Endogenous protein marking method used for Chip-seq genome-wide binding spectrum

Abstract

The invention relates to an endogenous protein marking method applied to genome-wide binding spectrum analysis. The method comprises the steps of designing sgRNA for upstream and downstream 50bp regions of an encoding termination site of an NCOA1 gene; verifying cutting efficiency of sgRNA mediated Cas9 by an experiment; building an anti-estrogen therapy tolerant MCF-7 breast cancer cell model; by utilizing a CRISPR/Cas9 homologous recombination technology, selecting and using effective sgRNA and DNA donors, and introducing a Flag tag in the 3' end of the ER alpha co-transcription factor NCOA1 gene; and establishing a stable cell line with the Flag tag. Compared with the prior art, the method has the advantages that gene editing is performed by utilizing the CRISPR/Cas9 technology, so that the tag insertion efficiency of the gene editing is greatly improved; more importantly, chromatin structure parameters are introduced for designing sgRNA, so that effective cutting of Cas9 at a target site of a genome is ensured; and positive clone screening is performed by adopting DNA sequencing and RT-PCR sequencing methods, so that the positive clone identification step is simplified and the manpower and material resources for screening are reduced.

Description

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AI Technical Summary

Benefits of technology

This patented method allows for better control over how different types of nucleic acid can enter living organisms by controllably deliver them inside target organs such as humans or animals without causing harmful side effects like immune responses from innate defense mechanisms. It also includes various ways to measure these properties accurately during research studies.

Problems solved by technology

This patented technical solution describes how CRIS PR/CAS plays important roles in controlling gene expression through modifying proteins involved in cellular processes such as metabolism or signal transmission. However, there remains room for improvement over current methods based solely on enzymatic activity alone without considering other aspects like regulators and drug discovery capabilities.

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