Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs

Kale Kundert; James E Lucas; Kyle E Watters; Christof Fellmann; Andrew H Ng; Benjamin M Heineike; Christina M Fitzsimmons; Benjamin L Oakes; Jiuxin Qu; Neha Prasad; Oren S Rosenberg; David F Savage; Hana El-Samad; Jennifer A Doudna; Tanja Kortemme

doi:10.1038/s41467-019-09985-2

Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs

Nat Commun. 2019 May 9;10(1):2127. doi: 10.1038/s41467-019-09985-2.

Authors

Kale Kundert¹, James E Lucas², Kyle E Watters³, Christof Fellmann³, Andrew H Ng², Benjamin M Heineike⁴, Christina M Fitzsimmons^{5

6}, Benjamin L Oakes³, Jiuxin Qu⁷, Neha Prasad⁷, Oren S Rosenberg^{7

8}, David F Savage³, Hana El-Samad^{8

9}, Jennifer A Doudna^{3

10}, Tanja Kortemme^{11

12}

Affiliations

¹ Graduate Group in Biophysics, University of California San Francisco, San Francisco, CA, 94158, USA. kale@thekunderts.net.
² UC Berkeley - UCSF Graduate Program in Bioengineering, University of California San Francisco, San Francisco, CA, 94158, USA.
³ Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, 94704, USA.
⁴ Bioinformatics Graduate Program, University of California San Francisco, San Francisco, CA, 94158, USA.
⁵ Chemistry and Chemical Biology Graduate Program, University of California San Francisco, San Francisco, CA, 94158, USA.
⁶ Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
⁷ Department of Medicine, University of California San Francisco, San Francisco, CA, 94158, USA.
⁸ Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA.
⁹ Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, 94158, USA.
¹⁰ Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, 94704, USA.
¹¹ Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA. tanjakortemme@gmail.com.
¹² Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, 94158, USA. tanjakortemme@gmail.com.

Abstract

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.

Publication types

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

MeSH terms

Aptamers, Nucleotide / genetics*
CRISPR-Associated Protein 9 / genetics*
CRISPR-Cas Systems / genetics*
DNA / genetics
Gene Editing / methods*
Ligands
RNA, Guide, CRISPR-Cas Systems / genetics*

Substances

Aptamers, Nucleotide
Ligands
RNA, Guide, CRISPR-Cas Systems
DNA
CRISPR-Associated Protein 9

Abstract

Publication types

MeSH terms

Substances

Grants and funding