Improving Editing Efficiency Of CRISPR/Cas9 System In Tomato Genome By Inducing P19m And Optimizing The Structure Of GRNA

In this study,tomatoes are used to be genetically modified by CRISPR/Cas9system,for exploring the possibility of improving the efficiency of genome editing.CRISPR/Cas9 system consists of a single gRNA and a Cas9 protein.It is easy to be composed,while having the high accuracy of editing,which makes it popular in the genome editing field.However,the efficiency is not satisfying in the actual application.In the plant field,the main problems are due to the low editing efficiency and the difficulty of tissue culture.In this study,to promote the genome editing,the improvement of editing efficiency is focused,while the tissue culture is hard to improve.The methods are as followed:?1?It has been proved in mammals that the editing efficiency can be significantly improved by optimizing the scaffold structure of gRNA.And in this study,the results are verified in tomatoes,and extending to all plants.We have extended the double strain of gRNA's scaffold by 5 bp?UGCUG?,and mutated the forth successive Thymine into Cytosine.One CRISPR system?GOG?is made of a original gRNA containing spacer1 and a optimized gRNA containing spacer2,another CRISPR system?OGOG?is made of a optimized gRNA containing the same spacer1 and a optimized gRNA containing the same spcaer2.Then,based on the number of the mutant in spacer2 site,we can make a comparison of the efficiency of original gRNA and optimized gRNA.?2?The previous study has found that the introduction of modified RNA silencing suppressor P19 in the editing of PDS gene can interfere in PTGS while enabling the plant to grow instead of dying in abnormality caused by P19.And we cofined the results in this research.In P19,the 43^th amino acid of Arg was turned to Trp,by mutating the127^th Cytosine into Thymine in the sequence,which resulting in P19m.We intergrate the sequence of P19m in the expressing vector of CRISPR/Cas9 system.By comparison of the efficiency of CRISPR/Cas9 without P19 element,we confirmed the function of P19m.?3?CRISPR/Cas9 system was built by optimized GoldenBraid.And for simplifying the operational process as well as improve the speed of our experiment,we have modified the genomic DNA extraction process and the construction of Peasy-ZERO vector.The self-made E.coli competence is also bring the possibility of cutting the budget.In this research,the genes related to miRNA are knockout by targeting the first excons,generating the transgenic T0 plants,which are detected for editing efficiency.Now,we have certified the efficiency of CRISPR/Cas9 system in tomato genome is improved to 75%by the introduction of P19m and the optimation of gRNA scaffold in a small-sample scale.From the above,this study,to improve the efficiency of genome editing,the structure of gRNA's scaffold is optimized and P19m is introduced for making a efficient and convenient genome editing system.