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

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.

SUBMITTER: Harayama T 

PROVIDER: S-EPMC5460891 | biostudies-other | 2017

REPOSITORIES: biostudies-other