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

• Published in: ACS synthetic biology Vol. 10; no. 6; pp. 1320 - 1327
• Main Authors: Jain, Surbhi, Xun, Guanhua, Abesteh, Shireen, Ho, Sherri, Lingamaneni, Manasi, Martin, Teresa A, Tasan, Ipek, Yang, Che, Zhao, Huimin
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18.06.2021
• Subjects: Clustered Regularly Interspaced Short Palindromic Repeats - genetics; Containment of Biohazards - methods; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems; DNA - metabolism; DNA-Binding Proteins - genetics; Gene Editing - methods; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; Humans; Ligands; Protein Binding; RNA, Guide, CRISPR-Cas Systems - genetics; Streptococcus pyogenes - enzymology; Transfection; Trimethoprim - metabolism; synthetic biology tools; anti-CRISPR; CRISPR/Cas9; off-target; destabilization domains
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.0c00548

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.