Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.
Sci Rep. 2020 Sep 8;10(1):14779. doi: 10.1038/s41598-020-71690-8.
The CRISPR/Cas9 system is a powerful method of editing genes by randomly introducing errors into the target sites. Here, we describe a CRISPR-based test for gene essentiality (CRISPR-E test) that allows the identification of essential genes. Specifically, we use sgRNA-mediated CRISPR/Cas9 to target the open reading frame of a gene in the genome and analyze the in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations in the targeted region of the gene in surviving cells. If the gene is non-essential, the cells would carry both in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations. In contrast, the cells would carry only in-frame (3n) mutations if the targeted gene is essential, and this selective elimination of frameshift (3n + 1 and 3n + 2) mutations of the gene indicate its essentiality. As a proof of concept, we have used this CRISPR-E test in the model organism Dictyostelium discoideum to demonstrate that Dync1li1 is an essential gene while KIF1A and fAR1 are not. We further propose a simple method for quantifying the essentiality of a gene using the CRISPR-E test.
CRISPR/Cas9 系统是一种通过随机在靶位点引入错误来编辑基因的强大方法。在这里，我们描述了一种基于 CRISPR 的基因必需性测试（CRISPR-E 测试），该测试可用于鉴定必需基因。具体来说，我们使用 sgRNA 介导的 CRISPR/Cas9 靶向基因组中基因的开放阅读框，并分析靶基因区域中存活细胞的框架内（3n）和移码（3n+1 和 3n+2）突变。如果基因是非必需的，那么细胞将同时携带框架内（3n）和移码（3n+1 和 3n+2）突变。相比之下，如果靶基因是必需的，那么细胞将仅携带框架内（3n）突变，这种对基因的移码（3n+1 和 3n+2）突变的选择性消除表明其必需性。作为概念验证，我们已经在模式生物盘基网柄菌中使用这种 CRISPR-E 测试证明了 Dync1li1 是一个必需基因，而 KIF1A 和 fAR1 则不是。我们进一步提出了一种使用 CRISPR-E 测试量化基因必需性的简单方法。
