Materials and Methods
Escherichia coli (TOP10 or Mach1-T1, Biomed) strains were used for plasmid amplification and interference assays. Escherichia coli (T7 Express, Biomed) strains were used for protein expression and phage plaque assays. Escherichia coli were routinely (unless otherwise indicated) cultured at 37°C in lysogeny broth (LB) medium with appropriate antibiotics (when required): ampicillin (50 μg/ml), kanamycin (50 μg/ml) or chloramphenicol (25 μg/ml).
HEK293T and HEK293T-BFP cells were cultured in DMEM (Gibco) medium supplemented with 10% (vol/vol) fetal bovine serum (FBS, Gibco) at 37°C and 5% CO2 in an incubator. U2OS cells were cultured in McCoy’s 5A (modified) medium (Gibco) supplemented with 10% FBS at 37°C and 5% CO2 in an incubator.
BLASTp program was used to search for AcrIIA6 (accession: AVO22749.1) homologs in the nonredundant protein database to manually examine the possible novel acr and aca genes from neighboring candidate genes. A gene was designated as an aca according to previous methods (33) by the following criteria: (i) directly upstream or downstream of an Acr homolog in the same orientation; (ii) containing a DNA-binding domain predicted by HHpred of MPI Bioinformatics Toolkit (34); and (iii) this gene can associate with more than two distinct types of Acrs.
BLASTp searches using aca or acr genes were conducted to screen acr gene candidates using the ‘guilt-by-association’ method, which were subject to further validation through biochemical analysis. A gene was considered as the Acr candidate by the following criteria: (i) small protein size (<300 amino acids); (ii) direct upstream or downstream of Acr or Aca proteins in the same orientation; and (iii) fusion feature of Acr with Aca as a more confident marker.
For phylogenetic analysis of Acr proteins, homologous protein sequences of Acrs were obtained by BLASTp program using the nonredundant protein database. Sequences with high homology (E-value <0.001, query coverage >70%) were determined to generate distance trees based on BLAST using the fast minimum-evolution tree method, 0.85 maximum sequence difference and the Grishin (protein) distance model (24). Further labels were edited using the MEGA X (35) and Illustrator (Adobe).
Plasmid interference assays in E. coli
Plasmids (pB001–pB008 and pB017–pB050) used in the interference assays were designed based on our previous report (36) and are listed in Supplementary Table S5. DNA sequences encoding Acr proteins were synthesized by Biomed or GenScript and ligated into the pBAD24 vector. The spacer sequences of Cas9 orthologs for targeting pT are shown in Supplementary Figure S2A and Supplementary Table S5. Plasmids were transformed into E. coli using the CaCl2 heat-shock procedure as described previously with slight modifications (37). Briefly, E. coli TOP10 or Mach1-T1 strains carrying Acr plasmids were cultured overnight in LB medium with 0.2% arabinose and then used as competent cells for subsequent transformation with 25 ng of pT and 25 ng of Cas9 (with matching spacer or mismatching spacer) plasmids. After recovery for 2 h in LB medium with 0.2% arabinose, cells were plated on LB agar with antibiotics (50 μg/ml ampicillin, 50 μg/ml kanamycin and 25 μg/ml chloramphenicol) and inducers (1 mM IPTG and 0.2% arabinose) and incubated at 37°C for 24–32 h. Clones were photographed using a gel scanner (Tanon 3500) and counted via ImageJ software. Inhibitory activity of each Acr was shown in percentage by calculating the ratio of cfu (colony forming units) between E. coli transformed with Cas9 plasmid with matching spacer and that of the mismatching spacer, which was the average of at least three biological replicates.
Phage plaque assays
Plasmids (pB009–pB016 and pB018–pB032) used in phage plaque assays were designed based on our plasmid interference assays in E. coli and are listed in Supplementary Table S5. Phage plaque assays were performed according to previous reports with some modifications (38,39). Briefly, E. coli (T7 Express, Biomed) cells were co-transformed with a plasmid expressing Cas9–sgRNA combinations targeting phage T4 and a compatible plasmid encoding Acr proteins. Both Cas9 plasmid for nontargeting phage T4 and an empty pBAD24 plasmid (no Acr) served as controls. The E. coli containing both Acr and Cas9 plasmids were cultured in LB medium supplemented with antibiotics (50 μg/ml ampicillin and 25 μg/ml chloramphenicol) and grown overnight at 37°C. The next morning, overnight cultures were inoculated in fresh LB medium with antibiotics and grown at 37°C for 2 h. Subsequently, 1 mM IPTG was added to induce the expression of Cas9 proteins. Two hours later, 0.2% arabinose was added to induce the expression of Acr proteins. After another 2 h, 200 μl culture was mixed with 4 ml molten top LB agar (0.7%) supplemented with 10 mM MgSO4 and poured over the prewarmed bottom LB agar (1.5%) plates containing 10 mM MgSO4, 0.2% arabinose, 1 mM IPTG and both antibiotics. Next, 10-fold serial dilutions of phage T4 lysate were spotted on the lawn surface. The plates were incubated overnight at 37°C and photographed using a gel scanner (Tanon 3500).
Protein expression and purification
DNA sequences encoding St1Cas9, St3Cas9 or Acr proteins were incorporated into the pET28a vector for protein expression in E. coli (T7 Express, Biomed) (pC003–pC017, Supplementary Table S5). Escherichia coli cells were routinely induced for protein expression in LB medium with 1 mM IPTG for 16 h at 18°C supplemented with 50 μg/ml kanamycin. Cells were harvested and resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 10 mM imidazole, 0.5 mM TCEP–NaOH and 500 mM NaCl) supplemented with 1 mM PMSF and lysozyme. After sonication and centrifugation, the supernatant of cells was bound to Ni-NTA agarose (QIAGEN), and bound protein was eluted with 500 mM imidazole. Amicon Ultra centrifugal filter (Millipore) was used to concentrate proteins and exchange buffer into storage buffer 20 mM HEPES–NaOH, pH 7.5, 5% (v/v) glycerol, 300 mM NaCl and 1 mM DTT. For Acr proteins, the second round of Ni-NTA purification was conducted to isolate untagged Acr proteins after incubation with tobacco etch virus protease (Sangon Biotech) overnight at 4°C. To minimize protein degradation, we prepared aliquots of all purified proteins stored at −80°C, and avoided repeated freezing and thawing.
In vitro DNA cleavage assays
All sgRNAs in the assays were prepared using in vitro T7 transcription kit (Invitrogen) according to the manufacturer’s manual, and the transcription templates were generated with linearized sgRNA plasmids (pC018–pC023, Supplementary Table S5). SpyCas9 protein was purchased from Invitrogen.
For Figure Figure3,3, and Supplementary Figures S4 and S5, the plasmid pC002 was constructed and further linearized through NotI restriction endonuclease (NEB) (Supplementary Table S5). For Figure Figure33 and Supplementary Figure S4, cleavage reactions were conducted in a total volume of 10 μl with NEBuffer 3.1, along with Cas9 proteins (500 nM), sgRNA (500 nM), target DNA substrate (30 ng/μl) and excess Acr proteins (10 μM). In addition, we conducted assays with SpyCas9 ribonucleoprotein (RNP) (256 nM) and Acr titrations (0, 128, 256, 512, 1024, 2048, 4096 and 8192 nM) in Supplementary Figure S5. For Figure Figure33 and Supplementary Figure S5, Cas9 protein was complexed with sgRNA for 10 min at 37°C. The Acr proteins were then added and incubated at room temperature for another 20 min. For Supplementary Figure S4, Cas9 protein was incubated with Acr protein at room temperature for 20 min. The sgRNA was then added and incubated for 10 min at 37°C. Subsequently, for all assays, target DNA was added and incubated for 10 min at 37°C. Reactions were stopped after the addition of 1 μl of Proteinase K. Products were analyzed on 1% agarose/1× TAE (Tris–acetate–EDTA) gels, which were visualized by the gel scanner (Tanon 3500).
AcrIIA24–32 inhibit the Streptococcus type II-A Cas9 orthologs in vitro. (A) Schematic overview of DNA cleavage assays, in which Cas9 RNP was used to target a DNA substrate in the presence of Acr proteins. DNA cleavage assays using SpyCas9 (B), St3Cas9 (C) and St1Cas9 (D) RNPs to target a linearized plasmid DNA in the presence or absence of Acr proteins. Acr subtypes and numbers are indicated. A, AcrIIA; C, AcrIIC. Hollow arrowheads indicate the uncleaved linearized plasmid DNA. Solid arrowheads indicate cleaved products. The gel images shown for DNA cleavage assays are representative of three independent replicates.
For Figure Figure6H6H and I, and Supplementary Figure S8E, fluorescently labeled substrate DNA was used for cleavage assays, which was prepared by annealing the synthetic oligos of target and nontarget strands labeled with Cy5 or Cy3 (Supplementary Table S3). The process of cleavage assays is shown in Figure Figure6G.6G. Briefly, Cas9 proteins (500 nM) and sgRNA (500 nM) were mixed to form Cas9 RNP complex for 10 min at 37°C in 1× binding buffer 20 mM Tris–HCl, pH 7.6, 150 mM KCl, 5 mM EDTA, 5 mM MgCl2, 1 mM DTT, 5% (v/v) glycerol, 50 μg/ml heparin, 0.01% Tween 20 and 100 μg/ml BSA to abrogate the cleavage activity of Cas9. Then, Acr proteins (10 μM) and substrate DNA (50 nM) were added in a different order, and incubated for 20 min at room temperature, respectively. Subsequently, MgCl2 (10 mM) was added to restore the DNA cleavage activity of Cas9, and then incubated for another 20 min at room temperature. The reactions were stopped through the addition of Gel Loading Buffer II (Invitrogen) and incubated at 85°C for 6 min. Products were analyzed on 12% denaturing PAGE gel and visualized by Typhoon 7000 (GE).
Acrs exhibit diverse mechanisms to inhibit type II-A Cas9 orthologs in vitro. EMSAs were conducted to analyze the effect of different Acr proteins on DNA binding of Cas9 RNP, when Acrs were added prior to or after the addition of target DNA, including AcrIIA25.1 (A), AcrIIA26 (B), AcrIIA27 (C), AcrIIA32.1 (D), AcrIIA24 (E), AcrIIA30 and AcrIIA31 (F). Assays were conducted with Cas9 RNP (256 nM) and Acr titrations (0.125, 0.25, 0.5, 0.1, 0.2, 0.4, 0.8 and 1.6 μM). The assays were analyzed on the nondenaturing gel with target DNA labeled by Cy5. The gels are representative of three independent replicates. (G) DNA cleavage assays to recover DNA cleavage activity of Cas9 through adding extra Mg2+ in EMSAs. The assays were used to examine whether Acrs affect the DNA cleavage activity of Cas9, when added prior to or after the addition of target DNA (see the ‘Materials and Methods’ section for details). RT, room temperature. (H, I) DNA cleavage assays were conducted to analyze the effect of Acrs on DNA cleavage activity of Cas9 under different conditions shown in panel (G). Acr subtypes and numbers are indicated. A, AcrIIA; C, AcrIIC. Assays were conducted using SpyCas9 RNP (500 nM), St1Cas9 RNP (500 nM), Acrs (10 μM) and substrate DNA (50 nM) with the nontarget strand labeled by Cy3. Experiments were repeated three times and the representative gel figures were shown. (J) Summary of different inhibitory mechanisms of anti-CRISPR proteins identified in this study. AcrIIA26, AcrIIA27, AcrIIA30 and AcrIIA31 block Cas9 binding to DNA, while AcrIIA24 abrogates the DNA cleavage activity of Cas9. Remarkably, AcrIIA25.1 and AcrIIA32.1 can inhibit both DNA binding and DNA cleavage of Cas9.
Construct of intein–Acr plasmids
DNA sequences encoding Acr proteins (AcrIIA4, AcrIIA5, AcrIIA25.1 and AcrIIA32.1) were cloned into the pCDNA3.1 vector for expression in human cells. Intein 37R3-2 sequence (40) was synthesized and inserted into the described positions of Acr proteins for construction of intein–Acr plasmids (pM043–pM052, Supplementary Table S5).
T7 endonuclease 1 assay
Plasmids (pM001–pM024, pM038–pM042, pM049 and pM051) expressing Cas9 orthologs, Acr or iAcr proteins, and sgRNAs used in T7 endonuclease 1 (T7E1) assays are listed in Supplementary Table S5. Target sequences in AAVS1, EMX1 and DYRK1A loci and primer sequences for PCR amplification are provided in Supplementary Table S2. For Figure Figure4,4, HEK293T cells cultured in the 24-well plate were transfected with 1 μg of Cas9 plasmid, 0.5 μg of sgRNA plasmid and 0.5 μg of Acr plasmid per well, using the Lipofectamine LTX reagent (Invitrogen) according to the manufacturer’s protocol. For Figure Figure7D7D–F, HEK293T cells were transfected with 1 μg of Cas9 plasmid, 0.5 μg of sgRNA plasmid, and 0.5 or 0.25 μg of Acr or iAcr plasmid per well in the 24-well plate, with or without 4-hydroxytamoxifen (4-HT, 1 μM, Selleck S7827). At 72 h post-transfection, genomic DNA from cells was extracted with the DNeasy Blood and Tissue Kit (QIAGEN) and amplified for PCR with Q5 High-Fidelity Polymerase (NEB). PCR products mixed with NEBuffer 2 were denatured and annealed before T7E1 (NEB) was added and incubated at 37°C. Samples were fractionated in a 3% agarose/1× TAE gel. T7 bands were quantified using the ImageJ software. The efficiency of genome editing in mammalian cells was calculated with the following formula: indel (%) = 100 .
AcrIIA24–32 inhibit different Cas9-mediated gene editing in human cells. (A) Schematic view of T7E1 assay to examine Acr inhibition against Cas9 orthologs in HEK293T cells. Cells were co-transfected with plasmids encoding Cas9, sgRNA and Acr and subsequently analyzed through T7E1 assay. Representative gel images of T7E1 assay to manifest the inhibitory activities of Acrs against SpyCas9 (B), St3Cas9 (D) and St1Cas9 (F). The target sites of human AAVS1 (targeted by SpyCas9) and DYRK1A (targeted by St1Cas9 and St3Cas9) are shown at the top of each gel and PAMs are highlighted in purple. Acr subtypes and numbers are indicated. A, AcrIIA; C, AcrIIC. Hollow arrowheads indicate the T7E1-undigested bands (unedited). Solid arrowheads indicate T7E1-digested bands (edited). The editing efficiencies indel (%) are labeled at the bottom of each lane. Quantification of gene editing efficiencies of SpyCas9 (C), St3Cas9 (E) and St1Cas9 (G) is shown in the presence of different Acrs. Error bars represent the mean ± SEM with three biological replicates.
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.
Fluorescence imaging for telomeric foci
Plasmids (pM009–pM012, pM025–pM027, pM032 and pM033) encoding Nme_dCas9-(sfGFP)3, Spy_dCas9-(mCherry)3, Spy_Cas9-(mCherry)3, their respective sgRNAs targeting telomeres and Acrs (AcrIIC1, AcrIIA4 and AcrIIA5) were used from our previous study (36). Vectors (pM013–pM024, pM028–pM031, pM034 and pM035) expressing St1_dCas9-(mCherry)3, St1_Cas9-(mCherry)3, St3_dCas9-(mCherry)3, St3_Cas9-(mCherry)3, their respective sgRNAs targeting telomeres and other Acrs are listed in Supplementary Table S5. For imaging, U2OS cells were cultured on 15-mm glass-bottom dishes (Electron Microscopy Sciences) in 24-well plates and co-transfected with 60 ng of each (d)Cas9 plasmid, 300 ng of each sgRNA-telomere plasmid and 300 ng of individual Acr plasmid using Lipofectamine LTX reagent (Invitrogen) according to the manufacturer’s manual. At 24 h post-transfection, cells were fixed with 4% paraformaldehyde (Beyotime) and imaged using a Nikon A1R+ confocal microscope with a 60× oil objective lens.
The ‘blind’ experiments were performed according to the previous studies (41). One experimenter coded the cells from each condition by labeling numbers. Another experimenter observed and scored the cells under the microscope, who did not know these conditions. For quantifications, only the cells expressing TagBFP and mCherry fluorescence as well as NmedCas9-(sfGFP)3 telomeric foci were assessed in the presence or absence of co-localizing S**(d)Cas9-(mCherry)3 telomeric foci.
Electrophoretic mobility shift assays
RNA electrophoretic mobility shift assays (EMSAs) were performed by incubating Cas9 protein (256 nM) and sgRNA (256 nM) in the presence or absence of Acrs (5 μM) with the indicated order in figure legends. Reactions were incubated in 1× binding buffer 20 mM Tris–HCl, pH 7.6, 150 mM KCl, 5 mM EDTA, 5 mM MgCl2, 1 mM DTT, 5% (v/v) glycerol, 50 μg/ml heparin, 0.01% Tween 20 and 100 μg/ml BSA. The sgRNAs and Acr proteins were added in different order and incubated for 10 min at 37°C, respectively. Samples were analyzed on 6% Tris–borate–EDTA (TBE) polyacrylamide gels and visualized using SYBR Gold (Invitrogen) stain by Typhoon 7000 (GE). We performed DNA EMSAs as previously described (42). Briefly, Cas9–sgRNA complexes were incubated in 1× binding buffer for 10 min at 37°C with the indicated concentration in figure legends. Subsequently, Acr proteins with various concentrations were added and incubated for 20 min at room temperature, and then 20 nM fluorescently labeled substrate DNA (Cy5-labeled target strand) was added to the mix followed by incubation for 10 min at 37°C. In a parallel experiment, fluorescently labeled substrate DNA was added and incubated for 10 min at 37°C before Acr proteins with various concentrations were added and incubated for 20 min at room temperature. Samples were analyzed by biphasic polyacrylamide (the upper half of the gel is 6% and the lower half of the gel is 12%)/0.5× TBE gel electrophoresis. Gels were visualized by Typhoon 7000 (GE). All assays were conducted in triplicates.
Gene editing efficiencies were assessed by next-generation sequencing (NGS) using a two-step PCR-based method. Briefly, genomic DNA from cells was extracted with the DNeasy Blood and Tissue Kit (QIAGEN) and amplified for first-step PCR with Q5 High-Fidelity Polymerase (NEB). Target sequences in EMX1 and DYRK1A loci and primer sequences carrying 5′ Illumina sequencing adaptors for PCR amplification are provided in Supplementary Table S2. PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and serve as template for second-step PCR with primer sequences carrying Illumina barcodes by Q5 High-Fidelity Polymerase (NEB). The PCR products were sequenced on an Illumina MiSeq machine by PE150 via commercial sequencing service (Tsingke Biotechnology) and the efficiency of genome editing was determined from the sequencing data using CRISPResso2 (43).
PE-mediated BFP-to-GFP gene editing in HEK293T-BFP cells
First, we established HEK293T-BFP cells with a chromosomally integrated BFP at AAVS1 locus (Supplementary Figure S9A) in HEK293T cells based on a previous report (44). Briefly, HEK293T cells were transfected with Cas9, AAVS1-targeting sgRNA plasmids and a donor vector containing homologous sequences and cassette sequences with BFP and puromycin resistant genes. HEK293T-BFP cells were selected by puromycin (2 μg/ml) treatment and flow cytometry (FACSAriaIII, BD).
To examine the activity of intein–Acr hybrids against Cas9 in human cells, we performed prime editing (PE)-mediated BFP-to-GFP gene editing in HEK293T-BFP cells in the presence or absence of intein–Acr variants under the condition of 4-HT treatment or not. The plasmid encoding prime editor PE2 was purchased from Addgene (#132775). The BFP-targeting pegRNA plasmid was constructed by synthesizing DNA sequences containing the target, sgRNA scaffold, PBS and RT template incorporated into the U6-sgRNA vector (Supplementary Figure S9C and Supplementary Table S5). HEK293T-BFP cells were cultured in 24-well plates prior to transfection with plasmids encoding prime editor PE2 (1 μg), BFP-targeting pegRNA (0.5 μg) and Acr (0.25 μg) per well using Lipofectamine LTX reagent (Invitrogen), with or without 4-HT (1 μM, Selleck S7827). At 72 h post-transfection, cells were collected and the percentage of GFP-positive cells by flow cytometry (FACSAriaIII, BD) was calculated.