Proxies of CRISPR-Cas9 activity to aid in the identification of mutagenized Arabidopsis plants

Abstract

CRISPR-Cas9 has become the preferred gene editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprograming of Cas9 specificity using customizable small non-coding RNAs, and to the ability to target several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource consuming. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function mutation produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxies. We found all three proxies predicted the occurrence of mutations in either or both of the other two proxies with efficiencies ranging from 40% to 100%, dramatically reducing the number of plants that need to be screened to identify CRISPR mutants. This selection strategy provides a framework to facilitate gene function studies of gene families as well as the function of single copy genes in polyploid plant species where the identification of multiplex mutants remains challenging.