AbstractCRISPR-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.

24CRISPR/Cas9 has become the preferred gene-editing technology to obtain loss-of-25 function mutants in plants, and hence a valuable tool to study gene function. This is 26 mainly due to the easy reprogramming of Cas9 specificity using customizable small 27 non-coding RNAs, and to the possibility of editing several independent genes 28 simultaneously. Despite these advances, the identification of CRISPR-edited plants 29remains time and resource-intensive. Here, based on the premise that one editing event 30in one locus is a good predictor of editing event/s in other locus/loci, we developed a 31 CRISPR co-editing selection strategy that greatly facilitates the identification of 32 CRISPR-mutagenized Arabidopsis thaliana plants. This strategy is based on targeting 33 the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed 34 mutagenesis. The proxy is an endogenous gene whose loss-of-function produces an 35 easy-to-detect visible phenotype that is unrelated to the expected phenotype of the 36 gene/s under study. We tested this strategy via assessing the frequency of co-editing of 37 three functionally unrelated proxy genes. We found that each proxy predicted the 38 occurrence of mutations in each surrogate gene with efficiencies ranging from 68% to 39