Screening Regulatory Element Function with CRISPR/Cas9-based Epigenome Editing

Methods Mol Biol. 2018:1767:447-480. doi: 10.1007/978-1-4939-7774-1_25.

Abstract

Genomic regulatory elements that control gene expression play an important role in many traits and diseases. Identifying the regulatory elements associated with each gene or phenotype and understanding the function of that element remain a significant challenge. To address this technological need, we developed CRISPR/Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. This protocol includes detailed instructions for design and cloning of gRNA libraries, construction of endogenous reporter cell lines via CRISPR/Cas9-mediated knock-in of fluorescent proteins, overall screen design, and recovery of the gRNA library for enrichment analysis. This protocol will be generally useful for implementing genome engineering technologies for high-throughput functional annotation of putative regulatory elements in their native chromosomal context.

Keywords: CRISPR/Cas9; Epigenetics; Epigenome editing; Gene expression profiling; High-throughput screening.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Cas Systems*
  • Cloning, Molecular / methods
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Epigenesis, Genetic*
  • Gene Editing / methods*
  • Gene Expression Profiling / methods
  • Gene Knock-In Techniques / methods
  • Gene Library
  • HEK293 Cells
  • High-Throughput Screening Assays / methods
  • Humans
  • K562 Cells
  • Luminescent Proteins / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • Red Fluorescent Protein
  • Regulatory Sequences, Nucleic Acid

Substances

  • Luminescent Proteins
  • RNA, Guide, CRISPR-Cas Systems