Study On The Recognition Of NGAC Protospacer Adjacent Motif By Cas9 Protein Variant VQR

Currently,Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9(CRISPR-Cas9)system,the third generation of genome editing techniques,can apply in various species for genome editing.In the process of gene editing,beside guide RNA(single guide RNA,sgRNA)and the target sequence bases complement each other in the genome,CRISPR-Cas9 requires several specific bases near the target sequence named Protospacer Adjacent Motif(PAM)in the genome.The widely used SpCas9 mainly recognizes NGG PAM,which limits the number of potential target sites within genome.The recently reported VQR variant protein basing on SpCas9 recognized NGAA,NGAG and NGAT PAMs in rice,which expanded the range of gene editing.However,it has not been verified that the VQR can now identifies NGAC PAM in rice.In this study,the target sites of gene exons with low gene editing efficiency in the original CRISPR-VQR system,was used for gene editing with improved CRISPR-VQR system.The corresponding gene editing vector is constructed to knock out the sequence of target sites,and transgenic plants were obtained by agrobacterium transfection.Analysis of sequencing showed that the editing rate of exons was significantly improved compared to the original CRISPR-VQR system.Then,the gene editing vector was successfully used to knock out the target sites containing PAM as NGAC in rice by using the improved CRISPR-VQR system,which obtained 57 transgenic plants.Analysis of sequencing results showed that VQR can effectively identify NGAC PAM,which suggests the scope of CRISPR-VOR system is further supplemented and the practical application value of VQR is improved.Since Gene mutation types are also extremely important for the study of gene function,the gene mutation types of the 5 target sites of CRISPR-VQR system were statistically analyzed.The results show that the main types of genetic mutation are heterozygotic mutations and biallelic mutations.Subsequently,in order to evaluate the editing capability of SpCas9 on the NGA PAM type target site,we selected the target site of NGAC PAM for editing with SpCas9.We successfully constructed the gene editing vector of SpCas9 knockout NGAC target site.The vector was transferred into rice by agrobacterium infection.The transgenic callus was obtained through the screening of hygromycin.The DNA of transgenic callus was extracted and sequenced.Analysis of sequencing results showed the editing ability of SpCas9 on the NGAC PAM type target site is relatively low.These results indicate that the improved CRISPR-VQR system can efficiently edit the NGAC PAM sites of the rice and produce abundant mutant types.This study not only laid a foundation for genome editing of the NGAC PAM sites in rice,but also provided important reference for genome editing in other plants related sites.