Research On Methods Of Improving Editing Efficiency Of CRISPR/Cas9 System In Tomato Genome

Modification of genomes in a site-directed manner to improve traits of crop is described to be critical for plant breeding and genetic engineering.The genome edit strategies were first proved to be effective in bacteria and human cell lines,but they have been successfully adapted in plant systems.In recent years,with the rapid development of SSN(sequence specific nuclease),CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9)system has become the most widely adopted gene editing technology.This is due to its straightforward construct design and assembly since CRISPR/Cas9 is a complex guided by short RNA sequences to complimentary target DNA that is then cleaved by Cas9.In animals,by introducing sufficient amount of sgRNA and Cas9 RNA through microinjecting,biallelic mutations can be generated in one-cell-stage embryos efficiently.However,Agrobacterium-mediated transformation of calli is a common method for generating transgenic plants.The transformed cells soon divide allowing relatively short time for generating the germline mutation.On the contrary,it takes several weeks even months to regenerate transgenic crop plants from embryogenic cells,and the sgRNA/Cas9 complex is continuously being expressed during this time period.Such long expression stage might result in the high risk of chimeric and heterozygous mutation in the first generation.Compared with animal,the probability of double-strand breaks repaired genome modifications via homologous directed in plants is rather low.Due to the posttranscriptional gene silencing(PTGS)mechanism caused by RNA Induced Silencing Complex(RISC)in plants system,Cas9 protein could not be accumulated excessively.Furthermore,non-transgenic plants could be generated by backcrossing T1 heterozygous mutants to eliminate unwanted parts of T-DNA,but if the efficacy of CRISPR /Cas9 is strong enough,T1 biallelic mutations can be generated effectively.Therefore,it is an urgent issue that how to increase the expression level of Cas9 protein so as to improve the genome editing efficiency.Tomato is one of the most important crops worldwide and is regarded as an important model plant for studying flower and fruit development,because of the availability of its entire genome sequence and its well-studied genomics.In this study,we use tomato plants for testing the efficiency of P19m(suppressor of RNA silencing)assembled sgRNA/Cas9 system.Our aim is to make it available for generating homozygous mutants in short time period and even manipulate the target region precisely in genome editing by HDR.(1)According to bioinformatics analysis,we designed 2 CRISPR/Cas9 target sites in the second exon of SlPDS(Solyc03g123760.2.1),2 CRISPR/Cas9 target sites in the third exon of SlCNR(Solyc02g077920.2.1),1 CRISPR/Cas9 target site in the second exon of SlHD-ZIP6500(Solyc08g066500),3 CRISPR/Cas9 target sites in the second exon of SlBES1(Solyc02g063010.2.1),3 CRISPR/Cas9 target sites in the second exon of SlBES4(Solyc01g094580.2.1),3 CRISPR/Cas9 target sites in the second exon of SlBES7(Solyc03g005990.2.1);(2)Mutagenesis of P19 by overlap-PCR then domesticated it by GB(GoldenBraid)to generate P19m;(3)Using the GoldenBraid cloning strategy designed and constructed SlPDS gRNA-Cas9 vectors with or without P19 m,through Agrobacterium-mediated transient leaf infiltration method,the mutation rate of leaf sample infiltrated by P19 m was higher.By Agrobacterium-mediated stable transformation,we obtained an albino T0 mutant plant;(4)Using the GoldenBraid cloning strategy designed and constructed SlBES1,SlBES4,SlBES7,and SlHD-ZIP6500 gRNA-Cas9 vectors with P19 m and transformed them into Agrobacterium for stable transformation of tomato plants and analyzed the target region of T1 progeny.We obtained 20 SlBES7 gRNA-Cas9 T1 progeny plants whch all contained mutation,among them,30% were mutants with 100% mutation frequency.3 mutants were homozygous mutants.The rest were 7 heterozygous mutants and 8 chimeric mutants.Through the analysis of the type of mutations,most of the mutation occurred at gRNA3 target region,including 8 mutation types,the majority of the detected mutations were 2-nucleotide deletions,followed by 1-nucleotide insertions.Surprisingly,there were 4 non-transgenic mutants among 20 mutants.We also obtained 4 SlHD-ZIP6500 transgenic mutants with 100% mutation frequency,they all were homozygous mutants and were 8-nucleotide deletions mutants.(5)Designed the donor DNA fragment of SlCNR for precise modification by HDR,designed and constructed SlCNR-U6-gRNAs--donorDNA-Cas9 vectors using the GoldenBraid cloning strategy.By Agrobacterium-mediated transient infiltration as well as stable transformation,application of RS-1 seemed to be useless in improve HDR probability in plants.(6)Cloned CPMV RNA-2 5' UTR and 3' UTR fragments from PEAQ-HT vector,domesticated them and constructed SlPDS gRNA-Cas9 vector,in which Cas9 was flanked by CPMV-HT.