Fluorescent Guide RNAs Facilitate Development of Layered Pol II-Driven CRISPR Circuits

ACS Synth Biol. 2018 Aug 17;7(8):1929-1936. doi: 10.1021/acssynbio.8b00153. Epub 2018 Jul 31.

Abstract

Efficient clustered regularly interspaced short palindromic repeat (CRISPR) guide RNA (gRNA) expression from RNA Polymerase II (Pol II) promoters will aid in construction of complex CRISPR-based synthetic gene networks. Yet, we require tools to properly visualize gRNA directly to quantitatively study the corresponding network behavior. To address this need, we employed a fluorescent gRNA (fgRNA) to visualize synthetic CRISPR network dynamics without affecting gRNA functionality. We show that studying gRNA dynamics directly enables circuit modification and improvement of network function in Pol II-driven CRISPR circuits. This approach generates information necessary for optimizing the overall function of these networks and provides insight into the hurdles remaining in Pol II-regulated gRNA expression.

Keywords: CRISPR; computational biology; fgRNA; genetic circuit; synthetic biology.

Publication types

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

MeSH terms

  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Computational Biology / methods
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism
  • RNA, Guide / chemistry*
  • RNA, Guide / metabolism*
  • Synthetic Biology / methods

Substances

  • RNA, Guide
  • RNA Polymerase II