Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX, USA.
School of Medicine, Ophthalmology, Visual and Anatomical Sciences, Wayne State University, Detroit, MI, USA.
Methods Mol Biol. 2023;2575:241-260. doi: 10.1007/978-1-0716-2716-7_11.
The CRISPR-Cas9 system is becoming an imperative tool to edit the genome of various organisms. The gene-editing study by the CRISPR-Cas9 system has revolutionized the diverse field of biomedical research, genome engineering, and gene therapy. CRISPR-Cas9 system has been modified to induce genome editing by small-guide RNAs, which function together with Cas9 nuclease for sequence-specific cleavage of target sequences. Here, we describe the simplified protocol of CRISPR-Cas9-mediated DNA editing in multicellular eukaryotes, including the construction of gRNA plasmids into vectors, screening of positive clones, transfections into 293FT cell line, and transduction into Jurkat cells. We also describe different bioinformatic tools to design suitable gRNAs with increased efficiency and decreased off-target events. Further, we describe the assessments of DNA editing by indel mutations and sequencing in transduced cells.
CRISPR-Cas9 系统正成为编辑各种生物基因组的必要工具。CRISPR-Cas9 系统的基因编辑研究彻底改变了生物医学研究、基因组工程和基因治疗的多个领域。CRISPR-Cas9 系统已被修改为通过小向导 RNA 诱导基因组编辑，这些小向导 RNA 与 Cas9 核酸酶一起作用，对靶序列进行特异性切割。在这里，我们描述了在多细胞真核生物中进行 CRISPR-Cas9 介导的 DNA 编辑的简化方案，包括将 gRNA 质粒构建到载体中、筛选阳性克隆、转染 293FT 细胞系以及转导 Jurkat 细胞。我们还描述了不同的生物信息学工具，以设计具有更高效率和更低脱靶事件的合适 gRNA。此外，我们还描述了通过转导细胞中的插入缺失突变和测序来评估 DNA 编辑。
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