T7 assays and TIDE sequencing

For transduction-based T7 assays, HeLa and Huh-7 cells were seeded at a density of 3000 cells per well, HEK293T cells were seeded at a density of 3500 cells per well, and HL-1 cells were seeded at a density of 12 000 cells per well into 96-well plates (Eppendorf) using 100 μl culture medium per well. Sixteen hours post-seeding, cells were co-transduced with AAV lysates encoding Cas9, a sgRNA, and the respective anti-CRISPR variant. For experiments targeting the EMX1, CCR5 or AAVS1 locus Figure ​Figure2B2B–D), Huh-7 and HeLa cells were transduced with 33 μl of SpyCas9 vector lysate, 33 μl of EMX1, CCR5 or AAVS1 sgRNA vector lysate, and either 1, 2.5, 5, 7.5 or 10 μl of either AcrIIA4-scaffold or AcrIIA4-2xmiR-122 vector lysate (as indicated in the figure legends). For experiments targeting the VEGFA locus (Figure ​(Figure4),4), Huh-7 and HEK293T cells were transduced with 40 μl of the NmeCas9 AAV lysate and either 5, 10 or 20 μl of AcrIIC1 AAV lysate or 2.5, 5 or 10 μl of AcrIIC3 AAV lysate (as indicated in the figure legends). HL-1 cells were co-transduced with AAV lysates comprising 10 μl of the SpyCas9 vector, 10 μl of the Rosa-26 sgRNA vector, and 3 μl of either AcrIIA4-scaffold or AcrIIA4-2xmiR-1 vector (Figure ​(Figure2G2G and Supplementary Figure S10).

Hepatocyte-specific NmeCas9 activity. (A, B) Huh-7 or HEK293T cells were co-transduced with AAV vectors expressing NmeCas9, a sgRNA targeting the human VEGFA locus and either AcrIIC1 (A) or AcrIIC3 (B) carrying two miR-122 target sites or not, followed by T7 endonuclease assay. During transduction, the Acr vector dose was varied as indicated. Data are means ± s.e.m. (n = 3 independent experiments). n.s. = not significant, **P < 0.01, ***P < 0.001, by two-sided Student's t-test with Bonferroni correction. Precise P-values are shown in Table ​Table11 (Materials and Methods).

The volume of the AAV lysate used per well was adjusted with PBS to 100 μl for SpyCas9 experiments in HeLa and Huh-7 cells, to 80 μl for NmeCas9 experiments in Huh-7 and HEK293T cells, and to 23 μl for T7 assays in HL-1 cells, so that the total volume per well was identical in all samples, including the positive (Cas9 plus sgRNA, but no Acr) and negative (Cas9 only) controls. Medium was replaced 24 h (for HEK293T, HeLa and Huh-7 cells) or 4 h (for HL-1) post-infection and the transduction was repeated 24 h after the first transduction started.

For transfection-based T7 assays (Supplementary Figure S11C and D), HEK293T cells were seeded at a density of 12 500 cells per well into 96-well plates (Eppendorf) using 100 μl culture medium per well. Sixteen hours post-seeding, cells were co-transfected with (i) 25 ng of SpyCas9, (ii) 25 ng of sgRNA construct targeting either the EMX1 or CCR5 locus, (iii) 25 ng of AcrIIA4-scaffold, AcrIIA4-2xmiR-122 or AcrIIA4-2xmiR-1 and (iv) 125 ng of either miR-122 or miR-1 expression plasmid, or empty vector using jetPRIME^® (Polyplus-transfection) according to the manufacturers’ protocol.

Seventy-two hours after the (first) transduction or transfection, cells were lysed using DirectPCR lysis reagent supplemented with proteinase K (Sigma-Aldrich). The genomic target locus was PCR-amplified with primers flanking the target site (Supplementary Table S3) using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). For TIDE sequencing analysis (Figure ​(Figure2F2F and Supplementary Figures S6 and S12), the amplicon was purified using gel electrophoresis followed by gel extraction using the QIAquick Gel Extraction Kit (Qiagen) and by Sanger sequencing (Eurofins). Data analysis was performed using the TIDE web tool (https://tide.deskgen.com/) (41). To assess the indel frequency by T7 assay, we employed a rapid T7 protocol (26). Ten microliters of the target locus amplicons were diluted 1:4 in 1× buffer 2 (New England Biolabs), heated up to 95°C, and slowly cooled down to allow re-annealing and formation of hetero-duplexes using a nexus GSX1 Mastercycler (Eppendorf) and the following program: 95°C/5 min, 95–85°C at −2°C/s, 85–25°C at −0.1°C/s. Subsequently, 0.5 μl T7 endonuclease (New England Biolabs) was added, samples were mixed and incubated at 37°C for 15 min followed by analysis on a 2% Tris–borate–EDTA agarose gel. The Gel iX20 system equipped with a 2.8 megapixel/14 bit scientific-grade CCD camera (INTAS) was used for gel documentation. To calculate the indel percentages from the gel images, the background was subtracted from each lane and T7 bands were quantified using the ImageJ (http://imagej.nih.gov/ij/) gel analysis tool. Peak areas were measured and percentages of insertions and deletions (indel(%)) were calculated using the formula indel (%) = 100 × (1 – (1 – fraction cleaved)^1/2), whereas the fraction cleaved = ∑(cleavage product bands)/∑(cleavage product bands + PCR input band). Full-length T7 assay gel images are shown in Supplementary Figure S2.