The Application Of CRISPR/Cas9 System In Plant Genome Editing

CRISPR/Cas9 has rapidly become a focus in the field of genome editing due to its easiness to use and high efficiency since early 2013.The principles of the technology are:guided by 20-nt target sequence fused to sgRNA(single guide RNA,sgRNA),Cas9 cleaves DNA,resulting in a double-strand break(DSB).DSBs at specific genomic sites can introduce mutations at the DNA break sites via the error-prone non-homologous end-joining(NHEJ)pathway or homologous recombination(HR)pathway.In this study,we developed a CRISPR/Cas9 toolkit for multiplex genome editing in plants.We validated the toolkit in maize protoplastsand transgenic lines,and Arabidopsis transgenic lines.In order to generate CRISPR/Cas9 binary vectors applicable for monocots and dicots,this study used maize-codon optimized SpCas9 with nuclear localization signals added at both ends.To highly efficiently express Cas9,we used the strong promoters which confer high expression level and were fequently used in plants:maize Ubil promoter in monocots and 2x35S promoter in dicots.To efficiently express sgRNA,we used promoters from rice and wheat U3 genes,and Arabidopsis U6-26 gene in monocots,and three Arabidopsis U6 gene promoters in dicots.In order to transiently and stably express CRISPR/Cas9 system,we used the pGreen and pCambia backbones.Additionally,we used three selection markers for their applications in a variety of plant species.Based on Golden Gate or Gibson Assembly strategies,this study established a method for obtaining the CRISPR/Cas9 binary vector set in only one step of cloning reaction.Using such a method,we were able to generate the binary vectors harboring one or more sgRNA expression cassettes.To validate the toolkit and compare the mutation efficiencies of three Cas9 variants and three Pol? promoters,we generated two sets of test vectors targeting the same maize genomic DNA site.The results indicated that maize codon-optimized Cas9 performed considerably better than the two human codon-optimized Cas9 genes,and the rice U3 promoter,wheat U3 promoter and Arabidopsis U6-26 promoter all worked well in maize.In order to verify potentials of this toolkit in creating mutants in plants,this study selected three Arabidopsis reporter genes related to trichome development:TRY,CPC and ETC2 for mutation analysis,the results showed that the frequency of genome edited T1 plants is up to 90%.The phenotypes and mutation analysis showed that the mutants in T1 Arabidopsis lines were all mosaics,and the frequency of transmission of T1 mutations to the next generation was very low according to mutation analysis of separated T2 non-transgenic plants.To solve these problems,we used egg cell-specific promoters to drive the expression of Cas9.We obtained non-mosaic T1 mutants for multiple target genes at a frequency of 8.3%and found the Cas9 terminator was important for the success.In an attempt to further improve the efficiency of generating T1 homozygous mutants,this study tested several strategies of fusion of enhancers to egg cell-specific promoters.The results showed that the composite promoter generated by fusing two egg cell-specific promoters(EC1.2en-EC1.1p)worked best.The T1 mutants could reach above 24.7%,nearly three times of that generated with the single promoter.Analysis of mutations in the 15 T1 mutant lines unexpectedly found high frequency off-target mutations:13%(2/15)and 87%(13/15)T1 lines harboring off target mutations in TRY and CPC,respectively.In order to increase specificity of CRISPR/Cas9 system,we tested six mutant Cas9 variants with enhanced specificity in Arabidopsis.These Cas9 variants include eSpCas9(1.0)/(1.1),SpCas9-HFl and three combinations of the two strategies for improving the Cas9 specificity.The results showed that eSpCas9(1.1)induced mutations in similar efficiencies in comparison with wild-type SpCas9 in T2 plants,although eSpCas9(1.1)had much lower mutation efficiencies than wild-type SpCas9 in T1 plants,suggesting dependence of efficient eSpCas9(1.1)on its expression level.Analysis of off-target mutations found that they were undetectable.Interestingly,we also found high frequency of T-DNA insertions into cleavage sites of CRISPR/Cas9 targets.In conclusion,this study developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species,which will facilitate plant research,as it enables high efficiency generation of mutants bearing multiple gene mutations.