Gene Targeting Research Based On CRISPR/Cas9 System

Gene targeting is a technology of fixed-point changing the genetic information of genome by recombing of exogenous DNA with the chromosome DNA of the receptor cells. The emergence of gene targeting techology is a major breakthrough in the field of molecular biology. So far, gene targeting technology has been successfully applied to the research of gene’s structure and function,gene expression and regulation, animal and plant transgenic, gene therapy, and many other aspects.This study is to conduct gene targeting by CRISPR/Cas9 system. CRISPR/Cas9 system is currently the most advanced and most efficient mediated genome editing method. It is more excellent compared with ZFN system and TALEN system with its concrete manifestation in: the carrier structure of CRISPR/Cas9 system is easier and less costly. CRISPR/Cas9 system can realize targeting of multiple sites and multiple gene. The most important thing about gene targeting is the targeting efficiency, and high targeting efficiency is more likely to achieve the modification of genetic material and is of higher research value. This study by using the CRISPR/Cas9 system and setting up related experimental platform in the 293 T cell lines and ES cell lines of mice, is to study the gene targeting of the system with the main results as follows:1. Based CRISPR / Cas9 system 293 T cells gene targeting, won 63% of the effective targeting efficiency.2. Through the research of structure and function of gRNA in CRISPR/Cas9, four gRNA targeting carriers(Meg3-3’-gRNA,Meg3-5’-gRNA,Kcnq1ot1-3’-gRNA,Kcnq1ot1-5’-gRNA) are built by the method of three synthesis combination.3. To create a new detection method of targeting efficiency: adding the termination codon targeting point in the two fluorescent labeled( cherry red fluorescence and green fluorescence) promoting regions, in order to make the successfully targeted cells to express the two fluorescence, and the non-targeted cells only expressed the red fluorescence. Finally the fluorescent quantitative analysis in the collaborative flow FCM came up with the result of targeting efficiency.4. By the flow cytometry instrument analysis, the selected three gRNA testing sites had higher targeting efficiency with shooting rates of 50.3%, 73.5% and 84.5%, further and better illustrating the high efficiency of CRISPR/Cas9 gene targeting.5. Finally, through constructing the isogenesis of mouse ES cells to replace the target vector and the gRNA vector of corresponding target sites, the fixed-point knockout of the mouse Meg3 genes and Kcnq1ot1 genes was realized.6. Through the technologies of fluorescence selecting, medicine selecting and PCR, ES cells of mice which have been knocked out the single gene of Meg3 and Kcnq1ot1 were successfully selected, and obtained the targeting efficiency of Meg3 with 14.3% and Kcnq1ot1 with 11.5%.