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CRISPR/Cas9-mediated genome editing in Hevea brasiliensis

文献类型： 外文期刊

作者： Dai, Xuemei 1 ; Yang, Xianfeng 1 ; Wang, Chun 2 ; Fan, Yueting 1 ; Xin, Shichao 1 ; Hua, Yuwei 1 ; Wang, Kejian 2 ; Huang 1 ;

作者机构： 1.Chinese Acad Trop Agr Sci, State Key Lab Incubat Base Cultivat & Physiol Tro, Key Lab Biol & Genet Resources Rubber Tree, Rubber Res Inst,Minist Agr & Rural Affairs, Haikou 571101, Hainan, Peoples R China

2.Chinese Acad Agr Sci, China Natl Rice Res Inst, State Key Lab Rice Biol, Hangzhou 311400, Zhejiang, Peoples R China

关键词： Hevea brasiliensis; CRISPR; Cas9; HbU6 promoter; Genome editing; Protoplast; Callus

期刊名称：INDUSTRIAL CROPS AND PRODUCTS （ 影响因子：4.244； 五年影响因子：4.583 ）

ISSN： 0926-6690

年卷期： 2021 年 164 卷

页码：

收录情况： SCI

摘要： Clustered regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) has not been completely established in Hevea brasiliensis. In the present study, firstly, five endogenous U6 promoters from H. brasiliensis were identified and exploited to drive single guide RNA (sgRNA) transcription for establishing CRISPR/Cas9 transient editing system in H. brasiliensis protoplast. All five promoters were functional, however, their corresponding editing efficiencies were different and varied from 8.47 % to 24.92 %. Secondly, mutation profiles were characterized using 10 sgRNAs targeted five flowering time related genes (HbFT1, HbFT2 and HbTFL1-1, HbTFL1-2, HbTFL1-3) in H. brasiliensis protoplast. Three mutation patterns i. e. deletion, insertion and base substitution were detected. Among these mutations, deletion was the most prominent one and the insertions were observed only in half of 10 target sites, in which the highest insertion frequency (15 %) occurred at TS6 target site. Base substitutions including transition and transversion were detected in 8 out of 10 target sites by deep sequencing. Lastly, stable transformation editing vector targeting phytoene desaturase gene (HbPDS) was constructed and transformed into rubber tree callus, and the ?+1? bp homozygous insertions were detected for 3 out of 16 calli at the target site, which turned to expected albino phenotype in the following subculture. This study pronounced the establishment of genome editing in H. brasiliensis by CRISPR/Cas9 plasmid system.

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