Protein Engineering of Cas9 for Enhanced Function

Methods Enzymol. 2014;546:491-511. doi: 10.1016/B978-0-12-801185-0.00024-6.


CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complementary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted within the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome-modifying tools.

Keywords: BH; CRISPR; Cas9; DNA; EM; FACS; GFP; Gene activation/repression; Genome editing; HR; IPTG; IT dCas9; LB; NHEJ; NUC; Nuclease; PAM; PDZ; PI; Protein engineering; REC; RFP; RNA; SOB; SOC; SpCas9; Synthetic biology; WT dCas9; aTC; crRNA; dCas9; sgRNA; ssDNA; tracrRNA.

MeSH terms

  • Amino Acid Sequence
  • Bacteria / chemistry
  • Bacteria / enzymology*
  • Bacteria / genetics
  • Bacteria / metabolism
  • Base Sequence
  • CRISPR-Associated Proteins / chemistry
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems
  • Deoxyribonuclease I / chemistry
  • Deoxyribonuclease I / genetics*
  • Deoxyribonuclease I / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • PDZ Domains
  • Protein Conformation
  • Protein Engineering / methods*
  • Streptococcus pyogenes / chemistry
  • Streptococcus pyogenes / enzymology
  • Streptococcus pyogenes / genetics
  • Streptococcus pyogenes / metabolism


  • CRISPR-Associated Proteins
  • Deoxyribonuclease I