CRISPR-edit point mutant allele detection (CEPAD)-PCR method for rapid screening of CRISPR edited point mutations.

Abstract

CRISPR-Cas9 mediated genome editing is widely used for generating genetic lesions in C. elegans. Detection of single-site mutations in F1 progeny after CRISPR-Cas9 injections is currently labor intensive due to lack of a single step PCR-based detection method. Here we present CEPAD-PCR, an allele-specific PCR detection method based on generating silent mutations around the site of the desired genetic lesion during the CRISPR-Cas9 genome editing process. Detection of the desired allele is then performed by taking advantage of the tetra primer PCR method, based on the principle described in the ARMS-PCR. In the CEPAD-PCR, however, unlike ARMS-PCR, presence of additional silent mutations near the desired site-specific mutation in the genome results in PCR priming with high specificity resulting in a low false positive rate. As proof of concept, the method was successfully tested on point mutations in two different genes, daf-15 and raga-1.