CRISPR/Cas9 generation of knock-out iPSCs

Accutase-dissociate iPSCs on the morning of transfection.

Plate 800,000 iPSCs onto one well of a Matrigel-coated 6-well plate in mTeSR medium supplemented with 10micromolar (µM) ROCK inhibitor (RI).

1 - 2 hours prior to transfection, change media to fresh, pre-warmed mTeSR + RI (2mL/well).

Prepare sgRNA:

Resuspend 1.5nanomolar (nM) sgRNA in 15µL of RNAse-free TE buffer to make a 100micromolar (µM) stock solution.

Dilute required amount of sgRNA to 1micromolar (µM) final concentration in TE buffer.

Flick tube of HiFi Cas9 enzyme (IDT) to mix and briefly spin.

Dilute required amount of HiFi Cas9 (62micromolar (µM) stock solution) to 1micromolar (µM) in Room temperature OptiMEM.

For transfecting a single well of a 6-well plate, set up Tube 1 with

A	B
OptiMEM	76 µL
1 µM HiFi Cas9	12 µL
1 µM sgRNA	12 µL

Mix by pipetting up and down gently, then incubate for 0h 5m 0s at Room temperature to allow RNP complexes to form.

Add 0.4µg GFP plasmid to RNP complex in Tube 1 and mix by pipetting.

Set up Lipofectamine Stem mix in Tube 2 by combining 100µL OptiMEM and 4µL Lipofectamine Stem.

Add Tube 2 solution to Tube 1 and flick tube to mix. Incubate mixture for 0h 10m 0s at Room temperature, then add mixture dropwise to iPSCs.

On the following day, individualize iPSCs with Accutase and plate onto 6 Matrigelcoated wells of a 6-well plate in mTeSR + RI.

Feed cells daily with mTeSR without RI until cells are about 70% confluent (after ~72h 0m 0s).

Coat 1-2x 10 cm dishes per condition with Matrigel and add 10mL mTeSR (+RI + Penicillin/Streptomycin ) on the day of FACS sorting. Keep 10 cm dishes in cell culture incubator at 37°C.

Collect transfected iPSCs with an Accutase split and resuspend into mTeSR (+RI +P/S).

Filter iPSCs through a 40 µm cell strainer into a 50 mL conical tube to individualize cells and avoid clumping.

Move single-cell suspension to a 15 mL tube.

Prepare an additional 15 mL conical tube with 1.5mL mTeSR (+P/S, +RI) for collecting cells after FACS sorting

.

Keep both tubes On ice until FACS sorting.

Perform FACS-sorting for GFP-expressing iPSCs using standard FACS parameters.

Plate ~10,000 GFP-expressing cells onto one 10 cm dish.

Move 10 cm dish back-and-forth and left to-right to distribute cells evenly.

Exchange media on the next day to mTeSR + P/S, but without RI. Keep feeding cells daily with mTeSR + P/S.

Pick clones when individual colonies reach a size of ~ 1 mm.

For the first attempt of CRISPR/Cas9 editing, we recommend picking 24 colonies per condition.

In two 96-well plates, coat 12 wells for each condition with Matrigel; then add 200µL mTeSR per well (+ P/S, + RI).

Put 96-well plates in cell culture incubator and allow some time to equilibrate.

Aspirate old mTeSR media off 10 cm dishes and replace with 15mL fresh media per dish.

To pick individual clones, visualize colonies under a tissue culture microscope.

Use a P200 pipette to scratch each colony from the dish and aspirate the colony in 100µL media.

Pipette up- and down four times in one well of an additional empty (third) 96-well plate to break up cell clusters.

Then add all 100µL to one well of one of the pre-equilibrated 96-well plates.

Pick 12 colonies into the first 96-well plate. Then return the first 96-well plate to the cell culture incubator and use the second 96-well plate for picking clones 13-24.

Feed iPSCs in 96-well plates daily with mTeSR + P/S. The cells are ready to be split when iPSCs in a well are almost confluent and/or individual colonies grow to a size >half of the well.

Splitting cells from 96-well plates to 24-well plates using Accutase:

Prepare 24-well plate(s) by coating with Matrigel GFR, then adding 500µL mTeSR + RI + P/S per well. Place 24-well plate(s) in cell culture incubator to pre-equilibrate.

• Aspirate mTeSR from all wells of the 96-well plate and wash with PBS.
• Then add 25µL Accutase per well. Return to incubator and let sit for 0h 6m 0s- 0h 8m 0s.
• Add 200µL mTeSR (+RI + P/S) to each well and break up bigger cell clusters by pipetting up and down 3-4 times in each well.
• Transfer cells from one well of the 96-well plate to one well of a 24-well plate.
  Note

  Optional: To obtain cellular DNA for screening successful CRISPR edits, pipette ~ ¼ of the volume into an Eppendorf tube.

Splitting cells from 24-well plates to 6-well plates using Accutase:

iPSCs should be ready to split within 2-4 days. Prepare 6-well plates by coating with Matrigel GFR, then adding 2mL mTeSR + RI per well.

Aspirate mTeSR from wells in 24-well plate and wash with PBS.* Add 100µL Accutase per well and incubate at 37°C for 0h 6m 0s- 0h 8m 0s.

• While cells are at 37°C, prepare one 1.5 mL Eppendorf tube with 200µL mTeSR + RI for each clone.

Aspirate cells in Accutase by pipetting up and down a few times in each well. * Then add cells to prepared Eppendorf tube.

Spin cells down using a tabletop centrifuge (0h 3m 0s at 200rcf,0h 0m 0s and Room temperature. * Resuspend each pellet in 1mL mTeSR + RI (taken from the prepared 6-well plate) and plate individual clones onto separate wells of 6-well plates.

Feed cells daily with mTeSR. Cells should be ready to be split and cryopreserved within 2-3 days.

For freezing down a higher number of clones, cells can again be Accutase-split into Eppendorf tubes, then resuspended in cryopreservation media:

A	B
DMSO	10%
Knockout Serum Replacement	20%
mTeSR	70%

Individual clones can then be thawed and expanded to confirm knock-out via Western Blot.