Preliminary Studies On Gene Editing In Brassica Oleracea L. Using CRISPR/Cas9 System

Cabbage(Brassica oleracea L.)is an important vegetable crop,the annual cultivation area is up to 900,000 hm2.The cabbage plays an important role in anniversary balanced supply of vegetables.As a typical cross pollinated plant,the cabbage show vigorous heterosis.However,the self-incompatibility characteristics that the cabbage had makes its reproduction requires time-consuming,low efficiency and high cost artificial pollination in bud stage,that greatly limit the application of male sterility in heterosis breeding.Due to increasingly understanding of the self-incompatibility genetic mechanism and self-incompatibility gene function,make it possible to edit key genes based genome editing technology,generate self-compatible lines.Recently,the emergence of CRISPR/Cas9 provides a convenient and efficient technology to precise modification of the target gene.Because only target sequence is modified by the CRISPR/Cas9,non-transgenic mutants without any exogenous DNA sequence can be obtained by self-cross the T0 transgenic plants,that are same as mutants constructed by traditional breeding technology.Now,agricultural products generated by using CRISPR/Cas9 technology had obtain the approval and are not subject to GMO constraints.In this study,we use this "magic" genome editing technology to accurately knock out cabbage BoPDS,SRK,BoGA4 gene and tobacco Nt PDS gene.In addition,in order to obtain a more simple and efficient gene editing system,the optimization of CRISPR/Cas9 gene editing system was carry out in tobacco and cabbage,the main results are as follows: 1.Knockout the genes in cabbage by CRISPR/Cas9 systemAccording to the BoPDS and SRK gene sequences,three target sites for each gene were selected and assembled into sgRNA driven by U6-26,U6-29,U6-1 promoter of Arabidopsis thaliana.The pCACas-BoPDS-ABC,pCACas-SRK-ABC expression vectors were introduced into cabbage respectively by Agrobacterium mediated transformation.The PCR products of the target site area from pCACas-BoPDS-ABC transgenic plants were sequenced,and the gene edited events were confirmed based on the overlapping sequence or different sequence showed at the target site,the results showed that mutations occurred in 3 plants at the target site,the mutation rate was37.5%.Then the PCR fragment was cloned and sequenced.It was found that the knockdown efficiency was 43.3%(13/30)at the target site 1,no knockdown was found at target site 2,31.3%(15/43)knockdown efficiency occurred at the target site 3.According PCR sequencing results of SRK gene from 43 pCACas-SRK-ABC T0 transgenic plants,we found knockdown occurred at the target site 2 in 10 plants,and 23 plants at the target site 3,the editing efficiency for SRK gene reached 53.5%.Further sequencing analysis of PCR fragments cloning showed that the knockdown efficiency was 0 at the target site 1,51.8%(28/54)at the target site 2,and 93.9%(77/83)at the target site 3.In addition,we found that the sequence of target site 1 in SRK gene and SLG gene were completely matched,and there were only two nucleotides mismatch in the seed sequence region at target site 2.However,the CRISPR/Cas9 system did not edit the potential missing sites of SLG gene after sequencing the PCR products of SLG gene in 30 pCACas-SRK-ABC plants.The results showed that CRISPR/Cas9 technology has a high specificity in cabbage gene editing.2.Optimization of CRISPR/Cas9 gene knockout systemThe previous research results showed extending the duplex by 5 bp combined with mutating the continuous sequence of Ts at position 4 to C or G of sgRNA significantly increased CRISPR-Cas9 gene knockout efficiency in TZM-bl cells.Using the t RNA processing system,a tandemly arrayed t RNA–gRNA architecture could be used to express multiple sgRNA,multiple gene or multiple loci of the same gene could be edited.pCACas-HsgRNA-NtPDS-AB,pCACas-HsgRNA-BoGA4-AB,pCACas-tRNANtPDS-AB,pCACas-tRNA-BoGA4-AB,pCACas-t RNA-NtPDS-ABC,pCACastRNA-NtPDS-ABD,pCACas-tRNA-SRK-ABCD,pCACas-t RNA-BoPDS-ABCD vectors were used to transform the hypocotyl and tobacco leaf discs by Agrobacterium mediated transformation.Mutation were found in 4 plants after investigated 9 pCACasHsgRN A-BoGA4-AB transgenic plants,and both target sites mutation simultaneously in 3 transgenic plants.All of 29 transgenic plants were found sequence change at the target sites in pCACas-t RNA-SRK-ABCD transgenic plants.Based on editing efficiency of pCACas-NtPDS-AB,pCACas-HsgRNA-NtPDS-AB,pCACas-tRN A-NtPDS-AB to Nt PDS gene in tobacco,we found HsgRN A/Cas9 mediated editing efficiency was significantly higher than that of unmodified sgRNA/Cas9 system,but lower editing efficiency of t RNA-HsgRNA /Cas9 system than that of sgRNA/Cas9 and HsgRN A/Cas9 system for the two loci was observed.The further analysis showed there were a long deletion between A and B loci in the Nt PDS gene coding sequence after sequenced amplified fragments.Despite the editing efficiency of pCACas-t RNANtPDS-ABC editing efficiency is higher than pCACas-tRN A-NtPDS-ABD,there was no obvious editing efficiency varied even the same target site sequence was inserted into different positions of tRNA-HsgRNA.The results show that the HsgRN A/Cas9 and tRNA-HsgRNA/Cas9 were stable and efficient gene knockdown system in tobacco and cabbage,and the t RNA-HsgRNA expression system depend on Gly-t RNA processing of Arabidopsis can be used to edit gene in both monocotyledon and dicotyledon.