An effective and inexpensive strategy to identify multiplexCRISPR-edited plants

Since its first introduction in 2013, CRISPR-Cas9 has become the preferred gene targeting tool to produce loss-of-function mutants in plants. In spite of the high specificity and ease of use, the identification of CRISPR-edited plants has remained a time consuming and onerous process. I have developed and tested an easy-to-use and inexpensive strategy to select for multiplex CRISPR mutagenized Arabidopsis plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy for CRISPR-Cas9 activity: an endogenous gene that produces an easy-to-detect visible phenotype. To test this strategy, I have chosen Arabidopsis gene JAR1, GL1, EIN2 as the candidate proxies. I have tested the T2 progeny of independent T1 plants harboring CRISPR/Cas9 and successfully identified plants where the visible marker and the genes of interest were simultaneously edited at a high frequency. The co-editing frequency ranged from 55.6% to 93.75% for two genes, and 14.3% to 50% for three genes, depending on the T1 progeny tested and the proxy gene of choice. The visual phenotype selection provides a narrow pool of plants to analyze, hence increasing the recovery frequency while decreasing the cost of identifying mutants. This selection strategy also offers a framework to similarly facilitate the identification of CRISPR-edited plants in other plant species with more complex polyploid genomes where multiplex mutants are essential for studying gene function.