Research On Optimizing CRISPR/Cas9 System To Improve Tobacco Gene Editing Efficiency

The CRISPR/Cas9 system is an adaptive immunity generated by archaea and bacteria during long-term evolution,which can resist the invasion of foreign DNA and viruses.The artificially modified CRISPR/Cas9 gene editing technology uses sg RNA to guide Cas9 protein directly cleavage of genes to produce DNA double-strand breaks,using the Homology directed repair(HDR)or Non-homologous end joining(NHEJ)to repair DNA.CRISPR/Cas9 system is one of the most widely used technologies in gene editing and modification.Tobacco(Nicotiana tabacum),as one of the most important economic crops and model plants,has important research value.However,because the cultivated tobacco is heterotetraploid and the genome is large and complex,gene editing efficiency is low when using current CRISPR/Cas9 system.In addition,the knock out plants would form to be chimera in T0 generation.If the large-scale gene editing in tobacco is wanted to be done,the existing gene knockout system will waste lots of manpower,material and financial resources.Therefore,this study is expected to optimize the CRISPR/Cas9 system to reduce the generation of chimeras,and improve the gene editing efficiency,by replacing Cas9 protein or replacing 35 S promoter and NOS terminator elements.The following results are currently obtained.??Construction of CRISPR/xCas9 vector based on xCas9 3.7 and detection of mutation efficiencyIn order to obtain xCas9 suitable for tobacco gene editing,the codon of xCas9 3.7 were optimized using tobacco codon,which was constructed to current knock out vector,named as 35S: nxCas9 vector.Nt PDS and Nt PDR genes were selected to detect gene editing efficacy in new vector.Two sg RNAs were designed in each PAM(NG,NGG,GAA,and GAT).The 16 vectors containing 8 sg RNAs basing on 35S: sp Cas9 and 8 sg RNAs constructed by35S: nxCas9,were delivered into agrobacterium respectively.The transgenic plants in T0 generation were obtained by Agrobacterium-mediated genetic transformation and molecular detection.The results of Sanger sequencing showed that the editing efficiency of xCas9 in the NTG PAM region of Nt PDR gene(30%-35%)was higher than sp Cas9(20%-25%).There was no significant change in the editing efficiency of xCas9(15%-25%)when compared to sp Cas9(20%-25%)in the NGG PAM region of Nt PDS gene.In the non-NGG PAM region,only one planet occurred gene editing(5%)in the NG PAM region of the Nt PDR gene.The above results showed that CRISPR/xCas9 knockout vector has a certain preference for gene and target site selection.In addition,xCas9 has the ability of genome editing in the PAM region of NG,which to a certain extent broaden the selectivity of gene editing in the PAM region of cas9.II.Construction of knockout vector of Cas9 protein promoted by NtEC1.2-1/2 and NtDMC1-1/2 specific promoter in tobacco and detection of mutation efficiency.Amino acid sequences of the germ cell-specific promoter genes(At EC1.2,AtDMC1)reported in A.thaliana were blasted in the tobacco genome database.Four matching genes were found,named as NtEC1.2-1,NtEC1.2-2.NtDMC1-1 and NtDMC1-2.Promoter element analysis results showed that both NtEC1.2 and NtDMC1 promoters contained cell division M-phase specific elements(AACGG)and targets involved in pollen-specific transcriptional activation motif(GAAA)and early activator(AACTTAA).The existence of these motifs indicated that the promoters we selected may be expressed during tobacco reproduction.RT-PCR results showed that NtEC1.2-1,NtEC1.2-2,NtDMC1-1 and NtDMC1-2 were all highly expressed in anthers.Especially NtEC1.2-1 was also highly expressed in ovary.The results of EGFP fluorescence analysis showed that the four promoters(NtEC1.2-1,NtEC1.2-2,NtDMC1-1 and NtDMC1-2)could drive the expression of EGFP in tobacco leaves.The results of GUS staining of transgenic plants showed that the NtEC1.2-1 promoter could drive gus expression in ovary and seed,with obvious blue appearance,while the other three promoters(NtEC1.2-2,NtDMC1-1 and NtDMC1-2)could not drive Gus expression without blue in the above tissues.The constructed knockout vectors containing the Cas9 protein expressed by the tobacco endogenous reproductive specific promoter(NtEC1.2-1: Cas9-rbcS-E9,NtEC1.2-2: Cas9-rbcS-E9,NtDMC1-1: Cas9,NtDMC1-2: Cas9)and the control plasmids(At EC1.2: Cas9-rbcS-E9)were transferred into Agrobacterium respectively.Transgenic plants of T0 generation were obtained through Agrobacterium-mediated genetic transformation and molecular detection.Because of the long growth cycle of tobacco,we injected Agrobacterium tumefaciens preserved in laboratory into plants of T0 generation instantaneously,so as to obtain T1 generation transgenic plants faster.By sequencing the T1 transgenic plants,we found that the mutation efficiency of pORE-At EC1.2-rbcS-E9 plants in the control group was 20%,and all of them were heterozygotes,without the production of chimeras and homozygotes.There was no chimera in the experimental group.The mutation efficiency of pORE-NtEC1.2-2-rbcS-E9,pORE-NtDMC1-1 and pORE-NtDMC1-2 was 5%-10%,lower than that of the control group.However,the mutation efficiency of pORE-NtEC1.2-1-rbcS-E9 was 26.7%,slightly higher than that of the control group.We constructed different knockout vectors by replacing cas9 protein and the promoter of cas9.We found that xcas9 has the preference of gene and target site,and the editing efficiency of Nt PDR gene was higher than that of spcas9,but whether it has universality needs further verification.The plants knock-out by endogenous reproductive specific promoter of tobacco,was found to be heterozygous without the occurrence of chimeras.Among them,the gene editing efficiency of the combination of the gene promoter of NtEC1.2-1 and the terminator of rbcs-e9 in tobacco was slightly higher that of the control group.Although in this study,we did not greatly improve the efficiency of tobacco gene editing,this research provided us with a good start.In the future,we can improve the efficiency of tobacco gene editing by optimizing promoters or selecting more active promoters.It is of great significance for the follow-up study of tobacco gene function and the creation of mutant materials.