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Isoform specific editing of the coxsackievirus and adenovirus receptor.
The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAREx7) exhibits basolateral localization, the eight exon-encoded isoform (CAREx8) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms, CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAREx8 cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized, and doxycycline-induction of CAREx8 restored sensitivity to adenovirus. These data reinforce the importance of CAREx8 in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.
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Tissue-specific (ts)CRISPR as an efficient strategy for in vivo screening in Drosophila.
Gene editing by CRISPR/Cas9 is commonly used to generate germline mutations or perform in vitro screens, but applicability for in vivo screening has so far been limited. Recently, it was shown that in Drosophila, Cas9 expression could be limited to a desired group of cells, allowing tissue-specific mutagenesis. Here, we thoroughly characterize tissue-specific (ts)CRISPR within the complex neuronal system of the Drosophila mushroom body. We report the generation of a library of gRNA-expressing plasmids and fly lines using optimized tools, which provides a valuable resource to the fly community. We demonstrate the application of our library in a large-scale in vivo screen, which reveals insights into developmental neuronal remodeling.
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CRISPR Takes the Front Seat in CART-Cell Development.
Chimeric antigen receptor T (CART)-cell immunotherapies have opened a door in the development of specialized gene therapies for hematological and solid cancers. Impressive response rates in pivotal trials led to the FDA approval of CART-cell therapy for certain hematological malignancies. However, autologous CART products are costly and time-intensive to manufacture, and most patients experience disease relapse within 1 year of CART administration. Additionally, CART-cell efficacy in solid tumors is extremely limited. CART-cell therapy is also associated with serious toxicities. Manufacturing difficulties, intrinsic T-cell defects, CART exhaustion, and treatment-associated toxicities are some of the current barriers to widespread adoption of CART-cell therapy. Genome editing tools such as CRISPR/Cas systems have demonstrated efficacy in further engineering CART cells to overcome these limitations. In this review, we will summarize the current approaches that use CRISPR to facilitate off-the-shelf CART products, increase CART-cell efficacy, and minimize CART-associated toxicities.
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Reducing phenolic off-flavors through CRISPR-based gene editing of the FDC1 gene in Saccharomyces cerevisiae x Saccharomyces eubayanus hybrid lager beer yeasts.
Today's beer market is challenged by a decreasing consumption of traditional beer styles and an increasing consumption of specialty beers. In particular, lager-type beers (pilsner), characterized by their refreshing and unique aroma and taste, yet very uniform, struggle with their sales. The development of novel variants of the common lager yeast, the interspecific hybrid Saccharomyces pastorianus, has been proposed as a possible solution to address the need of product diversification in lager beers. Previous efforts to generate new lager yeasts through hybridization of the ancestral parental species (S. cerevisiae and S. eubayanus) yielded strains with an aromatic profile distinct from the natural biodiversity. Unfortunately, next to the desired properties, these novel yeasts also inherited unwanted characteristics. Most notably is their phenolic off-flavor (POF) production, which hampers their direct application in the industrial production processes. Here, we describe a CRISPR-based gene editing strategy that allows the systematic and meticulous introduction of a natural occurring mutation in the FDC1 gene of genetically complex industrial S. cerevisiae strains, S. eubayanus yeasts and interspecific hybrids. The resulting cisgenic POF- variants show great potential for industrial application and diversifying the current lager beer portfolio.
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Engineering next-generation crops through CRISPR-mediated horizontal gene transfer.
Crops increasingly face overlapping stresses such as heat, drought, salinity, and pathogens that conventional breeding or genome editing rarely overcome in combination. To address this, we propose CRISPR-enabled horizontal gene transfer (CRISPR-HGT) as a programmable framework that recreates the evolutionary process by which plants historically acquired adaptive microbial genes. Microbial genes, refined under extreme environments, provide a naturally preadapted resource for multi-trait resilience. By integrating tools such as Cas12a, CasΦ, RNA-targeting, and dCas-based epigenome editors with AI-guided microbial gene discovery, CRISPR-HGT enables modular and inducible stress regulation. This approach shifts genome editing from allelic modification to evolution-guided design. We outline a conceptual pipeline spanning microbial gene mining to adaptive field deployment, highlighting the ecological, biosafety, and regulatory dimensions, from the European Union's cautious oversight to the UK's product-based framework. CRISPR-HGT thus introduces an evolution-informed paradigm for engineering crops that anticipate stress and sustain yield under climate uncertainty.
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CRISPRi repression of nonhomologous end-joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica.
In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical-producing yeast Yarrowia lipolytica. By using a multiplexed sgRNA targeting strategy, we demonstrate efficient repression of eight out of nine targeted genes to enhance HR. Strains with nonhomologous end-joining repressed were shown to have increased rates of HR when transformed with a linear DNA fragment with homology to a genomic locus. With multiplexed targeting of KU70 and KU80, and enhanced repression with Mxi1 fused to deactivated Cas9 (dCas9), rates of HR as high as 90% were achieved. The developed CRISPRi system enables enhanced HR in Y. lipolytica without permanent genetic knockouts and promises to be a potent tool for other metabolic engineering, synthetic biology, and functional genomics studies.
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SCEPTRE improves calibration and sensitivity in single-cell CRISPR screen analysis.
Single-cell CRISPR screens are a promising biotechnology for mapping regulatory elements to target genes at genome-wide scale. However, technical factors like sequencing depth impact not only expression measurement but also perturbation detection, creating a confounding effect. We demonstrate on two single-cell CRISPR screens how these challenges cause calibration issues. We propose SCEPTRE: analysis of single-cell perturbation screens via conditional resampling, which infers associations between perturbations and expression by resampling the former according to a working model for perturbation detection probability in each cell. SCEPTRE demonstrates very good calibration and sensitivity on CRISPR screen data, yielding hundreds of new regulatory relationships supported by orthogonal biological evidence.
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Detection and Modulation of DNA Translocations During Multi-Gene Genome Editing in T Cells.
Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes in human primary T cells and comprehensively evaluate the frequency and stability of the resulting translocations. A thorough translocation frequency analysis using three orthogonal methods (droplet digital PCR, unidirectional sequencing, and metaphase fluorescence in situ hybridization) yielded comparable results and an overall translocation rate of ∼7% between two simultaneous CRISPR-Cas9 induced edits. In addition, we show that chromosomal translocations can be reduced when using different nuclease combinations, or by the presence of a homologous single stranded oligo donor for multiplexed genome editing. Importantly, the two different approaches for translocation reduction are compatible with cell therapy applications.
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CRISPR-AsCas12a and dAsCas12a-Mediated Gene Knockout and Knockdown in Clostridioides difficile.
Clostridioides difficile (C. difficile) is a leading cause of antibiotic-associated diarrhea and severe colitis, yet its genetic manipulation has long been constrained by low DNA transfer efficiency and limited recombination systems. Recent advances in CRISPR-based technologies have revolutionized the genetic toolkit for this pathogen, enabling precise genome editing and transcriptional regulation. Among CRISPR nucleases, Cas12a offers distinct advantages over Cas9 for bacterial applications, including a smaller size, T-rich PAM recognition, single-crRNA requirement, and reduced toxicity, which enhances conjugation efficiency in genetically recalcitrant organisms. AsCas12a-based platforms have enabled large fragment deletions, multiplex editing, and rapid generation of marker-free mutants in C. difficile. Complementing these nuclease-active systems, nuclease-deactivated variants (dCas9 or dAsCas12a) support CRISPR interference (CRISPRi)-a reversible, tunable approach for transcriptional repression without altering genomic sequences. Compared to traditional mutagenesis, CRISPRi greatly accelerates functional genomics by enabling high-throughput screening and drug target discovery. Together, our lab has independently developed CRISPR-AsCas12a-mediated genome editing and dAsCas12a-based CRISPRi tools, providing complementary strategies to overcome longstanding genetic barriers in C. difficile. These tools open new avenues for system-level interrogation of virulence, antibiotic resistance, and host-pathogen interactions.
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Recent Advances and Therapeutic Strategies Using CRISPR Genome Editing Technique for the Treatment of Cancer.
CRISPR genome editing technique has the potential to target cancer cells in a precise manner. The latest advancements have helped to address one of the prominent concerns about this strategy which is the off-target integrations observed with dsDNA and have resulted in more studies being carried out for potentially safer and more targeted gene therapy, so as to make it available for the clinical trials in order to effectively treat cancer. CRISPR screens offer great potential for the high throughput investigation of the gene functionality in various tumors. It extends its capability to identify the tumor growth essential genes, therapeutic resistant genes, and immunotherapeutic responses. CRISPR screens are mostly performed in in vitro models, but latest advancements focus on developing in vivo models to view cancer progression in animal models. It also allows the detection of factors responsible for tumorigenesis. In CRISPR screens key parameters are optimized in order to meet proficient gene targeting efficiencies. It also detects various molecular effectors required for gene regulation in different cancers, essential pathways which modulate cytotoxicity to immunotherapy in cancer cells, important genes which contribute to cancer cell survival in hypoxic states and modulate cancer long non-coding RNAs. The current review focuses on the recent developments in the therapeutic application of CRISPR technology for cancer therapy. Furthermore, the associated challenges and safety concerns along with the various strategies that can be implemented to overcome these drawbacks has been discussed.
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