基于CRISPR-Cas9技术构建橡胶树胶孢炭疽菌的基因敲除系统

CRISPR-Cas9 ribonucleoprotein-mediated gene knock-out in Colletotrichum gloeosporioides from Hevea brasiliensis

摘要:

【背景】CRISPR-Cas9基因组编辑技术为病原真菌的基因敲除、敲入及定点编辑提供了新的思路。【目的】建立适用于橡胶树胶孢炭疽菌的CRISPR-Cas9基因敲除系统。【方法】通过大肠杆菌原核表达系统合成含有细胞核定位信号的Cas9蛋白；以URA5为靶标基因，预测该基因中Cas9的切割位点，并在体外转录合成相应的SgRNA；体外构建Cas9-SgRNA复合体，并将该复合体转入橡胶树胶孢炭疽菌原生质体；通过表型筛选及测序鉴定，筛选URA5的敲除突变体菌株。【结果】体外表达的Cas9蛋白与SgRNA能够形成复合体，并在体外对目标基因URA5的DNA序列进行切割；Cas9-SgRNA复合体能够成功转入橡胶树胶孢炭疽菌原生质体，并完成对URA5的敲除；敲除突变株表现出尿嘧啶缺陷表现型。【结论】建立了适用于橡胶树胶孢炭疽菌的基因敲除系统。

关键词:CRISPR-Cas9，橡胶树胶孢炭疽菌，基因敲除

Abstract:

[Background] Genome editing with CRISPR-Cas9 technique may provide some new ways for gene knock-out, knock-in and gene editing in fungal pathogens. [Objective] To construct a system for gene editing in Colletotrichum gloeosporioides from Hevea brasiliensis. [Methods] Cas9 containing the nuclear localization signal peptide was expressed in Escherichia coli and purified. Potential cleavage sites of URA5 was analyzed and an SgRNA was transcribed in vitro. The Cas9 protein and SgRNA were assembled in vitro to form a ribonucleoprotein. The ribonucleoprotein was transformed into the protoplast of C. gloeosporioides and the transformants were screened. [Results] The Cas9 protein and SgRNA could form a stable ribonucleoprotein and this complex showed high DNA cleavage activity in vitro. The ribonucleoprotein was transformed into the protoplast of C. gloeosporioides and URA5 was successfully knocked out. [Conclusion] The system is convenient for gene editing in C. gloeosporioides of H. brasiliensis.

Key words:CRISPR-Cas9, Colletotrichum gloeosporioides from Hevea brasiliensis, gene knock-out