Guide RNA functional modules direct Cas9 activity and orthogonality

Alexandra E. Briner, Paul D. Donohoue, Ahmed A. Gomaa, Kurt Selle, Euan M. Slorach, Christopher H. Nye, Rachel E. Haurwitz, Chase L. Beisel, Andrew P. May, Rodolphe Barrangou

Research output: Contribution to journalArticle

119 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)333-339
Number of pages7
JournalMolecular cell
Volume56
Issue number2
DOIs
StatePublished - Jan 1 2014

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

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    Briner, A. E., Donohoue, P. D., Gomaa, A. A., Selle, K., Slorach, E. M., Nye, C. H., Haurwitz, R. E., Beisel, C. L., May, A. P., & Barrangou, R. (2014). Guide RNA functional modules direct Cas9 activity and orthogonality. Molecular cell, 56(2), 333-339. https://doi.org/10.1016/j.molcel.2014.09.019