Method for identifying and analyzing specific locus interaction proteins on basis of CRISPR/cas9 and peroxidase APEX2 system

Abstract

The invention belongs to the technical field of biology, and relates to a method for identifying and analyzing specific locus interaction proteins on the basis of a CRISPR/cas9 and peroxidase APEX2 system. The method comprises the following steps: plasmid transformation; biotin labeling of specific locus binding protein complex; enrichment of biotin-labeled protein complex by utilizing streptomycin magnetic beads; and assay and analysis of enriched proteins. An experimental analysis result shows that the method is applicable to the research and analysis of protein at any given chromosomal location, and meanwhile, the idea that the method combines the CRISPR gene editing system with APEX2 to carry out specific locus protein analysis is also applicable to the combination of other genome editing methods (such as TALEN) and the APEX2. The method has the advantages of simplicity in operation, high sensitivity, high specificity, good repeatability and adaption to the requirement of the research of local chromatin interaction at any given chromosomal location.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for identifying and analyzing specific site interaction proteins based on CRISPR / cas9 and peroxidase APEX2 systems. Background technique [0002] The prior art discloses that in eukaryotic cells, DNA molecules are highly organized and tightly wrapped around repeating units of nucleosomes to form chromatin. However, in living cells, the structure of chromatin is dynamically changing, locally Chromatin is accessible by regulatory elements such as transcription factors and non-coding RNAs. In recent years, many mechanisms have been proposed to regulate the organization of chromatin. For example, each chromosome in the nucleus of eukaryotic cells is located in a specific region called a chromosome region, which contains many The domains of DNA are called topological domains; in topological domains, distal DNA elements regulate gene expression through dynamic interactions. Rese...

AI Technical Summary

Problems solved by technology

However, a comprehensive understanding of chromatin function requires the identification of DNA, RNA, and transcription factors present at specific genomic loci, however, technical difficulties and challenges remain

[0003] Although many techniques to study the local chromatin composition have been proposed, for example, chromatin immunoprecipitation (ChIp) is a classic and widely used technique to study the genome-wide distribution of a given protein, however, none has been proposed so far. Widely accepted method for studying locally interacting molecules at a given genomic site; locked nucleic acid probes are used to identify proteins that bind to telomeric regions, but this method is limited to highly repetitive genomic regions; LexA DNA binding site Genetically integrated into the yeast genome for site-specific chromatin purification; however, this method requires genomic modification of the target genome, thus it alters the natural environment of chromatin and is inefficient; improved Genome editing technologies such as transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR / cas9) have been used to enrich desired genomic loci, however, based on TALEN The method requires the design of an amino acid sequence for each site, and the CRISPR-based method needs to cross-link the cells with formaldehyde, and requires the availability of high-affinity and specific antibodies; Chromatin or genome-wide functional analysis

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