Rational design of a split-Cas9 enzyme complex
- 23 February 2015
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 112 (10), 2984-2989
- https://doi.org/10.1073/pnas.1501698112
Abstract
Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications. Significance Bacteria have evolved clustered regularly interspaced short palindromic repeats (CRISPRs) together with CRISPR-associated (Cas) proteins to defend themselves against viral infection. RNAs derived from the CRISPR locus assemble with Cas proteins into programmable DNA-targeting complexes that destroy DNA molecules complementary to the guide RNA. In type II CRISPR-Cas systems, the Cas9 protein binds and cleaves target DNA sequences at sites complementary to a 20-nt guide RNA sequence. This activity has been harnessed for a wide range of genome-engineering applications. This study explores the structural features that enable Cas9 to bind and cleave target DNAs, and the results suggest a way of regulating Cas9 by physical separation of the catalytic domains from the rest of the protein scaffold.Keywords
Funding Information
- Howard Hughes Medical Institute ((HHMI))
This publication has 26 references indexed in Scilit:
- Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 deliveryeLife, 2014
- A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial ImmunityScience, 2012
- Complete subunit architecture of the proteasome regulatory particleNature, 2012
- Split-protein systems: beyond binary protein–protein interactionsCurrent Opinion in Chemical Biology, 2011
- Structures of the RNA-guided surveillance complex from a bacterial immune systemNature, 2011
- The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coliNucleic Acids Research, 2011
- Engineering the ABA Plant Stress Pathway for Regulation of Induced ProximityScience Signaling, 2011
- Rapid blue-light–mediated induction of protein interactions in living cellsNature Methods, 2010
- Targeted genome editing in human cells with zinc finger nucleases constructed via modular assemblyGenome Research, 2009
- A New Generation of the IMAGIC Image Processing SystemJournal of Structural Biology, 1996