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Short Communication | Open Access

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

Xiaojing Liu, Jiangtao Yang, Yaya Song, Xiaochun Zhang, Xujing Wang( ), Zhixing Wang( )
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences/MARA Key Laboratory on Safety Assessment (Molecular) of Agri-GMO, Beijing 100081, China
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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-Cas9sgRNA numbersgRNA lengthEditing efficiency

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The Crop Journal
Volume 10 Issue 2,
April 2022
Pages 577-581
DOI: 10.1016/j.cj.2021.05.015
Cite this article:
Liu X, Yang J, Song Y, et al. Effects of sgRNA length and number on gene editing efficiency and predicted mutations generated in rice. The Crop Journal, 2022, 10(2): 577-581. https://doi.org/10.1016/j.cj.2021.05.015

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Received: 09 March 2021
Revised: 30 April 2021
Accepted: 11 June 2021
Published: 17 July 2021
© 2021 Crop Science Society of China and Institute of Crop Science, CAAS.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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