TITLE: Cas9 RNP nucleofection (CD34+ HSPCs)
AUTHORS: gregoire.cullot, Mandy Boontanrart, Mark Dewitt

[DESCRIPTION]
Protocol for nucleofection of human HSPCs with Cas9 RNP. 

[STEPS]
SECTION: HSPCs thawing
4. Prepare a 37 °C water bath (in a beaker) in the bead bath. 

SECTION: Preparation of RNP mix
14. For one reaction (200,000 HSPCs in small cuvettes) mix following components:

** first, dilute sgRNA in Cas9 Buffer 3X and then, slowly add Cas9 protein while mixing **

 


 

SECTION: Prepare Cells
18. For one reaction, transfer 200,000 cells into a 15 mL Falcon tube. 


 

SECTION: Prepare Cells
20. While the cells are spinning, prepare plate and cuvette. Place a 24-well plate with 1 mL per well of supplemented SFEMII in the incubator.
SECTION: Nucleofection
23. After centrifugation, discard supernatant completely and resuspend cells in the nucleofection solution

** The pellet is very soft so be careful **
SECTION: Nucleofection
24. Resuspend cells in the nucleofection solution. 
SECTION: Nucleofection
25. Mix 20 uL of cells with the RNP mix
SECTION: Nucleofection
26. Add 24.5 µL of cells/RNP electroporation mix to the multiwell cuvette, and cap. 

** Pay attention to the orientation of the cap and cuvette in the nucleofector, which is noted in the manufacturer’s instructions ** 
SECTION: Nucleofection
27. Choose P3 cell solution and ER-100 code. 
SECTION: Nucleofection
28. Insert cuvette into nucleofector and zap.
SECTION: Nucleofection
31. Culture cells for several days in SFEM II/PenStrep/CC110 before genotying by desired method. Consider including un-zapped controls to test viability.
SECTION: HSPCs thawing
6. After 2 min at 37°C, remove the vial from the water bath every 15-20 seconds and gently invert 3-4 times to check the level of thawing.
SECTION: HSPCs thawing
5. Check that the vial is tightly capped and place in a 37 °C water bath.

SECTION: HSPCs thawing
8. Gently invert 5 times to mix the cells and transfer the vial content to a 15mL tube at Room temperature.

SECTION: HSPCs thawing
7. When the vial contents are 50-75% liquid, remove from the water bath and continue to gently invert the vial until the entire contents are liquid.
SECTION: HSPCs thawing
9. Add 1 mL of media to rinse the vial and transfer any remaining cells to the 15mL tube. Also rinse the cap.

SECTION: HSPCs thawing
10. Slowly add 7 mL media (unsupplemented SFEMII) to the cells. Securely cap the tube and gently invert 3-5 times after adding all the media.

SECTION: HSPCs thawing
11. Centrifuge the tube at 200g x 8 minutes at Room temperature.

SECTION: HSPCs thawing
12. Resuspend the cells (pellet) in 2.5 mL of supplemented media. 

** Resuspend in 1ml first, transfer to the plate, wash the tube further with 1ml and then 500 ul to make sure you are transferring as many cells as possible **
SECTION: HSPCs thawing
13. Count the cells using hemacytometer, adjust volume to plate 250,000 CD34+ per well of 6-well plate.

** Generally, the count will decline slightly from day 0 to day 1, and recover from day 1 to day 2. The total number of cells on day 2 will be slightly higher than on day 0 **
SECTION: Media preparation
1. Ideally,  medium at  4 °C  . Mix thoroughly before use.

** Do not refreeze. Store at 2 - 8°C for up to 1 month **
SECTION: Media preparation
2. Thaw  at Room temperature. Mix thoroughly before use.

SECTION: Media preparation
3. Prepare the appropriate amount of supplemented media by adding CC110 (100X ; 10 uL for 1 mL) and P/S  (100X ; 10 uL for 1 mL) to SFEM II media.
SECTION: Preparation of RNP mix
15. Incubate 15 min at RT
SECTION: Preparation of nucleofection solution
17. For one reaction (200,000 HSPCs in small cuvettes) mix following components:


 

SECTION: Preparation of RNP mix
16. (Optionnal) Slowly add HDR donor (ssODN) protein while mixing.

** 1 uL ssODN at 100 uM  = Final quantity: 100 pmol ssODN **
SECTION: Prepare Cells
19. Spin 300 x g for 6 minutes to pellet cells softly. 

SECTION: Prepare Cells
21. After centrifugation, discard supernatant and wash with PBS

** Cells can be transfered into a 1.5 mL Eppendorf tube to remove more supernatant in the next steps **
SECTION: Prepare Cells
22. Spin 300 x g for 6 minutes to pellet cells softly. 
SECTION: Nucleofection
29. Wait 5 minutes at room temperature and then add 100 uL of supplemented SFEMII media to each cuvette.
SECTION: Nucleofection
30. Transfer the nucleofected cells into the pre-warmed 24-well plate. Wash each cuvette with an additional 100 µL of medium, and add that as well.