Disabling Cas9 by an anti-CRISPR DNA mimic
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Open Access
- 7 July 2017
- journal article
- research article
- Published by American Association for the Advancement of Science (AAAS) in Science Advances
- Vol. 3 (7), e1701620-1701620
- https://doi.org/10.1126/sciadv.1701620
Abstract
CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9–single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo–electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.Keywords
Funding Information
- National Institutes of Health (award331586, T32 AG000266)
- National Institutes of Health (award331588, DP5-OD021344)
- Howard Hughes Medical Institute (award331587)
- Howard Hughes Medical Institute (award331589)
This publication has 31 references indexed in Scilit:
- CTFFIND4: Fast and accurate defocus estimation from electron micrographsJournal of Structural Biology, 2015
- Easy quantitative assessment of genome editing by sequence trace decompositionNucleic Acids Research, 2014
- Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cellsNature Biotechnology, 2014
- Crystal Structure of Cas9 in Complex with Guide RNA and Target DNACell, 2014
- Quantifying the local resolution of cryo-EM density mapsNature Methods, 2013
- CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activityNucleic Acids Research, 2013
- CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineeringNature Biotechnology, 2013
- Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune systemNature, 2012
- Features and development of CootActa Crystallographica Section D-Structural Biology, 2010
- MolProbity: all-atom structure validation for macromolecular crystallographyActa Crystallographica Section D-Structural Biology, 2009