Rewiring Calcium Signaling for Precise Transcriptional Reprogramming

ACS Synth Biol. 2018 Mar 16;7(3):814-821. doi: 10.1021/acssynbio.7b00467. Epub 2018 Mar 6.

Abstract

Tools capable of modulating gene expression in living organisms are very useful for interrogating the gene regulatory network and controlling biological processes. The catalytically inactive CRISPR/Cas9 (dCas9), when fused with repressive or activating effectors, functions as a versatile platform to reprogram gene transcription at targeted genomic loci. However, without temporal control, the application of these reprogramming tools will likely cause off-target effects and lack strict reversibility. To overcome this limitation, we report herein the development of a chemical or light-inducible transcriptional reprogramming device that combines photoswitchable genetically encoded calcium actuators with dCas9 to control gene expression. By fusing an engineered Ca2+-responsive NFAT fragment with dCas9 and transcriptional coactivators, we harness the power of light to achieve photoinducible transcriptional reprogramming in mammalian cells. This synthetic system (designated CaRROT) can also be used to document calcium-dependent activity in mammals after exposure to ligands or chemicals that would elicit calcium response inside cells.

Keywords: CRISPR/Cas9; NFAT; calcium signaling; genome editing; optogenetics; transcriptional reprogramming.

Publication types

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

MeSH terms

  • CRISPR-Cas Systems / genetics
  • Calcium / metabolism
  • Calcium Release Activated Calcium Channels / metabolism
  • Calcium Signaling / genetics*
  • Cell Nucleus / metabolism
  • Gene Expression Regulation / radiation effects
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Light
  • Optogenetics
  • Protein Transport
  • Synthetic Biology / methods*
  • Transcription, Genetic*

Substances

  • Calcium Release Activated Calcium Channels
  • Calcium