C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9–APEX2

• DOI: 10.1038/s41592-018-0006-2
• Publication Date: 2018-05-04T09:23:23Z
• Authors: Xin D. Gao; Li-Chun Tu; Aamir Mir; Tomás Rodriguez; Yuehe Ding; John Leszyk; Job Dekker; Scott A. Shaffer; Lihua Julie Zhu; Scot A. Wolfe; Erik J. Sontheimer
• Keywords: Proteomics; 570; Proteome; Protein Engineering; Biochemistry; Article; 576; Structural Biology; CRISPR-Associated Protein 9; Cell Line, Tumor; DNA-(Apurinic or Apyrimidinic Site) Lyase; molecular biology; Humans; Amino Acids; Molecular Biology; Genome; Systems Biology; and Proteins; Chromosome Mapping; Genetics and Genomics; Genomics; Endonucleases; Multifunctional Enzymes; proteomes; Gene Expression Regulation; dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging; Peptides; C-BERST; genomic loci

Mapping proteomic composition at distinct genomic loci and subnuclear landmarks in living cells has been a long-standing challenge. Here we report that dCas9-APEX2Biotinylation at genomicElements byRestrictedSpatialTagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. By combining the spatially restricted enzymatic tagging enabled by APEX2 with programmable DNA targeting by dCas9, C-BERST has successfully identified nearly 50% of known telomere-associated factors and many known centromere-associated factors. We also identified and validated SLX4IP and RPA3 as telomeric factors, confirming C-BERST’s utility as a discovery platform. C-BERST enables the rapid, high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear and chromosome biology.