Internal guide RNA interactions interfere with Cas9-mediated cleavage.
The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9-gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9-gRNA complex formation.
Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, BIOL 1020, Cambridge, Massachusetts 02138, USA.
Summer B
0000-0003-3593-4148
SB
Thyme
Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, BIOL 1020, Cambridge, Massachusetts 02138, USA.
Laila
L
Akhmetova
Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, BIOL 1020, Cambridge, Massachusetts 02138, USA.
Tessa G
TG
Montague
Computational Biology Unit, Department of Informatics, University of Bergen, 5020 Bergen, Norway.|Sars Centre for Marine Molecular Biology, University of Bergen, Thormøhlensgaten 55, 5008 Bergen, Norway.
Eivind
E
Valen
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.|Center for Brain Science, Harvard University, Cambridge, Massachusetts 02138, USA.|Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.|Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA.|FAS Center for Systems Biology, Harvard University, Harvard, Massachusetts 02138, USA.
Alexander F
AF
Schier
D000818:Animals; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D016678:Genome; D009690:Nucleic Acid Conformation; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000378:metabolism*; D015027:Zebrafish
D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems