Phage targeting assays
E. coli BB101 cells were transformed with a plasmid expressing the relevant Cas9 protein with an sgRNA containing a spacer that targets phage Mu, and a second plasmid expressing anti-CRISPR. The cells were grown in lysogeny broth (LB) supplemented with 25 μg ml−1 chloramphenicol and 50 μg ml−1 streptomycin for 2 h at 37 °C. A final concentration of 0.01 mM isopropyl-1-thio-Beta-D-galactopyranoside (IPTG) was added to induce anti-CRISPR expression, and the cells were incubated for 3 h. 200 μL of cells were mixed into 0.7 % LB top agar and poured onto plates of LB supplemented with chloramphenicol, streptomycin, 0.2 % arabinose, and 10 mM MgSO4. Serial dilutions of phage Mu were applied to the plates, and the plates were incubated at 37 °C overnight. Assays were performed at n ≥ 3, with representative replicates shown in the figures.
Nickel affinity co-purification assays
Co-purification experiments with 6His-tagged HpaCas9 constructs and untagged AcrIIC4 were performed as previously described.13 For the REC2 co-purification experiments with AcrIIC3 and AcrIIC4, a plasmid expressing HpaCas9 REC2 with an N-terminal 6His-tag (pMCSG7) was transformed into E. coli BL21 (DE3) cells. An overnight culture of these cells grown at 37 °C was subcultured in fresh LB media supplemented with 100 μg ml−1 ampicillin. The culture was grown at 37°C until the OD600 reached 0.8-1.0. Then a final concentration of 1 mM IPTG was added to the culture to induce REC2 expression. After overnight incubation at 16°C, cells were harvested and resuspended in the cold binding buffer containing 50 mM HEPES pH 7.0, 300 mM NaCl, 5 % glycerol and 5 mM imidazole. The cell resuspension was lysed by sonication and centrifuged at 17000 rpm for 30 min to remove the cell debris. The supernatant was mixed with Ni-NTA agarose beads (Qiagen) and incubated at 4°C for 30 min. After several washes with the purification buffer containing 50 mM HEPES pH 7.0, 300 mM NaCl and 30 mM imidazole, REC2 was eluted from the Ni-NTA using the elution buffer with 50 mM HEPES pH 7.0, 300 mM NaCl, 5 % glycerol, and 300 mM imidazole. The protein was dialyzed into 50 mM HEPES pH 7.0, 300 mM NaCl, and 5% glycerol. Plasmids (pCDF1-b) expressing untagged AcrIIC3 or AcrIIC4 were individually transformed into E. coli BL21 (DE3) cells. The cells were grown as described above in LB media supplemented with 50 μg ml−1 streptomycin. After adding 1 mM IPTG and overnight induction at 16 °C, the cells were harvested and resuspended in cold binding buffer. The cells were lysed by sonication and centrifuged at 17000 rpm for 30 min to remove the cell debris. Purified 6His-tagged REC2 was applied to a Ni-NTA column and the anti-CRISPR supernatants were added and the samples were incubated at 4°C for 30 min. Unbound protein was removed through the application of purification buffer to the column and 6His-tagged REC2 and the bound anti-CRISPRs were eluted from the column using elution buffer. The co-eluting proteins were analyzed using SDS-PAGE on a 15 % Tris-Tricine gel visualized by Coomassie staining.
Purification of AcrIIC4
A plasmid (pHAT4) expressing AcrIIC4 with an N-terminal cleavable 6His-tag was transformed into E. coli BL21 (DE3) cells. The cells were grown and AcrIIC4 was purified as described above using buffers with 50 mM Tris pH 7.5, 500 mM NaCl, and 5, 30, or 300 mM imidazole. AcrIIC4 was dialyzed with 6His-tagged TEV protease in 1:100 (w/w) ratio into 20 mM Tris pH 7.5, 250 mM NaCl and 1 mM TCEP followed by another incubation with Ni-NTA at 4°C to remove His-tagged TEV protease. Finally, the protein was injected onto Superdex 200 Increase 10/300 GL size exclusion column (GE Healthcare, USA) equilibrated in Assay buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 5 mM EDTA, 1 mM TCEP, 5 % glycerol) and the purified fractions were collected.
Purification of Nme1Cas9
A plasmid (pMCSG7) expressing Nme1Cas9 with an N-terminal 6His-tag34 was transformed into E. coli BL21 (DE3) cells. The cells were grown in Terrific broth (TB) and Nme1Cas9 was purified as described above using buffers with 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, and 5, 30, or 300 mM imidazole. The protein was injected onto Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare, USA) equilibrated in Assay Buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 5 mM EDTA, 1 mM TCEP, 5 % glycerol).
HpaCas9 REC and AcrIIC4 cross-linking and mass spectrometry
HpaCas9 REC was purified as described above (Purification of Nme1Cas9). The cross-linking experiment followed the protocol described previously with modifications XLMS. A protein concentration of 1 mg mL−1 was used and a final concentration of 375 μM of disuccinimidyl suberate (DSS) was added for cross-linking. The cross-linked products were visualized by SDS-PAGE on an 8% polyacrylamide gel and Coomassie staining. The band of interest was excised from the gel and sent for mass spectrometry analysis at the Southern Alberta Mass Spectrometry Facility.
Luminescence transcriptional repressor assays
A plasmid encoding the J23119 artificial promoter, which drives constitutive expression of the luxCDABEoperon from Photorhabdus luminescens,35 was transformed into E. coli BL21 cells together with the pNme1Cas9sgRNA plasmid containing an spacer targeting the constitutive promoter region of J2311913 and pCDF-1b plasmid expressing the anti-CRISPR proteins. Kanamycin, chloramphenicol and streptomycin resistant transformants were grown in liquid LB medium at 37°C until they reached an optical density at 600 nm (OD600) of 0.6. The cells were then further diluted to an optical density of 0.1 in LB, and 100 μl was dispensed into the wells of a 96-well plate. Both luminescence and the OD600 were monitored for 24 h using a Synergy H1 reader controlled by Gen5 2.09 software (BioTek Instruments Inc.). The assay was performed at n ≥ 3, with representative replicates shown in the figures.
Purification of Nme1Cas9-sgRNA RNP
A plasmid expressing Nme1Cas9 with an N-terminal 6His-tag and sgRNA (pMCSG7) was transformed into E. coli BL21 (DE3) cells.11 Nme1Cas9-sgRNA ribonucleoprotein complex was purified as described above (Purification of Nme1Cas9) with the exception that the cells were incubated at 37°C for 3 h after the addition of IPTG. The protein was loaded onto a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare, USA) and purified using Assay Buffer or Assay Buffer without EDTA (for use in DNA cleavage assays). The presence of bound sgRNA was assessed by running the protein complex on a 12.5 % polyacrylamide/8 M urea gel and visualizing the nucleic acid using SYBR Gold staining.
Electrophoretic mobility shift assays
1 μM of Nme1Cas9-sgRNA ribonucleoprotein complex was incubated for 30 min at room temperature with AcrIIC4 or AcrIIC5N10 at concentrations ranging from 0 to 50 μM in Assay Buffer supplemented with 0.01 % Tween-20. 100 nM of fluorescein-labelled target DNA was added and incubated at 37°C for 1 h. The resulting complexes were analysed using a 6% native polyacrylamide gel, and fluorescein-labelled DNA bound to the complex was visualized using a ChemiDoc imager (BioRad).
DNA cleavage assays
150 nM of Nme1Cas9-sgRNA ribonucleoprotein complex was incubated with 0.1, 1, or 5 μM wild type or mutant AcrIIC4 in cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol) at room temperature for 30 min. 300 ng of linearized plasmid DNA containing the target protospacer sequence was added. After a 1 h incubation at 37 °C, the reactions were quenched by adding EDTA and incubating with 1 U proteinase K at 50°C for 10 min. The reaction products were visualized on a 1% agarose gel using SYBR Safe stain (ThermoFisher Scientific).
Nme1Cas9-sgRNA-DNA-AcrIIC4 size-exclusion chromatography
50 μM of purified Nme1Cas9-sgRNA RNP was incubated with 150 μM of unlabelled target DNA for 30 min at room temperature. 150 μM of AcrIIC4 was added and the mixture was incubated for an additional 30 min. The sample was then injected onto Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare, USA) equilibrated in Assay buffer. Samples of fractions with peaks at A280 were analyzed for protein components using SDS-PAGE (15% Tris-Tricine gel) and were visualized by Coomassie staining, and for nucleic acids using a 12.5 % polyacrylamide/8 M urea gel visualized by SYBR Gold staining (ThermoFisher Scientific).
Fluorescence polarization assays
Site-directed mutagenesis was used to engineer a single cysteine residue at the C-terminus of AcrIIC4. This AcrIIC4 variant was purified as described above. AcrIIC4 was then fluorescently labelled using fluorescein-5-maleimide (ThermoFisher Scientific). 4 nM of the labelled AcrIIC4 was incubated with Nme1Cas9 or Nme1Cas9-sgRNA RNP at concentrations ranging from 0 to 4 μM in Assay buffer supplemented with 0.01 % Tween-20 for 30 min at room temperature. Fluorescence polarization values were measured using a TECAN Spark reader.
For the DNA fluorescence polarization assay, 4 nM of fluorescein labelled target DNA was incubated with Nme1Cas9-sgRNA ribonucleoprotein complex at concentrations ranging from 0 to 4 μM in Assay buffer supplemented with 0.01 % Tween-20 for 30 min at room temperature. Fluorescence polarization values were measured using a TECAN Spark reader.
Crystallization and structure determination of AcrIIC4
Purified 6His-tagged AcrIIC4 was dialyzed overnight at 4°C in 10 mM Tris pH 7.5, 250 mM NaCl and 5 mM βME with 6His-tagged TEV protease in 1:100 (w/w) ratio. The cleaved 6His-tag and 6His-tagged TEV protease were removed by incubation with Ni-NTA beads. The protein was then further purified by size exclusion chromatography using a HiLoad Superdex 75 16/600 gel filtration column (GE Healthcare, USA) equilibrated in 10 mM Tris pH 7.5, 100 mM NaCl.
Crystallization trials of AcrIIC4 were established using sitting-drop vapour diffusion. Purified AcrIIC4 was concentrated to 16 mg/mL, and mixed with the precipitants from MCSG2 and 4, JCSG+, Rigaku Wizard Cryo and Index commercial screens (Hampton Research) in a 1:1 ratio by the Gryphon robot (Art Robbins Instrument). The first crystals were observed in 40% PEG 600, 0.1 M Na2HPO4/citric acid pH 4.2. The condition was further optimized by altering the polyethylene glycol (PEG) percentage and buffer pH. The crystals formed in 35% PEG 600, 0.1M Na2HPO4/citric acid pH 4.2 were cryo-protected in the mother liquor with 20% glycerol and flash-frozen in liquid nitrogen. Diffraction data for crystals was collected at the Structural Genomics Consortium (SGC) (Toronto, Canada) using a copper rotating anode X-ray source (lambda = 1.54 Å) and R-AXIS IV++ detector (Rigaku). The phase information of AcrIIC4 was solved by iodide—single-wavelength anomalous diffraction (I-SAD). The data was collected by soaking crystals in the mother liquor supplemented with 20% glycerol and 0.5 M NaI. Diffraction images were processed with mosflm.36 The model auto-building and automated refinement was performed using Phenix.37 Manual refinement was performed using Coot.38
Far-UV circular dichroism scans and temperature-induced unfolding experiments
Circular dichroism experiments were performed as described previously13 with modifications. For the temperature-induced unfolding experiments, the state of protein folding was measured at 207 nm. Assays were performed at n ≥ 3.
Nme1Cas9 mutants phage targeting assay
In PyMOL, the electrostatics surface map of the Nme1Cas9 structure in complex with sgRNA (PDB: 6JDQ)19 was generated using the Adaptive Poisson-Boltzmann Solver (APBS) plugin. Asp392, Glu393, Asp394, and Asp418 were identified to in the highly electronegative pocket of the REC2 domain. Point mutations at these positions were introduced in Nme1Cas9 by PCR using oligonucleotides that replace the original amino acid codon with the codon for either alanine or lysine. The introduction of D392A, E393K, D394K, and D418K mutations in Nme1Cas9 were verified by sequencing. The phage targeting assays were conducted as described above.
Modelling of AcrIIC4 onto Nme1Cas9
In the HADDOCK web server,28 the Nme1Cas9 structure in complex with sgRNA and target DNA in a seed pairing state (PDB: 6KC7)19 and the AcrIIC4 structure were used. Nme1Cas9 Asp394 and AcrIIC4 Arg46 were input as the active residues and default parameters were used. The resulting five modelled clusters were analyzed using PyMOL, and the steric clashes were visualized using the show_bumps script written by Thomas Holder.
Article TitleStructural and mechanistic insight into CRISPR-Cas9 inhibition by anti-CRISPR protein AcrIIC4
Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPRs have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal struture of AcrIIC4 and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4 likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.
Competing Interest Statement
The authors have declared no competing interest.