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

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

Yan You¹, Sharmila G Ramachandra¹, Tian Jin²

¹Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.

Scientific Reports|September 9, 2020

View abstract on PubMed

Summary

We developed a CRISPR-E test to identify essential genes by analyzing mutation types after CRISPR/Cas9 gene editing. This method distinguishes essential genes by observing the selective elimination of frameshift mutations in surviving cells.

Area of Science:

Genetics
Molecular Biology
Gene Editing

Background:

CRISPR/Cas9 is a gene editing tool that introduces errors at target sites.
Identifying essential genes is crucial for understanding cellular functions and disease mechanisms.

Purpose of the Study:

To develop and validate a CRISPR-based test for identifying essential genes.
To differentiate between essential and non-essential genes based on mutation outcomes.

Main Methods:

Utilized sgRNA-mediated CRISPR/Cas9 to target gene open reading frames.
Analyzed in-frame (3n) and frameshift (3n+1, 3n+2) mutations in surviving cells.
Applied the CRISPR-E test to Dictyostelium discoideum to assess gene essentiality.

Main Results:

Non-essential genes accumulate both in-frame and frameshift mutations.
Essential genes show a selective elimination of frameshift mutations.
Demonstrated Dync1li1 as essential, while KIF1A and fAR1 were identified as non-essential in Dictyostelium discoideum.

Conclusions:

The CRISPR-E test effectively identifies essential genes by detecting the absence of frameshift mutations.
The study proposes a method for quantifying gene essentiality using CRISPR-E.
This approach offers a novel strategy for large-scale essential gene identification.

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A Crispr-based Method For Testing The Essentiality Of A Gene