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Generation of Genetically Modified Mice Using CRISPR/Cas9.
Using CRISPR-based genome-editing techniques, we are able to generate a variety of new mouse models of several types of diseases. These animal models will be instrumental not only for enabling the comprehension of a particular disease and its underlying molecular mechanism but also as unique recipients for testing novel and innovative therapeutic approaches that are being currently explored. This chapter describes detailed step-by-step protocols, reagents, and equipment required for successful generation of genome-edited mice using CRISPR tools.
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Efficient breeding of low glutelin content rice germplasm by simultaneous editing multiple glutelin genes via CRISPR/Cas9.
Chronic kidney disease (CKD) and phenylketonuria (PKU) patients need to eat rice with low glutelin content. Therefore, breeding low glutelin content rice varieties with high yield and delicious taste is one of the major goals of rice breeders due to the high demand for the product. In this study, we designed three sgRNAs targeting nine glutelin genes and generated nine T-DNA-free homozygous editing lines with reduced glutelin content compared with the wild-type due to simultaneous mutation(s) in 5-7 glutelin genes. The glutelin content of two lines is even significantly lower than that of the low glutelin content cultivar, LGC-1. Compared to the wild-type, these low glutelin lines showed similar agronomic traits, including yield components and viscosity properties, and can be used as new varieties or parental materials for further breeding.
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CRISPR-Cas9 based gene editing of the immune checkpoint NKG2A enhances NK cell mediated cytotoxicity against multiple myeloma.
Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity, and the possibility to be applied as 'off-the-shelf' product makes them highly attractive for cell-based immunotherapies. In patients with multiple myeloma (MM), an elevated number of NK cells has been correlated with higher overall-survival rate. However, NK cell function can be impaired by upregulation of inhibitory receptors, such as the immune checkpoint NKG2A. Here, we developed a CRISPR-Cas9-based gene editing protocol that allowed us to knockout about 80% of the NKG2A-encoding killer cell lectin like receptor C1 (KLRC1) locus in primary NK cells. In-depth phenotypic analysis confirmed significant reduction in NKG2A protein expression. Importantly, the KLRC1-edited NK cells showed significantly increased cytotoxicity against primary MM cells isolated from a small cohort of patients, and maintained the NK cell-specific cytokine production. In conclusion, KLRC1-editing in primary NK cells has the prospect of overcoming immune checkpoint inhibition in clinical applications.
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eVLP compound delivery breaks the prime editing efficiency ceiling.
Prime editing could resolve gene variant function at scale, but the editing machinery needs to be delivered efficiently and reproducibly. Langley, Baudrier, et al.1 show that timing of editor delivery is rate limiting and introduce PRIME-VLP, a repeated dosing strategy with engineered virus-like particles that improves editing efficiency and screening performance.
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Production and Validation of Lentiviral Vectors for CRISPR/Cas9 Delivery.
Genetic information transferred by HIV-1-based lentiviral vectors as single-stranded RNA is converted to double-stranded DNA by reverse transcription and subsequently inserted into the genome of recipient cells. Integration into the genome allows stable, long-term expression of genes-of-interest driven by promoter sequences contained within the vector. This technology can be used as a standard method for production of cells stably expressing Cas9 protein and single guide RNA (sgRNA), the key components of the CRISPR genome editing system. Here, we provide a protocol for production and validation of VSV-G-pseudotyped lentiviral vectors for delivery of the CRISPR system and generation of knockout cell lines.
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Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast.
Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual heterozygous loci in the yeast genome. Moreover, Cas9-mediated introduction of a DSB resulted in large scale loss of heterozygosity affecting DNA regions up to 360 kb and up to 1700 heterozygous nucleotides, due to replacement of sequences on the targeted chromosome by corresponding sequences from its non-targeted homolog. The observed patterns of loss of heterozygosity were consistent with homology directed repair. The extent and frequency of loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, which would have to be taken into account in eukaryotic gene editing. In addition to contributing to the limited genetic amenability of heterozygous yeasts, Cas9-mediated loss of heterozygosity could be particularly deleterious for human gene therapy, as loss of heterozygous functional copies of anti-proliferative and pro-apoptotic genes is a known path to cancer.
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Identification of Ziziphus jujuba cv. Dongzao DNA Demethylase ZjROS1 Gene Family and Construction of CRISPR/Cas9-Mediated Gene-Editing Vector.
DNA methylation is one of the earliest and most extensively studied epigenetic regulatory mechanisms. The ROS1 (Repressor of Silencing 1) gene was first discovered in Arabidopsis thaliana, and it is a DNA demethylase that can remove 5-methylcytosine from DNA, thereby affecting DNA methylation levels and gene expression. Objectives: The objective of this study was to investigate the role of ROS1 in the development and maturation of Ziziphus jujuba cv. "Dongzao" fruit. Methods: We cloned the ROS1 gene and conducted bioinformatics and expression characteristics analyses on it. Results: Three ROS1 genes, named ZjROS1-1~3, was identified, and each member protein was localized in the nucleus, cytoskeleton, chloroplast, and vacuole. The promoter contained cis-elements such as light response, plant hormone signal transduction, and stress response cis-elements, and it interacted with many proteins such as CMT, MET, and ZDP. The results of the real-time fluorescence quantitative PCR show that ZjROS1 has specific expression patterns in different tissues of Z. jujuba cv. Dongzao, and the expression of ZjROS1-2 in flowers and fruits is high. At the same time, CRISPR/Cas9 technology was used to construct a gene-editing vector for ZjROS1, which provided a basis for the subsequent genetic transformation. Conclusions: In this study, the biological function of ZjROS1 was clarified and a gene-editing vector was constructed, which provided a theoretical basis for the regulation mechanism of demethylase ZjROS1 in the fruit ripening and development of Z. jujuba cv. Dongzao.
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Genome editing‑based strategies to combat geminiviruses: CRISPR/Cas9 and emerging high‑fidelity tools.
Plant virus diseases constitute a major constraint to agriculture and have adversely affected crop productivity worldwide. Over the past few decades, geminiviruses of the family Geminiviridae have emerged as some of the most destructive plant pathogens. Conventional. approaches for virus management have demonstrated varying degrees of success, their deployment is often hindered by prolonged timelines, limited availability of resistant sources, and reduced durability against rapidly evolving geminiviral pathogens. With the rapid evolution of viruses and the extensive damage they cause, it is imperative to develop rapid, cost-effective, and efficient antivirus resistance strategies. In recent years, CRISPR/Cas-based genome editing has made it possible to precisely target viral genomes, offering new avenues for developing antiviral resistance, although long-term durability remains under investigation. Derived from prokaryotic adaptive immune systems, the CRISPR/Cas platform confers resistance either by expressing genome-editing constructs that directly cleave viral genomes or by modifying host susceptibility (S) genes, representing mechanistically distinct strategies with different implications for durability of resistance. Recent advances have further expanded this toolkit with Cas-CLOVER, a dual guide RNA-dependent nuclease system designed to mitigate off-target effects associated with conventional Cas9. This review summarizes the principles of CRISPR/Cas-mediated antiviral resistance, critically evaluates viral escape, resistance durability, compares Cas9 with emerging platforms, and outlines conceptual frameworks for developing durable and field-ready genome-editing strategies.
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An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique.
Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.
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Genome Editing in a Wide Area of the Brain Using Dendrimer-Based Ternary Polyplexes of Cas9 Ribonucleoprotein.
A preassembled Cas9/single-guide RNA complex (Cas9 ribonucleoprotein; Cas9 RNP) induces genome editing efficiently, with small off-target effects compared with the conventional techniques, such as plasmid DNA and mRNA systems. However, penetration of Cas9 RNP through the cell membrane is low. In particular, the incorporation of Cas9 RNP into neurons and the brain is challenging. In the present study, we have reported the use of a dendrimer (generation 3; G3)/glucuronylglucosyl-β-cyclodextrin conjugate (GUG-β-CDE (G3)) as a carrier of Cas9 RNP and evaluated genome editing activity in the neuron and the brain. A Cas9 RNP ternary complex with GUG-β-CDE (G3) was prepared by only mixing the components. The resulting complex exhibited higher genome editing activity than the complex with the dendrimer (G3), Lipofectamine 3000 or Lipofectamine CRISPRMAX in SH-SY5Y cells, a human neuroblastoma cell line. In addition, GUG-β-CDE (G3) enhanced the genome editing activity of Cas9 RNP in the whole mouse brain after a single intraventricular administration. Thus, GUG-β-CDE (G3) is a useful Cas9 RNP carrier that can induce genome editing in the neuron and brain.
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