Development Of Plant Genome And Base Editing Toolkits And Their Validation

As the exploration of laws of life depends to a large extent on the interpretation of gene function,powerful genome editing tools are needed more urgently than ever before along with the rapid development of whole-genome sequencing and functional genomics.In early 2013,CRISPR/Cas9 genome editing technology has quickly became the focus of genome editing field due to its simple design principle,low cost,convenient operation and robust versatility.In order to test if the CRISPR/Cas9 system would work in plants,and to set up a versatile CRISPR/Cas9 tools facilitating the research of plant gene function and speeding up the discovery of plant genes with impotant function,in this study we first developed a plant CRISPR/Cas9 genome editing toolkit involved in a variety of binary vectors,sgRNA module vectors and sgRNAs assembly methods.The toolkit includes binary vectors pHSE401,pBSE401 and pKSE401 for dicotyledons,and binary vectors pHUE411 and pBUE411 for monocots.We then tested the efficiency of the toolkit in Zea mays and Arabidopsis thaliana.The results demonstrated this CRISPR/Cas9 toolkit could efficiently induce genome editing both in dicot and monocot plants,and thus could provide researchers with a more simple and convenient tool platform for efficient generation of plant mutants.During the application of CRISPR/Cas9 tools,we found that the monocot maize which use embryogenic calli as transformation acceptor could efficiently produce homozygous or biallelic mutants in TO generation,but the model plant Arabidopsis which has the simplest transformation method(floral dip method)generated a vast mount of chimeric T1 mutants,seriously affecting the efficiency of mutation passing to the next generation.Based on the supposition that the formation of chimeras in transgenic Arabidopsis T1 generation may be due to the weak activity of constitutive promoter in egg cell or one cell stage embryo,we used an egg cell specific promoter to drive the expression of Cas9and established EPC(egg cell-specific promoter controlled)CRISPR/Cas9 system and tested the performance of the EPC CRISPR/Cas9 system.Results showed that using the EPC CRISPR/(as9 system in Arabidopsis we has successfully achieved homozygous or biallelic mutations simultaneosly in two genes(CHLI1 CHLI2)or three genes(TRY CPC ETC2)in the first transgenic generation.Tus,we solved the serious problem of chimeric mutations in Arabidopsis in the first transgenic generation.The efficiencies of homozygous or biallelic mutations in three genes and two genes were 8.3%and 13.1%,respectively,in the T1 plants.By comparing the mutation efficiencies of the six combination of three promoters(EC1.2p/35Sp/EC1.lp)and two terminators(E9t/Nost),we concluded that the choia of Cas9 terminator plays a vital role for the success of EPC CRISPR/Cas9 system.In addition,in order to further improve the efficiency of EPC CRISPR/Cas9 system,IPC CRISPR/Cas9 was furture optimized by promoter fusion strategy.By trying various enhancersind different fusion forms we found the fusion form of EC1.2 enhancer with EC1.1 promoter significstly improved the efficiency of EPC CRISPR/Cas9 system with high frequency of homozygous or biablic mutations(17.0%),and thus we achieved the purpose of optimization.CRISPR/Cas9 and cytosine deaminase mediated base editing system was first successfully applied in animals.In order to test the application potentials of the base editing system in plant,we created a toolkit for plant base editing.This base editing tookit includes binary vectors pHSE901,pBSE901,pKSE901 and pHEE901 for dicotyledons,and binary vectors pHUE911 and pBUE911 for monocots.We tested the performance of the base editing tookit in Arabidopsis.By analyzing the editing effect of target gene AtALS in T1 generation and T2 generation,we found the edited plants accounted for 1.7%(4/240)of the total T1 plants and the edited targets could efficiently passed to T2 generation.Of the three T1 edited lines,the average proportion of T2 herbcide resistant plants confered by the edited base reached to 84.1%.Morover,these results suggested that the PAM region could also be a novel"deamination window".Taken together,the plant genome editing toolkit and the base editing toolkit displayed very good prospects for applications both in basic research and crop improvement.