TITLE: CRISPR/Cas9 generation of knock-out iPSCs
AUTHORS: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur

[DESCRIPTION]
This protocol describes RNP-based CRISPR/Cas9 gene-editing to generate knock-out iPSCs. iPSCs are transfected with sgRNA, recombinant Cas9, and GFP using Lipofectamine Stem. After FACS sorting to enrich for successfully transfected cells, individual clones are picked and expanded for further analysis.

[STEPS]
SECTION: Transfection with sgRNA and recombinant Cas9
1. Accutase-dissociate iPSCs on the morning of transfection. 
SECTION: Transfection with sgRNA and recombinant Cas9
2. 1 - 2 hours prior to transfection, change media to fresh, pre-warmed mTeSR + RI (2 mL/well). 
 


SECTION: Transfection with sgRNA and recombinant Cas9
3. Prepare sgRNA: 

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

SECTION: Transfection with sgRNA and recombinant Cas9
4. Flick tube of HiFi Cas9 enzyme (IDT) to mix and briefly spin.
SECTION: Transfection with sgRNA and recombinant Cas9
5. For transfecting a single well of a 6-well plate, set up Tube 1 with

 

SECTION: Transfection with sgRNA and recombinant Cas9
6. Add 0.4 µg GFP plasmid to RNP complex in Tube 1 and mix by pipetting.

SECTION: Transfection with sgRNA and recombinant Cas9
7. Set up Lipofectamine Stem mix in Tube 2 by combining 100 µL OptiMEM and 4 µL Lipofectamine Stem. 

SECTION: Transfection with sgRNA and recombinant Cas9
8. Add Tube 2 solution to Tube 1 and flick tube to mix. Incubate mixture for 10 min at Room temperature, then add mixture dropwise to iPSCs.

SECTION: Transfection with sgRNA and recombinant Cas9
9. On the following day, individualize iPSCs with Accutase and plate onto 6 Matrigelcoated wells of a 6-well plate in mTeSR + RI.
SECTION: FACS-sorting
10. Coat 1-2x 10 cm dishes per condition with Matrigel and add 10 mL mTeSR (+RI +Penicillin/Streptomycin) on the day of FACS sorting. Keep 10 cm dishes in cell culture incubator at 37 °C.
 
 



SECTION: FACS-sorting
11. Collect transfected iPSCs with an Accutase split and resuspend into mTeSR (+RI +P/S). 
 


SECTION: FACS-sorting
12. Filter iPSCs through a   cell strainer into a 50 mL conical tube to individualize cells and avoid clumping. 

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

SECTION: FACS-sorting
14. Exchange media on the next day to mTeSR + P/S, but without RI. Keep feeding cells daily with mTeSR + P/S. 
SECTION: Picking Colonies
15. Pick clones when individual colonies reach a size of ~ 1 mm. 
 

SECTION: Picking Colonies
16. For the first attempt of CRISPR/Cas9 editing, we recommend picking 24 colonies per condition. 
 

SECTION: Picking Colonies
17. To pick individual clones, visualize colonies under a tissue culture microscope.
 

SECTION: Picking Colonies
18. Use a P200 pipette to scratch each colony from the dish and aspirate the colony in 100 µL media. 

SECTION: Picking Colonies
19. 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.


 

 

SECTION: Expanding individual clones
20. 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.
 

SECTION: Expanding individual clones
21. Splitting cells from 96-well plates to 24-well plates using Accutase: 

SECTION: Expanding individual clones
22. Splitting cells from 24-well plates to 6-well plates using Accutase:


SECTION: Expanding individual clones
23. Feed cells daily with mTeSR. Cells should be ready to be split and cryopreserved within 2-3 days.

SECTION: Expanding individual clones
24. Individual clones can then be thawed and expanded to confirm knock-out via Western Blot.
 

SECTION: Transfection with sgRNA and recombinant Cas9
1.1. 
Plate 800,000 iPSCs onto one well of a Matrigel-coated 6-well plate in mTeSR medium supplemented with 10 micromolar (µM)  ROCK inhibitor (RI).

SECTION: Transfection with sgRNA and recombinant Cas9
3.1. 
Dilute required amount of sgRNA to 1 micromolar (µM) final concentration in TE buffer.

SECTION: Transfection with sgRNA and recombinant Cas9
4.1. Dilute required amount of HiFi Cas9 (62 micromolar (µM) stock solution) to 1 micromolar (µM) in Room temperature OptiMEM.
 

SECTION: Transfection with sgRNA and recombinant Cas9
5.1. Mix by pipetting up and down gently, then incubate for 5 min at Room temperature to allow RNP complexes to form.
SECTION: Transfection with sgRNA and recombinant Cas9
9.1.  
Feed cells daily with mTeSR without RI until cells are about 70% confluent (after ~4320 min).

SECTION: FACS-sorting
12.1.  Move single-cell suspension to a 15 mL tube.
SECTION: FACS-sorting
12.2.  Prepare an additional 15 mL conical tube with 1.5 mL mTeSR (+P/S, +RI) for collecting cells after FACS sorting
.
SECTION: FACS-sorting
12.3. Keep both tubes on ice until FACS sorting.

SECTION: FACS-sorting
13.1. Plate ~10,000 GFP-expressing cells onto one 10 cm dish.
SECTION: FACS-sorting
13.2.  Move 10 cm dish back-and-forth and left to-right to distribute cells evenly. 
SECTION: Picking Colonies
16.1. In two 96-well plates, coat 12 wells for each condition with Matrigel; then add 200 µL  mTeSR per well (+ P/S, + RI). 

SECTION: Picking Colonies
16.2. Put 96-well plates in cell culture incubator and allow some time to equilibrate. 
SECTION: Picking Colonies
16.3.  Aspirate old mTeSR media off 10 cm dishes and replace with 15 mL fresh media per dish.
 

 


SECTION: Picking Colonies
18.1. 
Pipette up- and down four times in one well of an additional empty (third) 96-well plate to break up cell clusters.
SECTION: Picking Colonies
18.2. 
Then add all 100 µL to one well of one of the pre-equilibrated 96-well plates. 

SECTION: Expanding individual clones
21.1. 
Prepare 24-well plate(s) by coating with Matrigel GFR, then adding 500 µL mTeSR + RI + P/S per well. Place 24-well plate(s) in cell culture incubator to pre-equilibrate. 

SECTION: Expanding individual clones
21.2. 
Aspirate mTeSR from all wells of the 96-well plate and wash with PBS.
Then add 25 µL Accutase per well. Return to incubator and let sit for 6 min- 8 min. 
Add 200 µL 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.
 

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

SECTION: Expanding individual clones
22.2. Aspirate mTeSR from wells in 24-well plate and wash with PBS.
Add 100 µL Accutase per well and incubate at 37 °C for 6 min- 8 min. 
While cells are at 37 °C, prepare one 1.5 mL Eppendorf tube with 200 µL  mTeSR + RI for each clone.





SECTION: Expanding individual clones
22.3. Aspirate cells in Accutase by pipetting up and down a few times in each well. 
Then add cells to prepared Eppendorf tube.
SECTION: Expanding individual clones
22.4. Spin cells down using a tabletop centrifuge (3 min at 200 rcf and Room temperature. 
Resuspend each pellet in 1 mL mTeSR + RI (taken from the prepared 6-well plate) and plate individual clones onto separate wells of 6-well plates.



SECTION: Expanding individual clones
23.1. 
For freezing down a higher number of clones, cells can again be Accutase-split into Eppendorf tubes, then resuspended in cryopreservation media: