Using The CRISPR/Cas9 Technology For Editing MiR167 Precursor Sequence

As one of the important producers of citrus,China has been a long history of citrus cultivation and the germplasm resources are very abundant.The seedless breeding of citrus still have been gotten high priority in conventional citrus breeding program,but the conventional breeding is time-consuming and difficult to meet the needs of directional improvement.The bud mutation and mutation selection have low efficiency due to the randomness of mutation.Recent years the gene engineering breeding has been applied in citrus genetic improvement.As an important auxiliary method,it could effectively improve the target genetic characteristics of plant according to the expectation of breeder,and innovate germplasm.But the transgenic technology raised a lot of controversy as the introduction of the exogenous genes into plants,so the safety of genetically modified crops has been gotten high concerns by public.CRISPR/Cas9 technology also called the third generation of gene editing techniques,can produce accurate point mutation at the target genes site at the genome level,moreover the mutation could be transmitted to the next generation.In recent years CRISPR/Cas9 gradually become a new approach to study the function of genes.MicroRNAs(miRNAs)are kinds of endogenous noncoding single-stranded RNA molecules,its length range from 19 nt to 26 nt.miRNAs are widely exists in animal and plant cells.The plant miRNAs could regulate the target genes expression through directly shearing of mRNA at post-transcription stage or inhibit its translation.Previous research already found some miRNAs were conserved not only at nucleotide sequence but also its function among different plant species.These miRNAs usually participate in the regulation of organs differentiation and development,stress response,hormone metabolization,signaling transduction and other important biological processes in plants.The miR167 is a kind of conservative mi RNA,its target ARF6 and ARF8,both transcription factors mainly involved in the regulation of flowers and fruit development process in plant.In this study we use the CRISPR/Cas9 technology to edit the precursor gene of miRNA167 a in citrus and tomato respectively,in aiming to create seedless citrus germplasms and study the function of miRNA167 a.The main results of this study were listed as follows:1.Firstly we used mi RBase database to query and download the sly-mi R167 a and csi-miR167 a mature nucleotide sequence and its precursor nucleotide sequences respectively for citrus and tomato,By BLAST with NCBI database we got their corresponding genomic DNA sequences.Through alignment analysis between csi-miR167 a and sly-miR167 a,both miRNA sequences were highly similarity,so it suggested that miR167 a mature sequence were conservative in citrus and tomato,while their precursor sequence were less conservative.2.Using the online website,we designed csi-gRNA and sly-gRNA for citrus csi-miR167 a precursor and sly-miR167 a precursor sequences,we successfully constructed two types of CRISPR/Cas9 recombinant expression vector named respectively as pKSE401 and pP1 C.1C.Transforming of citrus epicotyl stem section and tomato cotyledons respectively were performed with agrobacterium mediated genetic transformation system.In the transform tomato plants with pKSE401 vector,67 resistant plants were tested,PCR product sequencing showed that no-gene editing occurred at the target site or its proximity in all plants.We transformed the callus with pP1 C.1C vector express fluorescent tags,PCR product sequencing also indicated no gene editing at the target site.Further we performed the gRNA efficiency detection by cutting trial in vitro,the results showed that cutting ability of gRNA was miss in vitro.So gRNA defect might be a factor explaining no gene editing in genetic transformation tomato.3.In next step we improve the trials to design high efficient gRNA,we used the online website to design gRNA for another homologous sequence of the sly-miR167 a precursor.We successfully obtained a gRNA abbreviated as G1 through detection of the cutting ability in vitro trial.We further constructed a recombinant expression pP1 C.1C vector,and transformed tomato by agrobacterium mediated genetic transformation system.Finally three transformation materials with fluorescent tags were got.By PCR products sequencing analysis,the results showed that three mutant materials had nucleotide deletion and insertion at the target sites,and the mutation types were different.The research showed that CRISPR/Cas9 was feasible to edit specific gene through feasible designed gRNA.In later work,we can use this system for editing citrus miR167 a precursor sequences,in aimed to get citrus mutant materials and study their functions.