Endozoicomonas and its genetic adaptations to diverse hosts.

RevDate: 2025-10-16

Kim I, JY Suh (2025)

Old and new tactics of CRISPR-centric competition between bacteria and bacteriophages.

Current opinion in structural biology, 95:103168 pii:S0959-440X(25)00186-1 [Epub ahead of print].

The CRISPR-Cas system provides adaptive immunity for prokaryotes against mobile genetic elements (MGEs) such as bacteriophages and plasmids. As a countermeasure, MGEs have evolved various anti-CRISPR (Acr) mechanisms that neutralize the CRISPR-mediated immunity. Canonical Acr proteins block target binding of Cas proteins in a stoichiometric or enzymatic manner. New findings reveal that Acr also disintegrates functional Cas complexes, induces promiscuous target binding, and mimics Cas proteins and crRNA with defective mutations. Here, we summarize a broad repertoire of structural and functional mechanisms underlying CRISPR-centric competition, highlighting recent discoveries of molecular machinery that modulates CRISPR function.

RevDate: 2025-10-16

Liao X, Li Y, Wu Y, et al (2025)

TEMC-Cas: Accurate Cas Protein Classification via Combined Contrastive Learning and Protein Language Models.

ACS synthetic biology [Epub ahead of print].

The accurate classification of Cas proteins is crucial for understanding CRISPR-Cas systems and developing genome-editing tools. Here, we present TEMC-Cas, a deep learning framework for accurate classification of Cas proteins that combines a finely tuned ESM protein language model with contrastive learning. Unlike traditional methods that rely on sequence similarity (e.g., BLAST, HMMs) or structural prediction, TEMC-Cas leverages evolutionary-scale modeling to capture distant homology while employing contrastive learning to distinguish closely related subtypes. The framework incorporates LoRA for efficient parameter adaptation and addresses class imbalance through weighted loss functions. TEMC-Cas achieves superior performance in classifying the Cas1-Cas13 families and 17 Cas12 subtypes, demonstrating particular strength in identifying remote homology. This approach provides a robust tool for the discovery of the CRISPR system and expands the toolbox for genome engineering applications. TEMC-Cas is now freely accessible at https://github.com/Xingyu-Liao/TEMC-Cas.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Shafi Z, Shahid M, Ilyas T, et al (2025)

Next-generation perspectives on microbially synthesized siderophores: molecular engineering, multi-omics insights, and applications for smart climate-resilient crops.

World journal of microbiology & biotechnology, 41(10):393.

Siderophores, low-molecular-weight iron-chelating compounds synthesized by microbes, play a crucial role in iron (Fe) acquisition under Fe-limited conditions. In recent years, their significance in sustainable agriculture has gained increasing attention due to their multifaceted roles in plant growth promotion, stress alleviation, and disease suppression. This review presents next-generation insights into the biosynthesis, regulation, and applications of microbial siderophores, with a focus on advanced molecular and omics-based approaches. Innovations in synthetic biology and CRISPR/Cas-mediated genome editing have enabled precise manipulation of siderophore biosynthetic gene clusters, enhancing their production and functionality. Multi-omics platforms-genomics, transcriptomics, proteomics, and metabolomics-have revealed complex regulatory networks, unveiling cryptic pathways and inter-microbial variability in siderophore synthesis. Furthermore, the use of siderophore-producing plant growth-promoting rhizobacteria (PGPR) has shown promise in improving nutrient uptake, inducing systemic resistance, and mitigating abiotic stresses in crops. The integration of nano-formulations and encapsulation technologies has enhanced the stability and field efficacy of siderophore-based bioinoculants. This review also explores emerging strategies for developing microbial consortia and smart delivery systems to meet the challenges of climate-resilient agriculture. By bridging molecular insights with field-level applications, this article underscores the potential of siderophores as eco-friendly tools for next-generation sustainable farming practices.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Lye SH, Polycarp N, Durojaye TJ, et al (2025)

Functional Heterogeneity and Context-Dependent Roles of LncRNAs in Breast Cancer.

Cancers, 17(19):.

As with other non-coding RNAs (ncRNAs), the aberrant expression of long non-coding RNAs (lncRNAs) can be associated with different forms of cancers, including breast cancer (BC). Various lncRNAs may either promote or suppress cell proliferation, metastasis, and other related cancer signaling pathways by interacting with other cellular machinery, thus affecting the expression of BC-related genes. However, lncRNAs are characterized by features that are unlike protein-coding genes, which pose unique challenges when it comes to their study and utility. They are highly diverse and may display contradictory functions depending on factors like the BC subtype, isoform diversity, epigenetic regulation, subcellular localization, interactions with various molecular partners, and the tumor microenvironment (TME), which contributes to the intratumoral heterogeneity and phenotypic plasticity. While lncRNAs have potential clinical utility, their functional heterogeneity coupled with a current paucity of knowledge of their functions present challenges for clinical translation. Strategies to address this heterogeneity include improving classification systems, employing CRISPR/Cas tools for functional studies, utilizing single-cell and spatial sequencing technologies, and prioritizing robust targets for therapeutic development. A comprehensive understanding of the lncRNA functional heterogeneity and context-dependent behavior is crucial for advancing BC research and precision medicine. This review discusses the sources of lncRNA heterogeneity, their implications in BC biology, and approaches to resolve knowledge gaps in order to harness lncRNAs for clinical applications.

RevDate: 2025-10-17
CmpDate: 2025-10-17

Jiang W, Georgiadis I, Fumagalli T, et al (2025)

In Vivo DNA Assembly in Yarrowia lipolytica.

ACS synthetic biology, 14(10):4116-4121.

The oleaginous yeast Yarrowia lipolytica is an important platform organism for biotechnology applications. In this study, we established an in vivo DNA assembly system leveraging CRISPR-Cas9 for efficient genomic integration of multiple DNA fragments into the genome of Y. lipolytica. Using the green fluorescent protein mNeonGreen as a model, we demonstrated 53% correct assembly of three DNA fragments with homology arms as short as 50 bp. The system was further validated by constructing 2-3 step biosynthetic pathways for pigments betaxanthin and betanin. To improve the homologous recombination efficiency of Y. lipolytica, we expressed S. cerevisiae RAD52 (ScRAD52) or a Cas9-hBrex27 fusion protein. While ScRAD52 expression impaired growth, the cas9-hBrex27 fusion enhanced integration efficiency, particularly for multifragment pathway assemblies. The in vivo assembly method simplifies pathway construction and gene overexpression in Y. lipolytica.

RevDate: 2025-10-17
CmpDate: 2025-10-17

Golla SA, Abo-Hashesh M, Gupta D, et al (2025)

Model-Based Optimization of a qCRISPRi Circuit for Dynamic Control of Metabolic Pathways.

ACS synthetic biology, 14(10):3890-3898.

Metabolic engineering enables sustainable chemical production but often imposes metabolic burdens that reduce cellular viability and productivity. Dynamic control strategies, such as quorum sensing (QS)-based circuits, can mitigate these effects by autonomously regulating gene expression in response to cell density. In this study, we investigated a QS-regulated CRISPR interference (qCRISPRi) circuit for the dynamic control of metabolic pathways, focusing on the role of leaky expression and regulator stringency. Using a combination of mathematical modeling and experiments, we evaluated how promoter leakiness and LuxR stringency influence key switching characteristics including maximum gene expression, switching density, fold repression, and transition time. Our results show that high leaky expression of dCas9 reduces switching density and represses GFP prematurely, whereas a high-stringency LuxR variant enhances switching precision by reducing leakiness and enabling sharper transitions. These model predictions were validated experimentally in E. coli, confirming that LuxR stringency improves dynamic circuit performance. Together, this work provides a quantitative framework for optimizing QS-based regulatory systems and offers generalizable design insights for implementing dynamic control in metabolic engineering.

RevDate: 2025-10-17
CmpDate: 2025-10-17

Xiao G, Shi H, Lin Q, et al (2026)

A rapid CRISPR-Cas12a/T7EI integrated workflow for high-throughput screening of homozygous mutant cell lines.

Journal of pharmaceutical and biomedical analysis, 267:117152.

Efficient screening for homozygous mutant cell lines, particularly those resulting from low-efficiency CRISPR-Cas9 editing, remains challenging. Here, we developed HomoSelect-CT, an integrated workflow combining CRISPR-Cas12a nucleic acid detection with T7 Endonuclease I (T7EI) genotyping, designed to streamline the screening process for homozygous mutant cell lines. This method requires no specialized instrumentation, enhancing accessibility and efficiency. We validated HomoSelect-CT by successfully identifying homozygous mutants in CRISPR-Cas9-edited THP-1 cells, which was confirmed by Sanger sequencing and Western blot (WB). These findings demonstrate that HomoSelect-CT is a robust and efficient alternative for the rapid isolation of genome-edited cell lines. The entire screening workflow, from monoclonal cultures to confirmed homozygous mutants, is completed in under 4 h, requiring only standard PCR equipment and routine reagents. Thus, HomoSelect-CT represents a significant advancement in CRISPR screening methodology, offering remarkable simplicity and enabling high-throughput screening that is particularly suitable for mutants arising from low-efficiency editing events.

RevDate: 2025-10-17
CmpDate: 2025-10-17

Xu C, Niu X, Sun H, et al (2025)

Conversion of IscB and Cas9 into RNA-guided RNA editors.

Cell, 188(21):5847-5861.e11.

RNA-guided RNA editing represents an attractive alternative to DNA editing. However, the prevailing tool, CRISPR-Cas13, has collateral RNA cleavage activity that causes undesirable cytotoxicity in human cells. Here, we report an ultracompact RNA-editing platform engineered from IscB, which has comparable or higher activity than Cas13 but without cytotoxicity concerns. We show that IscB, the evolutionary ancestor of Cas9, has an intrinsic affinity for complementary single-stranded (ss)DNA and RNA. This activity becomes dominant when its double-stranded DNA binding activity is switched off through the deletion of its target-adjacent motif domain. The resulting R-IscB is comparable to or better than Cas13, can efficiently alter splicing outcomes in human cells, and can further mediate trans-splicing to correct any mutation at the mRNA level. R-IscB also drives efficient A-to-I editing on mRNA when fused to adenosine deaminase acting on RNA 2 (ADAR2) and mediates cleavage-based mRNA knockdown upon HNH engineering. Finally, we show that the same approach converts some Cas9s to RNA-targeting tools.

RevDate: 2025-10-17
CmpDate: 2025-10-17

Zhou Z, Zhu S, Hong Y, et al (2025)

Composite transposons with bivalent histone marks function as RNA-dependent enhancers in cell fate regulation.

Cell, 188(21):5878-5894.e18.

Discrete genomic units can recombine into composite transposons that transcribe and transpose as single units, but their regulation and function are not fully understood. We report that composite transposons harbor bivalent histone marks, with activating and repressive marks in distinct regions. Genome-wide CRISPR-Cas9 screening, using a reporter driven by the hominid-specific composite transposon SVA (SINE [short interspersed nuclear element]-VNTR [variable number of tandem repeats]-Alu) in human cells, identified diverse genes that modify bivalent histone marks to regulate SVA transcription. SVA transcripts are critical for SVA's cis-regulatory function in selectively contacting and activating long-range gene expression. Remarkably, a subset of bivalent SVAs is activated during erythropoiesis to boost multiple erythroid gene expression, and knocking down these SVAs leads to deficient erythropoiesis. The RNA-dependent cis-regulatory function of SVA activates genes for myelopoiesis and can contribute to aging-associated myeloid-biased hematopoiesis. These results reveal that the cis-regulatory functions of composite transposons are bivalently regulated to control cell fate transitions in development and aging.

RevDate: 2025-10-17
CmpDate: 2025-10-17

Park JC, Uhm H, Kim YW, et al (2025)

AI-generated MLH1 small binder improves prime editing efficiency.

Cell, 188(21):5831-5846.e21.

The prime editing (PE) system consists of a Cas9 nickase fused to a reverse transcriptase, which introduces precise edits into the target genomic region guided by a PE guide RNA. However, PE efficiency is limited by mismatch repair. To overcome this limitation, transient expression of a dominant-negative MLH1 (MLH1dn) has been used to inhibit key components of mismatch repair. Here, we designed a de novo MLH1 small binder (MLH1-SB) that binds to the dimeric interface of MLH1 and PMS2 using RFdiffusion and AlphaFold 3. The compact size of MLH1-SB enabled its integration into existing PE architectures via 2A systems, creating a PE-SB platform. The PE7-SB2 system significantly improved PE efficiency, achieving an 18.8-fold increase over PEmax and a 2.5-fold increase over PE7 in HeLa cells, as well as a 3.4-fold increase over PE7 in mice. This study highlights the potential of generative AI in advancing genome editing technology.

RevDate: 2025-10-17
CmpDate: 2025-10-17

Marzook NB, Song OR, Baumgärtel L, et al (2025)

The essential host genome for Cryptosporidium survival exposes metabolic dependencies that can be leveraged for treatment.

Cell, 188(21):5947-5961.e15.

Cryptosporidium is a leading cause of diarrheal disease, yet little is known regarding the infection cell biology of this intracellular intestinal parasite. To this end, we implemented an arrayed genome-wide CRISPR-Cas9 knockout screen to microscopically analyze multiple phenotypic features of a Cryptosporidium infection following individual host gene ablation. We discovered parasite survival within the host epithelial cell hinges on squalene, an intermediate metabolite in the host cholesterol biosynthesis pathway. A buildup of squalene within intestinal epithelial cells creates a reducing environment, making more reduced glutathione available for parasite uptake. Remarkably, the Cryptosporidium parasite has lost the ability to synthesize glutathione and has become dependent on this host import. This dependency can be leveraged for treatment with the abandoned drug lapaquistat, an inhibitor of host squalene synthase that shifts the redox environment, blocking Cryptosporidium growth in vitro and in vivo.

RevDate: 2025-10-17
CmpDate: 2025-10-17

Qin W, Lin SJ, Zhang Y, et al (2025)

Rationally Designed TadA-Derived Cytosine Editors Enable Context-Independent Zebrafish Genome Editing.

Advanced science (Weinheim, Baden-Wurttemberg, Germany), 12(39):e09800.

CRISPR base editors are crucial for precise genome manipulation. Existing APOBEC-based cytosine base editors (CBEs), while powerful, exhibit indels and sequence context limitations, and editing CC and GC motifs is challenging and inefficient. To address these challenges, existing tRNA adenine deaminase (TadA)-derived CBEs are evaluated in zebrafish, and a series of zTadCBE variants is developed that demonstrate high editing efficiency, minimized off-target effects, and an expanded targeting range compared to existing tools. The approach integrates beneficial mutations from TadA-based adenine base editors (ABEs) with SpRYCas9n-enhanced protospacer-adjacent motif (PAM) compatibility. The expanded window zTadCBE variants enable the targeting of cytosines at a broader range of nucleotide positions relative to the PAM sequence, further enhancing the versatility of this tool. Using zTadCBEs, four zebrafish disease models affecting the auditory, nervous, metabolic, and muscular systems are generated directly in the F0 generation-models that cannot be efficiently produced using earlier CBE tools. Together, zTadCBE variants provide a robust and flexible toolkit for efficient and precise C-to-T base editing in zebrafish, facilitating rapid in vivo functional assessment of genetic variants.

RevDate: 2025-10-17
CmpDate: 2025-10-17

Zhou J, Zhou C, Jiang G, et al (2025)

Engineering an Escherichia coli with performance-enhanced switch utilizing CRISPR-Cas9 system as living quorum quencher for biofilm formation inhibition.

Environmental research, 285(Pt 2):122383.

Quorum quenching (QQ) of signal molecules plays a critical role in disrupting bacterial communication, thereby suppressing biofilm formation. However, the wild-type QQ bacteria lacks the regulatory capacity to modulate gene expression levels. In this study, the QQ gene aiiO and reporter gene GFP were chromosomally integrated into Escherichia coli BW25113 using the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. The performance-enhanced switch in the engineering bacteria (EB) allowed it to express aiiO weakly without the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) and express aiiO strongly with IPTG, and 1.00 mM IPTG induction enhanced EB's QQ activity by 2.34-fold. In activated sludge systems, the inoculation of EB reduced biofilm formation by 18.56 % versus controls after 168 h, with the performance-enhanced switch enhancing inhibition to 24.72 %. EB reduced biofilm thickness by 22.96 %, total microbial biomass by 57.68 %, and significantly decreased extracellular polymeric substances secretion and adhesion strength of the biofilm (maximum reductions: 29.88 % and 34.31 %, respectively) across all sampling points versus controls. 1.00 mM IPTG addition sustainedly intensified these biofilm-inhibitory effects by EB, demonstrating the genetic switch's persistent functionality under environmentally relevant conditions. Furthermore, the genetically modified strain exhibited minimal environmental impact according to standardized assessments. Therefore, this study successfully constructed an implementable strategy for engineering bacteria-mediated biofilm control, with demonstrated applicability in complex environmental systems.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Chen C, Wu T, Liu J, et al (2025)

Threat and Control of tet(X)-Mediated Tigecycline-Resistant Acinetobacter sp. Bacteria.

Foods (Basel, Switzerland), 14(19): pii:foods14193374.

Tigecycline is regarded as one of the last-resort antibiotics against multidrug-resistant (MDR) Acinetobacter sp. bacteria. Recently, the tigecycline-resistant Acinetobacter sp. isolates mediated by tet(X) genes have emerged as a class of global pathogens for humans and food-producing animals. However, the genetic diversities and treatment options were not systematically discussed in the era of One Health. In this review, we provide a detailed illustration of the evolution route, distribution characteristics, horizontal transmission, and rapid detection of tet(X) genes in diverse Acinetobacter species. We also detail the application of chemical drugs, plant extracts, phages, antimicrobial peptides (AMPs), and CRISPR-Cas technologies for controlling tet(X)-positive Acinetobacter sp. pathogens. Despite excellent activities, the antibacterial spectrum and application safety need further evaluation and resolution. It is noted that deep learning is a promising approach to identify more potent antimicrobial compounds.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Wu KC, Chang YH, Chiang RY, et al (2025)

CAP-LAMP2b-Modified Stem Cells' Extracellular Vesicles Hybrid with CRISPR-Cas9 Targeting ADAMTS4 to Reverse IL-1β-Induced Aggrecan Loss in Chondrocytes.

International journal of molecular sciences, 26(19): pii:ijms26199812.

Extracellular vesicles (EVs) from mesenchymal stem cells hold therapeutic promise for inflammatory and degenerative diseases; however, limited delivery and targeting capabilities hinder their clinical use. In this study, we sought to enhance the anti-inflammatory and chondroprotective effects of EVs through CAP-LAMP2b (chondrocyte affinity peptide fused to an EV membrane protein) engineering and ADAMTS4 gene editing hybrid vesicle formation. Human umbilical cord MSCs (hUCMSCs) were characterized via morphology, immunophenotyping, and trilineage differentiation. EVs from control and CAP-LAMP2b-transfected hUCMSCs were fused with liposomes carrying CRISPR-Cas9 ADAMTS4 gRNA. DiI-labeled EV uptake was assessed via fluorescence imaging. CAP-LAMP2b was expressed in hUCMSCs and their EVs. EVs exhibited the expected size (~120 nm), morphology, and exosomal markers (CD9, CD63, CD81, HSP70). CAP-modified hybrid EVs significantly enhanced chondrocyte uptake compared to control EVs and liposomes. IL-1β increased ADAMTS4 expression, whereas CAP-LAMP2b-ADAMTS4 EVs, particularly clone SG3, reversed these effects by reducing ADAMTS4 and restoring aggrecan. Western blotting confirmed suppressed ADAMTS4 and elevated aggrecan protein. CAP-LAMP2b-ADAMTS4 EVs, therefore, showed superior uptake and therapeutic efficacy in inflamed chondrocytes, attenuating inflammatory gene expression and preserving matrix integrity. These results support engineered EVs as a promising cell-free approach for cartilage repair and osteoarthritis treatment.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Jeong SK, Park JR, Kim EG, et al (2025)

Development of Resistance to Damping-Off in Rice, Oryza sativa L., Using CRISPR/Cas9.

International journal of molecular sciences, 26(19): pii:ijms26199761.

Damping-off disease hinders rice seedling growth and reduces yield. Current control methods, such as seed or soil sterilization, rely on chemicals that cause environmental pollution and promote pathogen resistance. As a sustainable alternative, we targeted the damping-off resistance-related gene OsDGTq1 using CRISPR/Cas9. Field experiments first verified OsDGTq1's significance in resistance. The CRISPR/Cas9 system, delivered via Agrobacterium-mediated transformation, was used to edit OsDGTq1 in rice cultivar Ilmi. Lesions from major damping-off pathogens, Rhizoctonia solani and Pythium graminicola, were observed on G0 plants. All 37 regenerated plants contained T-DNA insertions. Among them, edits generated by sgRNA1-1, sgRNA1-2, and sgRNA1-3 resulted in the insertion of two thymine bases as target mutations. Edited lines were assigned names and evaluated for agronomic traits, seed-setting rates, and pathogen responses. Several lines with edited target genes showed distinct disease responses and altered gene expression compared to Ilmi, likely due to CRISPR/Cas9-induced sequence changes. Further studies in subsequent generations are needed to confirm the stability of these edits and their association with resistance. These results confirm that genome editing of OsDGTq1 alters resistance to damping-off. The approach demonstrates that gene-editing technology can accelerate rice breeding, offering an environmentally friendly strategy to develop resistant varieties. Such varieties can reduce chemical inputs, prevent pollution, and minimize seedling loss, ultimately enhancing food self-sufficiency and stabilizing rice supply.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Kapitonova MA, Shabalina AV, Dedkov VG, et al (2025)

CRISPR-Cas12a-Based Isothermal Detection of Mammarenavirus machupoense Virus: Optimization and Evaluation of Multiplex Capability.

International journal of molecular sciences, 26(19): pii:ijms26199754.

Bolivian hemorrhagic fever (BHF) is a zoonotic disease caused by Mammarenavirus machupoense (MACV) featuring severe neurological and hemorrhagic symptoms and a high mortality rate. BHF is usually diagnosed by serological tests or real-time polymerase chain reaction (RT-PCR); these methods are often inaccessible in endemic regions due to a lack of laboratory infrastructure, creating a demand for sensitive and rapid equipment-free alternatives. Here, we present an isothermal method for MACV nucleic acid detection based on the Cas12a-based DETECTR system combined with recombinase polymerase amplification (RPA) in a single tube: the RT-RPA/DETECTR assay. We demonstrate the possibility of using more than one primer set for the simultaneous detection of MACV genetic variants containing multiple point mutations. The method was optimized and tested using specially developed virus-like armored particles containing the target sequence. The multiplex RT-RPA/DETECTR method achieved a limit of detection of approximately 5 × 10[4] copies/ mL (80 aM) of armored particles. The method was validated using clinical samples spiked with virus-like particles. The assay proved to be selective and reliable in detecting certain nucleotide substitutions simultaneously.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Xu J, Pan M, Zhu Y, et al (2025)

CRISPR/Cas9-Mediated Targeted Mutagenesis of GmAS1/2 Genes Alters Leaf Shape in Soybean.

International journal of molecular sciences, 26(19): pii:ijms26199657.

ASYMMETRIC LEAVES1 (AS1) and AS2 play essential roles in regulating leaf development in plants. However, their functional roles in soybean remain poorly understood. Here, we identified two members of the soybean AS1 gene family, GmAS1a and GmAS1c, which exhibit high expression levels in stem and leaf tissues. Using the CRISPR/Cas9 system, we targeted four GmAS1 and three GmAS2 genes, generating mutant lines with distinct leaf development phenotypes, including wrinkling (refers to fine lines and creases on the leaf surface, like aged skin texture), curling (describes the inward or outward rolling of leaf edges, deviating from the typical flat shape), and narrow. We found that functional redundancy exists among the four GmAS1 genes in soybean. GmAS1 and GmAS2 cooperatively regulate leaf curling, leaf crinkling phenotypes, and leaf width in soybean, with functional redundancy also observed between these two genes. Transcriptome sequencing analysis of w3 mutant (as1b as1c as1d as2a as2b as2c) identified 1801 differentially expressed genes (DEGs), including 192 transcription factors (TFs). Gene ontology enrichment analysis revealed significant enrichment of DEGs in pathways associated with plant hormone biosynthesis and signal transduction. A detailed examination of the DEGs showed several genes involved in the development of leaf lateral organs, such as KNOX (SHOOT MERISTEMLESS (STM), KNAT1, KNAT2, and KNAT6), LOB (LBD25, LBD30), and ARP5, were down-regulated in w3/WT (wild-type) comparison. CRISPR/Cas9-mediated targeted mutagenesis of the GmAS1/2 genes significantly impairs leaf development and polarity establishment in soybean, providing valuable germplasm resources and a theoretical framework for future studies on leaf morphogenesis.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Huang C, Liu M, J Kok (2025)

Chromosomal and Plasmid-Based CRISPRi Platforms for Conditional Gene Silencing in Lactococcus lactis.

International journal of molecular sciences, 26(19): pii:ijms26199516.

Inducible CRISPR interference (CRISPRi) systems were established in Lactococcus lactis using both plasmid and chromosomal approaches. Expression of nuclease-deficient Cas9 (dCas9) from Streptococcus pyogenes was placed under the control of the nisin-inducible promoter PnisA, while sgRNAs were transcribed from the constitutive Pusp45 promoter. To monitor expression, dCas9 was fused with superfolder GFP. Plasmid-based constructs successfully repressed a luciferase reporter gene and silenced the gene of the major autolysin, AcmA, leading to the expected morphological phenotype. However, plasmid systems showed leaky expression, producing mutant phenotypes even without induction. Chromosomal integration of dCas9 reduced its expression level by approximately 20-fold compared with plasmid-based expression, thereby preventing leaky activity and ensuring tight regulation. This chromosome-based (cbCRISPRi) platform enabled controlled repression of the essential gene ybeY, which resulted in severe growth defects. Restoration of wild-type phenotypes was achieved by introducing a synonymous codon substitution in the sgRNA target region. Transcriptome analysis of ybeY-silenced cells revealed downregulation of ribosomal protein genes and widespread effects on membrane-associated proteins, ATP synthase subunits, and various transporters. These inducible CRISPRi platforms provide robust and tunable tools for functional genomics in L. lactis, particularly for studying essential genes that cannot be deleted.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Peng H, Li J, Sun K, et al (2025)

Advances and Applications of Plant Base Editing Technologies.

International journal of molecular sciences, 26(19): pii:ijms26199452.

Base editing represents a major breakthrough in the field of genome editing in recent years. By fusing deaminases with the CRISPR/Cas system, it enables precise single-base modifications of DNA. This review systematically summarizes the development of base editing technologies, including cytosine base editors (CBEs), adenine base editors (ABEs), and glycosylase base editors (GBEs), with a particular focus on their applications in crop improvement as well as future trends and prospects. We highlight advances in the creation of novel germplasm with enhanced stress resistance and desirable agronomic traits through base editing in rice, wheat, maize, potato, and other crops, particularly for improving herbicide resistance, disease resistance, and grain quality. Furthermore, we analyze factors that influence base editing efficiency, noting that challenges remain, such as PAM sequence constraints, limited base conversion types, off-target effects, narrow editing windows, and efficiency variation. Future efforts should aim to optimize deaminase activity, expand PAM compatibility, and develop versatile tools to facilitate the broad application of base editing in molecular breeding. This review provides a timely reference for researchers and breeders, offering theoretical guidance and practical insights into harnessing base editing for crop genetic improvement.

RevDate: 2025-10-16
CmpDate: 2025-10-16

He J, Shi N, Yao H, et al (2025)

Genome Editing in the Chicken: From PGC-Mediated Germline Transmission to Advanced Applications.

International journal of molecular sciences, 26(19): pii:ijms26199426.

Avian genome editing has historically lagged behind mammalian research. This disparity is primarily due to a unique reproductive biology that precludes standard techniques like pronuclear injection. A pivotal breakthrough, however, came from the development of efficient in vitro culture systems for primordial germ cells (PGCs). This has established the chicken as a tractable and powerful model for genetic engineering. Our review chronicles the technological evolution this has enabled, from early untargeted methods to the precision of modern CRISPR-based systems. We then analyze the broad applications of these tools, which are now used to engineer disease resistance, enhance agricultural traits, and develop novel platforms such as surrogate hosts and oviduct bioreactors. Collectively, these advances have established PGC-based genome editing as a robust and versatile platform. Looking forward, emerging precision editors and the expansion of these techniques to other avian species are poised to drive the next wave of innovation in poultry science and biotechnology.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Haldrup SB, McClements ME, Cehajic-Kapetanovic J, et al (2025)

Gene Therapy Strategies for the Treatment of Bestrophinopathies.

International journal of molecular sciences, 26(19): pii:ijms26199421.

The BEST1 gene encodes a transmembrane protein in the retinal pigment epithelium (RPE) in the eye, that functions as a calcium-dependent chloride channel (CaCC). Pathogenic variants in BEST1 are the underlying cause for bestrophinopathies, a group of inherited retinal disorders that vary in their pattern of inheritance, clinical appearance, and underlying molecular disease mechanisms. Currently, there are no treatments available for any of the bestrophinopathies, and gene therapy represents an attractive strategy due to the accessibility of the eye and slow disease progression. While gene augmentation may be effective for a subset of bestrophinopathies, others require allele-specific silencing or correction of the disease-causing variant to reconstitute expression of the BEST1 protein. This review aims to give an overview of the clinical diversity of bestrophinopathies and proposes the molecular disease mechanism of the pathogenic BEST1 variant as an important parameter for the choice of treatment strategy. Furthermore, we discuss the potential of different mutation-specific and mutation-independent CRISPR/Cas9-based gene editing strategies as a future treatment approach for bestrophinopathies.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Șerban M, Toader C, RA Covache-Busuioc (2025)

CRISPR and Artificial Intelligence in Neuroregeneration: Closed-Loop Strategies for Precision Medicine, Spinal Cord Repair, and Adaptive Neuro-Oncology.

International journal of molecular sciences, 26(19): pii:ijms26199409.

Repairing the central nervous system (CNS) remains one of the most difficult obstacles to overcome in translational neurosciences. This is due to intrinsic growth inhibitors, extracellular matrix issues, the glial scar-form barrier, chronic neuroinflammation, and epigenetic silencing. The purpose of this review is to bring together findings from recent developments in genome editing and computational approaches, which center around the possible convergence of clustered regularly interspaced short palindromic repeats (CRISPR) platforms and artificial intelligence (AI), towards precision neuroregeneration. We wished to outline possible ways in which CRISPR-based systems, including but not limited to Cas9 and Cas12 nucleases, RNA-targeting Cas13, base and prime editors, and transcriptional regulators such as CRISPRa/i, can be applied to potentially reactivate axon-growth programs, alter inhibitory extracellular signaling, reprogram or lineage transform glia to functional neurons, and block oncogenic pathways in glioblastoma. In addition, we wanted to highlight how AI approaches, such as single-cell multi-omics, radiogenomic prediction, development of digital twins, and design of adaptive clinical trials, will increasingly be positioned to act as system-level architects that allow translation of complex datasets into predictive and actionable therapeutic approaches. We examine convergence consumers in spinal cord injury and adaptive neuro-oncology and discuss expanse consumers in ischemic stroke, Alzheimer's disease, Parkinson's disease, and rare neurogenetic syndromes. Finally, we discuss the ethical and regulatory landscape around beyond off-target editing and genomic stability of CRISPR, algorithmic bias, explainability, and equitable access to advanced neurotherapies. Our intent was not to provide a comprehensive inventory of possibilities but rather to provide a conceptual tool where CRISPR acts as a molecular manipulator and AI as a computational integrator, converging to create pathways towards precision neuroregeneration, personalized medicine, and adaptive neurotherapeutics that are ethically sound.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Simoni S, Fambrini M, Pugliesi C, et al (2025)

Genome Editing by Grafting.

International journal of molecular sciences, 26(19): pii:ijms26199294.

Grafting is the process of joining parts of two plants, allowing the exchange of molecules such as small RNAs (including microRNAs and small interfering RNAs), messenger RNAs, and proteins between the rootstock and the scion. Genome editing by grafting exploits RNAs, such as tRNA-like sequences (TLS motifs), to deliver the components (RNA) of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system from transgenic rootstock to wild-type scion. The complex Cas9 protein and sgRNA-TLS produced in the scion perform the desired modification without the integration of foreign DNA in the plant genome, resulting in heritable transgene-free genome editing. In this review, we examine the current state of the art of this innovation and how it helps address regulatory problems, improves crop recovery and selection, exceeds the usage of viral vectors, and may reduce potential off-target effects. We also discuss the promise of genome editing by grafting for plants recalcitrant to in vitro culture and for agamic-propagated species that must maintain heterozygosity for plant productivity, fruit quality, and adaptation. Furthermore, we explore the limitations of this technique, including variable efficiency, graft incompatibility among genotypes, and challenges in large-scale application, while highlighting its considerable potential for further improvement and future broader applications for crop breeding.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Moon J, Zhang J, Guan X, et al (2025)

CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a.

Nature communications, 16(1):9142.

The CRISPR/Cas13a enzyme serves as a powerful tool for RNA detection due to its RNA-targeting capabilities. However, simple and highly sensitive detection using Cas13a faces challenges, such as the need for pre-amplification and elevated reaction temperatures. In this study, we investigate the allosteric regulation mechanism of Cas13a activation by target RNAs with various structures containing the CRISPR anti-tag sequence. We discover that the target RNA secondary structure and anti-tag sequences inhibit the trans-cleavage reaction of Cas13a. By designing and introducing a specific CRISPR anti-tag hairpin, we develop CRISPR Anti-tag Mediated Room-temperature RNA Detection (CARRD) using a single CRISPR/Cas13a enzyme. This method enables one-step cascade signal amplification for RNA detection without the need for pre-amplification. We apply the CARRD method to detect human immunodeficiency virus (HIV) and hepatitis C virus (HCV), achieving a detection sensitivity of 10 aM. Furthermore, we validate its clinical feasibility by detecting HIV clinical plasma samples, demonstrating a simple, affordable, and efficient approach for viral RNA detection. Due to its simplicity, sensitivity, and flexible reaction temperature, the CARRD method is expected to have broad applicability, paving the way for the development of field-deployable diagnostic tools.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Zhou SK, Luo JT, Chen YF, et al (2025)

Muscle-specific gene editing therapy via mammalian fusogen-directed virus-like particles.

Nature communications, 16(1):9145.

Muscle genetic defects can lead to impaired movement, respiratory failure, and other severe symptoms. The development of curative therapies is challenging due to the need for the delivery of gene-editing tools into skeletal muscle cells throughout the body. Here, we use muscular fusogens (Myomaker and Myomerger) to engineer muscle-specific virus-like particles (MuVLPs) for the systemic delivery of gene-editing tools. We demonstrate that MuVLPs can be loaded with diverse payloads, including EGFP, Cre and Cas9/sgRNA ribonucleoproteins (Cas9 RNPs), and can be delivered into skeletal muscle cells via targeted membrane fusion. Systemic administration of MuVLPs carrying Cas9 RNPs enables skeletal muscle-specific gene editing, which excised the exon containing a premature terminator codon mutation in a mouse model for Duchenne muscular dystrophy (DMD). This treatment restores dystrophin expression in various skeletal muscle tissues, including the diaphragm, quadriceps, tibialis anterior, gastrocnemius, and triceps. As a result, the treated mice exhibit a significantly increased capacity for exercise and endurance. This study established a platform for precise gene editing in skeletal muscle tissues.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Shang M, Li Y, Cao Q, et al (2025)

A motif preferred adenine base editor with minimal bystander and off-targets editing.

Nature communications, 16(1):9153.

47% of hereditable diseases are caused by single C•G-to-T•A base conversions, which means efficient A-to-G base editing tools (ABEs) have great potential for the treatment of these diseases. However, the existing efficient ABEs, while catalyzing targeted A-to-G conversion, cause high A or C bystander editing and off-target events, which poses safety concerns for their clinical applications. To overcome this shortcoming, we have developed ABE8e-YA (ABE8e with TadA-8e A48E) for efficient and accurate editing of As in YA motifs with YAY > YAR (Y = T or C, R = A or G) hierarchy through structure-oriented rational design. Compared with ABE3.1, which is currently the only ABE version with a YAC motif preference, ABE8e-YA exhibits an average A-to-G editing efficiency improvement of an up to 3.1-fold increase in the indicated YA motif while maintaining reduced bystander C editing and minimized DNA or RNA off-targets. Additionally, we demonstrate that ABE8e-YA efficiently and precisely corrects pathogenic mutations in human cells, suggesting its high suitability for addressing 9.3% of pathogenic point mutations, higher than that of ABE8e and ABE9. Moreover, by using ABE8e-YA, we efficiently and precisely generate hypocholesterolemia and tail-loss mouse models mimicking human-associated disease, as well as performed in vivo mouse proprotein convertase subtilisin/kexin type 9 (Pcsk9) base editing for hypercholesterolemia gene therapy. Together these data indicate its great potential in broad applications for disease modeling and gene therapy.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Shibue K, Kahraman S, Castillo-Quan JI, et al (2025)

Genome-wide CRISPR Screen Identifies Sec31A as a Key Regulator of Alpha Cell Survival.

Nature communications, 16(1):9159.

Glucagon, secreted by pancreatic alpha cells, is essential for maintaining normal blood glucose levels. In type 1 and advanced type 2 diabetes, alpha cells often fail to respond to low glucose, yet the mechanisms underlying their stress resistance remain unclear. To investigate this, we performed a genome-wide CRISPR screen and identify Sec31A, a gene involved in transporting proteins from the endoplasmic reticulum (ER), as a key regulator of alpha cell survival under stress. We show that loss of Sec31A enhances survival in stressed mouse alpha cells and in C. elegans. In human islets, SEC31A expression increases in alpha cells under inflammatory stress, and this upregulation is reversed by reducing ER stress. Functional studies in lab-grown human islet clusters reveal distinct responses in alpha versus beta cells following Sec31A suppression. We also find that Sec31A interacts with the insulin receptor, suggesting a link between stress adaptation and insulin signaling in alpha cells.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Wei Z, Luo H, Huang D, et al (2025)

Structure-specific electrochemiluminescent biosensor for FEN1 detection via dumbbell probe-mediated transcription and CRISPR/Cas13a-induced G-quadruplexes cleavage.

Analytica chimica acta, 1377:344662.

Flap endonuclease 1 (FEN1) is crucial for DNA replication, repair, and telomere maintenance. Its dysregulation is linked to various cancers and diseases. Accurate detection of FEN1 is essential for early diagnosis and therapeutic monitoring. Thus, a novel electrochemiluminescent (ECL) biosensor has been developed for the structure-specific and detection of FEN1. The strategy integrates dumbbell DNA probe-mediated transcription and CRISPR/Cas13a-induced trans-cleavage of RNA G-quadruplexes. In the presence of FEN1, the 5'-flap structure of the probe was selectively cleaved and subsequently ligated by T4 DNA ligase to form a closed circular template. This enabled T7 RNA polymerase to transcribe crRNA-encoded RNA strands, which activated Cas13a to cleave surface-tethered G-quadruplexes/hemin complexes on a Ru(II)/Ti3C2/AuNPs-modified electrode, thereby restoring the quenched ECL signal. The biosensor exhibited an ultralow detection limit of 4.82 × 10[-9] U μL[-1] and a wide dynamic range (1 × 10[-8] to 1 × 10[-5] U μL[-1]), along with excellent specificity and stability. Successful application in human serum validated its reliability for complex biological samples. This work presents a powerful platform for sensitive FEN1 monitoring, holding potential for clinical diagnostics and enzymatic analysis.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Wei Z, Huang D, Luo H, et al (2025)

A multi-level signal conversion architecture for enzyme sensing: Integrating MXene nanoplatforms with CRISPR-driven electrochemiluminescence.

Analytica chimica acta, 1377:344636.

Precise and ultrasensitive detection of flap endonuclease 1 (FEN1), a key DNA repair enzyme implicated in cancer diagnostics, remains challenging due to its subtle structural cleavage activity. Herein, we present a cascade-amplified electrochemiluminescence (ECL) biosensor based on a Ti3C2 MXene-supported Ru (bpy)3[2+]/Au nanocomposite integrated with a CRISPR-Cas13a system and DNA walker circuitry. Upon specific recognition and cleavage of a 5'-flap substrate by FEN1, a nicked DNA product is circularized and transcribed via T7 RNA polymerase, yielding RNA activators that trigger Cas13a-mediated collateral cleavage. This event releases a blocked DNA walker, which reorganizes Fc-labeled DNA on the electrode surface and restores the ECL signal suppressed by resonance energy transfer. The system achieves a detection limit as low as 1.48 fU/mL and exhibits a dynamic range spanning five orders of magnitude. Compared to fluorescence-based CRISPR detection systems, the ECL-based platform offers low background, high signal-to-noise ratios, and operational simplicity using standard electrochemical instrumentation, supporting practical deployment in clinical diagnostics. Furthermore, the platform demonstrates high selectivity against other nucleases and proteins, along with excellent performance in spiked human serum samples. This work presents a robust and modular strategy for accurate enzyme activity profiling with promising applications in early-stage disease diagnostics.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Yin K, CJ Tsai (2025)

Turbo-charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond.

The Plant journal : for cell and molecular biology, 124(1):e70527.

Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene-free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High-throughput sequencing technologies, including long-read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements-often missed by standard genotyping-when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user-friendly, synthetic biology-compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue-specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next-generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Yew WN, Dean CJ, DKH Chan (2025)

STAG2 mutations in the normal colon induce upregulation of oncogenic pathways in neighbouring wildtype cells.

PloS one, 20(10):pone.0332499.exml.

While driver mutations in the normal colon have been described, characterizing the role and function of these driver mutations in relation to colorectal oncogenesis remains incomplete. Here, we investigated the role of STAG2 mutants in the normal colon using patient-derived wildtype organoids. Using CRISPR-Cas9 gene editing, we generated STAG2 mutants, and co-cultured these mutants with wildtype organoids, mimicking the presence of such STAG2 mutants in the normal colon. We sought to determine the transcriptional impact of co-culture using scRNAseq. Surprisingly, we uncovered a possible cell-cell interaction between STAG2 mutants and wildtype organoids, in which wildtype organoids in co-culture with STAG2 mutants upregulated known oncogenic pathways. This included the upregulation of TNFα-signaling, as well as KRAS-signaling in wildtype organoids. These results suggested that STAG2 mutant cells exert a pro-oncogenic effect in a cell interactive manner, instead of via a cell autonomous approach. In conclusion, our findings demonstrate a novel mechanism of colorectal oncogenesis which can support further investigation.

RevDate: 2025-10-15

Yang S, Jiao X, Liu J, et al (2025)

CRISPR-Cas opens a new era of antimicrobial therapy as a powerful gene editing tool.

World journal of microbiology & biotechnology, 41(10):388.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Slattery JR, Naung NY, Kalinna BH, et al (2025)

CRISPR-Powered Liquid Biopsies in Cancer Diagnostics.

Cells, 14(19):.

Liquid biopsies promise major advantages for cancer screening and diagnosis. By detecting biomarkers in peripheral blood samples, liquid biopsies reduce the need for invasive techniques and provide important genetic information integral to the emerging molecular classification of cancers. Unfortunately, the concentrations of most biomarkers, particularly circulating tumour nucleic acids, are vanishingly small-beyond the sensitivity and specificity of most assays. Clustered Regularly Interspaced Short Palindromic Repeats diagnostics (herein labelled 'CRISPR-Dx') use gene editing tools to detect, rather than modify, nucleic acids with extremely high specificity. These tools are commonly combined with isothermal nucleic acid amplification to also achieve sensitivities comparable to high-performance laboratory-based techniques, such as digital PCR. CRISPR assays, however, are inherently well suited to adaptation for point-of-care (POC) use, and unlike antigen-based POC assays, are significantly easier and faster to develop. In this review, we summarise current CRISPR-Dx platforms and their analytical potential for cancer biomarker discovery, with an emphasis on enhancing early diagnosis, disease monitoring, point-of-care testing, and supporting cancer therapy.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Wei W, Zhu W, Silver S, et al (2025)

Single-dose genome editing therapy rescues auditory and vestibular functions in adult mice with DFNA41 deafness.

The Journal of clinical investigation, 135(20):.

Genome editing has the potential to treat genetic hearing loss. However, current editing therapies for genetic hearing loss have shown efficacy only in hearing rescue. In this study, we evaluated a rescue strategy using adeno-associated virus (AAV) type 2-mediated delivery of Staphylococcus aureus Cas9-sgRNA in the mature inner ear of the P2rx2V61L/+ mouse model of autosomal dominant deafness-41 (DFNA41), a dominant, delayed-onset, and progressive hearing loss in humans. We demonstrate that local injection in adult mice results in efficient and specific editing that abolishes the mutation without notable off-target effects or AAV genome integration. Editing effectively restores long-term auditory and vestibular function. Editing further protects P2rx2V61L/+ mice from hypersensitivity to noise-induced hearing loss, a phenotype also observed in patients with DFNA41. Intervention in mice at a juvenile stage broadens the frequency range rescued, highlighting the importance of early intervention. An effective and specific gRNA for the human P2RX2 V60L mutation has been identified. Our study establishes the feasibility of editing to treat DFNA41 caused by P2RX2 V60L mutation in humans and opens an avenue for using editing to rescue hearing and vestibular function while mitigating noise-induced hearing loss.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Zhang Y, Zhao Z, Liu M, et al (2025)

Asymmetric volume-mediated buffer control overcomes sensitivity limits in one-pot RAA-CRISPR/Cas12a visual detection.

Analytical and bioanalytical chemistry, 417(26):5971-5981.

Rapid, low-cost, and visual nucleic acid detection methods are highly attractive for curbing colistin resistance spread through the food chain. CRISPR/Cas12a combined with recombinase-aided amplification (RAA) offers a one-pot, aerosol-free approach for visual detection. However, traditional one-pot systems often run Cas12a trans-cleavage in a buffer suitable for RAA, thus limiting Cas12a cleavage efficiency. This study proposes an asymmetric volume-optimized RAA-CRISPR/Cas12a assay for ultrasensitive visual detection of mobile colistin resistance gene mcr-1. Unlike conventional one-pot systems constrained by buffer incompatibility, our design spatially segregates a minimal-volume RAA-MIX (lid) from a CRISPR-dominant buffer microenvironment (tube bottom). This architecture leverages RAA's exponential amplification power to ensure sufficient product yield from minimal reaction volumes, while enabling subsequent enhancement of Cas12a trans-cleavage through automatic buffer assimilation upon mixing. The results were able to be visually observed under UV light, achieving 63.1% cost reduction compared to standard one-pot methods. The sensitivity of the proposed method for the mcr-1 gene was 2.5 copies/reaction, with anti-interference against other plasmids or bacteria. This method was applied to the detection of mcr-1 in animal-derived foods, showing satisfactory practical performance. By fundamentally reengineering buffer microenvironments through volume asymmetry, this work provides a general strategy for one-pot molecular diagnostics, achieving dual optimization of amplification and cleavage without trade-offs.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Liu X, Fu Y, Li M, et al (2025)

Biofortification of tomatoes with beta-carotene through targeted gene editing.

International journal of biological macromolecules, 327(Pt 2):147396.

Vitamin A deficiency is one of the most severe micronutrient-related health issues worldwide. Tomatoes, a widely cultivated crop for their adaptability, nutritional value, and lycopene content (a beta-carotene precursor), are ideal candidates for biofortification. In this study, CRISPR-mediated knockout mutants (cr-SlLCYe and cr-SlBCH) were generated to enhance the precursor supply to the β-carotene biosynthetic pathway and reduce its degradation. Carotenoids profiling showed that β-carotene levels in the mutants were 1.7 to 2.5-fold higher than in the wild-type, whereas lycopene levels remained unaltered without altering lycopene content. To evaluate potential trade-offs, the characteristics of the mutant fruits were comprehensively assessed, including appearance quality (color, firmness), nutritional quality (sugars, organic acids, vitamin C), and postharvest traits (shelf life, resistance to Botrytis cinerea). These results provide a new strategy for elevating β-carotene without compromising fruit quality and offer new insights into combating vitamin A deficiency through targeted tomato breeding programs.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Bi J, Mo W, Liu M, et al (2025)

Systematic decoding of functional enhancer connectomes and risk variants in human glioma.

Nature cell biology, 27(10):1838-1847.

Genetic and epigenetic variations contribute to the progression of glioma, but the mechanisms underlying these effects, particularly for enhancer-associated genetic variations in non-coding regions, still remain unclear. Here we performed high-throughput CRISPR interference screening to identify pro-tumour enhancers in glioma cells. By integrating genome-wide H3K27ac HiChIP data, we identified the target genes of these pro-tumour enhancers and revealed the essential role of enhancer connectomes in promoting glioma progression. Through systematic analysis of enhancers carrying glioma risk-associated single-nucleotide polymorphisms (SNPs), we found that these SNPs can promote glioma progression through the enhancer connectome. Using CRISPR-Cas9-mediated enhancer interference and SNP editing, we demonstrated that glioma-specific enhancer carrying the risk SNP rs2297440 regulates SOX18 expression by specifically recruiting transcription factor MEIS1 binding, thereby contributing to glioma progression. Our study sheds light on the molecular mechanisms underlying glioma susceptibility and provides potential therapeutic targets to treat glioma.

RevDate: 2025-10-16
CmpDate: 2025-10-16

Rodschinka G, Forcelloni S, Kühner FM, et al (2025)

Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control.

Nature structural & molecular biology, 32(10):1932-1946.

The translation of mRNA into proteins in multicellular organisms needs to be carefully tuned to changing proteome demands in development and differentiation, while defects in translation often have a disproportionate impact in distinct cell types. Here we used inducible CRISPR interference screens to compare the essentiality of genes with functions in mRNA translation in human induced pluripotent stem cells (hiPS cells) and hiPS cell-derived neural and cardiac cells. We find that core components of the mRNA translation machinery are broadly essential but the consequences of perturbing translation-coupled quality control factors are cell type dependent. Human stem cells critically depend on pathways that detect and rescue slow or stalled ribosomes and on the E3 ligase ZNF598 to resolve a distinct type of ribosome collision at translation start sites on endogenous mRNAs with highly efficient initiation. Our findings underscore the importance of cell identity for deciphering the molecular mechanisms of translational control in metazoans.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Cherdantsev AI, Kulagin KA, Polyakova AN, et al (2025)

[DNA Double-Strand Break Repair System by a Mechanism of Non-Homologous End Joining Provides Resistance to DNA-Damaging and Oxidizing Stresses in the Yeast Debaryomyces hansenii].

Molekuliarnaia biologiia, 59(4):616-628.

The unconventional halotolerant yeast Debaryomyces hansenii is of great importance in biotechnology and the food industry, and in basic research it serves as a model for studying the molecular mechanisms of resistance to increased salinity and osmotic stress. We have previously established an efficient method for editing the D. hansenii genome using the CRISPR/Cas9 system. In turn, this has stimulated further investigation of the structure and physiological role of DNA double-strand break repair pathways in D. hansenii. The aim of the present work was to evaluate the involvement of key components of the DNA double-stranded break repair system by the non-homologous end joining (NHEJ) mechanism in the resistance of D. hansenii to DNA-damaging compounds and compounds that induce oxidative, high salinity, and osmotic stress. Using the CRISPR/Cas9 system, mutant strains with knockout of the DEHA2F10208g (DhKU70), DEHA2B01584g (DhKU80) , and DEHA2G04224g (DhLIG4) genes encoding key components of NHEJ were obtained. It was found that mutant strains, unlike the wild-type strain, are sensitive to chemical compounds that damage DNA, as well as to compounds that cause oxidative stress. Osmotic and high salinity stresses and vanillin do not cause significant changes in the rate of colony formation of mutant strains. Unexpectedly, mutant strains exhibit increased resistance to caffeine compared to the wild-type strain. The data indicate that the NHEJ systems of D. hansenii play a significant role in the response to DNA-damaging and oxidative types of stress. The importance of the NHEJ system in the processes of maintaining yeast cell homeostasis should be taken into account when creating strains producing valuable substances.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Li JM, Huang J, Liao Y, et al (2025)

Gene and RNA Editing: Revolutionary Approaches to Treating Diseases.

MedComm, 6(10):e70389.

Gene editing and RNA editing technologies are advancing modern medicine by enabling precise manipulation of genetic information at the DNA and RNA levels, respectively. The third-generation gene editing tools, particularly Clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) system, have transformed genetic disease treatment with high efficiency, precision, and cost effectiveness, while RNA editing, via adenosine deaminase acting on RNA (ADAR) enzymes and CRISPR-Cas13, offers reversible regulation to avoid genomic integration risks. Despite advancements, challenges persist in delivery efficiency, tissue specificity, and long-term safety, limiting their clinical translation. This review systematically discusses the molecular mechanisms and technological evolution of these tools, focusing on their promising applications in treating nervous system disorders (e.g., Alzheimer's, Parkinson's), immune diseases (e.g., severe combined immunodeficiency, lupus), and cancers. It compares their technical attributes, analyzes ethical and regulatory issues, and highlights synergies between the two technologies. By bridging basic research and clinical translation, this review provides critical insights for advancing precision medicine, reshaping disease diagnosis, prevention, and treatment paradigms.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Mo T, Ren HY, Zhang XX, et al (2025)

Phenotypic Function of Legionella pneumophila Type I-F CRISPR-Cas.

Biomedical and environmental sciences : BES, 38(9):1105-1119.

OBJECTIVE: CRISPR-Cas protects bacteria from exogenous DNA invasion and is associated with bacterial biofilm formation and pathogenicity.

METHODS: We analyzed the type I-F CRISPR-Cas system of Legionella pneumophila WX48, including Cas1, Cas2-Cas3, Csy1, Csy2, Csy3, and Cas6f, along with downstream CRISPR arrays. We explored the effects of the CRISPR-Cas system on the in vitro growth, biofilm-forming ability, and pathogenicity of L. pneumophila through constructing gene deletion mutants.

RESULTS: The type I-F CRISPR-Cas system did not affect the in vitro growth of wild-type or mutant strains. The biofilm formation and intracellular proliferation of the mutant strains were weaker than those of the wild type owing to the regulation of type IV pili and Dot/Icm type IV secretion systems. In particular, Cas6f deletion strongly inhibited these processes.

CONCLUSION: The type I-F CRISPR-Cas system may reduce biofilm formation and intracellular proliferation in L. pneumophila.

RevDate: 2025-10-14
CmpDate: 2025-10-14

Libri AB, Wang J, Marton T, et al (2025)

Senataxin promotes recombination fidelity during antigen receptor gene diversification.

Science signaling, 18(908):eadv8801.

Antigen receptor diversity depends on the assembly of variable (V), diverse (D), and joining (J) exons in genes encoding immunoglobulins (Igs) and T cell receptors (TCRs). During V(D)J recombination, DNA double-strand breaks (DSBs) introduced by the RAG1/2 nuclease complex are repaired by the process of nonhomologous end-joining (NHEJ). We hypothesized that functional redundancies between NHEJ and the chromatin DSB response, which depends on the kinase ATM, potentially masked the activity of additional factors that regulate V(D)J recombination. We performed targeted CRISPR-Cas9 knockout screens for genes implicated in V(D)J recombination in pro-B cells that were either untreated or treated with an ATM inhibitor. We found that loss of the RNA/DNA helicase senataxin (SETX) impaired V(D)J recombination and led to the formation of aberrant hybrid joints between coding ends and signal ends, both in vitro and in mice. The loss of SETX in a background deficient in the NHEJ factor XLF or in which ATM was inhibited led to substantial impairment of V(D)J recombination and to the presence of unsealed coding ends. SETX limited aberrant activation-induced cytidine deaminase (AID)-induced DNA end-joining between Igh-containing alleles during the process of class-switch recombination. Together, our findings reveal a previously uncharacterized role for SETX in promoting recombination fidelity during antigen receptor gene diversification.

RevDate: 2025-10-14
CmpDate: 2025-10-14

de Souza HCA, Panzenhagen P, Portes AB, et al (2025)

CRISPR-Cas systems in combating antimicrobial resistance: which system to choose? A systematic review.

World journal of microbiology & biotechnology, 41(10):381.

Antimicrobial resistance (AMR) poses a growing threat to global public health, progressively compromising the efficacy of available antimicrobials. Technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) have emerged as promising tools for controlling resistant pathogens, offering high specificity and versatility. However, a comprehensive and systematic synthesis of CRISPR strategies applied to AMR remains limited. From February 12, 2025, we conducted a systematic review of the PubMed, Embase, and Scopus databases, using the following search strategy: Population (resistant bacteria or plasmid-mediated resistance), Intervention (CRISPR, including variants such as CRISPR-Cas9, Cas3, Cas12, Cas13, and CRISPR interference [CRISPRi]), and Outcomes (bacterial resensitization or plasmid curing). The CRISPR-Cas9 system was the most frequently employed (75.7%), with conjugation identified as the primary delivery method. We identified the advantages and limitations of each system, highlighting CRISPRi and CRISPR-Cas13a as alternatives to overcome the constraints of direct genome editing. Delivery efficiency remains a central challenge, although nanocarrier- and bacteriophage-based methodologies show promising potential. We also propose a decision map that guides the selection of the most appropriate CRISPR-Cas system and delivery strategy, considering factors such as therapeutic objective, gene location, methodology efficiency, application environment, and clinical feasibility. This review provides an updated and structured synthesis of CRISPR strategies applied to AMR, emphasizing their potential translational and clinical applications. The findings can inform the development of CRISPR-based therapeutics, guide the design of preclinical studies, and support future strategies for combating multidrug-resistant infections in clinical settings.

RevDate: 2025-10-14

Levassor L, Whitford CM, Petersen SD, et al (2025)

StreptoCAD: An Open-Source Software Toolbox Automating Genome Engineering Workflows in Streptomycetes.

ACS synthetic biology [Epub ahead of print].

Streptomycetes hold immense potential for discovering novel bioactive molecules for applications in medicine or sustainable agriculture. However, high-throughput exploration is hampered by the current Streptomyces genetic engineering methods that involve the manual design of complex experimental molecular biological engineering strategies for each targeted gene. Here, we introduce StreptoCAD, an open-source software toolbox that automates and streamlines the design of genome engineering strategies in Streptomyces, supporting various CRISPR-based and gene overexpression methods. Once initiated, StreptoCAD designs all necessary DNA primers and CRISPR guide sequences, simulates plasmid assemblies (cloning) and the resulting modification of the genomic target(s), and further summarizes the information needed for laboratory implementation and documentation. StreptoCAD currently offers six design workflows, including the construction of overexpression libraries, base-editing, including multiplexed CRISPR-BEST plasmid generation, and genome engineering using CRISPR-Cas9, CRISPR-Cas3, and CRISPRi systems. In addition to automating the design process, StreptoCAD further secures compliance with the FAIR principles, ensuring reproducibility and ease of data management via standardized output files. To experimentally demonstrate the design process and output of StreptoCAD, we designed and constructed a series of gene overexpression strains, and performed CRISPRi knockdowns in Streptomyces Gö40/10, underscoring the tool's efficiency and user-friendliness.. This tool simplifies complex genetic engineering tasks and promotes collaboration through standardized workflows and design parameters. StreptoCAD is set to transform genome engineering in Streptomyces, making sophisticated genetic manipulations accessible for all and accelerating natural product discovery.

RevDate: 2025-10-14

Han J, Ganguly R, Yi JY, et al (2025)

Osmotically Tunable Microdroplets Enable Amplification-Free CRISPR Detection of Gene Doping.

Advanced science (Weinheim, Baden-Wurttemberg, Germany) [Epub ahead of print].

Gene doping is an increasing challenge in sports, demanding highly sensitive and specific detection tools beyond the limitations of the current amplification-dependent methods. Here, an innovative amplification-free clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) 12a assay integrated with osmotically tunable double emulsion (DE) droplets is reported for rapid and ultrasensitive gene doping detection. Target DNA and CRISPR/Cas12a complexes are encapsulated within DE droplets, where osmotic shrinkage rapidly concentrates the reaction components, thereby enhancing the fluorescent signal intensity without nucleic acid amplification. This platform enables the detection of the human erythropoietin (hEPO) gene at unprecedented attomolar levels within 30 min, achieving a 25-fold improvement in sensitivity compared with that of nonshrinkable formats. Notably, the assay demonstrated a robust and specific performance in complex serum samples with minimal matrix interference. This novel approach offers a rapid, reliable, and inherently contamination-free solution for gene doping surveillance with broad potential for versatile amplification-free nucleic acid diagnostics.

RevDate: 2025-10-14
CmpDate: 2025-10-14

Menelih A, Girma A, A Aemiro (2025)

Advancing nutritional quality in oilseed crops through genome editing: a comprehensive review.

GM crops & food, 16(1):709-732.

Genome editing has emerged as a powerful approach to enhancing the nutritional quality of oilseed crops. Clustered regularly interspaced short palindromic repeats case9 (CRISPR/Cas9) is the predominant editing tool, while transcription activator-like effector nucleases (TALENs) and base editors are used less commonly. Key fatty acid desaturase genes such as FAD2 and FAD3 are prime targets because of their critical functions in fatty acid desaturation. This review summarizes recent progress in editing genes associated with oil composition and related traits across oilseed species. Visual data representations including, Sankey diagrams, heat maps, and crop-trait matrices illustrate shared editing priorities and emerging trait targets across crops. Despite its promise, genome editing still faces challenges in transformation efficiency, field-level validation, and regulatory acceptance. This review underscores the increasing impact of target gene editing on nutritional trait improvement and its potential to accelerate the development of healthier, more sustainable oilseed varieties.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Clark MB, Funk AT, Paporakis A, et al (2025)

Efficient CRISPR-Cas9-Mediated Genome Editing of the Cane Toad (Rhinella marina).

The CRISPR journal, 8(5):321-332.

Invasive species inflict major ecological, economic, and cultural harm worldwide, highlighting the urgent need for innovative control strategies. Genome editing offers exciting possibilities for targeted control methods for invasive species. Here, we demonstrate CRISPR-Cas9 genome editing in the cane toad (Rhinella marina), one of Australia's most notorious invasive species, by targeting the tyrosinase gene to produce albino phenotypes as visual markers for assessing editing efficiency. Microinjection of Cas9 protein and guide RNAs into one-cell zygotes resulted in 87.6% of mosaic larvae displaying nearly complete albinism, with 2.3% exhibiting complete albinism. For completely albino individuals, genomic analysis confirmed predominantly frameshift mutations or large deletions at the target site, with no wild-type alleles detected. Germline transmission rates reflected the extent of albinism in the mosaic adult, with maternal transmission approaching 100%. This first application of CRISPR-Cas9 in the Bufonidae family opens possibilities for exploring basic research questions and population control strategies.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Skipper TS, Dickson KA, Denes CE, et al (2025)

Revealing genetic drivers of ovarian cancer and chemoresistance: insights from whole-genome CRISPR-knockout library screens.

Cellular oncology (Dordrecht, Netherlands), 48(5):1245-1265.

Understanding genetic dependencies in cancer is key to identifying novel actionable drug targets to advance precision medicine. Whole-genome CRISPR-knockout library screening methods have facilitated this goal. Pooled libraries of single guide RNAs (sgRNAs) targeting over 90% of the annotated protein coding genome are used to induce gene knockouts in pre-clinical cancer models. Novel genes of interest are identified by evaluating sgRNA dropout or enrichment following selection pressure application. This method is particularly beneficial for researching cancers where effective treatment strategies are limited. One example of a commonly chemoresistant cancer, particularly at relapse, is the low survival malignancy epithelial ovarian cancer (EOC), made up of multiple histotypes with distinct molecular profiles. CRISPR-knockout library screens in pre-clinical EOC models have demonstrated the ability to predict biomarkers of treatment response, identify targets synergistic with standard-of-care chemotherapy, and determine novel actionable targets which are synthetic lethal with cancer-associated mutations. Robust experimental design of CRISPR-knockout library screens, including the selection of strong pre-clinical cell line models, allows for meaningful conclusions to be made. We discuss essential design criteria for the use of CRISPR-knockout library screens to discover genetic dependencies in cancer and draw attention to discoveries with translational potential for EOC.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Sun X, Li M, Wang H, et al (2025)

Possible Reversion of CRISPR-Cas9-Edited Sequences in Octoploid Strawberry.

The CRISPR journal, 8(5):375-389.

Gene editing is more challenging in octoploids due to the presence of multiple copies of each gene. However, the ability to edit genes in these plants would allow editing in commercial varieties. Here, we delivered sequences targeting FaMYB9 into octoploid strawberry "Honeoye" and identified several gene-edited lines. Among them, the heterozygous gene-edited line FaMYB9[CR]-15 had curved and wrinkled leaves at 3 months, whereas leaves of 3-month-old wild-type (WT) strawberry seedlings were elliptical with a smooth surface. At that stage, FaMYB9[CR]-15 leaves also had large patches of wax. We identified 11,402 differentially expressed genes, divided into four clusters, between WT and FaMYB9[CR]-15 seedlings at 3 months. Notably, cluster 4 genes-related to nonhomologous end joining, microhomology-mediated end joining repairs, homologous recombination, nucleotide excision repair, and mismatch repair-were more highly expressed in the gene-edited line than in the WT. Surprisingly, by 6 months of age, FaMYB9[CR]-15 leaves had become smooth with small patches of wax, and expression levels of cluster 4 genes were significantly lower than at 3 months. Over the same period, the percentage of FaMYB9 loci harboring the mutant allele decreased from 70.2% to 43.7%. These findings lead us to conclude that there could be reversion of mutated sequences in octoploid strawberry, emphasizing the challenges of gene editing high-ploidy materials.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Sherkow JS (2025)

A "Bare Hope of A Result": The Second CRISPR Patent Appeal.

The CRISPR journal, 8(5):317-320.

On May 12, 2025, the US Court of Appeals for the Federal Circuit issued its second decision in the long-running CRISPR patent dispute between the Regents of the University of California and related institutions (CVC) and the Broad Institute. This Perspective recounts the principal dispute to date, reviews the Federal Circuit's recent opinion, and provides a critique of its analysis. In particular, this Perspective highlights how the decision is self-contradictory and in tension with patent law's conception doctrine-when an inventor has formed a "definite and permanent" idea of an invention in the mind or whether the invention was little more than a "bare hope" of a result. This Perspective briefly concludes with the implications of this recent decision and where the underlying dispute is likely headed.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Manuvera VA, Bobrovsky PA, Kharlampieva DD, et al (2025)

Bacterial Expression System with Deep Repression and Activation via CRISPR-Cas9.

The CRISPR journal, 8(5):353-365.

Incomplete repression of recombinant genes encoding toxic polypeptides can suppress cell growth even in the absence of a transcription inducer. To address this issue, we developed a CRISPR-Cas9-based genome editing approach that directly modifies the plasmid encoding the toxic peptide during Escherichia coli cultivation. The constructed plasmids contained a transcription terminator between the promoter and coding region, preventing full gene expression through abortive transcription. Upon CRISPR-Cas9 activation, this region is excised, thus restoring the functional gene. To implement this approach, we modified widely used pET-series expression plasmids by adding extra terminators in the 5'-untranslated region of the recombinant gene. Four antimicrobial peptides with strong bactericidal properties served as toxic gene products, while green fluorescent protein was used to assess the efficiency of expression repression. As a result, we developed an expression system with strong repression, which is activated by CRISPR-Cas9-mediated excision of a DNA fragment from the plasmids.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Han J, Yu B, Jing J, et al (2025)

EGFR blockade confers sensitivity to pan-RAS inhibitors in KRAS-mutated cancers.

Cellular oncology (Dordrecht, Netherlands), 48(5):1317-1335.

INTRODUCTION: KRAS is one of the most commonly occurring mutated oncogene in human cancers. Development of KRAS G12C or G12D inhibitors exhibit promising clinical activities, but patients harboring other hotspot KRAS mutations cannot benefit from those strategies. Recent development in pan-RAS inhibitors have broad therapeutic implications and merit clinical investigation. However, intrinsic and acquired drug resistance caused by tumor heterogeneity greatly limit the clinical application, posing a significant challenge in this field.

RESULTS: In this study, through CRISPR/Cas9 sgRNA screening using a human kinome sgRNA library, EGFR was discovered to correlate with the sensitivity of KRAS-mutated tumors to pan-RAS inhibitor RMC-7977. Through multiple in vitro cell proliferation or viability assays, EGFR loss or pharmacological EGFR inhibition significantly enhances the effectiveness of pan-RAS inhibitors in multiple KRAS[G12C] or KRAS[G12D] cancer cell lines, disregarding their cellular origins. Mechanistically, co-inhibition of EGFR and pan-RAS may further dampen the RTK-RAS-RAF-MEK-ERK pathway activation than either alone, thereby enhancing the anti-tumor activity of pan-RAS inhibitors. Strikingly, with the LL/2 syngeneic mice tumor model, the combination of pan-RAS inhibitors and EGFR inhibitors demonstrated more significant in vivo therapeutic efficacy compared to either single agent.

CONCLUSION: In conclusion, this study employed high-throughput CRISPR/Cas9 sgRNA screening to identify the enhanced anti-cancer effects when combining EGFR inhibitors with pan-RAS inhibitors in multiple human KRAS-mutated cancer cell lines as well as a mouse syngeneic tumor model. This synergy underscores the potential for a combinational therapy strategy, leveraging EGFR and pan-RAS inhibitors to improve treatment outcomes for patients with KRAS-driven cancers.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Guerra I, Jensen K, P Perez-Pinera (2025)

Implementation of an Undergraduate Laboratory-Based Mammalian Genome Editing Course.

The CRISPR journal, 8(5):366-374.

Genome engineering methods can be utilized to perform complex genetic manipulations in living cells with remarkable efficiency and precision. Given the transformative potential of these enabling technologies, their applications are steadily expanding into most biology and biomedical fields where they play a central role in many experimental frameworks. For these reasons, in order to effectively prepare future generations of biologists and bioengineers for successful careers, there is a high need to incorporate courses teaching genome editing fundamentals into existing curricula. To accomplish this objective, lecture-based courses are rapidly integrating genome editing concepts; however, there are few laboratory courses that teach the practical skills needed to successfully perform genome editing experiments. Here, we describe the development and implementation of a semester-long laboratory course that teaches students not only the techniques needed to perform gene knockout, gene activation, gene repression, and base editing in mammalian cells but also prepares them to design and troubleshoot experiments, write scientific manuscripts, as well as prepare and deliver scientific presentations. Course evaluations demonstrate that this class effectively equips students with the knowledge and hands-on experience needed to succeed in careers related to genome engineering, cell and tissue engineering, and, more broadly, biology.

RevDate: 2025-10-13
CmpDate: 2025-10-13

McCallion O, Du W, Glaser V, et al (2025)

HLA matching or CRISPR editing of HLA class I/II enables engraftment and effective function of allogeneic human regulatory T cell therapy in a humanized mouse transplantation model.

Nature communications, 16(1):9090.

Regulatory T cells (Tregs) hold promise for treating autoimmune disease and transplant rejection, yet generation of autologous products for adoptive transfer can suffer donor variability and slow turnaround, limiting their use in urgent indications. We therefore examine whether allogeneic, pre-manufactured ('off-the-shelf') Tregs could overcome these barriers. In a human skin-xenograft model, HLA-mismatched Tregs are swiftly eliminated by recipient CD8[+] T cells and fail to protect grafts. Stringent matching of HLA class I and II restores efficacy but is clinically impractical. Using non-viral CRISPR editing we disrupt B2M and CIITA while inserting an HLA-E-B2M fusion, generating hypo-immunogenic Tregs that evade both T and NK cell attack. Engineered cells retain FOXP3 stability and potent in vitro suppression, and after a single low-dose infusion, prolong human skin graft survival in a humanized mouse model comparably to autologous Tregs. Histology and spatial transcriptomics reveal minimal cytotoxic infiltration and enrichment of immunoregulatory and tissue-repair programmes. Multiplex HLA engineering thus enables ready-to-use allogeneic Tregs that withstand host immune attack for adoptive transfer.

RevDate: 2025-10-13

Yang L, Fang Y, Lian Y, et al (2025)

Aloe-Emodin Targeting FOXC2 Disrupts NETs Formation and EMT-Driven Postoperative Peritoneal Adhesion Through TGF-β1-Smad2/3 Pathway.

Advanced science (Weinheim, Baden-Wurttemberg, Germany) [Epub ahead of print].

Postoperative peritoneal adhesion (PPA) develops through TGF-β1-driven fibrotic remodeling, characterized by neutrophil extracellular trap (NETs)-induced aberrant epithelial-to-mesenchymal transition (EMT) deposition. Although aloe-emodin (AE) exhibits anti-fibrosis potential, its molecular mechanisms remain elusive. Forkhead box protein C2 (FOXC2) is a critical regulator of fibrotic tissue formation, yet its role in PPA is unknown. Here, it is demonstrated that FOXC2 expression is elevated in human ileostomy tissue, PPA rodent model, and TGF-β1-exposed peritoneal mesothelial cells (PMCs), where it orchestrates NETs formation and extracellular matrix (ECM) remodeling. Mechanically, CRISPR/Cas-based knockdown and overexpression of FOXC2 alter EMT changes in PMCs, which is achieved via TGF-β1-Smad2/3 signaling. FOXC2 functions as a dual mediator and amplifier through the TGF-β1-Smad2/3 pathway feedback loop to drive EMT alterations. Its overexpression further induces neutrophil recruitment and NETs formation, exacerbating EMT in PMCs. Notably, AE ameliorates FOXC2-driven peritoneal fibrosis by impeding NETs formation and EMT changes through the TGF-β1-Smad2/3 pathway. Moreover, AE binds directly to FOXC2, and the Ser125 residue is critical for the binding of FOXC2 to AE. These findings identify FOXC2 as a pivotal effector in fibrotic responses during PPA formation and reveal that AE targeting the Ser125 residue of FOXC2 may be a promising therapeutic approach to attenuate PPA.

RevDate: 2025-10-13
CmpDate: 2025-10-13

Kim WD, RJ Huber (2026)

Modeling Lysosomal Disease in Dictyostelium discoideum: Examining the Trafficking and Secretion of Lysosomal Enzymes.

Methods in molecular biology (Clifton, N.J.), 2976:189-207.

Non-mammalian models are powerful systems for enhancing our understanding of lysosomal function and lysosomal storage diseases. The social amoeba Dictyostelium discoideum is an excellent model organism for studying lysosomal function, as its genome encodes many proteins associated with lysosomal disease. Methods for gene knockout are straightforward in D. discoideum and include restriction enzyme-mediated integration (REMI) mutagenesis, homologous recombination via the Cre-loxP system, and CRISPR/Cas9-mediated gene editing, which collectively allow researchers to study protein function (e.g., lysosomal enzymes) in a genetically tractable biomedical model system. Additionally, activity assays for conserved lysosomal enzymes are well-established in D. discoideum. In this chapter, we outline methods for studying the intracellular localization and secretion of conserved lysosomal proteins in D. discoideum.

RevDate: 2025-10-13
CmpDate: 2025-10-13

Chear S, Chiam A, Talbot J, et al (2026)

Generation of Donor-Specific iPSC for Modelling Lysosomal Storage Disorders.

Methods in molecular biology (Clifton, N.J.), 2976:151-173.

iPSC technology has enabled the generation of human cell-based models of lysosomal storage disorders and has provided disease-relevant systems to undertake drug discovery or pre-clinical testing of gene- or cell-based therapies. Here, we provide a protocol to generate iPSCs derived from people with lysosomal storage disorders and illustrate expected results using a CLN2 disease donor-specific skin fibroblast culture. Protocol steps include lipofection of episomal plasmids, picking of putative iPSC colonies following live cell TRA-1-60 immunofluorescence, and quality control steps such as immunofluorescence for expression of undifferentiated cell markers, germ layer differentiation, and confirmation of pathological variant genotype. The iPSC generated by this protocol can be differentiated to several cell lineages and can be used with CRISPR/Cas technology to generate isogenic disease models.

RevDate: 2025-10-13
CmpDate: 2025-10-13

Kim HJ, Kwon MY, Song S, et al (2025)

Engineered Lactiplantibacillus plantarum and Levilactobacillus brevis utilizing ribonucleoprotein-mediated editing for inactivation of hemolysin gene.

World journal of microbiology & biotechnology, 41(10):373.

Lactiplantibacillus plantarum and Levilactobacillus brevis are widely used probiotics with significant potential as chassis organisms for probiotic engineering. However, their bioengineering remains underdeveloped compared to that of other probiotic bacteria due to the limited availability of genetic tools. Although CRISPR-Cas systems have shown promise for genome editing in Lactobacillus species, strain- or site-specific targeting challenges must be overcome to enhance their broader applicability. This study aimed to develop a novel editing system with reduced dependency on plasmids and antibiotics in L. plantarum WCFS1, L. plantarum SPC 72 - 1 and L. brevis SPC-SNU 70 - 2 using a Cas9-gRNA ribonucleoprotein (RNP) complex. Although the hlyIII gene has been annotated as a hemolysin-related gene in several Lactobacillus genomes, no functional hemolytic activity has been definitively demonstrated to date. In this study, hlyIII was selected as a target to evaluate genome editing efficiency and to assess its potential relevance to strain safety. To construct ΔhlyIII strains, the RNP complex targeting hlyIII was separately transformed with recombinase RecE/T and double-stranded donor DNA. As a result, ΔhlyIII mutants were obtained under optimized electroporation conditions. Sequencing analysis revealed a 50 bp deletion and the introduction of a stop codon in hlyIII across all mutant strains. The hemolytic activity test showed a reduction in free hemoglobin levels in the ΔhlyIII strains compared to the wild type: 27.0%, 74.3%, and 5.0% in L. plantarum WCFS1, L. plantarum SPC 72 - 1, and L. brevis SPC-SNU 70 - 2, respectively. These results suggest strain-dependent differences in hemolytic activity and indicate that inactivation of hlyIII may contribute to reduced hemolysis, although further validation is needed to clarify its functional role. In conclusion, the hlyIII gene was successfully edited in L. plantarum and L. brevis using Cas9-gRNA ribonucleoprotein-mediated editing, demonstrating the feasibility of this genome editing platform for application in probiotic strains.

RevDate: 2025-10-14
CmpDate: 2025-10-14

Xiao J, Hu X, Chen H, et al (2025)

CRISPR-Programmed CuO Nanocatalyst Release for Ultrasensitive Detection of Pathogens in Sterile Body Fluids.

Analytical chemistry, 97(40):22427-22435.

Treatment of sterile body fluids (SBFs) infections is delayed by conventional methods that require up to 72 h to detect pathogens. Here, we present a CRISPR-associated protein 12a (Cas12a)-programmed nanocatalyst release (CNR) method for culture-free diagnostics. To enhance both sensitivity and coverage, three starter DNA (sDNA)-complementary DNA (cDNA) probe pairs were designed for conserved regions and additional three pairs for variable regions of bacterial 16S or fungal 18S rRNA. Upon target recognition, cDNA undergoes strand displacement, releasing sDNA to activate Cas12a. The activated Cas12a cleaves copper oxide nanoparticle (CuONPs)-loaded magnetic probes, releasing tandem CuONPs. Upon acid dissolution, each CuONP generates Cu[2+] ions that catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), producing a visible colorimetric signal. This quadruple signal amplification strategy integrates high-copy rRNA targets, multi-cDNA recognition, Cas12a-mediated continuous release of tandem CuONPs, and Cu[2+]-driven chromogenic amplification. This nucleic acid amplification-free assay detects pathogens at 0.69 CFU mL[-1] in original SBFs samples (after 10-fold centrifugation) within 70 min. In 64 clinical samples, it achieved 100% sensitivity and 100% specificity versus culture. Notably, one culture-negative but clinically confirmed case was correctly identified. Overall, the CNR method offers a rapid, ultrasensitive, and accessible diagnostic solution for resource-limited settings.

RevDate: 2025-10-14
CmpDate: 2025-10-14

Deng L, He X, Zhou S, et al (2025)

A sticky end-driven PAM-free RPA-CRISPR/Cas12a dual amplification system for ultrasensitive detection of KRAS G12C.

Chemical communications (Cambridge, England), 61(83):16282-16285.

Herein, a fluorescent biosensing platform was constructed for KRAS G12C single base mutation detection by CRISPR/Cas12a-coupled RPA without the PAM site. The KRAS G12C gene sequence was cleaved into double-stranded DNA containing a sticky end using HindIII enzyme cleavage site specificity. Sticky end dsDNA activated the trans-cleavage activity of Cas12a and generates an intense fluorescent signal. This strategy detected KRAS G12C targets in a linear range of 10 aM-10 pM with a detection limit of 1.5 aM. What's more, the method was able to distinguish 0.1% KRAS G12C mutation in a total of 10 pM gene concentration and demonstrated excellent detection performance in real samples.

RevDate: 2025-10-14
CmpDate: 2025-10-14

Rybarikova M, Rey M, Hasanovic E, et al (2025)

Gene editing for Spinocerebellar ataxia type 3 taking advantage of the human ATXN3L paralog as replacement gene.

Gene therapy, 32(5):462-474.

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by a CAG expansion of the ataxin-3 gene (ATXN3). SCA3 patients suffer from ataxia, spasticity and dystonia in mid-adulthood, with spinocerebellar dysfunction and degeneration. As a monogenic disease for which only symptomatic treatment is available, ATXN3 is an attractive target for gene editing. We used the KamiCas9, a self-inactivating gene editing system, to explore gene editing strategies suitable for all SCA3 patients. We first tested the deletion of exon 10 or the introduction of a premature stop codon into exon 9. High editing events were observed in vitro, but efficiency was very low in SCA3 transgenic mice. We then evaluated an ablate-and-replace strategy. The ablate experiments resulted in 55 ± 18% cerebellar editing of the ATXN3 gene. A human ATXN3L paralog, expressed in the brains of SCA3 patients, may act as a natural, CRISPR-resistant replacement gene. In a proof-of-principle study, ablate and ablate-and-replace strategies were evaluated in SCA3 transgenic mice. Two months after injection, similar editing efficiencies were obtained in the ablate and ablate-and-replace groups. Immunofluorescence and RT-qPCR analyses of cerebellar markers support the development of this strategy for SCA3 treatment.

RevDate: 2025-10-14
CmpDate: 2025-10-14

Luo Y, Zhan X, Zhang Y, et al (2025)

CRISPR-Cas9-mediated knockup of OsDREB1C enhances rice yield without compromising grain quality.

Plant communications, 6(10):101433.

This study presents a CRISPR-Cas9-based strategy for engineering structural variations in the OsDREB1C gene in rice, leading to a yield increase of over 20% without compromising grain quality. The resulting homozygous plants are transgene-free, highlighting the potential of this approach for precise and effective crop improvement.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Yan RE, Corman A, Katgara L, et al (2025)

Pooled CRISPR screens with joint single-nucleus chromatin accessibility and transcriptome profiling.

Nature biotechnology, 43(10):1628-1634.

Pooled single-cell CRISPR screens have profiled either gene expression or chromatin accessibility but not both modalities. Here we develop MultiPerturb-seq, a high-throughput CRISPR screening platform with joint single-nucleus chromatin accessibility, transcriptome and guide RNA capture using combinatorial indexing combined with droplet microfluidics to scale throughput and integrate all three modalities. We identify key differentiation genes in a rare pediatric cancer and establish ZNHIT1 as a potential target for cancer reprogramming therapy.

RevDate: 2025-10-14
CmpDate: 2025-10-14

Jabalera Y, Tascón I, Samperio S, et al (2025)

A resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection.

Nature biotechnology, 43(10):1663-1672.

The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.

RevDate: 2025-10-15
CmpDate: 2025-10-15

Gould SI, Wuest AN, Dong K, et al (2025)

High-throughput evaluation of genetic variants with prime editing sensor libraries.

Nature biotechnology, 43(10):1648-1662.

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.

RevDate: 2025-10-13
CmpDate: 2025-10-13

Serreze DV, Tousey-Pfarrer M, JJ Racine (2025)

Humanized Mouse Models for Type 1 Diabetes.

Current protocols, 5(10):e70224.

T cell-mediated autoimmune type 1 diabetes (T1D) is under complex polygenic control in both humans and the NOD mouse model. However, in both species, particular major histocompatibility complex (MHC; designated HLA in humans) haplotypes provide the primary T1D risk factor. Both MHC/HLA class I and II variants interactively contribute to T1D by respectively driving autoreactive CD8 and CD4 T cell responses that cooperatively destroy insulin-producing pancreatic β cells. While NOD mice have provided important insights to the pathogenic basis of T1D, the model has so far provided only a limited means to identify possible clinically translatable disease intervention approaches. This highlights a need to humanize NOD mice in ways that their pathogenic basis of T1D development becomes more similar to that characterizing the disease course in patients. In this review, we discuss the use of CRISPR/Cas9-generated murine-MHC-deficient NOD mice as a platform for introduction of patient-relevant HLA and T cell receptor molecules. These mice provide ever-improving models for development of clinically applicable interventions for T1D and other autoimmune diseases. © 2025 The Author(s) Current Protocols published by Wiley Periodicals LLC.

RevDate: 2025-10-13

Ma F, Zheng Q, Sun Y, et al (2025)

Toward Tissue-Free Plant Engineering: Emerging Platforms for Sustainable Horticultural Transformation.

Journal of agricultural and food chemistry [Epub ahead of print].

Genetic transformation in horticultural crops is being reshaped by the emergence of nontissue culture technologies that bypass entrenched barriers of genotype dependence, regeneration inefficiency, and sterile culture requirements. This review surveys recent in planta methods, including regenerative activity-dependent in Planta injection delivery (RAPID), cut-dip-budding (CDB), virus-based delivery, nanoparticle-mediated transformation, and Agrobacterium rhizogenes-induced regeneration, and evaluates their operational versatility across species. We further examine their integration with developmental regulators (BABY BOOM [BBM], WUSCHEL [WUS]), visual markers (RUBY), and CRISPR/Cas systems to enhance transformation efficiency and precision. Case studies across fruit, vegetable, and ornamental crops illustrate broad applicability and growing technical maturity. Despite these advances, unresolved challenges in biosafety, reproducibility, and regulatory alignment remain. We advocate a new transformation paradigm that is rapid, genotype-independent, and environmentally compatible, enabling scalable and more accessible broadly applicable crop improvement in horticultural biotechnology.

RevDate: 2025-10-13
CmpDate: 2025-10-13

Chaturvedi A, R Ranjan (2025)

Strategies for plant-virus disease management from gene editing to nanotechnology.

Physiology and molecular biology of plants : an international journal of functional plant biology, 31(8):1293-1308.

Plant viruses are a global agricultural threat and can result in large financial losses. The globalization of agriculture and its international trading are the major causes of viruses and their vectors expanding to new environmental niches. Conventional methods are not effective in managing virus infection. To mitigate the virus spread, one of the cutting-edge biotechnological approaches, CRISPR/Cas is a robust tool. CRISPR/Cas is a powerful genome editing technology, and provides a highly specific viral genome targeting. Additionally, nanotechnology is a cutting-edge method for mitigating plant viruses. Nanoparticles in biosensors aid in the early identification of plant viruses, hence preventing the spread of disease in the future. Moreover, nanoparticles can also be used as a flexible delivery system. Nanoparticle-mediated delivery of dsRNA ensures minimal off-target while maintaining biosafety. This review explores the genome editing approach and nanotechnological strategies for ensuring sustainable agriculture practices for virus disease management, focusing on biosafety, efficacy, and practical applicability. It also aims to provide a clear insight into the limitations and strengths of each approach.

RevDate: 2025-10-11
CmpDate: 2025-10-11

Wu M, Wang F, Wang Y, et al (2025)

Mismatch-introduced crRNA guided PCR-CRISPR/Cas12a platform improves EGFR point mutation detection in single tumor cell.

Mikrochimica acta, 192(11):727.

Dynamic monitoring of epidermal growth factor receptor (EGFR) mutations is essential for the early identification of resistance and treatment adaptation. Single-cell heterogeneity analysis is crucial for precision cancer medicine, yet sensitive and specific detection methods for individual tumor cells remain challenging. Here, we develop a PCR-CRISPR/Cas12a platform enhanced by the incorporation of mismatched base in crRNA at specific site for single-cell point mutation detection. This platform demonstrated high specificity and sensitivity, detecting point mutation at a frequency of 0.1% and in as low as 1.02 ng of genomic DNA, which represents an improvement over the amplification-refractory mutation system PCR (ARMS-PCR). Notably, the accuracy of the platform is highly consistent with next-generation sequencing (NGS), as evidenced by Kappa test values surpassing 0.9. By utilizing a conical-pore membrane with optimized porosity for single circulating tumor cell (CTC) enrichment, our platform enables point mutations detection in individual tumor cells, offering potential enhancements in precision and reliability for EGFR mutation analysis. This novel methodology holds potential for more accurate and personalized cancer treatment strategies.

RevDate: 2025-10-11

Zhang J, Dai P, Weng Z, et al (2025)

Efficient CRISPR/Cas-based gene editing in cotton induced by cotton leaf crumple virus.

Journal of biotechnology pii:S0168-1656(25)00247-0 [Epub ahead of print].

Plant viral vectors can replicate autonomously and spread within host cells, making them an ideal tool for the delivery of CRISPR/Cas gene-editing elements. Here, we constructed a cotton CRISPR/Cas system mediated by cotton leaf crumple virus (CLCrV) as a delivery vector. We first inoculated Pro35s::Cas9 and ProUbi::Cas9 cotton with sgRNAs designed to knock out GhAGL16, GhPDS, and GhCLA1 target genes via the CLCrV vector and then compared the effects of these two transformation receptors on the editing efficiency of the same target genes. We next explored the feasibility of simultaneous multi-target editing in cotton via pooled virus inoculation. Finally, we used a cotton line overexpressing nCas9-TadA7.10 as the transformation receptor to explore the feasibility of CLCrV-mediated adenine base editing and verify the specificity of gene editing in this system. Mutation detection and deep sequencing revealed that the Pro35s::Cas9 and ProUbi::Cas9 cotton lines did not differ significantly in editing efficiency, and both could be used as successful receptors for the CLCrV-mediated Cas9 system. Pooled inoculation with CLCrV-sgRNAs enabled the simultaneous editing of multiple target genes in Pro35s::Cas9 and ProUbi::Cas9 cotton, although this approach had somewhat lower editing efficiency than inoculation with single sgRNAs. The CLCrV-mediated adenine base-editing system enabled A-to-G conversion at target sites in cotton GhPEBP and showed high gene-editing specificity. In summary, this study establishes an efficient CLCrV-mediated CRISPR system in cotton, providing a powerful technical tool for editing of multiple target genes and base editing.

RevDate: 2025-10-11
CmpDate: 2025-10-11

Gupta I, Sharma JG, T Kaul (2025)

Nanoparticle-driven CRISPR-Cas9 genome editing: a new frontier in crop improvement.

Molecular biology reports, 52(1):1015.

The agricultural sector has experienced unpredictable and extreme climatic aberrations, which have severely hampered food production. However, applying advanced nanotechnological approaches in agriculture will be crucial for ensuring more secure and sustainable food production. The revolutionizing phyto-nanotechnology enables the precise delivery of biomolecules i.e., nucleotides and proteins, and the modulated release of agrochemicals, including fungicides and pesticides. In addition, CRISPR-Cas-based genetic engineering holds great promise for food security, agriculture, and environmental sustainability. However, its application in plants faces challenges, including cargo delivery, germline transformation, species independence, HDR efficiency, and overall editing effectiveness. Nanomaterials offer innovative and effective solutions to overcome these challenges by enhancing genome-editing tools precision, efficiency, and delivery mechanisms. This review examines the key limitations of CRISPR-mediated plant genome editing and how nanoparticle technologies can overcome them. We highlight essential nanotech innovations that enhance genome modification, paving the way for a faster, more versatile genomic toolbox in plant biotechnology.

RevDate: 2025-10-13
CmpDate: 2025-10-11

Frey T, Kandolf-Zumpf C, Kaempf A, et al (2025)

T cell receptor associated transmembrane adaptor 1 (TRAT1) modulates human Th17 and Treg responses via PI3-kinase and STAT dependent mechanisms.

Cell communication and signaling : CCS, 23(1):431.

BACKGROUND: Adaptor proteins associated with the T cell receptor (TCR) play critical roles in regulating immune responses by Translating receptor engagement into intracellular signals. T cell Receptor Associated Transmembrane Adaptor 1 (TRAT1) has been implicated in modulating TCR complex stability, but its functional role in human effector and regulatory CD4[+] T cell subsets remains poorly understood. This study aimed to elucidate the role of TRAT1 in regulating T cell activation and differentiation, particularly in helper T cells function and regulatory T cells.

METHODS: Primary human CD4⁺ T cells, including thymus-derived and induced regulatory T cells (Treg), were genetically modified by CRISPR/Cas9-mediated gene deletion or retro-/lentiviral overexpression of TRAT1. Functional assays, flow cytometry, cytokine quantification, and RNA sequencing were performed to evaluate modulation of T cell functions. Mechanistic studies included pathway inhibition using small molecules and phospho-protein analysis. The influence of TRAT1 on Treg function was further assessed in a CAR Treg context in an immune organoid model of allo-rejection.

RESULTS: Thymus-derived, TGFb-induced and FOXP3-transgenic Treg displayed reduced expression of TRAT1 compared to effector T cells, which showed pronounced up-regulation of TRAT1 following activation. In effector T cells, deletion of TRAT1 led to increased signaling through the phosphoinositide 3-kinase pathway resulting in enhanced proliferation and increased expression of activation markers. However, this was accompanied by reduced production of interleukin-17, which was linked to elevated activity of STAT6 as shown by inhibition experiments using small molecule inhibitors. Overexpression and CRISPR/Cas9-mediated knockout of TRAT1 in Treg enhanced suppression of CD4⁺ target cells via up-regulation of LAP/GARP but reduced suppression of CD8⁺ target cells, an effect confirmed in HLA-A2-specific CAR Treg in a human organoid model of allo-rejection.

CONCLUSIONS: TRAT1 acts as a dual regulator of human CD4⁺ T cell function, limiting effector activation through modulation of intracellular signaling and supporting regulatory T cell-mediated suppression. These findings reveal a novel mechanism of immune regulation with potential implications for the development of cell-based immunotherapies.

RevDate: 2025-10-10
CmpDate: 2025-10-10

Das A, Debnath S, Pramanik S, et al (2025)

Bio-digital feedback loop systems: a synergistic integration of predictive genomics, genome editing, and AI-driven phenomic synthesis for next-generation edible and medicinal mushroom breeding.

Antonie van Leeuwenhoek, 118(11):168.

Edible mushrooms face persistent challenges in yield optimization, bioactive compound production, and climate resilience that conventional breeding methods struggle to address. Traditional approaches such as cross-breeding, protoplast fusion, and mutagenesis are limited by genetic noise, laborious screening, and unstable trait inheritance. This review proposes a transformative paradigm built upon converging advances in molecular biology and data science: the bio-digital feedback loop (BDFL) framework, integrating multi-omics, CRISPR-engineered chassis strains, and predictive phenomics for precision mushroom breeding. Our framework employs multi-omics to decipher gene networks governing critical traits, such as substrate degradation enzymes, developmental synchrony regulators, and secondary metabolite pathways. CRISPR-Cas9 and synthetic biology tools then deploy these insights to verify and design modular gene circuits in pre-engineered "plug-and-play" chassis strains, enabling conflict-free stacking of desirable traits. Artificial intelligence serves as the linchpin, not only automating high-throughput phenotyping through advanced imaging but also accelerating the entire breeding cycle by predicting trait heritability from omics data and optimizing the design of CRISPR guide RNAs and genetic constructs for efficient editing. The BDFL we describe iteratively refines strains by feeding phenomics data back into AI algorithms, enabling rapid trait optimization cycles. This transcends the trial-and-error limitations of classical methods, accelerating development of climate-smart mushrooms for circular bioeconomies including strains engineered to thrive on agricultural waste, overproduce immunomodulatory compounds, or resist emerging pathogens. The integration of predictive genomics, AI-driven phenomics, and CRISPR-edited chassis strains heralds a new era of precision mycology, where mushrooms are computationally designed as sustainable solutions for global food security, pharmaceutical innovation, and ecological resilience, ultimately transforming fungi into programmable biological factories tailored to address pressing agricultural and ecological challenges.

RevDate: 2025-10-13
CmpDate: 2025-10-13

Chen T, Hu G, Fu J, et al (2025)

Structural Basis of PAM-Induced Conformational Changes in SpCas9: A Molecular Dynamics Study.

Journal of chemical information and modeling, 65(19):10624-10633.

As the most widely utilized CRISPR gene-editing enzyme, SpCas9 has been extensively studied and applied. However, its strict dependence on the canonical NGG PAM sequence significantly restricts its targeting scope. Although recent research has successfully engineered SpCas9 variants capable of recognizing noncanonical (non-NGG) PAMs, these variants still exhibit limitations when binding noncanonical PAMs, including substantially reduced cleavage efficiency. To elucidate the molecular mechanisms underlying noncanonical PAM recognition by SpCas9, we employed molecular dynamics simulations to compare the structural differences within the Cas9-gRNA-DNA ternary complex when bound to various PAM sequences. Our analysis revealed significant conformational changes within SpCas9 upon engagement with noncanonical PAMs and uncovered the regulatory mechanisms underpinning these changes. We further identified key dynamic determinants governing the extensive conformational transitions occurring during the noncanonical PAM binding process. These findings provide insights into the dynamic landscape of noncanonical PAM recognition, offering crucial mechanistic guidance for designing efficient, PAM-compatible Cas9 variants.

RevDate: 2025-10-13
CmpDate: 2025-10-13

Yamazaki M, Ueta A, Nakanishi T, et al (2025)

Involvement of impaired phosphate production and aberrant extracellular ATP signaling in the pathogenesis of hypophosphatasia: Analysis of ALPL-Knockout human iPS cell models.

Bone, 201:117629.

Hypophosphatasia (HPP) is caused by inactivating variants of ALPL, the gene encoding tissue non-specific alkaline phosphatase (TNSALP). In order to deepen our understanding of the pathogenic mechanisms of HPP, we herein generated ALPL-knockout (KO) human induced pluripotent stem (iPS) cells by applying CRISPR/Cas9-mediated gene deletion to an iPS clone derived from a healthy subject. We analyzed two ALPL-KO clones, one ALPL-hetero KO clone, and a control clone isogenic except for ALPL. In an osteogenic culture using β-glycerophosphate, which generates inorganic phosphate (Pi) by TNSALP-mediated degradation, ALPL-KO clones showed impaired mineralization, elevated levels of extracellular pyrophosphate (PPi), and reduced levels of extracellular Pi. Osteogenic induction using 3 mM Pi instead of β-glycerophosphate rescued the decreased content of hydroxyapatite in ALPL-KO cells despite the still high levels of extracellular PPi; however, abnormal distribution of hydroxyapatite was noted. Osteoblast lineage cells differentiated from ALPL-KO iPS clones showed the up-regulation of SPP1 and the down-regulation of ANKH and the genes for type III sodium/phosphate co-transporters in the culture using β-glycerophosphate, but not when 3 mM Pi was used. Extracellular ATP levels were elevated in osteoblast lineage cells derived from ALPL-KO iPS clones in both culture conditions, which was associated with the down-regulation of P2X7 encoding a purinergic receptor. Moreover, osteoblast lineage cells differentiated from ALPL-KO iPS clones in the culture using β-glycerophosphate showed a change in cellular response to extracellular Pi. These results suggest that the reduced local production of extracellular Pi and aberrant ATP signaling play substantial roles in the pathogenesis of HPP.

RevDate: 2025-10-13
CmpDate: 2025-10-13

Mikdar M, Shabani E, Grüring C, et al (2025)

Sialyl-T Antigen: A Novel Red Blood Cell Determinant for Plasmodium falciparum Invasion.

American journal of hematology, 100(11):1952-1962.

Malaria continues to pose significant health challenges globally despite advances in control measures. Plasmodium falciparum, the parasite responsible for most severe malaria cases, uses multiple redundant invasion pathways to enter the red blood cell (RBC) during the blood stage of infection. Through a combination of RNA interference screening in erythroid cells and validation by CRISPR/Cas9-mediated knockout in primary human hematopoietic stem cells, we identified the glycosyltransferase Core 1 Synthase Glycoprotein-N-Acetylgalactosamine 3-Beta-Galactosyltransferase 1 (C1GALT1) as a novel host determinant for P. falciparum invasion. Analyses of C1GALT1-deficient cultured reticulocytes and RBCs with the glycophorin A/B-null MkMk blood group phenotype demonstrated that the C1GALT1-dependent α(2-3) sialic acid structures within mucin-type O-glycans are crucial for efficient invasion of both sialic acid-dependent and sialic acid-independent P. falciparum strains, but not the primate malaria parasite Plasmodium knowlesi. However, different P. falciparum parasite strains exhibit variable dependencies on distinct sialic acid configurations on the RBC surface. Overall, our findings highlight a key role for RBC glycans in malaria infection.

RevDate: 2025-10-12
CmpDate: 2025-10-12

Greisle T, Kunze I, Wang X, et al (2025)

Generation of a Flattop-T2A-H2B-Venus x C-peptide-mCherry double reporter human iPSC line to monitor WNT/Planar cell polarity pathway activity.

Stem cell research, 88:103838.

Deriving functional β-cells from human induced pluripotent stem cells (hiPSCs) holds potential for cell replacement therapy, disease modeling, and drug testing in diabetes research. Wnt/Planar cell polarity (PCP) signaling is crucial for endocrine cell development and β-cell maturation in murine models and can be tracked by the expression of the tissue-specific effector gene Flattop. Here, we report the generation of a human fluorescent FLTP/CFAP126 (Flattop-T2A-H2B-Venus) and FLTP-Insulin (Flattop-T2A-H2B-Venus x C-peptide-mCherry) double reporter by CRISPR/Cas9 gene editing. These hiPSC reporter lines allow monitoring of WNT/PCP signaling during endocrine cell formation and studying its role in β-cells in a human model system.

RevDate: 2025-10-12
CmpDate: 2025-10-12

Madhusudhan K, Padmanaban A, VD Parvathi (2025)

Early detection of Parkinson's disease via aptamer-CRISPR platform.

Neuroscience, 586:163-195.

Parkinson's disease (PD) is a neurodegenerative disorder with a worldwide prevalence of around 9.4 million that is expected to double by 2040. It's extended prodromal phase allows irreversible neuronal loss to occur before manifestation of symptoms. Current diagnostic approaches, primarily based on clinical assessment and neuroimaging, are often delayed and lack sensitivity in the early stages, highlighting the need for an early, conclusive, and minimally invasive test. This review focuses on the integration of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) diagnostics with aptamers to detect PD-associated biomarkers. CRISPR systems utilising Cas12 and Cas13 enzymes offer high specificity and collateral cleavage activity that can be harnessed for signal amplification. Aptamers are short, single-stranded oligonucleotides that can be designed to identify nucleic and non-nucleic acid targets. Their fusion with CRISPR may enable the sensitive detection of key PD biomarkers such as α-Syn, dopa decarboxylase, glial fibrillary acidic protein, and neurofilament light chain in biological fluids like blood, CSF, urine, saliva, and sweat. We explore various strategies for aptamer-CRISPR integration, detection, and multiplexing techniques for parallel biomarker detection. We also examine existing diagnostic platforms and discuss barriers to clinical translation. Ultimately, aptamer-CRISPR diagnostics could represent a powerful, next-generation approach for early PD detection.

RevDate: 2025-10-12
CmpDate: 2025-10-12

Watts JL, Willeke L, RW Stottmann (2025)

Mouse variants in Taf1c result in reduced survival to birth.

Developmental biology, 528:143-151.

Ribosome biogenesis is a key cellular function and disruptions in this process can lead to congenital anomalies or "ribosomopathies" with varying phenotypes including craniofacial malformations and neurodevelopment symptoms. Classically, the mouse is a robust model to understand the molecular mechanisms underlying ribosomopathies to further elucidate human pathogenesis. We identified novel compound heterozygous missense variants in the TATA-box binding protein associated factor, RNA polymerase I subunit C (TAF1C) locus in a patient with some phenotypes consistent with ribosomopathies. TAF1C encodes a subunit of the SL1 complex which is critical for the RNA PolI complex to initiate ribosomal RNA transcription. We hypothesized that functional TAF1C is required at developmental stages critical for craniofacial and neurodevelopment. To test this hypothesis, we created mouse Taf1c variants orthologous to the human variants using CRISPR-CAS9 technology (Taf1c[R202Q] and Taf1c[S428A]). We also created an 11bp deletion to complement the missense variants (Taf1c[11bpdel]). We created multiple allelic combinations to determine the roles for Taf1c in survival and craniofacial development. Homozygous mice for any of these novel variants were underrepresented at organogenesis stages. We did not observe craniofacial anomalies in any surviving mice. Our results suggest that these specific TAF1C variants are not the cause of any human phenotype present in the patient motivating the study. However, we showed that Taf1c is required for embryonic survival and our studies contribute to knowledge about the role of ribosome biogenesis machinery throughout organogenesis.

RevDate: 2025-10-12
CmpDate: 2025-10-12

Herbrich S, Ramachandran H, Seibt A, et al (2025)

CRISPR/Cas9-mediated editing of COQ4 in induced pluripotent stem cells: A model for investigating COQ4-associated human coenzyme Q10 deficiency.

Stem cell research, 88:103825.

Pathogenic variants in the gene COQ4 cause primary coenzyme Q10 deficiency, which is associated with symptoms ranging from early epileptic encephalopathy up to adult-onset ataxia-spasticity spectrum disease. We genetically modified commercially available wild-type iPS cells by using a CRISPR/Cas9 approach to create heterozygous and homozygous isogenic cell lines carrying the disease-causing COQ4 variants c.458C > T, p.Ala153Val and c.437T > G, p.Phe146Cys, respectively. All iPSCs lines exhibited a normal cell morphology, expression of pluripotency markers, and the ability to differentiate into the three primary germ layers. The COQ4-deficient cell lines will provide a helpful tool to investigate the disease mechanism and to develop therapeutic strategies.

RevDate: 2025-10-12
CmpDate: 2025-10-12

Raabe J, Lewandowski V, Fuchs S, et al (2025)

Generation of a biallelic NRAP-knockout mutant from a human iPSC line.

Stem cell research, 88:103829.

Cardiomyopathies, a leading cause of mortality, are associated with dysfunctional intercalated discs, which connect neighbouring cardiomyocytes and ensure proper contractility. In human cardiac diseases, loss-of-function mutations of the intercalated disc-associated protein Nebulin-Related Anchoring Protein (NRAP) have been reported. NRAP plays a crucial role in myofibril assembly and mechanotransduction, however, its regulatory functions remain unclear. To investigate the effects of NRAP loss-of-function in cardiac disease, a human induced pluripotent stem cell (hiPSC) line was generated carrying a biallelic NRAP-knockout (KO) using the CRISPR-Cas9 technology. Control and mutant cell lines were assessed for karyotype integrity, pluripotency, off-target effects, mycoplasma contamination, and differentiation into ectoderm, mesoderm, and endoderm. This hiPSC line provides a valuable tool to study how NRAP modulates cardiac function and contributes to disease progression.

RevDate: 2025-10-12
CmpDate: 2025-10-12

Ran Y, Ruan J, Wang Y, et al (2025)

Generation of a PHF19 knockout human embryonic stem cell line by CRISPR/Cas9 system.

Stem cell research, 88:103824.

PHD finger protein 19 (PHF19) is a polycomb protein that promoted cardiac hypertrophy via epigenetic targeting SIRT2. To determine the role of PHF19 in myocardial hypertrophy, we established a large fragment knockout model of PHF19 gene in human embryonic stem cells (hESCs-H7) using the CRISPR/Cas9 system based on a vector. This PHF19-KO cell line has a normal karyotype, classical human pluripotent stem cell morphology, strong pluripotency, and significantly reduced PHF19 gene expression, which will become a useful tool for further in-depth research on the pathogenesis of PHF19 gene deficiency induced myocardial hypertrophy.

RevDate: 2025-10-12
CmpDate: 2025-10-12

Kim JW, Jo S, Kang EH, et al (2025)

Generation of human embryonic stem cell line expressing dCas9-TET1 fusion protein for epigenetic editing.

Stem cell research, 88:103811.

CRISPR-based epigenome editing systems can induce site-specific transcriptional activation or repression of target genes. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) is a transcriptional activation effector involved in the cytosine demethylation of CpG dinucleotides in gene regulatory regions. In this study, we generated a human embryonic stem cell line that stably expresses catalytically dead Cas9 (dCas9) fused to the catalytic domain of TET1 via lentiviral transduction. This cell line can be used for locus-specific transcriptional activation in combination with single guide RNAs and serves as a valuable tool for epigenetic regulation in stem cell and organoid models.

RevDate: 2025-10-12
CmpDate: 2025-10-12

Cota-Coronado A, Manning M, Kim DH, et al (2025)

Generation of two Betacellulin CRISPR-Cas9 knockout hiPSC lines to study the affected EGF system paradigm in schizophrenia.

Stem cell research, 88:103808.

Several members of the epidermal growth factor (EGF) family have been implicated in the biology of schizophrenia (Ketharanathan et al., 2024). The EGF-related ligand, Betacellulin (BTC), plays an important role in the proliferation and differentiation of neural stem cells and our group found markedly reduced BTC levels in patients with schizophrenia. Nevertheless, the interplay of affected BTC and its participation in neural specification and neurodevelopment remains elusive. We generated Knockout (KO) - BTC clones from an existing hiPSC line through CRISPR/Cas9-mediated modification. Furthermore, we validated BTC-KO through genotyping/sequencing, FACS and Western Blot. Finally, we demonstrated trilineage differentiation potential in vitro.

RevDate: 2025-10-10
CmpDate: 2025-10-10

Shi Z, TL Cheng (2025)

UGI relocation inside Cas9 reduces Cas9 dependent off target effects in cytosine base editors.

Scientific reports, 15(1):35518.

Cytosine base editors (CBEs) achieve precise C-to-T conversions by addition of uracil DNA glycosylase inhibitor (UGI) with Cas9 nickase (nCas9) and cytidine deaminase, and the conventional fusion at the nCas9 carboxyl terminus effectively inhibits uracil excision repair to enhance editing efficiency. However, despite potent on-target activity, classical CBEs exhibit significant Cas9-dependent DNA off-target effects that necessitate optimization for future applications. Here we present a strategic UGI relocation through internal fusion within the nCas9 architecture. This spatial reorganization maintains comparable on-target editing efficiency while substantially reducing Cas9-dependent DNA off-target activity. Our findings establish an alternative engineering paradigm to develop high-fidelity CBEs, offering an improved platform for widespread genome editing applications.

RevDate: 2025-10-10
CmpDate: 2025-10-10

Zhang H, Li M, Wang G, et al (2025)

Paired NLRs originated from Triticum dicoccoides coordinately confer resistance to powdery mildew in wheat.

Nature communications, 16(1):9040.

Wheat has evolved diverse resistance genes against powdery mildew, typically controlled by single-gene-encoded proteins. Here, we report the map-based cloning of PmWR183, a resistance locus encoding two adjacent NLR proteins (PmWR183-NLR1 and PmWR183-NLR2) from wild emmer wheat. Stable transformation and CRISPR/Cas9 knockout experiments demonstrate that the two NLRs function cooperatively: neither gene alone confers resistance, but their co-expression restores immunity, while disruption of either gene abolishes resistance. PmWR183 mediates a developmental stage-dependent response, with susceptibility at the seedling stage and strong resistance at the adult stage. Protein interaction assays reveal constitutive association of PmWR183-NLR1 and PmWR183-NLR2, supporting their cooperative role. Geographical and haplotype analyses show the locus originates from wild emmer and is rare in cultivated wheat, exhibiting at least nine haplotypes. Together, our findings uncover a rare NLR gene pair conferring effective resistance to powdery mildew, providing valuable resources for wheat breeding.

RevDate: 2025-10-10
CmpDate: 2025-10-10

Nie Y, Wang W, Wang N, et al (2025)

PCR-CRISPR/Cas12a-based fluorescence and lateral flow dipstick platforms for efficient screening of CD71 biallelic mutants.

Analytica chimica acta, 1376:344622.

CRISPR/Cas9 technology plays a pivotal role in gene editing and has been extensively utilized in gene function studies, disease modeling, and gene therapy. However, efficient and accurate detection of CRISPR/Cas9-induced mutants remains a challenge due to the complexity, time-consuming nature, and high cost of existing detection methods. Meanwhile, CRISPR/Cas12a systems have been widely applied in molecular diagnostics due to the non-specific trans-cleavage activity of Cas12a, yet their application in detecting CRISPR/Cas9-induced mutations remains limited. In this study, we developed a PCR-CRISPR/Cas12a-based method to enable the rapid and accurate screening of CD71 biallelic mutants. The detection system was mainly composed of CRISPR RNA specific to the CD71 gene-editing site, Cas12a protein, target DNA, and ssDNA probes for fluorescence or lateral flow dipstick assays. The system demonstrated high specificity in distinguishing CD71 biallelic mutants, with validation through TA cloning confirming its accuracy. Additionally, the method exhibited high sensitivity, establishing it as an efficient tool for biallelic mutated cell clone screening. These findings underscore the potential of PCR-CRISPR/Cas12a as a rapid, sensitive, and cost-effective approach for the precise identification of biallelic mutants, contributing to advancements in gene-editing research and molecular diagnostics.

RevDate: 2025-10-10
CmpDate: 2025-10-10

Cao R, Wang S, Guo Q, et al (2025)

DNAzyme-driven SDA reaction regulates CRISPR/Cas12a for highly sensitive and selective analysis of underexpressed miRNA.

Analytica chimica acta, 1376:344620.

Underexpressed microRNA (miRNA) exerts a pivotal influence across a spectrum of physiological and pathological processes, with their role being particularly pronounced in the incipient stages of tumorigenesis. However, there are several challenges to analyzing these underexpressed miRNAs for their lower abundance and relative decreases in some cancers. Here, we developed a novel fluorescence biosensor based on the DNAzyme-driven strand displacement amplification (SDA) regulating CRISPR/Cas12a for the sensitive and selective detection of underexpressed miRNA, using prostate cancer-associated miR-222 as a proof-of-concept. This study innovatively expanded the application of DNAzyme substrates, designed as templates to trigger SDA and CRISPR/Cas12a reaction, which could effectively generate a positive signal output for detecting underexpressed miRNA. In the absence of miR-222, DNAzyme formation was blocked, allowing the complete substrate to activate SDA, which generated ssDNA that triggered CRISPR/Cas12a trans-cleavage activity to produce a strong fluorescent signal. In contrast, intact DNAzymes (in the presence of miR-222) cleaved the substrates into short DNA fragments, preventing SDA and CRISPR/Cas12a activation, thereby maintaining the sensor in a low fluorescent state. The biosensor demonstrated a linear detection range spanning from 0.1 pmol/L to 1 nmol/L, accompanied by a detection limit of 33.5 fmol/L. Moreover, it exhibited excellent specificity and anti-interference capacity, enabling the successful detection of miR-222 in blood samples. This "DNAzyme-SDA-CRISPR" fluorescence strategy offers a effective, programmability and scalable solution for detecting underexpressed miRNAs in early cancer screening, which is expected to become a powerful tool in early tumor diagnostics and precision therapy.

RevDate: 2025-10-11
CmpDate: 2025-10-11

Hu M, Zhou C, Li M, et al (2026)

From 3D culture to clinical decision-making: Systematic innovations in breast cancer organoids.

Biomaterials advances, 179:214528.

Breast cancer is a malignant tumour with high heterogeneity. Traditional research models rely mainly on 2D cell culture and patient-derived tumour xenografts (PDXs). However, these models have limited use in clinical trials because of their shortcomings in mimicking the tumour microenvironment and preserving the genetic background. In recent years, organoids, emerging models capable of self-organizing to form 3D structures in vitro, have become key tools for overcoming the traditional dilemma and are promising alternatives for breast cancer research. This review integrates cutting-edge technologies such as organ-on-a-chip and CRISPR/Cas9 gene editing to summarize the multidimensional generation strategy of breast cancer organoids and discusses the clinical value of translation from diagnosis to therapy. Compared with existing studies, this review provides a systematic solution from "model generation" to "precision medicine" for breast cancer research, and the hope is that this review will pave the way for the further development of organoids.

RevDate: 2025-10-11
CmpDate: 2025-10-11

Deng D, Yi X, Wen W, et al (2025)

The role of the transformer gene in sex determination and its employment in CRISPR/Cas9-based homing gene drive in the global fruit pest Drosophila suzukii.

Insect biochemistry and molecular biology, 184:104406.

Sex determination of Diptera is established by the cascade genes such as transformer (tra), though the primary signals for sex determination differ among different insects. Here, we report the isolation, expression and function of tra gene in an invasive pest, Drosophila suzukii, and study the potential use of the D. suzukii tra (Dstra) gene in CRISPR/Cas9-based homing gene drive for genetic-based pest management. The Dstra gene is highly conserved in structure and has a sex-specific transcript. To test the function of this gene in sex determination, Dstra dsRNA was injected into embryos. Almost all XX embryos developed into masculinized phenotypic male adults with intersex morphology. Abnormal ovaries were revealed in XX pseudomales upon dissection. Based on the necessary role of Dstra for female development, we developed and evaluated a homing gene drive that targets Dstra in D. suzukii. The drive component consisting of multiplex Dstra single guide RNAs and Cas9 with Dsvasa promoter was introduced into the Dstra locus. Abnormal development of both the external genitalia and gonads was observed in G0 and G1 chromosomal female adults that expressed the male-specific doublesex (dsx) transcript. Interestingly, knocking out Dstra led to significantly reduced fertility in adults of corresponding sex and moderate transmission rates of the DsRed gene (63.54 %) were observed. Our results not only confirm the conserved function of the Dstra gene in sex determination, but also highlight the potential of sex conversion-based suppression gene-drive strategy targeting the Dstra gene in controlling of D. suzukii populations.

RevDate: 2025-10-10
CmpDate: 2025-10-10

Shi S, Qin F, Wu J, et al (2025)

Ultrasensitive single-particle collision electrochemical platform employing CRISPR/Cas12a for ctDNA biosensing.

Analytica chimica acta, 1376:344590.

Circulating tumor DNA (ctDNA) is a characteristic tumor biomarker used for cancer diagnosis, treatment, and prognosis. However, the low concentration of ctDNA in peripheral blood and the interference of complex matrices with signals make the detection of ctDNA extremely challenging. Single-particle collision electrochemistry (SPCE) has been widely used in bioanalysis due to its advantages such as high throughput, simple operation, high sensitivity, and low detection limit. In this work, a novel SPCE biosensor for the rapid detection of ctDNA was developed by combining CRISPR/Cas12a with excellent cleavage activity and magnetic beads (MBs) with good separation and enrichment capabilities. The trans-cleavage ability of CRISPR/Cas12a can only be triggered in the presence of target ctDNA to effectively cleave the ssDNA2 on the surface of Ag NPs-ssDNA2 within 1 h, thereby activating the collision activity of silver nanoparticles (Ag NPs). ctDNA was quantified by the collision frequency of Ag NPs. The detection limit of the developed SPCE biosensor for ctDNA was as low as 4.2 fM, and the linear range was 10 fM-1 nM. In addition, MBs allow the biosensor to detect ctDNA in complex samples by directly sampling from complex matrices, with excellent sensitivity and specificity, demonstrating the great potential of the developed SPCE biosensor in the detection of patient samples.

RevDate: 2025-10-10

Patel RR, Arun PP, Singh SK, et al (2025)

Overcoming Antimicrobial Resistance: Phage Therapy as a Promising Solution to Combat ESKAPE Pathogens.

International journal of antimicrobial agents pii:S0924-8579(25)00195-5 [Epub ahead of print].

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

RevDate: 2025-10-10

Henriques WS, Bowman J, Hall LN, et al (2025)

Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci.

Structure (London, England : 1993) pii:S0969-2126(25)00350-8 [Epub ahead of print].

Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify and determine structures of Cas1 and the Cas2/3 fusion proteins from Pseudomonas aeruginosa at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.

RevDate: 2025-10-10
CmpDate: 2025-10-10

Guo Y, Xu M, Xue H, et al (2025)

Genome-wide CRISPR screen identifies splicing factor SF3B4 in driving hepatocellular carcinoma.

Science advances, 11(41):eadw7181.

Although genome sequencings have recognized many cancer-associated genes in hepatocellular carcinoma (HCC), distinguishing their functional effect remains challenging. Leveraging on a genome-wide CRISPR knockout (KO) screening, we uncovered spliceosome factors as major survival essential genes in HCC and up-regulations of ferroptosis suppressors [particularly glutamate-cysteine ligase catalytic subunit (GCLC)] in lenvatinib resistance. Our KO screen in patient-derived HCC organoid showed splicing factor 3b subunit 4 (SF3B4) to be top-ranked, conferring prosurvival signal in HCC organoid and driving tumorigenic potentials in both hepatic progenitor organoids and hydrodynamic tail vein injection HCC murine model. The combined RNA immunoprecipitation sequencing, long-read isoform sequencing, and transcriptome revealed characteristic splicing landscape regulated by SF3B4 and identified T-box transcription factor 3 (TBX3) variant TBX3+2a as a potent downstream effector. Our findings highlighted vital roles of SF3B4 in HCC cell survival and tumor progression, and the phenomenon of ferroptosis resistance in patients unresponsive to first-line agent lenvatinib.

RevDate: 2025-10-10

Zhang Y, Wu Y, Guo A, et al (2025)

Fluorescent biosensors for the detection of foodborne pathogenic bacteria in food: a comprehensive review.

Analytical methods : advancing methods and applications [Epub ahead of print].

Foodborne pathogenic bacterial contamination poses a major challenge to global food safety and public health, making the development of rapid, sensitive, and specific detection technologies critically important. Conventional methods are limited by their long turnaround time, complex operations, and reliance on large-scale instruments, making them unsuitable for on-site rapid detection. Fluorescent biosensors, which combine highly specific biological recognition elements with highly sensitive fluorescent signal output, demonstrate significant advantages in detecting foodborne pathogens. This review systematically summarizes recent advances in fluorescent biosensors for the detection of common foodborne pathogenic bacteria, with a focus on the application of signal amplification strategies such as functional nanomaterials, amplification techniques, CRISPR/Cas systems, and Argonaute proteins. Furthermore, it analyzes performance metrics including multiplex pathogen detection, real-time quantification, anti-interference capability, and on-site applicability. Finally, future development trends and challenges are discussed, aiming to provide insights for the innovation of food safety monitoring technologies.

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RJR Experience and Expertise

Researcher

Robbins holds BS, MS, and PhD degrees in the life sciences. He served as a tenured faculty member in the Zoology and Biological Science departments at Michigan State University. He is currently exploring the intersection between genomics, microbial ecology, and biodiversity — an area that promises to transform our understanding of the biosphere.

Educator

Robbins has extensive experience in college-level education: At MSU he taught introductory biology, genetics, and population genetics. At JHU, he was an instructor for a special course on biological database design. At FHCRC, he team-taught a graduate-level course on the history of genetics. At Bellevue College he taught medical informatics.

Administrator

Robbins has been involved in science administration at both the federal and the institutional levels. At NSF he was a program officer for database activities in the life sciences, at DOE he was a program officer for information infrastructure in the human genome project. At the Fred Hutchinson Cancer Research Center, he served as a vice president for fifteen years.

Technologist

Robbins has been involved with information technology since writing his first Fortran program as a college student. At NSF he was the first program officer for database activities in the life sciences. At JHU he held an appointment in the CS department and served as director of the informatics core for the Genome Data Base. At the FHCRC he was VP for Information Technology.

Publisher

While still at Michigan State, Robbins started his first publishing venture, founding a small company that addressed the short-run publishing needs of instructors in very large undergraduate classes. For more than 20 years, Robbins has been operating The Electronic Scholarly Publishing Project, a web site dedicated to the digital publishing of critical works in science, especially classical genetics.

Speaker

Robbins is well-known for his speaking abilities and is often called upon to provide keynote or plenary addresses at international meetings. For example, in July, 2012, he gave a well-received keynote address at the Global Biodiversity Informatics Congress, sponsored by GBIF and held in Copenhagen. The slides from that talk can be seen HERE.

Facilitator

Robbins is a skilled meeting facilitator. He prefers a participatory approach, with part of the meeting involving dynamic breakout groups, created by the participants in real time: (1) individuals propose breakout groups; (2) everyone signs up for one (or more) groups; (3) the groups with the most interested parties then meet, with reports from each group presented and discussed in a subsequent plenary session.

Designer

Robbins has been engaged with photography and design since the 1960s, when he worked for a professional photography laboratory. He now prefers digital photography and tools for their precision and reproducibility. He designed his first web site more than 20 years ago and he personally designed and implemented this web site. He engages in graphic design as a hobby.

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CRISPR-Cas

By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

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Collection of publications by R J Robbins

Reprints and preprints of publications, slide presentations, instructional materials, and data compilations written or prepared by Robert Robbins. Most papers deal with computational biology, genome informatics, using information technology to support biomedical research, and related matters.

Research Gate page for R J Robbins

ResearchGate is a social networking site for scientists and researchers to share papers, ask and answer questions, and find collaborators. According to a study by Nature and an article in Times Higher Education , it is the largest academic social network in terms of active users.

Curriculum Vitae for R J Robbins

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Curriculum Vitae for R J Robbins

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