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Finkelstein Lab Wiki

KD's Cas12a (Cpf1) Purification

Used in our papers:123456

Contents

Section titled “Contents”
  1. Buffers
  2. Generating Pellets
    1. Day 0
    2. Day 1
    3. Day 2
    4. Day 3
    5. Day 4
  3. Purification
    1. Preparation
    2. Day 1
    3. Day 2
  4. Useful Numbers
  5. References

1. Buffers

Section titled “1. Buffers”
  • Make sure to degas all buffers by leaving them under vacuum after filtering, until the bubbles stop forming. Make sure to make the day before and keep at 4C.

Lysis Buffer (1L, pH 8.0)

  • 1 M NaCl
  • 20 mM HEPES
  • 1 mM EDTA
  • 5% glycerol
  • 0.1% Tween-20

Wash Buffer (1L, pH 8.0)

  • 1 M NaCl
  • 20 mM HEPES
  • 5% glycerol
  • 5 mM MgCl2
  • 1.0 mM PMSF (add just before use)

Elution Buffer (30 mL, pH 8.0)

  • Make fresh each time!
  • 1 M NaCl
  • 20 mM HEPES
  • 10 mM desthiobiotin
  • 5 mM MgCl2

S200 Buffer (1L, pH 8.0)

  • 500 mM NaCl
  • 20 mM HEPES
  • 5 mM MgCl2
  • 2 mM DTT - Add on day of use

2. Generating Pellets

Section titled “2. Generating Pellets”

2.1 Day 0

Section titled “2.1 Day 0”
  1. Make LB-Carb plates.
  2. Go through the protocol and make sure you have all the supplies.

2.2 Day 1

Section titled “2.2 Day 1”
  1. Transform the Cas12a plasmid (pIF254 is nuclease active, pIF255 has the nuclease-inactivating D908A mutation) into BL21 (DE3) cells:
    1. Thaw the cells on ice.
    2. Add 1-50 ng plasmid and flick 4-5 times to mix. The cells are quite sick and can be easily killed by vigorous shaking/centrifugation.
    3. Incubate for 30 minutes on ice. Turn the shaker-incubator on and set it to 37°C.
    4. Heat shock cells for 25-30 seconds at 42°C.
    5. Incubate the tubes on ice for 5 minutes.
    6. Add 950 µL room temperature SOC to each tube and shake/incubate for 1 hour at 37°C. Warm selection plates in 37°C incubator.
    7. Transfer 100 µL to an LB-Carb agar plate.
    8. Incubate overnight at 37°C.

2.3 Day 2

Section titled “2.3 Day 2”
  1. Once you’re sure the transformation worked, reserve an incubator for tomorrow and the next day.
  2. Make 2 L of LB.
  3. At the end of the day, start a 100 mL overnight culture at 37°C (using a single colony from the plate) into LB supplemented with carb.
  4. If an incubator is available, you can warm them without shaking overnight at 37°C.

2.4 Day 3

Section titled “2.4 Day 3”
  1. Add carbenicillin to the 4L flasks and mix.
  2. Take a 5mL sample of LB from one of the 4L flasks for blanking the Nanodrop.
  3. Inoculate each flask with 10 mL overnight culture.
  4. Grow at 37°C with shaking until the OD600 is around 0.6. Once you get that measurement, take a sample of culture and spin down the cells, and dilute it with some TE.
  5. Reduce the temperature of the incubator to 18°C. Cool the flasks in ice water before moving to next step.
  6. Add a final concentration of 1.0 mM IPTG to each flask to induce Cas12a production.
  7. Shake for 24 hours at 18°C.
  8. Make sure there is sufficient liquid nitrogen. If not, go buy some.

2.5 Day 4

Section titled “2.5 Day 4”
  1. Take a sample of cell culture and freeze.
  2. Spin down the cells in 1L centrifuge tubes and adapters at 8000 g for 15 minutes at 4°C.
  3. Take the centrifuge tubes to the cold room. Pour out most of the supernatant but keep ~40 mL.
  4. Resuspend pellets with ~10 mL supernatant and transfer to cold 50 mL conical tubes. Use cold serological pipettes.
  5. Spin down the pellets at 6800 g for 10 minutes at 4°C in the swinging bucket rotor.
  6. Return to the cold room and discard the supernatant.
  7. Immediately flash freeze the pellets in liquid nitrogen.
  8. Store at -80°C until needed.

3. Purification

Section titled “3. Purification”

3.1 Preparation (typically done same day that pellet is made)

Section titled “3.1 Preparation (typically done same day that pellet is made)”
  1. Make buffers.
  2. Make eight 8% SDS-PAGE gels.
  3. Put the following in the cold room:
    • 50 mL serological pipettes (there’s usually a bag of them in there already)
  4. Clean a yellow gravity column and mount on the column holder in the cold room.
  5. Add Strep Tactin resin up to a 5 mL bed volume and equilibrate with 50 mL cold wash buffer.
  6. Decide when you’ll do the purification and reserve the FPLC.

3.2 Day 1

Section titled “3.2 Day 1”

Preparation of lysate

  1. Thaw and resuspend the cells in:
    • lysis buffer, to 45mL (do not exceed 45 mL, or else lysate will leak out during centrifugation)
    • 1.0 mM PMSF (caution: PMSF is super toxic)
    • 450 µL 100X Bimake Protease Inhibitor Cocktail or HALT protease inhibitor (1.0X final concentration)
    • 2,000 units of DNAse I
  2. Take a 100 µL sample from the resuspended cells and freeze.
  3. Pre-chill the Ti45 rotor tubes for the ultracentrifuge.
  4. Lyse the cell pellets with sonication.
    1. Remove the microtip if it is on the sonicator.
    2. Wash sonicator tip with EtOH then water before use.
    3. Select program and use the following settings:
      • Amplitude 30
      • Process time 1 minute and 30 seconds
      • Pulse-ON time 30 seconds
      • Pulse-OFF time 1 minute and 30 seconds
    4. Wash sonicator tip with EtOH then water.
  5. Take a 100 µL sample from the lysate and freeze.
  6. Transfer lysate to the pre-chilled Ti45 rotor tubes and balance carefully. I wipe off any water on the tubes and make sure they are less than 0.0 g apart.
  7. Centrifuge the lysate at 35,000xg for 35 minutes at 4C.
  8. In the cold room, use a pipettor to transfer the lysate into a cold 50 mL tube and keep it on ice.
    • The purpose here is to get the lysate away from the pellets but keep it cold while you wait to put it on the column.
  9. Take a 100 µL sample of the clarified lysate and freeze it.

Purification on Strep Tactin Column

  1. Apply supernatant onto the pre-equilibrated Strep-Tactin Superflow resin and collect flow through.
    • If this looks like it’s going to take hours, you can cap the column and apply light pressure with a 10 mL syringe and plastic tubing.
  2. While you wait, get a 50 mL conical tube, label it as eluate and store on ice.
  3. Wash with 100 mL wash buffer. If time permits, use up to 200 mL. Collect the wash.
  4. Slowly add 20 mL elution buffer, being careful not to disturb the pellet bed.
  5. Wait five minutes.
  6. Elute the protein into a cold 50 mL tube.
  7. Take a 100 µL sample of the eluate and freeze it. This will be used to show if SUMO proteolysis worked.
  8. Spin concentrate the sample to 700 to 800 µL. Place sample into a chilled 1.7 mL tube.
  9. Add SUMO protease to the eluate. A final concentration of 2.4 µM SUMO protease led to 100% cleavage previously.
  10. Rotate the sample overnight at 4C in the cold room.

Preparation for tomorrow

  1. End the current run on the FPLC.
  2. Clean the injection loop with H2O, 3M NaCl, 0.1 M HCl, and 0.1M NaOH, then wash with H2O once more.
  3. Add the loop to the FPLC.
  4. Do a pump wash basic to get pumps into water and S200 buffer, respectively.
  5. Run the FPLC in inject mode with the column set to bypass for 5 mL using water, to clean all the tubes/pumps between the injection loop at the column. Then repeat with 5 mL of S200 buffer.
  6. Press End to ensure no program is running.
  7. Equilibrate S200 column overnight into S200 buffer using the program. Make sure the program starts from the beginning (the volume ticks on the X-axis should start at 0 mL).

3.3 Day 2

Section titled “3.3 Day 2”

S200 Purification

  1. Put a box of 0.5 mL snap tubes in the cold room.
  2. Put a 50 mL conical tube on ice in the cold room. When it’s cold enough, transfer the contents of the dialysis tubing to the 50 mL tube.
  3. Run a gel of pre- and post-SUMO proteolysis fractions to confirm that SUMO was cleaved.
  4. Purify the sample on the S200.
  5. There should be a large peak, potentially with a small shoulder, at around 62-63 mL. This is Cpf1. There should also be another peak in the ~84 mL range, which is His6-TwinStrep-SUMO.
  6. Run a gel of the peak fractions with BSA standards. While that’s running, you can aliquot the fractions.

Storage

  1. Do all of the following in the cold room!
  2. Pool the good fractions and invert gently to mix.
  3. Make 10µL aliquots of the protein with a repeat pipettor.
  4. Keep one aliquot for a gel.
  5. Flash freeze the protein in liquid nitrogen and store at -80°C until ready for use.
  6. Celebrate with high fives if successful.

4. Useful Numbers

Section titled “4. Useful Numbers”
  • MW:
    • His6-TS-SUMO-AsCpf1-3xFLAG: 171 kDa
    • AsCpf1-3xFLAG: 154 kDa
    • His6-TS-SUMO-LbCpf1-3xFLAG: 163 kDa
    • LbCpf1-3xFLAG: 146 kDa
    • His6-TS-SUMO: 16.5 kDa
    • SUMO Protease: 25 kDa (according to Kaylee and Jeff)
  • pI (calculated with ExPASy)
    • His6-TS-SUMO-AsCpf1-3xFLAG: 6.68
    • AsCpf1-3xFLAG: 6.92
    • His6-TS-SUMO-LbCpf1-3xFLAG: 6.76
    • LbCpf1-3xFLAG: 7.46
    • His6-TS-SUMO: 6.10

5. References

Section titled “5. References”

Footnotes

Section titled “Footnotes”

  1. Strohkendl et al. (2018). Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a. Mol. Cell. ↩

  2. Jones et al. (2021). Massively parallel kinetic profiling of natural and engineered CRISPR nucleases. Nature Biotechnology. ↩

  3. Strohkendl et al. (2021). Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin. Science Advances. ↩

  4. Sinan et al. (2024). Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a. RNA. ↩

  5. Nalefski et al. (2025). Unleashing high trans-substrate cleavage kinetics of Cas12a for nucleic acid diagnostics. Nucleic Acids Research. ↩

  6. Sinan et al. (2025). One-Pot Isothermal Linear Amplification and Cas12a-Based Nucleic Acid Detection. ACS Synthetic Biology. ↩