RNA-Guided Genome Editing in Plants Using a CRISPRCas System

Targeted gene mutation was successfully achieved in rice using the CRISPRCas9 system. Experimental analyses of mutation efficiency and off-target effect as well as genome-wide prediction of specific guide RNA seeds suggest that the CRISPRCas9 system is a simple and effective tool for plant functional genomics and crop improvement.Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPRCas9 system. Three guide RNAs (gRNAs) with a 2022-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif (PAM). The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation (insertion or deletion) by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 38%. In addition, the off-target effect of an engineered gRNACas9 was found on an imperfectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNACas9 targeting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPRCas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants.