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Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana through Viral Delivery of SpCas9 and sgRNA.
Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in Nicotiana benthamiana PHYTOENE DESATURASE. Transient downregulation of RNA silencing enhanced gene editing efficiency, resulting in an order of magnitude increase (0.8-13.2%) in the frequency of transgenerational gene editing. While the TRSV system had a preference for certain sgRNA sequences, co-inoculation of a TRSV vector carrying only Cas9 and a tobacco rattle virus vector carrying sgRNA successfully introduced systemic mutations with all five tested sgRNAs. Extensively gene-edited lateral shoots occasionally grew from plants inoculated with the virus vectors, the transgenerational gene editing frequency of which ranged up to 100%. This virus-mediated heritable gene editing method makes plant gene editing easy, requiring only the inoculation of non-transgenic plants with a virus vector(s) to obtain gene-edited individuals.
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Identification of Genes Regulating Hepatocyte Injury by a Genome-Wide CRISPR-Cas9 Screen.
Gene editing introduces stable mutations into the genome and has powerful applications extending from research to clinical gene therapy. CRISPR-Cas9 gene editing can be employed to study directly the functional impact of stable gene knockout, activation, and knockdown. Here, we describe the end-to-end methodology by which we employ genome-wide CRISPR-Cas9 knockout to study drug toxicity using acetaminophen (APAP) in a hepatocellular carcinoma liver model as an example. This methodology can be extended to other proliferative cell types and chemical metabolic and toxicity models. By employing a massively parallelized genome-wide knockout model, the genes responsible for cellular toxicity and proliferation may be assayed concurrently. Resultant data are interrogated in the context of existing gene expression data, pathway analysis, drug-gene interactions, and orthogonal confirmatory assays to better understand the metabolic mechanisms.
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Advanced domestication: harnessing the precision of gene editing in crop breeding.
Human population growth has increased the demand for food crops, animal feed, biofuel and biomaterials, all the while climate change is impacting environmental growth conditions. There is an urgent need to develop crop varieties which tolerate adverse growth conditions while requiring fewer inputs. Plant breeding is critical to global food security and, while it has benefited from modern technologies, it remains constrained by a lack of valuable genetic diversity, linkage drag, and an effective way to combine multiple favourable alleles for complex traits. CRISPR/Cas technology has transformed genome editing across biological systems and promises to transform agriculture with its high precision, ease of design, multiplexing ability and low cost. We discuss the integration of CRISPR/Cas-based gene editing into crop breeding to advance domestication and refine inbred crop varieties for various applications and growth environments. We highlight the use of CRISPR/Cas-based gene editing to fix desirable allelic variants, generate novel alleles, break deleterious genetic linkages, support pre-breeding and for introgression of favourable loci into elite lines.
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Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9.
Single-stranded DNA (ssDNA) templates along with Cas9 have been used for knocking-in exogenous sequences in the genome but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 12-19% of internal bases, which we denote as enhanced ssDNA (esDNA), improve knock-in (KI) by 2-3-fold compared to end-modified ssDNA in airway basal stem cells (ABCs), CD34 + hematopoietic cells (CD34 + cells), T-cells and endothelial cells. Over 50% of alleles showed KI in three clinically relevant loci (CFTR, HBB and CCR5) in ABCs using esDNA and up to 70% of alleles showed KI in the HBB locus in CD34 + cells in the presence of a DNA-PKcs inhibitor. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. The esDNA templates did not improve KI in induced pluripotent stem cells (iPSCs). This may be due to the absence of the nuclease TREX1 in iPSCs. Indeed, knocking out TREX1 in other cells improved KI using unmodified ssDNA. esDNA can be used to modify 20-30 bp regions in primary cells for therapeutic applications and biological modeling. The use of this approach for gene length insertions will require new methods to produce long chemically modified ssDNA in scalable quantities.
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CRISPR/Cas9-Mediated Genome Editing for Huntington's Disease.
This chapter describes the potential use of viral-mediated gene transfer in the central nervous system for genome editing in the context of Huntington's disease. Here, we provide protocols that cover the design of various genome editing strategies, the cloning of CRISPR/Cas9 elements into lentiviral vectors, and the assessment of cleavage efficiency, as well as potential unwanted effects.
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Current status of potential applications of repurposed Cas9 for structural and functional genomics of plants.
Redesigned Cas9 has emerged as a tool with various applications like gene editing, gene regulation, epigenetic modification and chromosomal imaging. Target specific single guide RNA (sgRNA) can be used with Cas9 for precise gene editing with high efficiency than previously known methods. Further, nuclease-deactivated Cas9 (dCas9) can be fused with activator or repressor for activation (CRISPRa) and repression (CRISPRi) of gene expression, respectively. dCas9 fused with epigenetic modifier like methylase or acetylase further expand the scope of this technique. Fluorescent probes can be tagged to dCas9 to visualize the chromosome. Due to its wide-spread application, simplicity, accessibility, efficacy and universality, this technique is expanding the structural and functional genomic studies of plant and developing CRISPR crops. The present review focuses on current status of using repurposed Cas9 system in these various areas, with major focus on application in plants. Major challenges, concerns and future directions of using this technique are discussed in brief.
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Optimized protocols for efficient gene editing in mouse hepatocytes in vivo using CRISPR-Cas9 technology.
We provide a protocol for gene editing in mouse hepatocytes in vivo using the CRISPR-Cas9 technology via AAV delivery. This protocol describes the construction of AAV plasmids, AAV packaging, injection, and the detection of in vivo knockout efficiency. Using this protocol, we can get up to 1014 AAV and knock out genes in hepatocytes efficiently within 15 days. Moreover, we describe an optimized protocol to simultaneously target two genes via AAV delivery of CRISPR-Cas9 materials in the liver. For complete details on the use and execution of this profile, please refer to Wei et al. (2020).
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Computationally guided high-throughput engineering of an anti-CRISPR protein for precise genome editing in human cells.
The application of CRISPR-Cas systems to genome editing has revolutionized experimental biology and is an emerging gene and cell therapy modality. CRISPR-Cas systems target off-target regions within the human genome, which is a challenge that must be addressed. Phages have evolved anti-CRISPR proteins (Acrs) to evade CRISPR-Cas-based immunity. Here, we engineer an Acr (AcrIIA4) to increase the precision of CRISPR-Cas-based genome targeting. We developed an approach that leveraged (1) computational guidance, (2) deep mutational scanning, and (3) highly parallel DNA repair measurements within human cells. In a single experiment, ∼10,000 Acr variants were tested. Variants that improved editing precision were tested in additional validation experiments that revealed robust enhancement of gene editing precision and synergy with a high-fidelity version of Cas9. This scalable high-throughput screening framework is a promising methodology to engineer Acrs to increase gene editing precision, which could be used to improve the safety of gene editing-based therapeutics.
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CRISPR-cas systems in pharmacology: functional pharmacogenomics, drug screening, resistance, and therapeutic translation.
CRISPR-Cas9 gene-editing technology has advanced pharmacological research by enabling targeted genetic modification for disease modeling, therapeutic development, and precision medicine. This review discusses the applications of CRISPR-Cas9 in drug discovery, personalized therapy, cancer drug resistance research, genetic disorders, and antimicrobial resistance. By editing disease-associated genes, CRISPR-Cas9 supports the development of patient-specific therapeutic strategies and more accurate preclinical models. In cancer, CRISPR-Cas9 is used to investigate the target genes involved in treatment resistance, while in genetic disorders, it offers potential mutation-correcting approaches, with the most robust clinical evidence currently seen in selected hemoglobinopathies. CRISPR-based strategies also hold promise for restoring antibiotic susceptibility by targeting genes that confer antibiotic resistance. Despite these advances, clinical translation remains limited by off-target effects, delivery challenges, immune responses, long-term safety concerns, and ethical and regulatory issues. Continued improvements in editing precision, delivery systems, and governance frameworks are essential for responsible clinical integration. Overall, CRISPR-Cas9 represents a vital platform for future pharmacological innovation, but its broad clinical use may require further validation of safety, efficacy, durability, and accessibility.
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Endogenous U6 promoters improve CRISPR/Cas9 editing efficiencies in Sorghum bicolor and show potential for applications in other cereals.
Endogenous U6 promoters increase CRISPR/Cas9 editing efficiency in sorghum and may be useful for gene editing applications in other cereals.
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