Precise Regulation of Cas9-Mediated Genome Engineering by Anti-CRISPR-Based Inducible CRISPR Controllers

Surbhi Jain, Guanhua Xun, Shireen Abesteh, Sherri Ho, Manasi Lingamaneni, Teresa A. Martin, Ipek Tasan, Che Yang, Huimin Zhao

Research output: Contribution to journalArticlepeer-review


CRISPR/Cas9 is a powerful genome editing tool, but its off-target cleavage activity can result in unintended adverse outcomes for therapeutic applications. Here we report the design of a simple tunable CRISPR controller in which a chemically inducible anti-CRISPR protein AcrIIA4 is engineered to disable Cas9 DNA binding upon the addition of trimethoprim. Dose-dependent control over Cas9 editing and dCas9 induction was achieved, which drastically improved the specificity and biosafety of the CRISPR/Cas9 system. We utilized the anti-CRISPR protein AcrIIA4 as a means to interfere with Cas9 DNA binding activity. By fusing AcrIIA4 to a ligand-inducible destabilization domain DHFR(DD), we show significantly reduced off-target activity in mammalian cells. Furthermore, we describe a new inducible promoter system Acr-OFF based on CRISPR controllers, which is regulated by an FDA-approved ligand trimethoprim.

Original languageEnglish (US)
Pages (from-to)1320-1327
Number of pages8
JournalACS synthetic biology
Issue number6
StatePublished - Jun 18 2021


  • CRISPR/Cas9
  • anti-CRISPR
  • destabilization domains
  • off-target
  • synthetic biology tools

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)


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