Gene Deletion And Replacement Using The Technique Of Gene Editing

Gene editing is anew technology using artificialengineering nuclease for genome modification,which could generate bases deletions,insertions and substitutions through the repair mechanism of HR(homologous recombination)or NHEJ(nonhomologous end-joining)after DNA double-stranded breaks produced by specificity identification and cutting of genome target sequence.It is of great significance in the identification of gene function and genetic improvement for its characteristics containing directed mutation,high efficiency,easy operation and strong specificity.Gene deletion is that the ends of two DSBs joint by the repair way of NHEJ and the gene between DSBs is deleted after the introduction of two DSBs by nuclease.Gene replacement is on the basis of gene deletion which can realize the introduction of any designed DNA sequence provided repair template by the repair way of HR.MicroRNAs(miRNA)are small RNAs which processed by pri-miRNA which has a hairpin structure.It plays an important role in the gene regulation at transcriptional and post-transcriptional levels in the biology.Gene deletion can be used to creat the null mutants of miRNA for the study of gene function.This study aims to realize gene deletion and gene replacement based on the gene editing technique of CRISPR(clustered regularly interspaced short palindromic repeats)in plant.The main results are as follows:1.The system of gene deletion technique had been established.To construct the vector based on CRISPR/Cas9 which guided by dual-sgRNA(single-guide RNA),target sites were selected in target area within both ends of pri-miR169 a and pri-miR827 a of the locus of AtMIR169aandAtMIR827 a.2.Mutants of gene deletion were screened,identified and its phenotypewas analyzed.The deletedefficiency of pri-miRNA of the locus of AtMIR169 a and AtMIR827 awere 24% and 20% using the technique of PCR.Homozygous mutants of gene deletion were selected through sequencing of its genotype.The result of Northern blot showed that expression level of mir169 aof the mutant of homozygous deletionwas weaker.The result oftreatedexperiment of drought stress showed mir169 a of the mutant of homozygous deletion had stronger drought resistance.3.The system of gene replacementwas designed and established.To construct the vector of gene deletion based on the system of gene deletion,target sites were selected in target area within the part gene fragment of AtTFL1.To construct the vector of repair template,two 800 bp homology arms in accordance withlateral sequences of target area of AtTFL1 were designed based on the repair way of HR.They were jointed with both ends of a express box of 35S:eGFP:NOS and two target sites in accordance with gene deletion were added.4.Mutants of gene replacement were screened,identified and its phenotype was analyzed.The mutants of replacement of the part gene fragment of AtTFL1 were screened by PCR using specific primers in transgenic plants.Statistical results showed the efficiency of gene replacement was 0.8%.The mutants that the part gene fragment of AtTFL1 had been replaced and repair template had been deleted were screened.Its expression of eGFP was observed.Experiments showed eGFPcould stable express in leaves and roots in arabidopsis thaliana.5.The heredity of target mutations of gene deletion and gene replacement was analyzed.Heterozygosis plants of gene deletion and plants of gene replacement were screened.Progeny plants were identified.The results showed that the type of mutation was in agreement with Mendel's law of segregation.The target mutations of gene deletion and gene replacement were stably in progeny.This study can realize gene deletion efficiently and creat null mutants of miRNA.This studycan realize gene replacement in AtTFL1 based on gene deletion by the repair way of HR.This study can providea new strategy for the study of function of miRNA in plant and the replacement and modification of DNA sequence.