CRISPR/Cas9-mediated Multiplex Genome Editing In Rice (Oryza Sativa L.) And The Application In Rice Breeding

As the completion of rice genome sequencing,it is urgent to clarify the function of important rice genes.A single gene knockout in plants had achieved by the ZFN and TALEN technologies.However,the gene functional analysis of the gene families with functional redundancy,the interaction between genes,and to clarify some complex agronomic traits regulated by lots of genes require multi-gene mutation for further research.The traditional hybrid methods were usually used to obtain multi-gene mutation mutants,because of the genetic linkage and gene introgression between rice varieties,the workload for screening mutants are burdensome and frivolous.Recently,the development of the CRISPR/Cas9 system has been demonstrated to be oriented for the introduction of mutations,the gene editing techniques is able to avoid the genetic linkage and gene introgression in traditional breeding method.Thus,CRISPR/Cas9 mediated multiplex genome editing create mutants not only provides a valuable resource for the function of genes but also for rice breeding.In this study,we established the multiple gene knockout system in rice based on the existing CRISPR/Cas9 knockout system.Then we optimized the CRISPR/Cas9 multiplex genome editing system and simplify the operating procedure and then using the optimized system to editing multi-genes in rice.The main results were as follows:Part 1 CRISPR/Cas9-mediated single gene editing in riceBased on the previous research on CRISPR/Cas9 system,we knock out the OsMKK4 gene by using the CRISPR/Cas9 system.We constructed the binary expression vector pC 1300-Cas9-gMKK4 and transformed it to Zhonghual1 via the Agrobacterium-mediated transformation method for generating transgenic rice.The PCR amplification/restriction enzyme digestion assay?PCR/RE?show that there are 29 mutants with gene mutations among 36 transgenic positive plants in T0 generation,and the mutagenic efficiency was 80.56%.Among the 29 mutants,there are 19 homozygous mutants and 10 heterozygous mutants.The sequencing results show that we obtained a lot of mutants with insertion mutants and deletion mutants,and most of these mutations were frame-shift mutation.This study indicates that we could efficiently obtain the knockout mutant by using the CRISPR/Cas9 system in rice.Part 2 CRISPR/Cas9-mediated multiplx gene knockout system in rice1.In order to simplify the steps of assemble sgRNAs to the pC1300-Cas9 expression vector,we joined the other two pair isocaudarners?Xba I and Nhe I,Xho I and Sal I?in the middle carrier SK-gRNA with only one pair isocaudarners?BamH I and Bgl II?,and the new intermediate vector namely SK-4G.The optimized CRISPR/Cas9 multiplex genome editing system can assemble 1-3 target sites into an intermediate vector?SK-4G?or an expression vector?pC1300-Cas9?by a single cloning method.2.We connected three target sites?OsPDS,Os02g23823 and OsMPK2?into the binary expression vector by a round of cloning experiments.By a genetic transformation experiment,we obtained 110 transgenic positive plants in T0 generation.3.PCR/RE assay and sequencing results showed that the mutation frequency of OsPDS,Os02g23823,OsMPK2 were 68.2%,66.4%,and 81.0%,respectively.There are many mutations around the target sites,such as small insertion,deletion of nucleotides and microhomology insertion and so on.The results suggest that the mutations among different sgRNAs are independent of each other.Anyhow,our optimized CRISPR/Cas9 multiplex genome editing system has high knockout efficiency in rice.Part 3 CRISPR/Cas9-mediated 8 genes editing in rice1.To test the potential of the CRISPR/Cas9 multiplex genome editing system we established,we use this system to assemble 8 sgRNAs of 8 agronomic traits related genes in rice?three genes DEP1,EP3 and Gn1a related to panicle;two gene GS3 and GW2 regulated the grain size;one gene LPA1 regulates the plant architecture;BADH2 and Hdl have a relationship with rice fragrance and photoperiod,respectively?into the expression vector,and the binary expression vecror named pC1300-Cas9-gDEP1-gGn1a-gGW2-gEP3-gBADH2-gLPAI-gGS3-gHd1.In this study,we co-transformed the sgRNAs and Cas9 protein by using the transient expression system in rice protoplast and found that,through PCR/RE assay,all eight targeting sites were modified in rice protoplast.We also found that the gene editing frequency of CRISPR/Cas9 system was not affected by the number of sgRNAs.In the same time,the results suggest that the target sequences we designed could successfully guide the Cas9 protein for cutting double-stranded DNA in vivo experiment.2.The binary vector loading eight sgRNAs was used for genetic transformation of Nipponbare via the Agrobacterium-mediated transformation method for generating transgenic rice.The sequencing results suggesting that the mutation frequency of EP3,GS3,GW2,BADH2,DEP1,Gn1a,LPA1,and Hd1 were 50%,100%,67%,81%,83%,97%,67%,and 78%,respectively.Further analyzing the genotypes of 8 target genes,we found 25 different gene mutation combinations mutants,even including two homozygous octuple mutations,suggesting that the optimized CRISPR/Cas9 multiplex genome editing system has high efficiency of gene knockout in rice.3.The results of sequence specificity analysis by NCBI BLAST indicated that,BADH2,DEP1 and EP3 have the potential off-target sites,and the sequencing results showed that the mutation frequency were 0%,11.1%,and 5.6%,respectively.This study showed that the system has very low off-target efficiency.4.Through the phenotypic characterization of T0 generation of mutant,we found that except the photoperiod gene Hdl,the phenotype of target genes were similar to the phenotype observed in a previous study.This result clearly demonstrated that CRISPR/Cas9 multiplex genome editing system can obtain simultaneous mutations of up to 8 genes.Part 4 CRISPR/Cas9-mediated yield related QTLs editing in rice1.We construct the expression vector for editing two yield-related QTLs,GS3 and Gn1a by using the CRISPR/Cas9 system.By the Agrobacterium-mediated transformation method,we obtained transgenic positive plants in five widely-cultivated Oryza sativa L.ssp.japonica varieties in Jiangsu and Zhejiang area,named Nanjing 9108,Wuyunjing 27,Yangjing 4227,Zhejing 22,and Zhejing 88.The sequencing results of target genes in T0 generation indicated that,the main mutations were insertion and deletion.As the high mutation frequency of GS3?up to 100%?,we only obtained 10 new rice lines?gs3-N9108,gs3gn1a-N9108,gs3-W27,gs3gn1a-W27,gs3-Y4227,gs3gn1a-Y4227,gs3-Z22,gs3gn1a-Z22,gs3-Z88,and gs3gn1a-Z88?in five rice varieties.2.By investigating the yield of 10 new lines in T₁ generation,three of them had higher grain yields than the WT,and the others had decreased grain yields.We also isolated transgenic-free mutants in Ti generation.Grain yield of rice is determined largely by the number of effective tillers or panicles per plant,the number of grains per panicle,the 1000-grain weight,and setting percentage.To elucidate the underlying causes of the observed reductions in grain yield,we divided tillers into two categories depending upon the grain number:major tillers and minor tillers.Further statistical analysis results showed that the number of major tillers determines the final yield changes.3.We then grew 10 new rice lines from T₁ generation with improvements in field management practices,and analysis the yield and tiller number per plant in T2 generation.Except the lines gs3gnla-Y4227,the grain yield per plant of the the rest 9 lines were enhanced compared with WT.These studies provide the practice of using the CRISPR/Cas9 technology in rice breeding.To sum up,our study provides practical reference for rice breeding.