Specificity and inheritance of rBE3 and rBE4 endonuclease-induced gene modifications in rice.

Bin Ren; Fang Yan; Yongjie Kuang; Na LI; Dawei Zhang; Honghui Lin; Huanbin Zhou

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Specificity and inheritance of rBE3 and rBE4 endonuclease-induced gene modifications in rice.

Author: Bin REN ¹ ; Fang YAN ¹ ; Yongjie KUANG ¹ ; Na LI ¹ ; Dawei ZHANG ² ; Honghui LIN ² ; Huanbin ZHOU ¹
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1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
2. Ministry of Education Key Laboratory of Bio-Resource and Eco-Environment, College of Life Sciences, Sichuan University, Chengdu 610065, Sichuan, China.
Publication Type:Journal Article
Keywords: CRISPR/Cas9n; T-DNA segregation; off-target; rat APOBEC1; rice
From: Chinese Journal of Biotechnology 2017;33(10):1776-1785
CountryChina
Language:Chinese
Abstract: To gain more insights into the rice base editor (rBE3 and rBE4), we evaluated the mutation efficiency, off-target and inheritance of OsSERK1(D428N) and pi-ta(S918F) genes modified with rBE endonucleases. We predicted and analyzed the putative off-target sites of the sgRNA designed for OsSERK1(D428N) and pi-ta(S918F) by PCR amplification and Sanger sequencing. Then we further characterized the inheritance and stability of targeted base mutations and T-DNA segregation in the progeny of the self-fertilized T0 plants. Analysis of the DNA sequencing data of T0 plants of OsSERK1(D428N) revealed no nucleotide change at any of the four potential off-target sites. For OsSERK1(D428N) and Os08g07774 carry the same sgRNA targeting sites, base substitution at both two loci were detected at a frequency of 41.67%. The targeted base mutations could be transmitted readily to T1 progeny. Furthermore, genetic segregation caused the loss of T-DNA at a frequency between 25.0% and 40.9% in the T1 transgenic plants of OsSERK1(D428N) and pi-ta(S918F). These results demonstrated that the rBE3 and rBE4 systems could mediate specifically targeted base editing in one- or multi-site, and the targeted base editing could be stably inherited to next generation.