Construction And Application Of CRISPR-Cas9-UBA Gene Editing System In Rice

CRISPR(Clustered regularly interspaced short palindromic repeat sequences)-Cas(CRISPR-associated proteins)system is an acquired immune system presently found in most bacteria and all archaea and it confers immunity against extraneous plastids or phages through identification,integration,expression and interference of foreign DNA.CRISPR-Cas system has been widely applied to genetic manipulation containing gene targeted deletion and addition,as well as activation and inhibition of gene expression.CRISPR-Cas9 based on the type II CRISPR system has become the third generation gene editing tool following the first generation zinc finger nucleases(ZFN)and the second generation transcriptional activation factor nucleases(TALEN).Compared with ZFN and TALEN gene editing tools,CRISPR-Cas9 system has the advantages of high efficiency,simple construction and low cost,but there are still some problems in the editing process,such as large differences in the editing efficiency of target genes and low editing efficiency of some target sites.In order to improve the editing efficiency of the CRISPR-Cas9 system at specific gene loci,this study further optimized the single transcript unit CRISPR-Cas9 system for genome editing in plants developed in our laboratory by fusing Cas9 protein to ubiquitin associated domain UBA1,UBA2,and UBA3,respectively,and the CRISPR-Cas9-UBA gene editing system was established in rice.The CRISPR-Cas9-UBA directed editing vectors were constructed to target different sites of three endogenous rice genes,and the cleavage activity and editing efficiency were measured by protoplast transient transformation system and agrobacterium-mediated stable genetic transformation system to evaluate the effects of different UBA domains on Cas9 protein activity and editing efficiency.The main contents and results of this research are summarized as follows:1.By comparing the different ubiquitin associated domains,UBA1,UBA2 and UBA3 domains from Arabidopsis thaliana were selected.Rice codon optimization was carried out and then three CRISPR-Cas9-UBA gene editing vectors pYLJ01,pYLJ02 and pYLJ03 were constructed based on the pTX172 of CRISPR-Cas9 system vector.2.Three endogenous genes OsPDS,OsDEP1,and OsROC5 related to rice albinism,dwarfing and leaf curl phenotype were selected as reporter genes.The four target sites OsPDS-sgRNA01,OsPDS-sgRNA02,OsDEP1-sgRNA02 and OsROC5-sgRNA01 with low relatively editing efficiency,which were demonstrated in previous experiments,were selected as the research objects.Based on three CRISPR-Cas9-UBA systems pYLJ01,pYLJ02,pYLJ03 and control CRISPR-Cas9 system pTX172,16 expression vectors of targeted genome editing were constructed for the above four target sites,respectively.3.The PEG-mediated rice protoplast transient transformation system was used to detect the cleavage activity of Cas9 protein fused to different UBA domains.The results showed that compared with the control group CRISPR-Cas9,three CRISPR-Cas9-UBA gene editing system showed shear activity at all four target sites,which indicated that the UBA1,UBA2,and UBA3 domains had no effect on the shear activity of Cas9 protein,and can be used for further study on gene editing efficiency.4.Further,OsPDS-sgRNA01 and OsDEP1-sgRNA02 were used as the research objects,and Agrobacterium-mediated stable genetic transformation was carried out in Nipponbare rice varieties.Mutations detection,phenotypic analysis and mutation efficiency comparison were performed on the target sites of regenerated transgenic plants to evaluate the difference in effectiveness,stability and editing efficiency of CRISPR-Cas9-UBA gene editing system.Based on PCR-RFLP and the genotyping by sequencing analysis,the cleavage sites of endogenous genes in rice in the three CRISPR-Cas9-UBA gene editing systems were consistent with those in the control CRISPR-Cas9 system,and in most cases,1bp base deletion or insertion occurred at the position 3-4 adjacent to the PAM of diallele or one of the alleles.This indicates that the fusion of UBA1,UBA2 or UBA3 domains have no significant effect on the shear mode of Cas9 protein.Phenotypic analysis of mutant plant showed that at the same gene targeting site,there was no difference in mutant phenotypes in plants induced by three CRISPR-Cas9-UBA gene editing systems and CRISPR-Cas9 system,These results suggest that the CRISPR-Cas9-UBA gene editing system has no obvious difference from the CRISPR-Cas9 system in the mechanism of inducing mutations,and can be used for targeted gene editing and mutant creation.5.Finally,compared with the control group CRISPR-Cas9,the three CRISPR-Cas9-UBA systems could effectively induce gene mutations at OsPDS-sgRNA01 and OsDEP1-sgRNA02,and their gene editing efficiency were significantly improved.Among them,the targeted editing of CRISPR-Cas9-UBA2 system incorporating the UBA2 domain was the most efficient.