Method for identifying and analyzing specific genomic locus interaction DNAs 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 genomic locus interaction DNAs 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 and DNA complex by utilizingstreptomycin magnetic beads and sequencing and analysis of enriched DNAs. An experimental analysis result shows that the method is applicable to the analysis of any genomic locus, and can obtain theinformation of genomic loci interacting with the genomic locus, and meanwhile, the idea that the method combines the CRISPR gene editing system with APEX2 to carry out specific locus DNA 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 DNAs at any given chromosomal location.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a method for identifying and analyzing DNA interacting with specific genomic sites 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, and localized chromatin can be accessed by regulatory elements such as transcription factors and non-coding RNAs. In recent years, several mechanisms for the regulation of chromatin organization have been proposed in the field. For example, each chromosome in the nucleus of eukaryotic cells is located in a specific region called a chromosome region, which contains many The domain called topological domain; in the topological domain, distal DNA elements regulate gene ...

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Problems solved by technology

These discoveries ushered the technical field into a new era of chromatin function research. However, a comprehensive understanding of chromatin function requires the identification of DNA, RNA, and transcription factors present at specific genomic loci. difficult and challenging

[0003] In recent years, studies have proposed techniques for local chromatin composition. For example, chromatin immunoprecipitation (ChIP) is one of the classic techniques widely used to study the genome-wide distribution of a given protein. However, there is no A 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 sites are genetically integrated into the yeast genome for site-specific chromatin purification; however, this method requires genomic engineering of the target genome, which alters the natural environment of chromatin and is inefficient ; modified 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, TALEN-based methods require the design of an amino acid sequence for each site, CRISPR-based methods require cells to be cross-linked with formaldehyde, and antibodies with high affinity and specificity are required to be available, etc.; research practices have shown that the above Methods do not yet specifically provide functional analysis of native chromatin or genome-wide

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