RRC ID 66881
Author You Y, Ramachandra SG, Jin T.
Title A CRISPR-based method for testing the essentiality of a gene.
Journal Sci Rep
Abstract 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.
Volume 10(1)
Pages 14779
Published 2020-9-8
DOI 10.1038/s41598-020-71690-8
PII 10.1038/s41598-020-71690-8
PMID 32901070
PMC PMC7478968
MeSH Amino Acid Sequence Base Sequence CRISPR-Cas Systems* Dictyostelium / genetics* Dictyostelium / growth & development Dictyostelium / metabolism Gene Editing* Genes, Essential* Protozoan Proteins / antagonists & inhibitors Protozoan Proteins / genetics* Protozoan Proteins / metabolism Sequence Homology
IF 3.998
Cellular slime molds pTM1285