Editing Efficiency And Application Research Of CRISPR/Cas9 Technique In Brassica Napus

Brassica napus(AACC)is one of the major oil crops in our country,which is an allotetraploid species formed by the hybridization of Brassica rapa(AA)and Brassica oleracea(CC).The highly function redundancies were observed in B.napus,because more paralogues are identified in B.napus genome,which hinders the functional genomics studies of B.napus.CRISPR/Cas9 technology is widely used to create mutants which are helpful for gene function studies.Many plant mutants were obtained by using CRISPR/Cas9 technology,but the mutation pattern and genetic characteristics are still unclear in B.napus.In this study,we examined the mutation efficiency,pattern,specificity and heritability of 12 B.napus genes by using CRISPR/Cas9 technology.The results showed that,in T0 generation,the average mutation frequency of singlegene targeted sg RNAs was 65.3%,while the mutation frequency of sg RNAs targeting multiple genes was 66.3%.The homozygotes,bi-alleles,and heterozygotes were stably inherited as classic Mendelian law in the next generation without any new mutations or reversions.Interestingly,one Cas9 free mutant was found in our mutants population,which probably due to the loss of Cas9 during callus regeneration.In addition,no mutations were detected at the expected off-target sites among the examed T0 and T1 plants,indicating that CRISPR/Cas9-mediated gene mutation is target specific in B.napus.Compared to the wildtype plants,we observed different phenotypes of mutant plants,such as dwarf phenotype of Bna A6.RGA gain-of-function mutants,and the longer stem in Bna RGA quadruple mutants.In summary,CRISPR/Cas9 is an efficient tool to create genome modification which can greatly accelerate the research of functional genomics in Brassica napus,also provide important mutant resources for genetic breeding of Brassica napus.These fndings open many doors for biotechnological applications in oilseed crops.