Efficient genome editing of rubber tree (hevea brasiliensis) protoplasts using CRISPR/Cas9 ribonucleoproteins
Efficient genome editing of rubber tree (hevea brasiliensis) protoplasts using CRISPR/Cas9 ribonucleoproteins
复制标题
使用 CRISPR/Cas9 核糖核蛋白对橡胶树 (hevea brasiliensis) 原生质体进行高效基因组编辑
DOI：
10.1016/j.indcrop.2020.112146
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发表时间：
2020-04-01
影响因子：
5.9
通讯作者：
Hua, Yuwei
中科院分区：
文献类型：
--
作者：
Fan, Yueting;Xin, Shichao;Hua, Yuwei
CRISPR/Cas9(簇状规则间隔短回文重复序列/CRISPR相关蛋白9)是一种强大的靶向突变工具，已在许多植物物种中实施。到目前为止，CRISPR/CAS9在橡胶树上的应用尚未见报道。在这里，我们描述了通过直接传递CRISPR/Cas9核糖核蛋白(RNPs)在橡胶树中进行有效的靶向突变。设计了5个针对橡胶树开花基因T(FT)和末端FLOWER1(TFL1)的sgRNAs。在Cas9/sgRNA比例为1：7的情况下，将Cas9蛋白与体外转录的sgRNAs进行预组装，然后导入橡胶树原生质体。在5个靶点成功地诱导了3.74%~20.11%的靶向突变。在所有靶点均检测到+1nt插入和缺失两种突变模式，其中-1nt缺失是所有病例中最常见的突变。此外，通过传递针对多个基因的sgRNAs组合，通过一个转化步骤在橡胶树原生质体中诱导了多个靶向突变。这个基于RNP的基因组编辑系统展示了对橡胶树进行精确遗传修改的潜力。此外，本研究还与橡胶树原生质体再生体系相结合，为利用原生质体获得无DNA基因组编辑的橡胶树植株提供了一条很有前途的途径。
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) is a powerful targeted mutagenesis tool that has been implemented in many plant species. To date, the application of CRISPR/Cas9 in rubber tree (Hevea brasiliesis) has not yet been reported. Here, we describe the efficient targeted mutagenesis in rubber tree by direct delivery of CRISPR/Cas9 ribonucleoproteins (RNPs). Five sgRNAs were designed to target FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) genes in rubber tree. Using a Cas9/sgRNA ratio of 1:7, the Cas9 proteins were preassembled with in vitro transcribed sgRNAs, and then introduced into rubber tree protoplasts. Targeted mutations were successfully induced at frequencies ranging from 3.74% to 20.11% at five target sites. Two mutation patterns including +1 nt insertions and deletions were detected at all target sites, and the -1 nt deletion was the most common mutation obtained in all cases. In addition, by delivering combinations of sgRNAs targeting multiple genes, multiple targeted mutations were induced in rubber tree protoplasts through one transformation step. This RNP-based genome editing system demonstrates the potential for precise genetic modifications of rubber tree. Furthermore, together with the rubber tree protoplast regeneration system, our study provides a promising approach for the production of DNA-free genome edited rubber tree plants from protoplast.