Determination of local chromatin interactions using a combined CRISPR and peroxidase APEX2 system

The architecture and function of chromatin are largely regulated by local interacting molecules, such as transcription factors and noncoding RNAs. However, our understanding of these regulatory molecules at a given locus is limited because of technical difficulties. Here, we describe the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and an engineered ascorbate peroxidase 2 (APEX2) system to investigate local chromatin interactions (CAPLOCUS). We showed that with specific small-guide RNA targets, CAPLOCUS could efficiently identify both repetitive genomic regions and single-copy genomic locus with high resolution. Genome-wide sequencing revealed known and potential long-range chromatin interactions for a specific single-copy locus. CAPLOCUS also identified telomere-associated RNA. CAPLOCUS, followed by mass spectrometry, identified both known and novel telomere-associated proteins in their native states. Thus, CAPLOCUS may be a useful approach for studying local interacting molecules at any given chromosomal location. Overall design: CAPLOCUS combined with NGS sequencing was carried out on a repetitive region on chromosome 13 (chr13: 112,930,173-112,968,847), a single-copy locus on chromosome 11 (chr11: 5,497,611-5,497,843), and sgGAL4 control in replicate. In this method, with sgRNAs targeting a specific genomic region, local proteins were labeled with biotin and can be captured by M-280 Steptavidin Dynabeads. Thus, sgC13-IP and sgC11-IP were transfected with different sgRNAs targeting C13 region or C11 region, but both were pulled down with M-280 Steptavidin Dynabeads.