College of Health and Life Sciences, Division of Biological and Biomedical Sciences, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha 34110, Qatar.
College of Health and Life Sciences, Division of Genomics and Precision Medicine, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha 34110, Qatar.
STAR Protoc. 2022 Mar 4;3(1):101216. doi: 10.1016/j.xpro.2022.101216. eCollection 2022 Mar 18.
Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verification to determine indels in one or both alleles by Sanger sequencing. This strategy is based on the efficiency of DNA editing, avoids antibiotic selection, and bypasses the need for cell sorting.
在这里，我们描述了一种使用 CRISPR-Cas9 系统敲除人 PRDX1 基因的细胞的方案。该方案按顺序描述了所有步骤：（1）单引导 RNA 的设计、克隆和转染；（2）通过 T7EI 测定评估基因编辑；（3）单细胞分离；以及（4）通过桑格测序确定一个或两个等位基因中的插入缺失来验证敲除。这种策略基于 DNA 编辑的效率，避免了抗生素选择，并绕过了细胞分选的需要。
