CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

Benjamin L Oakes; Christof Fellmann; Harneet Rishi; Kian L Taylor; Shawn M Ren; Dana C Nadler; Rayka Yokoo; Adam P Arkin; Jennifer A Doudna; David F Savage

doi:10.1016/j.cell.2018.11.052

CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

Cell. 2019 Jan 10;176(1-2):254-267.e16. doi: 10.1016/j.cell.2018.11.052.

Authors

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
² Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Gladstone Institutes, San Francisco, CA 94158, USA.
³ Graduate Group in Biophysics, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
⁵ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
⁶ Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
⁷ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Gladstone Institutes, San Francisco, CA 94158, USA; Graduate Group in Biophysics, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
⁸ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: savage@berkeley.edu.

Abstract

The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.

Keywords: CRISPR-Cas; Cas9-CP; ProCas9; circular permutation; fusion proteins; genome editing; protein engineering.

Publication types

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

MeSH terms

CRISPR-Associated Proteins / chemistry
CRISPR-Cas Systems / physiology*
Clustered Regularly Interspaced Short Palindromic Repeats / physiology*
DNA / chemistry
Gene Editing / methods*
Genome
Models, Molecular
RNA / chemistry
RNA, Guide, CRISPR-Cas Systems / genetics

Substances

CRISPR-Associated Proteins
RNA, Guide, CRISPR-Cas Systems
RNA
DNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding