Genome Editing and Targeted Mutagenesis in Plants Using the CRISPR-Cas9 System

Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, the bacterial cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) system is emerging as an efficient tool for genome editing. In this study, we demonstrated RNA-guided genome editing and targeted gene mutation in plants via the CRISPR-Cas9 system. Three guide RNAs (gRNAs) with a 20-22 nt spacer 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 genome editing events, the mutation efficiency at these target sites was estimated to be 3 - 8%. In addition, the off-target effect of an engineered gRNA-Cas9 was found on an imperfectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. To further assess the specificity and utility of the CRISPR-Cas9 system, we have conducted genome-wide prediction of specific gRNA spacer sequences and targetable transcription units in eight model plants and major crops. Our study suggests that the CRISPR-Cas9 system can be exploited as a powerful tool for precise genome editing and genetic modification of agricultural crops.