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Efficient genome editing of Brassica campestris based on the CRISPR/Cas9 system

文献类型： 外文期刊

作者： Xiong, Xingpeng 1 ; Liu, Weimiao 1 ; Jiang, Jianxia 4 ; Xu, Liai 1 ; Huang, Li 1 ; Cao, Jiashu 1 ;

作者机构： 1.Zhejiang Univ, Inst Vegetable Sci, Lab Cell & Mol Biol, Hangzhou 310058, Zhejiang, Peoples R China

2.Minist Agr, Key Lab Hort Plant Growth Dev & Qual Improvement, Hangzhou 310058, Zhejiang, Peoples R China

3.Zhejiang Prov Key Lab Hort Plant Integrat Biol, Hangzhou 310058, Zhejiang, Peoples R China

4.Shanghai Acad Agr Sci, Crop Breeding & Cultivat Res Inst, Shanghai 201403, Peoples R China

关键词： Brassica campestris; CRISPR; Cas9; Multiplex genome editing; PME; Heritable mutation

期刊名称：MOLECULAR GENETICS AND GENOMICS （ 影响因子：3.291； 五年影响因子：3.257 ）

ISSN： 1617-4615

年卷期： 2019 年 294 卷 5 期

页码：

收录情况： SCI

摘要： Conventional methods for gene function study in Brassica campestris have lots of drawbacks, which greatly hinder the identification of important genes' functions and molecular breeding. The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile tool for genome editing that has been widely utilized in many plant species and has many advantages over conventional methods for gene function study. However, the application of CRISPR/Cas9 system in B. campestris remains unreported. The pectin-methylesterase genes Bra003491, Bra007665, and Bra014410 were selected as the targets of the CRISPR/Cas9 system. A single-targeting vector and a multitargeting vector were constructed. Different types of mutations were detected in T-0 generation through Agrobacterium transformation. The mutation rate of the three designed sgRNA seeds varied from 20 to 56%. Although the majority of T-0 mutants were chimeric, four homozygous mutants were identified. Transformation with the multitargeting vector generated one line with a large fragment deletion and one line with mutations in two target genes. Mutations in Bra003491 were stable and inherited by T-1 and T-2 generations. Nine mutants which did not contain T-DNA insertions were also obtained. No mutations were detected in predicted potential off-target sites. Our work demonstrated that CRISPR/Cas9 system is efficient on single and multiplex genome editing without off-targeting in B. campestris and that the mutations are stable and inheritable. Our results may greatly facilitate gene functional studies and the molecular breeding of B. campestris and other plants.

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