Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity

Nat Commun. 2016 Jul 1;7:12009. doi: 10.1038/ncomms12009.

Abstract

Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants.

Publication types

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

MeSH terms

  • Bacterial Proteins / metabolism*
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Endonucleases / metabolism*
  • Epigenesis, Genetic
  • Estrogen Antagonists / pharmacology
  • Gene Editing / methods*
  • Gene Expression Regulation
  • Glucocorticoids / pharmacology
  • Plasmids
  • Protein Binding
  • Protein Domains*
  • Receptors, Cytoplasmic and Nuclear / drug effects
  • Receptors, Cytoplasmic and Nuclear / metabolism*
  • Receptors, Estrogen / drug effects
  • Receptors, Estrogen / metabolism*
  • Receptors, Glucocorticoid / drug effects
  • Receptors, Glucocorticoid / metabolism*
  • Staphylococcus aureus
  • Streptococcus pyogenes
  • Tamoxifen / analogs & derivatives
  • Tamoxifen / pharmacology

Substances

  • Bacterial Proteins
  • Estrogen Antagonists
  • Glucocorticoids
  • Receptors, Cytoplasmic and Nuclear
  • Receptors, Estrogen
  • Receptors, Glucocorticoid
  • glucocorticoid receptor alpha
  • Tamoxifen
  • afimoxifene
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases