A reagent for editing dnmt1 gene by CRISPR-Cas9 site-directed mutagenesis and its application

Abstract

The invention discloses a reagent for CRISPR-Cas9 site-directed mutation editing of DNMT1 gene and its application. The reagent contains a specific single-stranded guide RNA and an oligodeoxynucleotide template, and the target sequence is targeted by a specific single-stranded guide RNA sequence, and the oligodeoxynucleotide template is used as a gene repair template, thereby introducing a single site The mutation of the DNMT1 gene was subjected to single base editing to generate a cell line carrying the site-directed mutation of DNMT1; the mutated cell line can be further used to study the effect of the DNMT1 mutation on the expression level of the γ-globin gene (HBG) It has a great mechanism of action and has great application prospects.

Description

technical field [0001] The invention relates to the technical field of cell models and gene editing, and more specifically, to a reagent for CRISPR-Cas9 site-directed mutation editing of DNMT1 gene and its application. Background technique [0002] DNA methyltransferase 1 (DNMT1) is widely and highly expressed in many tissue structures of the human body. Under normal physiological conditions, it is used to maintain the methylation level of specific DNA sites and mediate the expression silencing of downstream genes. The DNMT1 protein includes a N-terminal regulatory domain (N-terminal regμLatory domain) and a carbon-terminal catalytic domain (C-terminal catalytic domain). These two domains form a certain spatial structure and interact to complete the catalytic function of DNMT1. The nitrogen-terminal regulatory region is composed of several different structural domains, including: PCNA binding domain (proliferating cell nuclear antigen (PCNA) binding domain, PBD), TS domain (...

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Problems solved by technology

Chinese patent CN109971755A discloses a sgRNA specifically targeting the human DNMT1 gene of a CRISPR / Cas9 system, and its CRISPR / Cas9 carrier, which can quickly, efficiently and specifically knock out the human DNMT1 gene and promote the inactivation of the DNMT1 gene in tumor cells. Inhibit the expression of DNMT1 and its methylation effect on DNA, thereby inhibiting the growth of tumor cells. However, it cannot realize the site-directed mutation of DNMT1, so it cannot be applied to the construction of DNMT1 (c.2633G>A, p.Ser878Phe) mutation model

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