一个CRISPR/Cas9-VQR基因编辑系统的构建

Establishment of a CRISPR/Cas9-VQR gene editing system

CRISPR/Cas9系统是一种广泛应用于细菌、酵母、动物和植物中的基因组定点编辑技术,但该编辑系统的使用范围受PAM(proto-spacer-motif)位点NGG的限制。本研究通过突变Streptococcus pyogenes Cas9(SpCas9)编码氨基酸(1135位的天冬氨酸D突变成缬氨酸V,1335位的精氨酸R突变为谷胱氨酸Q,1337位的苏氨酸T突变为精氨酸R,命名该突变子为Cas9-VQR)改造其识别PAM为NGA的位点以扩大其使用范围。并使用玉米Ubi启动子启动Cas9-VQR基因、优化SpCas9的密码子、加入保守的核定位信号序列、增加单子叶植物中保守的3′UTR序列和使用水稻U6启动子启动gRNA来修饰该编辑系统。结果表明Cas9-VQR系统能够识别PAM为NGA的位点,并进行有效的切割。体外酶切活性检测结果表明Cas9-VQR的切割效率为5%~70%。水稻转化检测结果表明Cas9-VQR的切割效率约为27.5%~70.5%,平均切割效率为46.23%。本研究拓宽了CRISPR/Cas9系统在作物中的使用范围,特别是NGA PAM位点较高的作物。

Clustered Regularly Interspaced Short Palindromic Repeat and Cas9(CRISPR/Cas9),a new generation of genomeediting technology,is widely applied among bacteria,yeast,animals and plants,however,the typical CRISRP/Cas9 cannot recognize the NGA proto-spacer-motif(PAM),which limits its application.In order to broaden the applications of CRIPSR/Cas9 system,we modified the Streptococcus pyogenes Cas9(SpCas9)sequence by the PCR site-direct mutagenesis,which encodes V(1135),Q(1335),and R(1337),to make the CRIPSR/Cas9-VQR able to recognize the NGA PAM motif.We also constructed a binary expression vector of CRISRP/Cas9-VQR with maize ubiquitin as the promoter to drive the Cas9-VQR,optimizing SpCas9-codon,adding conserved nuclear localization signal sequence,and increasing the conserved 3'UTR sequence of monocots,and using OsU6 transcripts of sRNA.CRISPR/Cas9-VQR could recognize the NGA motif and cut targeted sequence in vivo.We assembled the Cas9-VQR protein with the sRNAs in vitro.The Cas9-VQR could cleave the targeted fragments with about 5%–70%of mutation efficiency.In the transformation of rice,we detected about 27.50%–70.50%of mutation ratio,with an average of 46.23%.This system broadens the CRISPR/Cas9 applications in crops,especially in these with higher PAM locus of NGA.