Effects of sgRNA length and number on gene editing efficiency and predicted mutations generated in rice

Abstract

CRISPR-Cas9 is a common tool for gene editing, and appropriate sgRNAs are the key factor for successful editing. In this study, the effect of sgRNA length and number on editing efficiency was analyzed in rice using CYP81A6 as the target gene. A series of CRISPR-Cas9 plant expression vectors containing single sgRNAs with different lengths (17, 18, 19, 20, 21, 22, 23 nt) or two sgRNAs were constructed and introduced into rice cultivar Zhonghua11 by Agrobacterium-mediated transformation. Analysis of the editing status of 1283 transgenic rice plants showed that 371 were successfully edited with base preference. Single A or T insertions were the most frequent among the six edited types. The editing efficiency of transgenic rice with two sgRNAs was higher than that with a single sgRNA. Editing efficiency and sgRNA length showed a normal distribution with 20 nt sgRNA (25%) being the most efficient. The editing efficiency decreased slightly with decreases of 1–2 bases (19 nt 20%, 18 nt 21%), but decreased significantly with a decrease of 3 bases (17 nt 4.5%). Editing efficiency was significantly reduced by adding 1 to 3 bases (21 nt 16.8%, 22 nt 13%, 23 nt 13%) to the sgRNA. These results provide data for successful gene editing or rice by CRISPR-Cas9.

Keywords

• CRISPR-Cas9
• sgRNA number
• sgRNA length
• Editing efficiency