In vitro Enzymology of Cas9

Transform chemically competent Rosetta 2 DE3 cells (Novagen, Merck Millipore) according to the protocol supplied with the cells. Briefly, add ~200 ng of plasmid DNA (pMJ806) to 50 μl of freshly thawed competent cells and incubate on ice for 15 min. Heat-shock cells by incubation at 42 °C for 45 s, then place cells on ice for further 3 min. Add 500 μl of LB (Luria Broth) medium to the cells and incubate the culture at 37 °C for 1 h in a shaking incubator. Plate 100 μl of culture out on LB agar containing 50 μg ml^-1 kanamycin and 33 μg ml^-1 chloramphenicol. Incubate plates overnight at 37 °C.

Pick one colony from the agar plate to inoculate 50 ml LB medium containing 50 μg ml-1 kanamycin and 33 μg ml^-1 chloramphenicol. Incubate the preculture at 37 °C in a shaking incubator (250 rpm) for a minimum of 4–5 h or overnight.

Use 7.5 ml of the preculture to inoculate 750 ml prewarmed LB medium supplemented with 50 μg ml^-1 kanamycin and 33 μg ml^-1 chloramphenicol in a 2 l baffled flask. We typically express 6 x 750 ml total culture volume at a time. Incubate the cultures at 37 °C in a shaking incubator (90 rpm) while monitoring the cell growth by measuring optical density at 600 nm (OD600). Reduce the temperature to 18 °C at an OD of ~ 0.8 and continue shaking for additional 30 min. Induce protein expression by the addition of 150 μl 1 M isopropyl-β-D-1-thiogalactopyranoside to each flask (200 μM final concentration). Continue shaking at 18 °C overnight for another 12-16 h.

Harvest cells by centrifugation for 15 min at 3500 rpm (~2700 x g) in a swing-bucket rotor in 1 l bottles. Decant the supernatant and resuspend the cell pellets using ~15 ml ice-chilled lysis buffer (20 mM Tris-Cl, pH 8.0, 250 mM NaCl, 5 mM imidazole, pH 8.0, 1 mM phenylmethylsulfonyl fluoride) per cell pellet from 1 l culture. The resuspended cell pellets can either be used directly for further purification or flash frozen in liquid nitrogen and stored at -80 °C for several months without loss of Cas9 enzymatic activity.

Lyse the resuspended cell pellets using a cell homogenizer (Avestin Emulsiflex). Pass the cell suspension through the homogenizer three times at ~1000 bar to ensure complete lysis. The lysate should be cooled on ice between passes.

Clarify the lysate by centrifugation in 50 ml Nalgene Oak Ridge tubes at 18,000 rpm (~30,000 x g) in an SS-34 rotor (or equivalent) for 30 min at 4 °C. Collect the supernatant.

All chromatographic steps should be performed at 4 °C. Equilibrate 10 ml of His-Select Ni resin (Sigma-Aldrich) packed in a XK 16/20 column housing (GE Healthcare) with 20 ml lysis buffer. Load the cleared lysate on the column using a peristaltic pump at ~1.5 ml min^-1. Attach the column with bound protein to an FPLC system equilibrated in wash buffer (20 mM Tris-Cl, pH 8.0, 250 mM NaCl, 10 mM imidazole, pH 8.0).

Wash with ~50 ml wash buffer at 1.5 ml min^-1 until the absorbance nearly reaches the baseline again. Elute with ~50 ml elution buffer (20 mM Tris-Cl, pH 8.0, 250 mM NaCl, 250 mM imidazole, pH 8.0) and collect in 2 ml fractions. Analyze the peak fractions using SDS-PAGE and pool those containing Cas9 protein.

Estimate protein concentration by measuring the absorbance at 280 nm (use elution buffer as blank). Add 0.5 mg TEV protease per 50 mg of protein. Dilute the Cas9 sample to ~1 mg ml^-1 with dialysis buffer (20 mM HEPES-KOH, pH 7.5, 150 mM KCl, 10 % (v/v) glycerol, 1 mM dithiothreitol (DTT), 1 mM EDTA) and dialyze the sample in dialysis tubing with a molecular weight cut off (MWCO) of 12-14 kDa against 2 l dialysis buffer at 4 °C overnight. Dialysis buffer (without DTT and glycerol) can be prepared as a 10 x stock, but DTT should be added immediately prior to use.

Recover the dialyzed sample from the dialysis tubing. Typically, slight precipitation occurs after successful TEV protease cleavage. Centrifuge the sample at 3900 rpm (~3200 x g) for 5 min at 4 °C to remove the precipitate. Check the extent of TEV protease cleavage using SDS-PAGE.

Equilibrate a 5-ml HiTrap SP FF column (GE Healthcare) with IEX buffer A (20 mM HEPES-KOH, pH 7.5, 100 mM KCl) and load the cleaved protein onto the column using a peristaltic pump or a sample loading superloop at a flow rate of ~2 ml min^-1. Attach the column to the FPLC system. Set the flow rate to 2 ml min^-1 and the pressure limit to 0.3 MPa for further steps using the HiTrap column. Collect 2 ml fractions throughout. Wash the column with 10 ml IEX buffer A and elute bound protein by applying a gradient from 0% to 50% IEX buffer B (20 mM HEPES-KOH, pH 7.5, 1 M KCl) over 60 ml. Cas9 typically elutes in two peaks with different ratios of the absorbances at 260 and 280 nm (A₂₆₀/A₂₈₀). The first peak starts eluting at ~15% IEX buffer B with its maximum at ~20%, the second peak elutes at ~25-40%, with a maximum at ~30%. Analyze all peak fractions for the presence of Cas9 using SDS-PAGE. Pool Cas9-containing fractions that have an A₂₆₀/A₂₈₀ of less than ~0.6. The pooled sample can be stored at 4 °C overnight or can be flash frozen in liquid nitrogen and stored at -80 °C.

Exchange the buffer to SEC buffer (20 mM HEPES-KOH, pH 7.5, 500 mM KCl, 1 mM DTT) while concentrating the protein to <1.5 ml volume using a 30,000 MWCO centrifugal concentrator (Amicon) at 3900 rpm. The buffer exchange helps circumvent precipitation in the centrifugal concentrator. Recover concentrate in a 1.5 ml Eppendorf tube and centrifuge for 10 min at 14,000 rpm (16,900 x g) at 4 °C to remove any precipitated material.

In the meantime, equilibrate a HiLoad 16/600 Superdex 200 PG gel filtration column (GE Healthcare) with the SEC buffer. Inject concentrated Cas9 onto column using a 2 ml sample loop. Elute with 120 ml SEC buffer at a flow rate of 1 ml min^-1, collecting 2 ml fractions. Cas9 typically elutes at a volume of ~66 ml. Analyze the peak fractions using SDS-PAGE and pool those containing Cas9 protein.

Concentrate eluted Cas9 using a 30,000 MWCO centrifugal concentrator to a concentration required for further experiments. In the described SEC buffer, Cas9 can be concentrated up to ~30 mg ml^-1 (189.3 μM) without precipitation. The concentration is determined based on the assumption that 1 mg ml^-1 has an absorbance at 280 nm of 0.76 (based on a calculated extinction coefficient of 120,450 M^-1 cm^-1).

Divide concentrated protein sample into 50 μl aliquots and flash freeze in liquid nitrogen. Frozen Cas9 can be stored at -80 °C for several months without loss of activity. Prior to use, thaw an aliquot and dilute to the required concentration with SEC buffer. We typically dilute Cas9 to 15 μM for endonuclease activity assays. To avoid unnecessary freeze-thaw cycles that can result in loss of enzymatic activity, Cas9 can be stored on ice or at 4 °C for at least 2 days without loss of activity if used for several experiments. However, it is recommended to check the stored sample periodically for the absence of any precipitated material and to monitor protein integrity by SDS-PAGE.

A double-stranded transcription template is prepared by amplifying a single-stranded oligonucleotide by PCR amplification of the antisense template oligonucleotide with the T7 promoter sequence as forward primer and the 3’ end of the antisense template as reverse primer (Fig. 1.1C). Mix reagents for PCR according to Table 1.1 and split into 50 μl aliquots into a 96-well plate suitable for PCR. Perform PCR cycling according to Table 1.2. After PCR, combine all reactions into 500 μl fractions in 2 ml tubes. Precipitate the PCR product by adding 50 μl 3 M sodium acetate, pH 5.2 and 1.45 ml ice-chilled 100 % ethanol to each 500 μl fraction and incubate for 1 h at -20 °C. Centrifuge the precipitated PCR product at 14,000 rpm (16,900 x g) for 40 min at 4 °C. Remove supernatant and wash the DNA pellet with 200 μl ice-chilled 70% ethanol. Centrifuge for 5 min, remove supernatant and air-dry the DNA pellet for 30 min. Dissolve the DNA pellet in 100 μl water. Measure the absorbance at 260 nm and calculate the concentration of the double-stranded DNA template. Extinction coefficients can be calculated using the OligoCalc server (Kibbe, 2007). Dilute the double-stranded template with 5 x transcription buffer (150 mM Tris-Cl, pH 8.1, 125 mM MgCl₂, 0.05% Triton X-100, 10 mM spermidine) and water to a final concentration of 10 μM in a total volume of 1 ml. Incubate mixture at 75 °C for 5 min and allow it to cool down slowly to room temperature. Proceed with the transcription reaction described in step 3.

To prepare a partially single-stranded DNA template, mix 100 μl of 100 μM template oligonucleotide stock with 150 μl 100 μM (1.5-fold molar excess) complementary T7 promoter oligonucleotide (5’-TAATACGACTCACTATAGG-3’) and 200 μl 5 x transcription buffer. Add water to bring the total volume to 1 ml. Anneal the oligonucleotides at 75 °C for 5 min and let the mixture slowly cool down to room temperature.

Transcription reaction: Set up a transcription reaction by mixing the reagents according to Table 1.3. A 5 ml reaction is usually sufficient for ~0.5-1 mg of pure sgRNA. The reaction can be scaled down as needed.

Incubate the reaction at 37 °C for 1.5 h. During transcription, magnesium pyrophosphate precipitates from the reaction due to production of inorganic pyrophosphate upon NTP incorporation into nascent transcripts. To restore magnesium ion levels, add 25 μl 1 M MgCl₂ after 1.5 h and continue with incubation at 37 °C for another 1.5 h. The optimal reaction times may vary as a function of length and RNA sequence of the transcribed RNA and should be determined empirically.

Add 25 μl (25 U) of RNase-free RQ1 DNase (Promega) to the transcription reaction and incubate 15 min at 37 °C in order to remove the template DNA. Centrifuge the mixture for 5 min at 3900 rpm (~3200 x g) and 4 °C to pellet magnesium pyrophpsphate and remove the supernatant containing transcribed RNA. The supernatant can be stored at -20 °C at this point.

Add 5 ml 2 x RNA loading dye (5% glycerol, 2.5 mM EDTA, pH 8.0, 90% formamide, trace bromophenol blue) to the sample. Separate the RNA by electrophoresis on a prewarmed 8% polyacrylamide, 7 M urea denaturing gel in 0.5 x TBE (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH 8.0) until the bromophenol blue dye reaches the lower quarter of the gel. We typically use gels with the dimensions of 400 mm x 360 mm x 4 mm (h x w x d) and run them at 50 W for ~4-6 h. Visualize RNA by UV-shadowing over a silica glass TLC plate and excise the band corresponding to the correct RNA product using a disposable scalpel. Crush the gel slices in a 50-ml tube using a clean spatula or a plastic serological pipette and add water to 50 ml total volume. Incubate overnight at 4 °C while rocking.

Centrifuge the crushed gel suspension for 5 min at 3900 rpm (~3200 x g) and 4 °C and collect the supernatant. Filter supernatant through a 0.22-μm disposable filter (Steriflip, Millipore). Concentrate the eluted RNA in 3000 Da MWCO centrifugal concentrator to a final volume of ~2 ml. Aliquot the concentrate into 500 μl fractions in 2-ml tubes. Add 50 μl 3 M sodium acetate, pH 5.2 and 1.5 ml ice-chilled 100 % ethanol to each 500 μl fraction and incubate for 1 h at -20 °C. Centrifuge the precipitated guide RNA at 14,000 rpm (16,900 x g) for 40 min at 4 °C. Remove supernatant and wash the RNA pellet with 200 μl ice-chilled 70% ethanol. Air-dry the pellet and dissolve in 100 μl water. Determine RNA concentration by measuring absorbance at 260 nm.

For plasmid substrates, linearize the circular DNA by a restriction digest. Choose a restriction enzyme that cuts at a unique site away from the Cas9 target site so that Cas9-mediate cleavage of the linear DNA produces two well-separated fragments. For pUC19-based plasmids, use 50 units of SspI-HF (New England Biolabs) and 5 μg plasmid in 1 x CutSmart™ buffer in a total volume of 50 μl. Incubate the reaction for 1 h at 37 °C. Heat-inactivate SspI-HF by incubating for 20 min at 65 °C. Analyze plasmid cleavage by electrophoresis on a 1 % agarose gel in 1 x TAE buffer (40 mM Tris, pH 8.0, 20 mM glacial acetic acid, 1 mM EDTA, pH 8.0) stained with GelRed (Biotium) or similar nucleic acid stain. Complete digestion of pUC19-derived vectors yields a single band of ~2700 bp.

For oligonucleotide duplex substrate, the 5’-ATTO532-labeled target strand oligonucleotide should be PAGE-purified. To generate the oligonucleotide duplex, anneal target and nontarget strand by mixing the oligonucleotides in a molar ratio of 1:1.5. Prepare 100 μM stock solutions of the target and nontarget strand oligonucleotides. Mix 1.0 μl of the target strand with 1.5 μl of the nontarget strand and add water to a total volume of 25 μl. Heat the mixture to 75 °C for 5 min and cool slowly to room temperature (Table 1.5). Dilute the mixture with 225 μl DEPC-H₂O to obtain 400 nM stock solution of the oligonucleotide duplex substrate.

Prepare 5 x cleavage buffer (100 mM HEPES, pH 7.5, 500 mM KCl, 25% glycerol, 5 mM DTT, 2.5 mM EDTA, pH 8.0, 10 mM MgCl₂).

Dilute Cas9 to 15 μM with SEC buffer (see Seption 2). Dilute in vitro transcribed sgRNA to 15 μM with water.

Anneal the guide RNA by heating to 90 °C for 5 min and slowly cooling to room temperature.

Set up reaction mixes according to Table 1.4 (plasmid DNA substrates) or Table 1.5 (oligonucleotide DNA substrates). Add equimolar amounts of Cas9 protein and guide RNA in the reaction mix without DNA. Incubate for 10-15 min at room temperature.

Start the cleavage reaction by adding the DNA target to the reaction mix and incubate at 37 °C immediately.

Remove 10 μl aliquots at different time points and quench by adding to 1.0 μl of 500 mM EDTA, pH 8.0 (final concentration 50 nM) in a separate 1.5 ml tube. Mix by pipetting up and down and store at -80 °C until all time points are collected.

Thaw the samples and add 1.0 μl Proteinase K (20 mg.ml^-1) to digest DNA-bound Cas9. Incubate for 20 min at room temperature. Add 6 x plasmid DNA-loading buffer (10 mM Tris, pH 7.6, 60 mM EDTA, 60% glycerol, 0.03% bromophenol blue, 0.03% xylene cyanol FF) or 2 x oligonucleotide-loading buffer (90% formamide, 10% glycerol) to each sample.

Plasmid cleavage assay: Analyze each plasmid sample on a 1% agarose gel in 1 x TAE stained with GelRed (Biotium) or compatible nucleic acid stains. For visualization with a Typhoon FLA 9500 scanner, loading 2 μl of sample was sufficient.

Oligonucleotide cleavage assay: Resolve oligonucleotide duplex samples on a 16% polyacrylamide, 7 M urea denaturing gel (300 mm x 180 mm; 1 mm thick) in 0.5 x TBE buffer. Load 12.5 μl of each sample and run at 25 W for ~2 h until the bromophenol blue dye front has reached the bottom half of the gel. Scan the gel using a laser gel scanner (e.g., Typhoon FLA9500, GE Healthcare) with the appropriate excitation and emission wavelength settings (532 and 553 nm for ATTO532, respectively).

Plasmid-based assays: In the absence of Cas9-mediated cleavage, linearized plasmid yields a single band at 2702 bp. Cleavage by Cas9 leads to gradual emergence of two cleavage products at 2104 and 598 bp.

Oligonucleotide-based assays: Cleavage of the oligonucleotide substrate by Cas9 leads to a mobility shift of the fluorescent signal. The 5’-labeled target strand of the substrate has a length of 31 nt, while the cleaved product runs as a 14-nt band.