dictyNews
Electronic Edition
Volume 46, number 23
August 14, 2020

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

Yan You#, Sharmila G. Ramachandra#, Tian Jin*

Chemotaxis Signal Section, Laboratory of Immunogenetics, 
National Institute of Allergy and Infectious Diseases, 
National Institutes of Health, Rockville, MD.
#Authors contribute equally
*[log in to unmask]


Nature Scientific Report, accepted

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 on the target 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. 


submitted by: Sharmila Ramachandra [[log in to unmask]]
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[End dictyNews, volume 46, number 23]