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

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.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • 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


  • Protozoan Proteins