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Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6.
Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system. Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells. We describe a method to multiplex homologous recombination (HR) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins (RNPs). In addition, the use of reporter genes allows FACS-purification and tracking of cells that have had multiple alleles or loci modified by HR. We believe this method will enable broad applications not only to the study of human hematopoietic gene function and networks, but also to perform sophisticated synthetic biology to develop innovative engineered stem cell-based therapeutics.
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CRISPR-Cas9 Editing in Maize: Systematic Evaluation of Off-target Activity and Its Relevance in Crop Improvement.
CRISPR-Cas9 enabled genome engineering has great potential for improving agriculture productivity, but the possibility of unintended off-target edits has evoked some concerns. Here we employ a three-step strategy to investigate Cas9 nuclease specificity in a complex plant genome. Our approach pairs computational prediction with genome-wide biochemical off-target detection followed by validation in maize plants. Our results reveal high frequency (up to 90%) on-target editing with no evidence of off-target cleavage activity when guide RNAs were bioinformatically predicted to be specific. Predictable off-target edits were observed but only with a promiscuous guide RNA intentionally designed to validate our approach. Off-target editing can be minimized by designing guide RNAs that are different from other genomic locations by at least three mismatches in combination with at least one mismatch occurring in the PAM proximal region. With well-designed guides, genetic variation from Cas9 off-target cleavage in plants is negligible, and much less than inherent variation.
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The Template-Jumping Editing Approach in F9-Associated Hemophilia B Gene Therapy.
Hemophilia B is a hereditary bleeding disorder caused by mutations localized throughout the F9 gene. Existing gene therapy products containing AAV vectors have significant limitations. Replacement therapy with coagulation factor FIX infusions is not an optimal way of treatment, as patients still have periodic bleeding and require frequent transfusions. Moreover, approximately 5% of adult patients with hemophilia B develop inhibitory antibodies to recombinant forms of FIX. Therefore, it is important to develop universal CRISPR/Cas gene therapy approaches for F9 editing using non-viral delivery systems to enable gene reversion to a functional sequence at an early stage of disease development and establishment of the patients' immune system. In this study, a unique approach of F9 prime-editing was tested for the first time. This method is estimated to edit 7.3% of pathogenic F9 mutation types. Specifically, it targets the gene region encoding amino acids 374 V to 408 Q, which accounts for approximately 9.35% of patients with hemophilia B. An advantage of this gene therapy approach is the absence of the need to change Primer Binding Site (PBS) or Reverse Transcriptase Template (RTT) sequences until going from preclinical to clinical trials, as well as the introduction of gain of function mutations in order to compensate for the low prime-editing frequencies and enhance the effect of treatment in vivo.
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Gene Editing: Powerful New Tools for Nephrology Research and Therapy.
Biologic research is experiencing a transformation brought about by the ability of programmable nucleases to manipulate the genome. In the recently developed CRISPR/Cas system, short RNA sequences guide the endonuclease Cas9 to any location in the genome, causing a DNA double-strand break (DSB). Repair of DSBs allows the introduction of targeted genetic manipulations with high precision. Cas9-mediated gene editing is simple, scalable, and rapid, and it can be applied to virtually any organism. Here, we summarize the development of modern gene editing techniques and the biology of DSB repair on which these techniques are based. We discuss technical points in applying this technology and review its use in model organisms. Finally, we describe prospects for the use of gene editing to treat human genetic diseases. This technology offers tremendous promise for equipping the nephrology research community to better model and ultimately, treat kidney diseases.
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CRISPR/Cas9-directed epigenetic editing in colorectal cancer.
Colorectal cancer (CRC) remains a leading cause of cancer-related illness and death worldwide, arising from a complex interplay of genetic predisposition, environmental influences, and epigenetic dysregulation. Among these factors, epigenetic modifications-reversible and heritable changes in gene expression-serve as crucial regulators of CRC progression. Understanding these modifications is essential for identifying potential biomarkers for early diagnosis and developing targeted therapeutic strategies. Epigenetic drugs (epidrugs) such as DNA methyltransferase inhibitors (e.g., decitabine) and bromodomain inhibitors (e.g., JQ1) have shown promise in modulating aberrant epigenetic changes in CRC. However, challenges such as drug specificity, delivery, and safety concerns limit their clinical application. Advances in CRISPR-Cas9-based epigenetic editing offer a more precise approach to modifying specific epigenetic markers, presenting a potential breakthrough in CRC treatment. Despite its promise, CRISPR-based epigenome editing may result in unintended genetic modifications, necessitating stringent regulations and safety assessments. Beyond pharmacological interventions, lifestyle factors-including diet and gut microbiome composition-play a significant role in shaping the epigenetic landscape of CRC. Nutritional and microbiome-based interventions have shown potential in preventing CRC development by maintaining intestinal homeostasis and reducing tumor-promoting epigenetic changes. This review provides a comprehensive overview of epigenetic alterations in CRC, exploring their implications for diagnosis, prevention, and treatment. By integrating multi-omics approaches, single-cell technologies, and model organism studies, future research can enhance the specificity and efficacy of epigenetic-based therapies. Shortly, a combination of advanced gene-editing technologies, targeted epidrugs, and lifestyle interventions may pave the way for more effective and personalized CRC treatment strategies.
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Microinjection in Zebrafish for Genome Editing and Functional Studies.
A major strength of zebrafish as a model organism is their rapid, in vitro development. The easy access to embryos compared to mammals, allows larval molecular and cellular composition to be manipulated by microinjection, providing a powerful avenue for biological and translational studies. Here, we describe the essential steps and different applications of microinjection in zebrafish for genome editing and functional studies, along with some experimental tips that are critical for microinjection success.
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Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.
The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.
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Characterization of Two Polyketide Synthases Involved in Sorbicillinoid Biosynthesis by Acremonium chrysogenum Using the CRISPR/Cas9 System.
Acremonium chrysogenum is an important fungal strain used for cephalosporin C production. Many efforts have been made to develop versatile genome-editing tools to better understand the mechanism of A. chrysogenum. Here, we developed a feasible and efficient CRISPR/Cas9 system. Two genes responsible for the synthesis of yellow pigments (sorbicillinoids) were chosen as targets, and plasmids expressing both the Cas9 protein and single-guide RNAs were constructed. After introducing the plasmids into the protoplasts of A. chrysogenum, 83 to 93% albino mutants harboring the expected genomic alteration, on average, were obtained. We have generated two mutant strains that respectively disrupt sorA and sorB by flexible CRISPR/Cas9 system. We further confirmed that the sorbicillinoid biosynthetic gene cluster is regulated by an autoinduction mechanism. This work will lay a solid foundation for gene function research and regulation in the sorbicillinoid biosynthetic pathway.
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Overcoming barriers in CAR-NK immunotherapy: CRISPR-Driven advances in checkpoint editing and allogeneic design.
Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells are emerging as an exciting avenue in cancer immunotherapy due to their potent cytotoxicity to malignant cells and lower risk of graft-versus-host disease (GvHD) than conventional T cell therapies. The new technology of CRISPR/Cas9 genome editing has significantly expedited the engineering of CAR-NK cells by enabling easy, multiplex, and precise changes to enhance their efficacy, persistence, and specificity to tumors. This review focuses on the incorporation of CRISPR technology into CAR-NK cell development. It examines uses of knockout of inhibitory checkpoint genes (CISH, PD-1, and TGFBR2), as well as knock-in of CAR into safe genomic locations and multiplex editing of CAR-NK cells to improve cytotoxicity against cancer while resisting suppression from the tumor microenvironment (TME). We further explore immuno-cytokine armoring strategies by knock-in of IL-15 or IL-12, to ensure prolonged proliferation and survival of NK cells, and investigate CRISPR-mediated knockouts of immune inhibitors like NKG2A and TIGIT, to evade immune strategies used by the tumor to evade immune destruction. Furthermore, CRISPR-mediated upregulation of the homing receptor enhances NK cell tumor infiltration, addressing a major obstacle in treating solid tumors. It is significant to mention the progress in generating off-the-shelf products, which is a key step supporting the pursuit of allogeneic therapies. While substantial progress has been made, challenges remain related to optimizing CRISPR delivery, off-target effects, and enhancing in vivo persistence. Future directions of CAR-NK studies will likely capitalize on next-generation genome editing tools and synthetic biology for the development of tunable and logic-gated CAR-NK cells. Overall, this review illustrates the revolutionary capacity of combining CRISPR technology with CAR-NK immunotherapy to develop next-generation programmable and efficacious treatments for hematologic and solid malignancies.
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The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells.
Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.
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