Engineered CRISPR-Cas9 nuclease with expanded targeting space

Science. 2018 Sep 21;361(6408):1259-1262. doi: 10.1126/science.aas9129. Epub 2018 Aug 30.


The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.

Publication types

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

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics*
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems*
  • Crystallography, X-Ray
  • Endonucleases / chemistry*
  • Endonucleases / genetics*
  • Gene Editing*
  • HEK293 Cells
  • Humans
  • Protein Engineering


  • Bacterial Proteins
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases