修饰后的CRISPR-Cas9-VQR系统将成为基因编辑的强大工具

作为基因编辑的强大工具,CRISPR-Cas9的识别位点局限在以NGG为特征的PAM结构上,这大大限制了CRISPR-Cas9的应用范围。近期发现的CRISPR-Cas9的VQR (D1135V/R1335Q/T1337R) 变体可以识别以NGA为特征的PAM位点,一定程度上扩充了Cas9的应用范围。但是,VQR变体的编辑效率不高,极大地限制了其应用。

最近,中国农科院水稻所王克剑团队和中科院遗传所李家洋团队合作,通过改造sgRNA结构和利用内源强启动子(UBI1ACT1),显著提高了VQR变体的编辑效率。通过试验,水稻中的编辑效率最高可达到近80%,提升幅度最高可达8倍。尤其在多位点编辑时,效率提升幅度更加明显,可以达到15倍。因此,修饰后的CRISPR-Cas9-VQR系统将成为多NGA靶标位点编辑的强大工具。

 

Plant Biotechnol J. 2017 Jun 12.

Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice.

 

Author

Hu X, Meng X, Liu Q, Li J, Wang K*.

*: State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, China.

 

Abstract

Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is a revolutionary technology that enables efficient genomic modification in many organisms. Currently, the wide use of Streptococcus pyogenes Cas9 (SpCas9) primarily recognises sites harbouring a canonical NGG protospacer adjacent motif (PAM). The newly developed VQR (D1135V/R1335Q/T1337R) variant of Cas9 has been shown to cleave sites containing NGA PAM in rice, which greatly expanded the range of genome editing. However, the low editing efficiency of the VQR variant remains, which limits its wide application in genome editing. In this study,by modifying the single guide RNA (sgRNA) structure and using strong endogenous promoters, we significantly increased the editing efficiency of the VQR variant. The modified CRISPR-Cas9-VQR system provides a robust toolbox for multiplex genome editing at sites containing non-canonical NGA PAM.