Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • CRISPR-Associated Protein 9
  • CRISPR-Associated Proteins / chemistry*
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA / chemistry*
  • DNA Cleavage*
  • Endonucleases / chemistry*
  • Genetic Engineering / methods*
  • HEK293 Cells
  • Humans
  • Nucleic Acid Conformation
  • RNA, Guide / chemistry*
  • RNA, Guide / genetics
  • RNA, Guide / ultrastructure

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • RNA, Guide
  • DNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases