Construction Of OsGRF Knock-out Rice Lines Using CRISPR/Cas Systems

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is a programmable genome editing technology newly developed in recent years. In this study, the CRISPR/Cas9 technology was used to induce site-directed mutations in five putative Growth-Regulating Factor (GRF) genes (OsGRF3/7/8/9/11) in rice(Oryza sativa L.), creating gene knock-out plants of these genes and comparatively analyzing their mutation frequencies, mutation types and zygote types. Our main results are as follows.1. Mutation frequency. The proportions of mutated plants in the To generations of the five target genes OsGRF3/7/8/9/11 were 22/25,38/47,22/22,12/16 and 22/22, respectively, ranging 75.0%-100.0%. These results suggest that the CRISPR/Cas9 technology could efficiently mediate editing in the rice genome. Here, Cas9 coding sequence was codon-optimized for rice and its expression was driven by the maize ZmUbi promoter, while the expression of guide RNA was driven by the rice type Ⅲ promoter OsU6. We guess that it might be just because of these modifications of the core components in the expression cassette making the high mutation rates in rice.2. Mutation type. Most (～95%) of the mutations found in the To plants were short insertion/deletion (≤3 bp). Besides, the base of 1-bp insertions showed strong bias toward T and A (accounting for 89.3% in total). We infer from these results that the double-strand breaks created by Cas9/gRNA complexes often led to short mutations mainly due to the intrinsic nonhomologous end-joining repair, and that the percentages of different nucleotides around the loci might be a factor limiting the mutation types of the target sites.3. Zygote type. Among 132 To mutated plants,87 were bi-allelic mutants, of which 59 appeared to be compound-heterozygotes. These results suggest that most of the transformed embryogenic plant cells underwent target mutations before their first division. In addition, we investigated the segregation ratios of several Ti rice lines targeting OsGRF718, and found that they basically followed the Mendelian law.The results of this study demonstrate the efficiency of the CRISPR/Cas9 system in rice genome editing, and provide valuable materials for further functional studies of OsGRF genes.