水稻CRISPRa系统的优化及验证

Optimization and Validation of Rice CRISPRa System

CRISPR激活(CRISPR activation,CRISPRa)是通过dCas9-sgRNA复合体融合或招募转录激活域至启动子区以增强基因转录的CRISPR衍生技术。为提高水稻CRISPRa系统特异性及其激活效率,设计点突变引物将dCas9突变为特异性更强的HFdCas9,同时采用2×35S启动子表达密码子优化的水稻HFdCas9-TV。通过将HFdCas9-TV质粒和sgRNA质粒共转染水稻原生质体,基于OsGW7和OsF3H的相对表达量变化检测所构建的CRISPRa系统激活效率。结果表明,该系统可激活OsGW7至对照组的95倍,为CCTV系统的2.2倍;可同时激活OsGW7、OsF3H表达量分别至对照组的33倍和26倍。该系统的建立为开发特异性更强、效率更高的植物CRISPRa系统奠定前期试验基础,为水稻及其他作物分子育种提供“多重加法”的技术方案。

CRISPR activation(CRISPRa)is a CRISPR-derived technology for enhancing gene transcriptionthrough the dCas9-sgRNA complex or transcriptional activation domains to promoter regions.In order to improve the specificity and activation efficiency of the rice CRISPRa system,point mutation primers were designed to mutate dCas9 into a more specific HFdCas9,and the 2×35S promoter was used to express the codon-optimized rice HFdCas9-TV.The activation efficiency of the constructed CRISPRa system was determined by the relative expression changes of OsGW7 and OsF3H in rice protoplasts,which were co-transformed with HFdCas9-TV plasmid and sgRNA plasmid.The results showed that the CRISPRa system activated OsGW795 times higher than that of the control group and 2.2 times higher than that of the CCTV system;simultaneously,the CRISPRa system activated OsGW7 and OsF3H 33 times and 26 times higher than that of control group,respectively.This system lays a preliminary experimental foundation for the development of a more specific and efficient plant CRISPRa system,and provides a“multiple addition”technical solution for molecular breeding of rice and other crops.