Detection of genome-edited mutant clones by a simple competition-based PCR method.

Hodaka Fujii,Takeshi Harayama,Howard Riezman

doi:10.1371/journal.pone.0179165

Abstract

Genome editing by the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) system is a revolutionary strategy to study gene functions. Since the efficiency of gene disruption in cell culture does not reach 100% typically, cloning of mutant cells is often performed to obtain fully mutated cells. Therefore, a method to discriminate accurately mutated clones easily and quickly is crucial to accelerate the research using CRISPR/Cas9. Here, we show that knockout cells can be discriminated by a competition-based PCR, using a mixture of three primers, among which one primer overlaps with the Cas9 cleavage site. Together, we show how to optimize primer design in order to improve the effectiveness of the discrimination. Finally, we applied this method to show that mutations conferring drug resistance can be detected with high accuracy. The provided method is easy to perform and requires only basic laboratory equipment, making it suitable for almost all laboratories.

Highlights

Genome editing by the CRISPR/Cas9 system has became a widely used strategy to study gene functions in cells and in vivo [1]
In the widely-used CRISPR/Cas9 system derived from Streptococcus pyogenes, the Cas9 nuclease creates a double strand break in the genome at a site complementary to the guide RNA, which is repaired by various repair machineries of the cell
The indels caused by CRISPR/ Cas9 editing are often small, standard PCR with genomic DNA is not useful to detect the mutants since the amplicon sizes are not different enough

Summary

Introduction

Genome editing by the CRISPR/Cas system has became a widely used strategy to study gene functions in cells and in vivo [1]. In the widely-used CRISPR/Cas system derived from Streptococcus pyogenes, the Cas nuclease creates a double strand break in the genome at a site complementary to the guide RNA, which is repaired by various repair machineries of the cell This often leads to the introduction of indels, resulting in an efficient loss of function of gene. The usage of Cas nuclease with two guide RNAs targeting relatively distant sites enables an easy discrimination by inducing a bigger deletion detectable by PCR [4] (not to be confounded with the double-nickase strategy where the offset should be small enough to get a double strand break [5])

Methods

Results

Conclusion