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Mol Cell. 2014 Oct 23;56(2):333-339. doi: 10.1016/j.molcel.2014.09.019. Epub 2014 Oct 16.

Guide RNA functional modules direct Cas9 activity and orthogonality.

Author information

1
Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA.
2
Caribou Biosciences, Inc., Berkeley, CA 94710, USA.
3
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA; Chemical Engineering Department, Faculty of Engineering, Cairo University, Giza 12613, Egypt.
4
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA. Electronic address: cbeisel@ncsu.edu.
5
Caribou Biosciences, Inc., Berkeley, CA 94710, USA. Electronic address: andy.may@cariboubio.com.
6
Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA. Electronic address: rbarran@ncsu.edu.

Abstract

The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner and has been widely used for programmable genome editing. Cas9 activity is dependent on interactions with guide RNAs, and evolutionarily divergent Cas9 nucleases have been shown to work orthogonally. However, the molecular basis of selective Cas9:guide-RNA interactions is poorly understood. Here, we identify and characterize six conserved modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity. We show the bulge and nexus are necessary for DNA cleavage and demonstrate that the nexus and hairpins are instrumental in defining orthogonality between systems. In contrast, the crRNA:tracrRNA complementary region can be modified or partially removed. Collectively, our results establish guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.

PMID:
25373540
DOI:
10.1016/j.molcel.2014.09.019
[Indexed for MEDLINE]
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