Targeted Gene Activation Using RNA-Guided Nucleases

Methods Mol Biol. 2017;1468:235-50. doi: 10.1007/978-1-4939-4035-6_16.

Abstract

The discovery of the prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) system and its adaptation for targeted manipulation of DNA in diverse species has revolutionized the field of genome engineering. In particular, the fusion of catalytically inactive Cas9 to any number of transcriptional activator domains has resulted in an array of easily customizable synthetic transcription factors that are capable of achieving robust, specific, and tunable activation of target gene expression within a wide variety of tissues and cells. This chapter describes key experimental design considerations, methods for plasmid construction, gene delivery protocols, and procedures for analysis of targeted gene activation in mammalian cell lines using CRISPR-Cas transcription factors.

Keywords: CRISPR-Cas9; Gene activation; Gene expression; Genome engineering; RNA-guided nucleases; Synthetic biology; Transcription.

MeSH terms

  • CRISPR-Cas Systems
  • Gene Editing
  • Gene Targeting / methods*
  • Genetic Engineering
  • HEK293 Cells
  • Humans
  • Mutagenesis
  • RNA, Guide / metabolism
  • Ribonucleases / metabolism*
  • Transcriptional Activation*

Substances

  • RNA, Guide
  • Ribonucleases