Protocol to target a promoter region in human embryonic kidney cells using the CRISPR-dCas9 system for single-locus proteomics

Abstract

Summary: The unbiased identification of less-abundant transcription factors, which direct the expression of a target gene, is technically challenging. Here, we present a protocol to analyze the locus-specific chromatin-regulating proteome using in situ capture of chromatin interactions by an inactive Cas9 (dCas9). We describe steps for designing guide RNAs and transfection, followed by precipitation of chromatin and associated proteins. In the last step, we describe the elution of DNA and proteins for PCR and mass spectrometric analysis, respectively.For complete details on the use and execution of this protocol, please refer to Alkhayer et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Genomics
• ChIPseq
• CRISPR