New method for gene point mutation repair

Abstract

The present invention provides a new method for gene point mutation repair. Main steps are as follows: in a target genome sequence, cell gene mutation sites are selected as repaired gene sites; according to the selected gene repair sites, sgRNA of a CRISPR / Cas 9 system is designed; the sgRNA is constructed to an expression vector carrying CRISPR / Cas9 proteins; a segment of correct sequence is selected near upstream or downstream of the mutation sites, and a designed Donor DNA is used as a template for the gene mutation repair; in vitro culture of cells carrying the mutation site genes is conducted; an electrotransfection method is used to transfect the sgRNA, CRISPR / Cas9 expression vector and Donor DNA into the cells. The method fully mixes a Cas9 expression vector, the sgRNA and the DonorDNA with mouse fibroblast cell line carrying Msh5 point mutation established in vitro, then the cell line is electrically transfected to increase transfection efficiency of the cells, and the CRISPR / Cas9 system is used to conduct the repair of the mutation sites, thereby providing a scientific basis for treatment of primary amenorrhea in women.

Description

technical field [0001] The invention belongs to the technical field of gene repair, in particular to a new method for gene point mutation repair. Background technique [0002] CRISPR / CAS9 system is a new high-efficiency gene editing technology developed in recent years, which can delete and modify the genome at the cellular level. Since the CRISPR / CAS system was announced in early 2013, it has been widely used all over the world, and was named one of the top ten scientific breakthroughs in 2015 by the "SCIENCE" magazine. CRISPR / CAS9 is an RNA-protein complex. The commonly used CAS9 nuclease is composed of 1409 amino acids and has two important nuclease domains, namely RUVC and HNH domains. The HNH domain is responsible for cleavage by targeting complementary DNA single strands, and the cleavage site is located 3NT upstream of the PAM (PROTOSPACER-ADJACENT MOTIFS) sequence. The RUVC domain is responsible for breaking the other DNA strand, and the cutting site is located 3-8...

AI Technical Summary

Problems solved by technology

[0004] Our previous research found a genetic mutation that can lead to primary amenorrhea in women (TING GUO ETAL, 2017, HUMAN MOLECMLAR GENETICS), which is located at the G to C mutation at position 1459 of the Msh5 gene, which leads to the inability of Msh5 Normal driving function, so that the germ cell meiosis process cannot be completed, and eventually cause germ cell death

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