Exploration On RNA-templated Homologous Recombination And Comparison Ofdifferent CRISPR/Cas9 Systems

DNA double strand breaks(DSBs) is the most serious and fatal DNA damage compared with other different types, and the organism is to ensure the integrity of the genome through homologous recombination repair(HRR) mainly. Homologous recombination is a molecular process, which has multiple roles in various DNA metabolism. Under general circumstances,it occurs between the DNA molecules. The genome sequence can be edited preciselyand the target gene can be modified directionally by the homologous recombination repair mechanism, which can be used in the safe animal breeding. At the same time, it has great significance in prevention and treatment of cancer and other diseases, and obvious advantages in gene therapy.There are a large number of DNA and RNA molecules in the cells of all species in nature, furthermore the number and category of RNA molecules are much more than DNA molecules. Considering the abundance of RNA transcripts in cells, it may have an effect on the stability and plasticity of the genome. In protozoa, bacteria and yeast cells, there have been some related reports pointed out that RNA interacted with homologous DNA directly to repair the DNA damage. However, there is no relevant reference of research reports in mammalian cells. Therefore, this thesis mainly explored and discussed whether RNA mediated DNA editing and transformation process was existed in the mammalian cells. The RNA-DNA interaction found in the yeast cells could provide a new way for DNA replication and repair in the process of mammalian cell DNA damage.Secondly, in order to provide effective technical means for the exploration on RNA mediated recombination in the future,different CRISPR / Cas9 system nuclease activity were compared, resulting in a set of working efficiency was relatively higher targeting systemthat couldcreate more efficient double strand break effect, so as to improve the gene editing efficiency.In the reporter level, exon 3 & exon 4 and exon 3& exon 5 of β-actin gene were fused with eGFP reporter gene respectively to design and construct Actin E3&4 and Actin E3&5report system of homologous recombination in which exon3 &4 and exon3&5 were the template for the DNA recombination and repair. Cas9-sgRNA expression vector and reporterplasmids were co-transfected into HEK293 T cells, in the existence of the endogenous transcription product ofβ-actin m RNA to achieve precise repair of β-actin gene in cells.Extracted cell genome after real time fluorescence observation, set the PCR identification of different strategies. At the molecular level, the corresponding RNA editing results were not detected.Designed and constructedpiggyBac reporter vector, and co-transfected with transposase into 293 A cells to obtain random integrated cell line pB-293 A after G418 screeningsuccessfully.In the genome level, Cas9-sgRNA targeting vector and eGFP donor vectors that were expressed homologous mRNA template of eGFPin the intracellular were co-transfected into pB-293 A. Positive cells were enriched and screened by zeocin, then sequenced. The sequencing results showed that the precise editing positive results of the samples were not detected.Designed and constructed different CRISPR / Cas9 systems about sgRNA secondary structure, sgRNA 5’end enzyme cutting sites and hstCa9 and hspCas9 derived from different bacteria sources(Streptococcus thermophilus and Streptococcus pyogenes), thedifferent CRISPR / Cas9 expression vectors and SSA reporter plasmid co-transfected into HEK293 T cells to do the quantitative analysis of the CRISPR/Cas9 nuclease activity by flow cytometry.Concluded the results of different processing CRISPR / Cas9 system efficiency, sgRNA secondary structure of the stable of hst Cas9 system nuclease activity was 21.15% which was relatively high than others.In mammalian cells, due to the sensitivity of the system and other reasons,the RNA mediated homologous recombination repair was not detected in both the level of the vector and the level of the genome. Under the premise of no relevant reported reference, this exploration process about RNA and DNA direct homologous interactions and exchanges of genetic information in the mammalian cells is with certain scientific research characteristic and innovation. It can offer reference for the optimization design of the system in the future,and provide ideas for further research.