基于原生质体的谷子CRISPR/Cas9基因编辑系统优化

Optimization of CRISPR/Cas9 Genome Editing Systems in Protoplasts of Setaria italica

为了优化和筛选谷子中高效的CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISRP-associated nuclease 9)基因编辑系统,针对谷子八氢番茄红素脱氢酶基因(SiPDS)设计6种gRNA(gRNA1—gRNA2针对外显子1,gRNA3—gRNA6针对外显子12),构建多种CRISPR/Cas9基因编辑系统,通过聚乙二醇(PEG)介导的方法转入谷子原生质体中,然后利用开发的大规模平行测序技术快速检测它们对SiPDS的突变效率。结果表明,采用7 d谷子黄化苗幼茎建立的原生质体转化系统的转化效率较高,为50.44%~57.36%。将Super启动子(SP)、谷子内源的泛素启动子(Ubi)分别驱动Cas9基因的基因编辑系统转入谷子原生质体中,发现两者对SiPDS基因的突变效率分别为0.5%、5.5%。随后采用Ubi启动子驱动Cas9基因,比较单一gRNA、双gRNA和tRNA结构的基因编辑系统对SiPDS基因的突变效率,发现双gRNA基因编辑系统Ubi-dgRNAE1和Ubi-dgRNAE12的突变效率分别较单一gRNA基因编辑系统提高了1.66倍和1.11倍;tRNA基因编辑系统Ubi-tRNA的突变效率较单一gRNA基因编辑系统提高了5.87倍,为51.24%,且Ubi-tRNA可同时针对多个位点进行编辑,其多位点突变频率为3.23%。比较gRNA3、gRNA4、gRNA5体外转录产物分别与Cas9蛋白混合制成的核糖核蛋白(RNP)复合物的体外切割活性,发现RNP-gRNA5复合物体外切割活性最高,其对SiPDS基因的突变效率是2.0%,并且引起的突变类型主要为小于3 bp的缺失。

To optimize and screen efficient genome editing systems in millet(Setaria italica),multiple CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISRP-associated nuclease 9)genome editing systems were constructed with six kinds of gRNAs designed for phytoene desaturase(SiPDS)gene(gRNA1—gRNA2 for exon 1,gRNA3—gRNA6 for exon 12),and transformed into protoplasts of millet by polyethylene glycol-mediated method.Then the mutation efficiency against SiPDS gene was rapidly detected by large-scale parallel sequencing.The results showed that the protoplast transformation system established by the young stems of 7 day-old millet etiolated seedlings had high transformation efficiency from 50.44%to 57.36%.Using this method,the gene editing systems with Cas9 gene driven by Super and Ubi promoters were transferred into millet protoplasts,the mutation efficiencies of SiPDS gene were 0.5%and 5.5%respectively.Thus,the Cas9 gene of gene editing systems with single gRNA,double gRNA or tRNA cassettes were driven by Ubi promoter,and mutation efficiency of SiPDS gene induced by these systems was compared.It was found that the mutation efficiencies of gene editing system with double gRNA cassettes such as Ubi-dgRNAE1 and Ubi-dgRNAE12 were 1.66 and 1.11 times higher than that with single gRNA system.The mutation efficiency induced by t RNA-based system Ubi-tRNA was 51.24%,which was 5.87 times higher than that with single gRNA system.Moreover,Ubi-tRNA could simultaneously edit multiple sites of SiPDS gene,the multi-site mutation frequency was 3.23%.The cleavage activities in vitro of ribonucleoprotein(RNP)complexes which were mixture of gRNA3,gRNA4 or gRNA5 transcripts with Cas9 protein respectively were compared,it was found that RNP-gRNA5 complex had the highest cleavage activity in vitro,the mutation efficiency of SiPDS gene was 2.0%,and the dominant mutation type was deletion less than 3 bp.