Rheostatic Control of Cas9-Mediated DNA Double Strand Break (DSB) Generation and Genome Editing

ACS Chem Biol. 2018 Feb 16;13(2):438-442. doi: 10.1021/acschembio.7b00652. Epub 2017 Sep 15.

Abstract

We recently reported two novel tools for precisely controlling and quantifying Cas9 activity: a chemically inducible Cas9 variant (ciCas9) that can be rapidly activated by small molecules and a ddPCR assay for time-resolved measurement of DNA double strand breaks (DSB-ddPCR). Here, we further demonstrate the potential of ciCas9 to function as a tunable rheostat for Cas9 function. We show that a new highly potent and selective small molecule activator paired with a more tightly regulated ciCas9 variant expands the range of accessible Cas9 activity levels. We subsequently demonstrate that ciCas9 activity levels can be dose-dependently tuned with a small molecule activator, facilitating rheostatic time-course experiments. These studies provide the first insight into how Cas9-mediated DSB levels correlate with overall editing efficiency. Thus, we demonstrate that ciCas9 and our DSB-ddPCR assay permit the time-resolved study of Cas9 DSB generation and genome editing kinetics at a wide range of Cas9 activity levels.

Publication types

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

MeSH terms

  • Benzothiazoles / pharmacology
  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems / genetics*
  • DNA / genetics*
  • DNA Breaks, Double-Stranded*
  • Gene Editing
  • HEK293 Cells
  • Humans
  • INDEL Mutation / genetics
  • Isoquinolines / pharmacology
  • Polymerase Chain Reaction / methods
  • Streptococcus pyogenes / enzymology
  • bcl-X Protein / antagonists & inhibitors

Substances

  • A-1155463
  • BCL2L1 protein, human
  • Benzothiazoles
  • CRISPR-Associated Proteins
  • Isoquinolines
  • bcl-X Protein
  • DNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes