The Use Of DNA Shuffling Technology To Build GRATEN System For Genomic Coding Series Of Research

Gene mutation has been the most effective way to study gene function. With the rapid development of modern genome sequencing technology, the functions of a large number of genes or regulatory factors are required to be identified. Therefore, to establish a precise and efficient technology of site-directed genomic mutagenesis, the use of reverse genetics to study gene(or DNA) function is one of the hotspots of the current functional genomics research. Artificial zinc-finger nuclease technology(ZFN), transcription activator-like effector nuclease technology(TALEN) and RNA-mediated clustered regularly interspaced short palindromic repeats(CRISPR) systems can produce DNA double-strand breaks precisely at the specific sites of the genome, which achieves mutations of gene insertions, deletions, base substitutions by intracellular endogenous DNA repair mechanisms(homologous recombination and non-homologous end joining repair). Compared with the traditional gene editing techniques, the new technology not only retains characteristics of modifications at specific sites, but also can be applied to more species. It greatly improves the efficiency of the gene designated editing. The time of constructing vector is shortened. The costs are reduced. It also provides a broad platform for the realization of the designated genome editing. However, recent studies have found that CRISPR system can generate very high rate of off-target.In this study, we cloned multiple gene fragments and achieved mutations of two amino acid on specific site at once time by DNA shuffling. We also constructed an expression vector using DNA shuffling technology and executed genome editing using GRATEN(guided RNA and tethered endonuclease) for the first time in the plant. We explored the possibility of site-directed genome mutagenesis by GRATEN system in the plant and then transiently expressed in tobacco leaves, and conducted a preliminary study of its mutagenic effect.GRATEN system comprises two portions containing a protein and RNA and the two components were cloned into the same plant expression vector. The protein component comprises MS2 coat protein(MS2 coat protein, MCP) which is from bacteriophage, and the cleavage domain of endonuclease Fok I. The N- and C-terminal of MCP are fused with Fok I respectively, forming fusion protein(Fok I:MCP:Fok I) after expression. The components of RNA contain a series of guide RNA(guided RNA, g RNA) and MCP binding RNA. The g RNA recognize and bind to the target DNA by base pairing, MCP in the form of a homodimer combined a RNA that forms specific secondary structure. Thereby the dimeric Fok I can cut DNA at specific site, producing double-strand breaks(DSB). The DSBs are repaired by the introcelluler repair mechanisms(mainly Non-Homologous End Joining, NHEJ) and produce mutations of base substitutions, deletions, insertions.We chose phytoene desaturase(PDS) gene in Nicotiana benthamiana to be targeting gene for GRATEN system. The results show that it can produce higher(approximately 35.7%) mutation rate. Our preliminary findings illustrate GRATEN system can be used to make genomic site-directed mutagenesis in plant(possibly in other organisms), it offers a new way for functional genomics research and gene therapy.