Computational design of anti-CRISPR proteins with improved inhibition potency

Nat Chem Biol. 2020 Jul;16(7):725-730. doi: 10.1038/s41589-020-0518-9. Epub 2020 Apr 13.

Abstract

Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR-Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR-Cas9 inhibitor, to improve its efficacy on different targets. We first show that inserting exogenous protein domains into a selected AcrIIC1 surface site dramatically enhances inhibition of Neisseria meningitidis (Nme)Cas9. Then, applying structure-guided design to the Cas9-binding surface, we converted AcrIIC1 into AcrIIC1X, a potent inhibitor of the Staphylococcus aureus (Sau)Cas9, an orthologue widely applied for in vivo genome editing. Finally, to demonstrate the utility of AcrIIC1X for genome engineering applications, we implemented a hepatocyte-specific SauCas9 ON-switch by placing AcrIIC1X expression under regulation of microRNA-122. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard CRISPR technologies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Cell Line, Tumor
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Gene Editing / methods*
  • Genome, Human
  • HEK293 Cells
  • Hepatocytes / cytology
  • Hepatocytes / metabolism
  • Humans
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Models, Molecular
  • Mutagenesis, Insertional
  • Neisseria meningitidis / enzymology
  • Neisseria meningitidis / genetics
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Protein Domains
  • Protein Engineering / methods*
  • Protein Structure, Secondary
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Staphylococcus aureus / enzymology
  • Staphylococcus aureus / genetics

Substances

  • MIRN122 microRNA, human
  • MicroRNAs
  • RNA, Guide
  • CRISPR-Associated Protein 9