Transcriptional Regulation with CRISPR/Cas9 Effectors in Mammalian Cells

Methods Mol Biol. 2016;1358:43-57. doi: 10.1007/978-1-4939-3067-8_3.

Abstract

CRISPR/Cas9-based regulation of gene expression provides the scientific community with a new high-throughput tool to dissect the role of genes in molecular processes and cellular functions. Single-guide RNAs allow for recruitment of a nuclease-dead Cas9 protein and transcriptional Cas9-effector fusion proteins to specific genomic loci, thereby modulating gene expression. We describe the application of a CRISPR-Cas9 effector system from Streptococcus pyogenes for transcriptional regulation in mammalian cells resulting in activation or repression of transcription. We present methods for appropriate target site selection, sgRNA design, and delivery of dCas9 and dCas9-effector system components into cells through lentiviral transgenesis to modulate transcription.

Keywords: CRISPR/Cas9; Cas9-effectors; Gene activation; Gene repression; Transcriptional regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Computational Biology / methods*
  • Gene Expression Regulation / genetics
  • Genome
  • Lentivirus / genetics
  • Mammals
  • RNA, Guide / genetics*
  • Streptococcus pyogenes / genetics
  • Transcription, Genetic*

Substances

  • RNA, Guide