2. Academic Validation
  3. A chemically controlled Cas9 switch enables temporal modulation of diverse effectors

A chemically controlled Cas9 switch enables temporal modulation of diverse effectors

  • Nat Chem Biol. 2023 Mar 6. doi: 10.1038/s41589-023-01278-6.
Cindy T Wei 1 2 3 4,  Nicholas A Popp 2,  Omri Peleg 5,  Rachel L Powell 2,  Elhanan Borenstein 5 6 7,  Dustin J Maly 8 9,  Douglas M Fowler 10 11
Affiliations

Affiliations

  • 1 Molecular and Cellular Biology, University of Washington, Seattle, WA, USA.
  • 2 Department of Genome Sciences, University of Washington, Seattle, WA, USA.
  • 3 Department of Chemistry, University of Washington, Seattle, WA, USA.
  • 4 Novartis Institutes for BioMedical Research Inc, San Diego, CA, USA.
  • 5 The Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, Israel.
  • 6 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
  • 7 Santa Fe Institute, Santa Fe, NM, USA.
  • 8 Department of Chemistry, University of Washington, Seattle, WA, USA. djmaly@uw.edu.
  • 9 Department of Biochemistry, University of Washington, Seattle, WA, USA. djmaly@uw.edu.
  • 10 Department of Genome Sciences, University of Washington, Seattle, WA, USA. dfowler@uw.edu.
  • 11 Department of Bioengineering, University of Washington, Seattle, WA, USA. dfowler@uw.edu.
PMID: 36879061 DOI: 10.1038/s41589-023-01278-6
Abstract

CRISPR-Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies.

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