QEva-CRISPR: A method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites

Abstract

Genome editing technology based on engineered nucleases has been increasingly applied for targeted modification of genes in a variety of cell types and organisms. However, the methods currently used for evaluating the editing efficiency still suffer from many limitations, including preferential detection of some mutation types, sensitivity to polymorphisms that hamper mismatch detection, lack ofmultiplex capability, or sensitivity to assay conditions. Here, we describe qEva-CRISPR, a new quantitative method that overcomes these limitations and allows simultaneous (multiplex) analysis of CRISPR/Cas9-induced modifications in a target and the corresponding offtargets or in several different targets. We demonstrate all of the advantages of the qEva-CRISPR method using a number of sgRNAs targeting the TP53, VEGFA, CCR5, EMX1 and HTT genes in different cell lines and under different experimental conditions. Unlike other methods, qEva-CRISPR detects all types of mutations, including point mutations and large deletions, and its sensitivity does not depend on themutation type. Moreover, this approach allows for successful analysis of targets located in 'difficult' genomic regions. In conclusion, qEva-CRISPR may become amethod of choice for unbiased sgRNA screening to evaluate experimental conditions that affect genome editing or to distinguish homologydirected repair from non-homologous end joining.