Adapting dCas9-APEX2 for subnuclear proteomic profiling.

ABSTRACT

Genome organization and subnuclear protein localization are essential for normal cellular function and have been implicated in the control of gene expression, DNA replication, and genomic stability. The coupling of chromatin conformation capture (3C), chromatin immunoprecipitation and sequencing, and related techniques have continuously improved our understanding of genome architecture. To profile site-specifically DNA-associated proteins in a high-throughput and unbiased manner, the RNA-programmable CRISPR-Cas9 platform has recently been combined with an enzymatic labeling system to allow proteomic landscapes at repetitive and nonrepetitive loci to be defined with unprecedented ease and resolution. In this chapter, we describe the dCas9-APEX2 experimental approach for specifically targeting a DNA sequence, enzymatically labeling local proteins with biotin, and quantitatively analyzing the labeled proteome. We also discuss the optimization and extension of this pipeline to facilitate its use in understanding nuclear and chromosome biology.