Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins

Nucleic Acids Res. 2019 Jul 26;47(13):e75. doi: 10.1093/nar/gkz271.


The rapid development of CRISPR-Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR-Cas orthologue, for which a potent anti-CRISPR protein is known.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics
  • Binding Sites
  • CRISPR-Associated Protein 9 / antagonists & inhibitors
  • CRISPR-Associated Protein 9 / biosynthesis
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Cas Systems*
  • Dependovirus / genetics
  • Enzyme Activation
  • Enzyme Induction
  • Gene Editing / methods*
  • Gene Expression Regulation*
  • Genes, Reporter
  • HEK293 Cells
  • HeLa Cells
  • Hepatocytes / metabolism
  • Humans
  • Luciferases, Renilla / analysis
  • Luciferases, Renilla / genetics
  • MicroRNAs
  • Myocytes, Cardiac / metabolism
  • Organ Specificity
  • Protein Isoforms / antagonists & inhibitors
  • Transgenes*


  • 3' Untranslated Regions
  • MIRN1 microRNA, human
  • MIRN122 microRNA, human
  • MicroRNAs
  • Protein Isoforms
  • Luciferases, Renilla
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes