Establishment of a CRISPR/Cas9-VQR gene editing system
【Abstract】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 s RNAs 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.
【Keywords】 CRISPR/Cas9-VQR; site-direct mutagenesis; PAM; mutation rate;
(Translated by HE Z)
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