Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality

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....

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Published in:	Molecular cell Vol. 56; no. 2; pp. 333 - 339
Main Authors:	Briner, Alexandra E., Donohoue, Paul D., Gomaa, Ahmed A., Selle, Kurt, Slorach, Euan M., Nye, Christopher H., Haurwitz, Rachel E., Beisel, Chase L., May, Andrew P., Barrangou, Rodolphe
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 23-10-2014
Subjects:	Bacterial Proteins - chemistry Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-Associated Protein 9 CRISPR-Associated Proteins - chemistry CRISPR-Cas Systems DNA - chemistry DNA Cleavage Endonucleases - chemistry Genetic Engineering - methods HEK293 Cells Humans Nucleic Acid Conformation RNA, Guide, CRISPR-Cas Systems - chemistry RNA, Guide, CRISPR-Cas Systems - genetics RNA, Guide, CRISPR-Cas Systems - ultrastructure
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Summary:	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. [Display omitted] •Several modules within guide RNAs drive Cas9-mediated cleavage•These modules are universally relevant for Type II-A CRISPR-Cas systems•Guide RNAs can be altered to cross Cas9 orthogonality boundaries Briner et al. establish functional modules within the native and engineered guide RNAs that direct Cas9-mediated targeting and cleavage, with implications for development of next-generation CRISPR technologies and genome editing applications.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1097-2765 1097-4164
DOI:	10.1016/j.molcel.2014.09.019