Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

Nat Methods. 2013 Nov;10(11):1116-21. doi: 10.1038/nmeth.2681. Epub 2013 Sep 29.


The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas acquired immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but it can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences, and we identify an unexpectedly versatile Cas9 protein from Neisseria meningitidis. We provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / physiology*
  • Gene Expression Regulation*
  • Molecular Sequence Data
  • RNA / genetics*
  • RNA Editing*
  • Sequence Homology, Amino Acid
  • Streptomyces / physiology


  • Bacterial Proteins
  • RNA