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. 2022 Feb 21;50(5):2836–2853. doi: 10.1093/nar/gkac099

Figure 7.

Figure 7.

Chemically inducible anti-CRISPR systems for the control of CRISPR–Cas9-mediated genome editing. (A) A schematic view of iAcr systems. Insertion of a ligand-dependent intein into Acr protein renders Acr inactive. 4-HT binding can trigger intein protein splicing and restore Acr activity to inhibit Cas9. (B) Schematic view of the BFP-to-GFP reporter system for PE to examine the activity of intein–Acr hybrids against Cas9 in human cells. HEK293T cells with a chromosomally integrated BFP (HEK293T-BFP cells) were transfected with plasmids encoding prime editor, BFP-targeting pegRNA and intein–Acr hybrids in the presence or absence of 4-HT (1 μM). The percentage of GFP-positive cells was calculated via flow cytometry at 72 h post-transfection. The PE can switch BFP to GFP by replacing CC to GT, causing single H66Y amino acid substitution. The target sequence, PAM and replaced base are shown in blue, purple and red, respectively. (C) Comparison of BFP-to-GFP conversion efficiencies in the presence or absence of wild-type (WT) Acr or intein–Acr variants under the condition of 4-HT treatment or not. Intein–Acr variants are identified by the residue replaced by the intein. WT Acrs including C1 (AcrIIC1), A4 (AcrIIA4), A5 (AcrIIA5), A25.1 (AcrIIA25.1) and A32.1 (AcrIIA32.1) are used as controls. Error bars represent the mean ± SEM with three biological replicates. (D) Representative gel images of T7E1 assay to manifest the inhibitory activities of Acr and iAcr proteins against SpyCas9 in the presence or absence of 4-HT. HEK293T cells were transfected with Cas9 (1 μg), sgRNA (0.5 μg) and Acr (0.5 or 0.25 μg) plasmids (see the ‘Materials and Methods’ section for details). The editing efficiencies [indel (%)] are labeled at the bottom of each lane. The target sequence and PAM are highlighted in blue and purple, respectively. The gels are representative of three independent replicates. Representative gel images of T7E1 assay to investigate the inhibitory activities of Acr and iAcr proteins against SpyCas9 (E) or St3Cas9 (F) in the presence or absence of 4-HT. HEK293T cells were transfected with Cas9 (1 μg), sgRNA (0.5 μg) and Acr (0.25 μg) plasmids (see the ‘Materials and Methods’ section for details). The editing efficiencies [indel (%)] are labeled at the bottom of each lane. The target sites of human EMX1 (targeted by SpyCas9) and DYRK1A (targeted by St3Cas9) are shown at the top of each gel and PAMs are highlighted in purple. The gels are representative of three independent replicates.