Genome-wide prediction of highly specific guide RNA spacers for the CRISPR-Cas9 mediated genome editing in model plants and major crops

The CRISPR-Cas9 system is recently emerging as an efficient and versatile tool for genome editing in microbes, animals and plants. However, its potential off-target effect is a major concern for specific gene targeting and precise genome editing. In this study, genome-wide prediction of highly specific guide RNA (gRNA) spacer sequences was carried out in eight model plants and agricultural crops. The gRNA spacers were analyzed and classified based on the recent reports and criteria regarding gRNA specificity and off-target potential. Among seven of the eight plant species (except maize), highly specific gRNAs could be designed to target 85-98% of annotated transcript units. Therefore, we predict that the off-target effect of CRISPR-Cas9 could be minimized and will not constrain genome editing in plants. A plant gRNA database and web-based tools have been developed to guide the selection of specific gRNA spacers and to facilitate the CRISPR-Cas9 mediated genome editing and genetic engineering.