Materials and Methods

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

MATERIALS AND METHODSCloningA list of all constructs used and created in this study is shown in Supplementary Table S1. Annotated vector sequences are provided as Supplementary Data (GenBank files). Plasmids were created using classical restriction enzyme cloning, Golden Gate Assembly (33) or Gibson assembly (New England Biolabs). Oligonucleotides were obtained from Integrated DNA Technologies (IDT) or Sigma-Aldrich. Synthetic, double-stranded DNA fragments (gBlocks) were obtained from IDT.Luciferase knockdown reporters carrying miRNA binding sites within the 3′UTR of the Renilla luciferase gene (psiCheck-2 2xmiR-122, 2xmiR-1 or 2x scrambled target sites) were generated by inserting a DNA fragment encoding two miRNA target sites followed by a bovine growth hormone (BGH) polyA signal into the psiCheck2 vector (Promega) via XhoI/NotI. The CMV promoter-driven SpyCas9 expression vector (Addgene plasmid #113033) was previously developed by us (34). A SpyCas9-GFP fusion was cloned by PCR-amplifying EGFP from vector pSpCas9(BB)-2A-GFP (Addgene plasmid #48138, which was a kind gift from Feng Zhang) followed by insertion of the PCR amplicon into the SpyCas9 vector via EcoRI/HindIII. The AcrIIA4 and mCherry-AcrIIA4 coding sequences were obtained as human codon-optimized, synthetic DNA fragments from IDT and cloned into pcDNA3.1(–) (ThermoFisher) via NheI/NotI. 2xmiR-122 target sites, 2xmiR-1 target sites or a scaffold sequence identical in length but lacking the miR target sites were inserted into the resulting vectors by oligo cloning via EcoRI/HindIII, yielding vectors CMV-(mCherry)-AcrIIA4–2xmiR-122, CMV-AcrIIA4-2xmiR-1 and CMV-(mCherry)-AcrIIA4–scaffold.The luciferase cleavage reporter for measuring SpyCas9 activity was previously reported by us (34). It comprises an SV40 promoter-driven Renilla luciferase gene, a TK promotor-driven Firefly luciferase gene, and an H1 promoter-driven sgRNA targeting the Firefly luciferase gene. The pRL-TK vector encoding Renilla luciferase was obtained from Promega. AAV vectors encoding (i) SpyCas9 (Addgene #113034) or (ii) an H1 or U6 promoter-driven sgRNA (F+E scaffold (35)) and a RSV promoter-driven EGFP (Addgene #113039) were previously reported by us (36). Annealed oligonucleotides corresponding to the genomic target site were cloned into the sgRNA AAV vector via BbsI using Golden Gate cloning (33). All sgRNA target sites relevant to this study are shown in Supplementary Table S2. AAV vectors encoding CMV or EF1α promoter-driven AcrIIA4 variants were created by replacing the RSV promoter-driven GFP expression cassette from the sgRNA plasmids (36) with synthetic DNA fragments encoding CMV-FLAG-AcrIIA4-scaffold, CMV-FLAG-AcrIIA4-2xmiR-122, CMV-FLAG-AcrIIA4-2xmiR-1, EF1α-AcrIIA4-scaffold or EF1α-AcrIIA4-2xmiR-122, all succeeded by a BGH terminator sequence. AAV vectors encoding a CMV promoter-driven mCherry-AcrIIA4-scaffold or mCherry-AcrIIA4-2xmiR-122 were obtained by replacing the ITR-flanked transgene cassette in the sgRNA plasmids (36) with respective PCR fragments based on the mCherry-AcrIIA4 vectors described above. A vector for AAV-mediated expression of YFP (scAAV-YFP) was previously reported by us (37).An AAV vector co-encoding an N-terminal SpyCas9 fragment fused to a split-intein and a U6 promoter-driven sgRNA scaffold (F+E) was generated by inserting a DNA fragment encoding the U6-promoter-sgRNA scaffold via MluI/XbaI into vector pAAV-SMVP-Cas9N (kind gift from George Church (Addgene plasmid #80930)). An AAV vector co-encoding the corresponding C-terminal SpyCas9 fragment fused to a split-intein was a kind gift from George Church (Addgene plasmid #80931). CMV promoter-driven AcrIIA4 fragments with or without 2xmiR-122 target sites were introduced into this vector by first inserting unique XbaI and MluI sites behind the SV40 polyA. These were subsequently used to introduce CMV-AcrIIA4-scaffold and CMV-AcrIIA4-2xmiR-122 fragments generated by PCR from corresponding template vectors described above.The pAAV-pSi vector co-encoding Firefly and Renilla luciferase (pAAV-pSi) was previously reported by us (36). A single miR-1 binding site was introduced into the Renilla luciferase gene 3′UTR by oligo cloning via XhoI/NotI resulting in the vector pAAV-pSi 1xmiR target site.The CMV-miR-122 expression construct was previously reported by us (38). The CMV-miR-1 expression vector was created by replacing the miR-122 coding sequence in vector CMV-miR-122 by the miR-1 coding sequence, which we obtained via restriction digest of pTRE_Tight_miR-1 (kind gift from David Bartel, Addgene plasmid #14896) with BamHI/HindIII. The Tet-inducible luciferase reporter and corresponding sgRNA construct (sgRNA1_Tet-inducible Luciferase reporter) were kind gifts from Moritoshi Sato (Addgene plasmids #64127 and #64161). dCas9-VP64_GFP was a kind gift from Feng Zhang (Addgene plasmid #61422). The pEJS654 All-in-One AAV-sgRNA-hNmeCas9 vector was a kind gift from Erik Sontheimer (Addgene plasmid #112139). The VEGFA target site (NTS-33 (Ref. 39)) was introduced into this vector by oligo cloning via SapI. The AcrIIC1 and AcrIIC3 coding sequences were obtained as human codon-optimized, synthetic DNA fragment from IDT and cloned into the AAV CMV promoter-driven AcrIIA4-scaffold or AAV CMV promoter-driven AcrIIA4-2xmiR-122 vectors by replacing the AcrIIA4 sequences with the AcrIIC1 or AcrIIC3 coding sequences.In all cloning procedures, PCRs were performed using Phusion Flash High-Fidelity polymerase (ThermoFisher) or Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs) followed by agarose gel electrophoresis to analyse PCR products. Bands of the expected size were cut out and the DNA was extracted by using a QIAquick Gel Extraction Kit (Qiagen). Restriction digests and ligations were performed with enzymes from New England Biolabs and ThermoFisher and according to the manufacturer's protocols. Chemically-competent Top10 cells (ThermoFisher) were used for plasmid amplification and plasmid DNA was purified using the QIAamp DNA Mini, Plasmid Plus Midi or Plasmid Maxi Kit (all from Qiagen).Cell cultureCells lines were cultured at 5% CO2 and 37°C in a humidified incubator and passaged when reaching 70–90% confluency. HeLa and HEK293T cells were maintained in 1× DMEM without phenol red (ThermoFisher) supplemented with 10% (v/v) fetal calf serum (Biochrom AG), 2 mM l-glutamine and 100 U per mL penicillin/100 μg per mL streptomycin (both ThermoFisher). Huh-7 medium was additionally supplemented with 1 mM non-essential amino acids (ThermoFisher). HeLa, HEK293T and Huh-7 cells were authenticated and tested for mycoplasma contamination prior to use via a commercial service (Multiplexion). HL-1 cells were cultured in Claycomb medium supplemented with 10% (v/v) fetal calf serum, 1% penicillin/streptomycin, 0.1 mM norepinephrine, and 2 mM l-glutamine on plates pre-coated with 0.02% (w/v) gelatin and 5 μg/ml fibronectin (all Sigma-Aldrich).Cells were transfected with Lipofectamine™2000, Lipofectamine™3000 (both ThermoFisher) or jetPRIME® (Polyplus-transfection) according to the manufacturers’ protocols and as specified in the experimental sections below.Fluorescence microscopyFor the AcrIIA4 knockdown experiment in Figure ​Figure1C,1C, HeLa and Huh-7 cells were seeded into 8-well Glass Bottom μ-Slides (ibidi) at a density of 30 000 cells per well for HeLa cells and of 60 000 cells per well for Huh-7 cells in 300 μl media. The next day, cells were transfected using 1.85 μl Lipofectamine™2000 per well by following the manufacturer's protocol. The total amount of transfected DNA per well was 720 ng evenly split between plasmids encoding SpyCas9-GFP, either mCherry-AcrIIA4-scaffold or AcrIIA4-2xmiR-122, and the luciferase cleavage reporter plasmid (to provide a sgRNA and an exogenous target). The medium was exchanged 6 h post-transfection.Open in a separate windowFigure 1.Designing a cell-specific Cas-ON switch based on miRNA-regulated anti-CRISPR genes. (A) Schematic of the Cas-ON switch. Target sites for cell-specific, abundant miRNAs are inserted into the 3′UTR of an Acr-encoding transgene. Upon delivery, a knockdown of Acr expression occurs selectively within the target cells, thereby permitting CRISPR–Cas activity. In OFF-target cells lacking the miRNA signature, the Acr protein is translated and inhibits CRISPR–Cas. (B) Schematics of mCherry-AcrIIA4 fusion constructs with or without 2xmiR-122 target sites. (C, D) Hepatocyte-specific knockdown of mCherry-AcrIIA4 expression. Huh-7 and HeLa cells co-transfected with SpyCas9-GFP and either mCherry-AcrIIA4-scaffold or mCherry-AcrIIA4-2xmiR-122. (C) Representative fluorescence images and corresponding overlays from n = 2 independent experiments. Scale bar is 200 μm. (D) Complementary Western blot analysis of SpyCas9-GFP and mCherry-AcrIIA4 expression. Data represent a single experiment.Twenty-four hours post-transfection, HeLa and Huh-7 cells were treated with Hoechst 33342 solution at a final concentration of 5 μg per mL for 15 min at 37°C. Then, the medium was replaced and imaging was performed using a Leica SP8 confocal laser scanning microscope equipped with automated CO2 and temperature control, a UV, argon, and a solid state laser, as well as a HCX PL APO 20× oil objective (N/A = 0.7). mCherry fluorescence was recorded using the 552 nm laser line for excitation and the detection wavelength was set to 578–789 nm. GFP fluorescence was recorded using the 488 nm laser line for excitation and the detection wavelength was set to 493–578 nm.To investigate the transduction efficiency of AAV2 (Supplementary Figure S5), HeLa, Huh-7 and HEK293T cells were seeded into 96-well plates at a density of 4000 cells and using a volume of 100 μl media per well. Each well contained 10 μl of AAV2 lysate encoding a YFP reporter, i.e. cells were transduced while seeding (reverse transduction). Seventy-two hours post-transduction, cells were fixed with 4% PFA for 30 min and subsequently stained with Hoechst 33342 (ThermoFisher). Images were acquired with a 10× objective in nine positions per well with a fully automated epifluorescence Scan∧R screening microscope. To obtain quantitative values for transduction efficiencies (percentages of YFP-positive cells) and for mean expression intensities per cell, a previously established microscopy-based assay for quantitative analysis was used (40).Western blotFor Western blot analysis (Figure ​(Figure1D),1D), cells were seeded into 6-well plates (CytoOne) at a density of 300 000 cells per well for HeLa cells and 450 000 cells per well for Huh-7 cells. The following day, cells were co-transfected with 500 ng of SpyCas9-GFP and either 500 ng CMV-mCherry-AcrIIA4-scaffold or CMV-mCherry-AcrIIA4-2xmiR-122 per well using Lipofectamine™3000. Twenty-four hours post-transfection, the media was aspirated from the culture plates and the cells were washed with ice-cold PBS. Fifty microliters of protein lysis buffer (150 mM NaCl, 10 mM Tris, 1 mM EDTA, 0.5% NP-40 and 10% cOmplete Protease Inhibitor (Roche), pH 8.0) were added, and the cells were detached from the culture plate surface using a cell scraper. The cell suspension was then transferred into a 1.5 ml tube, incubated for 20 min on ice, and centrifuged for 10 min at 13 200 rpm (15 974 × g) and 4°C. The supernatant containing the protein lysate was transferred into a new 1.5 ml tube and protein concentrations were measured using the Bradford Reagent (Sigma-Aldrich) according to the manufacturer's protocol. Fifty microgram of protein lysate were then mixed with 4× Laemmli Sample Buffer (Bio-Rad), and the volume of each sample was adjusted to 30 μl using lysis buffer. The samples were denatured for 10 min at 95°C, cooled down on ice and loaded on a 10% Bis–Tris gel (Life Technologies). Proteins were then separated by molecular weight by applying 130 V for 120 min in 1× MOPS buffer (Life Technologies). Next, proteins were transferred onto a nitrocellulose membrane (pore size: 0.2 μm) (Millipore) by using 1× borate transfer buffer (20 mM boric acid, 1 mM EDTA, 6.25 mM NaOH) and applying 300 mA current overnight. The membrane was then cut at ∼72 and ∼45 kDa, and washed in TBS-T (Tris-buffered saline (20 mM Tris, 140 mM NaCl, pH7.6) supplemented with 0.1% Tween 20 (Sigma-Aldrich)) and blocked by incubation in 5% milk (skim milk powder, GERBU Biotechnik GmbH, diluted in TBS-T) at room temperature for 1 h. GFP antibody (ChromoTek, diluted 1:1500) was used for SpyCas9-GFP detection (190 kDa), α-tubulin antibody (Santa Cruz Biotechnology, diluted 1:500) was used for α-tubulin detection (55 kDa) and RFP antibody (ChromoTek, diluted 1:500) was used for mCherry-AcrIIA4 detection (38 kDa). All antibodies were diluted in 5% milk in TBS-T, added to the corresponding membrane piece and incubated overnight at 4°C while shaking. The next day, the membrane was washed three times for 5 min in TBS-T followed by incubation with HRP-(horse radish peroxidase-)linked secondary antibodies (anti-mouse antibody, 1:5000 in 5% milk in TBS-T (Dianova) or anti-rat antibody, 1:1000 in 5% milk in TBS-T (Jackson ImmunoResearch)) for 1 h at room temperature. The membrane was then washed three times for 5 min in TBS-T to remove unbound antibodies and SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher) was applied for 5 min. Finally, the luminescence signal was detected using a ChemoStar detector (INTAS). The full-length Western blot image is shown in Supplementary Figure S1.Luciferase assaysFor luciferase experiments, HeLa and Huh-7 cells were seeded at a density of 6000 cells per well, HEK293T cells were seeded at a density of 12 500 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 either transiently transfected or transduced with AAV vectors as specified below (all plasmid/vector amounts are per well).For miR-122- or miR-1-induced Renilla luciferase knockdown experiments (Supplementary Figures S3 and S9), Huh-7, HeLa and HEK293T cells were transfected with 20 ng of psiCheck-2 reporter (with or without 2xmiR-122 target sites within the Renilla luciferase 3′UTR) and 80 ng of an irrelevant stuffer DNA (pcDNA3.1(–), ThermoFisher). HL-1 cells were pre-treated with 0.5 μM of Bortezomib (Biomol) to improve transduction efficiency and then transduced with 10 μl pAAV-pSi vector per well (with or without miR-1 binding site within the Renilla 3′UTR; see below for AAV production).For SpyCas9 luciferase cleavage experiments (Figure ​(Figure2A2A and Supplementary Figure S4), cells were co-transfected with 20 ng luciferase cleavage reporter plasmid, 20 ng CMV-SpyCas9 expression vector, and different doses of AcrIIA4-scaffold or AcrIIA4-2xmiR-122 expression vectors (0.25, 1, 5 or 20 ng, indicated in the figure legends). For split-SpyCas9 luciferase cleavage experiments (Supplementary Figure S8), cells were co-transfected with 20 ng luciferase cleavage reporter plasmid, 20 ng of AAV N-SpyCas9-Intein and 20 ng of either (i) AAV Intein-C-SpyCas9, (ii) AAV Intein-C-SpyCas9-CMV-AcrIIA4-scaffold or (iii) AAV Intein-C-SpyCas9-CMV-AcrIIA4-2xmiR-122. To keep the total amount of DNA transfected constant between all samples, DNA was topped up to 100 ng per well using an irrelevant stuffer DNA (pcDNA3.1(–)).Open in a separate windowFigure 2.Hepatocyte- or cardiomyocyte-specific genome editing. (A) Hepatocyte-specific luciferase reporter cleavage. Huh-7 or HeLa cells were co-transfected with plasmids encoding SpyCas9, a luciferase reporter, a reporter-targeting sgRNA, and AcrIIA4-miR-122 or AcrIIA4-scaffold, followed by luciferase assay. During transfection, the Acr vector dose was varied as indicated. Data are means ± s.e.m. (n = 7 independent experiments). (B) Hepatocyte-specific indel mutation of the human EMX1 locus. Huh-7 and HeLa cells were co-transduced with AAV vectors encoding SpyCas9, an EMX-1-targeting sgRNA, and the indicated AcrIIA4 variant, 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). (C, D) Huh-7 cells were co-transduced with AAV vectors encoding SpyCas9, a sgRNA targeting the human CCR5 (C) or AAVS1 (D) locus and the indicated AcrIIA4 variant, 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). (A–D) n.s. = not significant, *P < 0.05, P < 0.01, *P < 0.001, by two-sided Student's t-test with Bonferroni correction. Precise P-values are shown in Table ​Table11 (Material and Methods). (E) Schematic of AcrIIA4 vectors for cardiomyocyte-specific genome editing. (F, G) MiR-1-dependent indel mutation of the Rosa-26 locus in cardiomyocytes. HL-1 cells were co-transduced with AAV vectors expressing SpyCas9, a sgRNA targeting the murine Rosa-26 locus, and AcrIIA4 either with or without miR-1 binding sites, followed by TIDE sequencing. (F) Detailed analysis of indels observed at the Rosa-26 locus. Data for a representative sample is shown. (G) Quantification of the overall editing efficiency. Data are means ± s.e.m. (n = 3 independent experiments).For miR-122- or miR-1-induced Renilla luciferase knockdown experiments (Supplementary Figure S11A), HEK293T cells were co-transfected with 20 ng psiCheck-2 reporter (with 2xmiR-122 target sites, 2xmiR-1 target sites or 2x scrambled target sites within the Renilla luciferase 3′UTR) and 80 ng of either miR-122 or miR-1 expression plasmid, or an irrelevant stuffer DNA (pcDNA3.1(-)). For SpyCas9 luciferase cleavage experiments (Supplementary Figure S11B), cells were co-transfected with (i) 20 ng luciferase cleavage reporter plasmid, (ii) 20 ng CMV-SpyCas9 expression vector, (iii) 20 ng of AcrIIA4-scaffold, AcrIIA4-2xmiR-122 or AcrIIA4-2xmiR-1 expression vectors and (iv) 80 ng of either miR-122 or miR-1 expression plasmid, or an irrelevant stuffer DNA.For SpydCas9-VP64-mediated luciferase activation experiments (Figure ​(Figure3B),3B), cells were co-transfected with 20 ng Tet-inducible luciferase reporter plasmid, 20 ng dCAS9-VP64_GFP expression vector, 20 ng sgRNA1_Tet-inducible luciferase reporter, 1 ng pRL-TK (encoding Renilla luciferase, Promega), and different doses (1, 5, 10, 20, 40 or 60 ng, indicated in the corresponding figure legends) AcrIIA4 expression vector. DNA was topped up to 101 ng per well using an irrelevant stuffer DNA (pcDNA3.1(–)).Open in a separate windowFigure 3.MiR-122-dependent gene activation via SpydCas9-VP64. (A) Schematic of miR-122-dependent activation of luciferase reporter expression. MiR-122-dependent knockdown of AcrIIA4 results in SpydCas9-VP64 activity and thus luciferase expression. (B) Huh-7 cells were co-transfected with vectors encoding SpydCas9-VP64, a luciferase reporter driven by a minimal promoter preceded by tet operator (TetO) sites, a TetO-targeting sgRNA and AcrIIA4-miR-122 or AcrIIA4-scaffold construct (as control), followed by a luciferase assay. During transduction, the Acr vector dose was varied as indicated. Data are means ± s.e.m. (n = 4 independent experiments). P < 0.01, *P < 0.001, by two-sided Student's t-test with Bonferroni correction. For all doses, precise P-values are shown in Table ​Table11 (Material and Methods).Six hours post-transfection or 4 h post-transduction, the medium was replaced. HeLa, Huh-7 and HEK293T cells were incubated for 48 h and HL-1 cells were incubated for 72 h before measuring Renilla and Firefly luciferase activity using a Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's recommendation. In brief, cells were harvested in the supplied lysis buffer and Firefly and Renilla luciferase activities were measured using a GLOMAX™ Discover or GLOMAX™ 96-microplate luminometer (both Promega). Integration time was 10 s, and delay time between automated substrate injection and measurement was 2 s. For the miRNA-dependent luciferase knockdown experiments based on the psiCheck-2 and pAAV-pSi vectors, Renilla luciferase photon counts were normalized to the Firefly luciferase photon counts (as miR target sites were inserted into the Renilla luciferase 3′UTR). For SpyCas9 luciferase cleavage experiments and SpydCas9-VP64-mediated luciferase activation experiments, Firefly luciferase photon counts were normalized to Renilla photon counts (as Cas9 is targeting the Firefly luciferase gene in these assays).AAV lysate productionFor production of AAV-containing cell lysates, low-passage HEK293T cells were seeded into 6-well plates (CytoOne) at a density of 350 000 cells per well. The following day, cells were triple-transfected with (i) the AAV vector plasmid, (ii) an AAV helper plasmid carrying AAV serotype 2 (AAV2) rep and either the AAV2 (when targeting Huh-7, HeLa or HEK293T cells) or AAV6 cap genes (when targeting HL-1 cells) and (iii) an adenoviral plasmid providing helper functions for AAV production, using 1.33 μg of each construct and 8 μl of TurboFect Transfection Reagent (ThermoFisher) per well. The AAV vector plasmid encoded either (i) SpyCas9 driven from an engineered, short CMV promoter, (ii) a U6 promoter-driven sgRNA (based on the improved F+E SpyCas9 scaffold), and a RSV promoter-driven GFP marker, (iii) a CMV promoter-driven AcrIIA4, AcrIIC1 or AcrIIC3 either with or without two miRNA binding sites in their 3′UTRs, (iv) U1a promoter-driven NmeCas9 co-encoding a U6 promoter-driven sgRNA (VEGFA or non-targeting control), or (v) a CMV promoter-driven YFP. Seventy-two hours post-transfection, cells were collected in 300 μl PBS and subsequently subjected to five freeze-thaw cycles by alternating between snap freezing in liquid nitrogen and a 37°C water bath. The cell debris was removed by centrifugation at ∼18 000 × g for 10 min and the AAV-containing supernatant was stored at −20°C until use.T7 assays and TIDE sequencingFor 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).Open in a separate windowFigure 4.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.Flow cytometryFor flow cytometry experiments (Supplementary Figure S7), Huh-7 cells were seeded at a density of 18 000 cells per well into 24-well plates (greiner bio-one) using 1 ml culture medium per well. Sixteen hours post-seeding, cells were transduced with 6, 30 or 60 μl AAV lysates encoding either mCherry-AcrIIA4-scaffold or mCherry-AcrIIA4-2xmiR-122. Of note, these volumes are equivalent to 1, 5 or 10 μl of AcrIIA4 AAV lysate applied in T7 assays in 96-well format (Figure ​(Figure2B2B–D), in which a sixth of the cell number was used (3000 cells per compartment) as compared to the 24-well format (18 000 cells per compartment). The volume of the AAV lysate used per well was adjusted to 100 μl with PBS. Twenty-four hours post-transduction, medium was replaced and the transduction repeated. Seventy-two hours after the (first) transduction, cells were washed with PBS, detached from the cell surface by trypsinization, and collected in 200 μl PBS. Flow cytometry was performed on a FACSCanto (BD Biosciences) system equipped with 405, 488 and 561 nm lasers. Per condition, 8000–10 000 events were recorded. Data analysis was performed using the FACSDiva 8.0 (BD Biosciences) software package, applying the gating strategy shown in Supplementary Figure S7A.Statistical analysisIndependent experiments correspond to cell samples seeded, transfected and treated independently and on different days. Uncertainties in the reported mean values are indicated as the standard error of the mean (s.e.m.). A two-sided Student's t-test was applied to test differences in reported mean values for statistical significance. P-values were Bonferroni corrected to account for the multiple, pair-wise comparisons made. Made comparisons (as indicated by the brackets in the figures) thereby always correspond to identical doses of Acr construct with and without miRNA binding sites present in their 3′UTR. We made these particular comparisons, as they enable assessing the miRNA-dependent regulation of Cas9 activity for statistical significance. Resulting P-values < 0.05, 0.01 and 0.001 are indicated by one, two or three asterisks, respectively. Table ​Table11 shows P-values, significance levels (asterisks), and corresponding effect sizes (fold changes) for all made comparisons.Table 1.Summary of statistical analysisFigureCell lineDose/miRNA P-valueSignificance levelFold changeFigure ​Figure22AHuh-720ng0.00043.25ng< 0.00018.5HeLa20ng1.7242n.s.1.05ng0.4322n.s.1.2Figure ​Figure22BHuh-75μl0.007510.57.5μl< 0.0001*11.410μl0.001816.1HeLa5μl0.8160n.s.1.37.5μl2.5254n.s.0.810μl2.4297n.s.1.3Figure ​Figure22CHuh-71μl0.00812.62.5μl0.00156.75μl0.0369*14.3Figure ​Figure22DHuh-71μl0.1149n.s.1.52.5μl0.00454.55μl0.008713.0Figure ​Figure33BHuh-760ng0.0080114.040ng< 0.000154.020ng< 0.000145.410ng0.003611.45ng0.3000n.s.3.71ng0.7338n.s.1.5Figure ​Figure44AHuh-75μl0.00722.910μl< 0.00015.720μl< 0.000124.2HEK293T5μl0.3279n.s.3.110μl0.2556n.s.5.420μl2.7279n.s.0.9Figure ​Figure44BHuh-72.5μl< 0.0001*3.45μl0.00877.810μl0.007812.5HEK293T2.5μl2.4603n.s.0.95μl2.4537n.s.0.910μl1.9962n.s.1.4 Supplementary Figure S8 Huh-720ng0.00184.2HeLa20ng0.8608n.s.1.0 Supplementary Figure S11B HEK293Tscaffold0.6523n.s.1.00.2221n.s.0.92xmiR-1220.00153.20.9901n.s.1.02xmiR-10.2232n.s.0.80.00125.4Open in a separate window

Article TitleCell-specific CRISPR–Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins

Abstract

Vectors are available via Addgene (#120293 - 120303). Annotated sequences (GenBank files) of all vectors created in this study are provided as Supplementary data. Flow cytometry data is available on FlowRepository (Repository ID: FR-FCM-ZYVK).


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