A CRISPR-based method for testing the essentiality of a gene

• Published in: Scientific reports Vol. 10; no. 1; p. 14779
• Main Authors: You, Yan, Ramachandra, Sharmila G., Jin, Tian
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 08.09.2020; Nature Publishing Group
• Subjects: 631/1647; 631/80; Alginic acid; Amino Acid Sequence; Base Sequence; Cartilage diseases; Cell number; Cellulose; Chondrocytes; Cloning; Collagen (type II); CRISPR; CRISPR-Cas Systems; Dictyostelium - genetics; Dictyostelium - growth & development; Dictyostelium - metabolism; Gene Editing; Genes; Genes, Essential; Humanities and Social Sciences; Hyaluronic acid; multidisciplinary; Mutation; Oct-4 protein; Osteoarthritis; Pluripotency; Printing; Proteins; Protozoan Proteins - antagonists & inhibitors; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Science; Science (multidisciplinary); Sequence Homology; Stem cells; Tissue engineering
• ISSN: 2045-2322; 2045-2322
• 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.