Blue Light-Operated CRISPR/Cas13b-Mediated mRNA Knockdown (Lockdown)

Adv Biol (Weinh). 2021 May;5(5):e2000307. doi: 10.1002/adbi.202000307. Epub 2021 Feb 11.

Abstract

The introduction of optogenetics into cell biology has furnished systems to control gene expression at the transcriptional and protein stability level, with a high degree of spatial, temporal, and dynamic light-regulation capabilities. Strategies to downregulate RNA currently rely on RNA interference and CRISPR/Cas-related methods. However, these approaches lack the key characteristics and advantages provided by optical control. "Lockdown" introduces optical control of RNA levels utilizing a blue light-dependent switch to induce expression of CRISPR/Cas13b, which mediates sequence-specific mRNA knockdown. Combining Lockdown with optogenetic tools to repress gene-expression and induce protein destabilization with blue light yields efficient triple-controlled downregulation of target proteins. Implementing Lockdown to degrade endogenous mRNA levels of the cyclin-dependent kinase 1 (hCdk1) leads to blue light-induced G2/M cell cycle arrest and inhibition of cell growth in mammalian cells.

Keywords: CRISPR/Cas13b; RNA downregulation; blue light-gene expression control; mammalian synthetic biology; optogenetics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems* / genetics
  • Light
  • Optogenetics*
  • RNA
  • RNA, Messenger / genetics

Substances

  • RNA, Messenger
  • RNA