Rationally Designed Anti-CRISPR Nucleic Acid Inhibitors of CRISPR-Cas9

Nucleic Acid Ther. 2019 Jun;29(3):136-147. doi: 10.1089/nat.2018.0758. Epub 2019 Apr 16.


Clustered regularly interspaced short palindromic repeat (CRISPR) RNAs and their associated effector (Cas) enzymes are being developed into promising therapeutics to treat disease. However, CRISPR-Cas enzymes might produce unwanted gene editing or dangerous side effects. Drug-like molecules that can inactivate CRISPR-Cas enzymes could help facilitate safer therapeutic development. Based on the requirement of guide RNA and target DNA interaction by Cas enzymes, we rationally designed small nucleic acid-based inhibitors (SNuBs) of Streptococcus pyogenes (Sp) Cas9. Inhibitors were initially designed as 2'-O-methyl-modified oligonucleotides that bound the CRISPR RNA guide sequence (anti-guide) or repeat sequence (anti-tracr), or DNA oligonucleotides that bound the protospacer adjacent motif (PAM)-interaction domain (anti-PAM) of SpCas9. Coupling anti-PAM and anti-tracr modules together was synergistic and resulted in high binding affinity and efficient inhibition of Cas9 DNA cleavage activity. Incorporating 2'F-RNA and locked nucleic acid nucleotides into the anti-tracr module resulted in greater inhibition as well as dose-dependent suppression of gene editing in human cells. CRISPR SNuBs provide a platform for rational design of CRISPR-Cas enzyme inhibitors that should translate to other CRISPR effector enzymes and enable better control over CRISPR-based applications.

Keywords: CRISPR-Cas9; RNA; anti-CRISPR; gene editing; inhibition; nucleic acid.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9 / antagonists & inhibitors
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / pharmacology
  • CRISPR-Cas Systems / drug effects
  • CRISPR-Cas Systems / genetics*
  • DNA / drug effects
  • DNA / genetics
  • DNA-Binding Proteins / drug effects
  • DNA-Binding Proteins / genetics*
  • Gene Editing*
  • Humans
  • Nucleotide Motifs / drug effects
  • Nucleotide Motifs / genetics
  • Oligonucleotides / genetics
  • Oligonucleotides / pharmacology
  • RNA, Guide, CRISPR-Cas Systems / adverse effects
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / pharmacology
  • Streptococcus pyogenes / enzymology
  • Tandem Repeat Sequences / drug effects
  • Tandem Repeat Sequences / genetics


  • DNA-Binding Proteins
  • Oligonucleotides
  • RNA, Guide, CRISPR-Cas Systems
  • DNA
  • CRISPR-Associated Protein 9