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

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.

journal_name

Sci Rep

journal_title

Scientific reports

authors

You Y,Ramachandra SG,Jin T

doi

10.1038/s41598-020-71690-8

subject

Has Abstract

pub_date

2020-09-08 00:00:00

pages

14779

issue

1

issn

2045-2322

pii

10.1038/s41598-020-71690-8

journal_volume

10

pub_type

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