TITLE: Creating iPSC lines with Ribonucleoprotein (RNP): Nucleofection, Single-cell Sorting, Genotyping, and Line Maintenance Protocol
AUTHORS: Kamaljot Gill, Aradhana  Sachdev, Bruce Conklin, Claire D Clelland

[DESCRIPTION]
This protocol describes how to perform gene editing on human induced pluripotent stem cells (iPSCs) via ribonucleoprotein (RNP) and how to the isolate lines with the desired excision. It describes nucleofection, single cell sorting via FACS, genotyping, and the maintenance of the cell lines throughout the process. This protocol is optimized for spCas9.

[STEPS]
SECTION: Nucleofecting
1. Cell Culture


SECTION: Nucleofecting
1.1. Grow iPSCs in 1 well of a 6 well-plate until ~70-80% confluency.
 

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2. Coating plates
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2.1. Coat 1 x 6 well-plate with Matrigel.

Matrigel is diluted in KO DMEM to make a working concentration of 80 μl/ml (keep the Matrigel ice-cold) 
Matrigel coating volumes

    6 well plate: 1 mL
   12 well plate: 0.5 mL
   24 well plate: 0.25 mL
   48 well plate 0.125 mL
   96 well plate: 0.100 mL

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2.2. Incubate the plate at 37 °C for 30 min.

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3. Turn on the Lonza 4D-Nucleofector.
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4. Prepare Media
  
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4.1. 
For 20 mL:

 

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5. Prepare P3 Buffer.
 
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5.1. For 12 reactions ( 250 µL ) in a 16 well nucleofector strip.


 

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5.2. Place on ice
 


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6. Reconstitute 1.5 nanomolar (nM) sgRNA in a BSC.

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6.1. Add 15 µL TE buffer to make 100 micromolar (µM) sgRNA.


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7. Prepare RNP

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7.1. For each nucleofection, mix a ratio of 1:3 (spCas9:guide)

sgRNA  (100 micromolar (µM)) = 1.2 µL, spCas9  (40 micromolar (µM)) = 1 µL

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6.2. Store -20 °C for long term storage.

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7.2. Incubate RNPs at Room temperature for 10-15 mins.

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8. Prepare cells (below are the instructions for 1 well of a 6-well plate)
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8.1. Wash the well with PBS.
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8.2. Detach cells from plate with Accutase.

Incubate cells in 0.5 mL Accutase. Incubate the plate for 10-15 mins at 37 °C. Quench the Accutase with 2.5 mL of PBS.
Transfer media into a 15 mL conical tube.
Spin down the cells in a 15 mL conical for 3 min at 800 rpm.
Remove supernatant.
Resuspend the cells in 1-2 mL of warm media.
 






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8.3. Count cells.
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8.4. Transfer ~350k cells in an Eppendorf tube (per reaction).
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9. Prepare reaction.


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9.1. Centrifuge the cells at 800 rpm for 3 min. Aspirate supernatant.

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9.2. Resuspend pellet in 20 µL of P3 buffer.

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9.3. Add 2 µL  of RNP (Cas9+sgRNA) to the Eppendorf tube.

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9.4. If using two different RNPs, only add 1 µL of each because only half of each RNP is needed.
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9.5. Transfer each sample to one well of a 16 strip nucleofector cuvette/strip. Check lid to make sure it is in the correct orientation.
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9.6. Tap nucleofector strip on surface to distribute sample and pop any bubbles.
If needed, use a P20 pipette tip to gently pop any bubbles.
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10. Nucleofecting
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10.1. Set up the reaction on 4D-Nucleofector Core X - Unit

Select wells being used in your script
Select P3 buffer setting
Select 16-well strip 
Select Pulse Code = DS138
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10.2. Place the nucleofector strip in the nucleofector and hit start.
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10.3. Green plus sign should appear if reaction is successful.
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11. Place the nucleofector strip in the hood (do not spray it with EtOH) and incubate at Room temperature for 5-10 mins.

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12. Prepare plate 
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12.1. Obtain the 6 well-plate that was coated with Matrigel earlier.
 
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12.2. Aspirate Matrigel.
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12.3. Replace with 2 mL of media.

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13. Recover Cells
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13.1. Add 80 µL of media to each well of the nucleofector strip to recover the cells.

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13.2. Slowly pipette up and down to ensure cells are properly dispersed.
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14. Transfer cells to plate.
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14.1. For each nucleofection, transfer 50 µL/well of the cells to 2 wells of a 6 well-plate.

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14.2. Gently rock the plate and incubate 3 min at 37 °C.

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15. Maintain cells.
SECTION: Harvesting Cells for Freezing and Genotyping:
16. At 70-80% confluency, collect the pooled population of cells.

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15.1. Grow the cells until they are 70-80% confluency (approximately 3-4 days).
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15.2. When cells are at 70-80% confluency, you may 

Harvest one well for sorting.
Harvest one well for freezing and genotyping (lift the well and pellet into 2 separate conical tubes).

SECTION: Harvesting Cells for Freezing and Genotyping:
16.1. Incubate cells in 0.5 mL Accutase. Incubate the plate for ~5-10 mins at 37 °C. 

SECTION: Harvesting Cells for Freezing and Genotyping:
16.5. Aspirate the supernatant.
SECTION: Harvesting Cells for Freezing and Genotyping:
16.4. Spin down the cells at 800 rpm for 3 min.

SECTION: Harvesting Cells for Freezing and Genotyping:
16.3. Transport 1.5 mL/conical tube of the cells to 2 x 15 mL conical tube.

SECTION: Harvesting Cells for Freezing and Genotyping:
16.2. Quench the Accutase with 2.5 mL of PBS.

SECTION: Harvesting Cells for Freezing and Genotyping:
17. Freezing the pool.
 
SECTION: Harvesting Cells for Freezing and Genotyping:
17.1. Resuspend 1 of the pellets in 1 mL of ice-cold CryoStor.

SECTION: Harvesting Cells for Freezing and Genotyping:
17.4. Place Mr. Frosty at -80 °C to freeze for 1440 min.
After 24-48 hours, move the vial to liquid nitrogen.

SECTION: Harvesting Cells for Freezing and Genotyping:
17.3. Transport vial to a Mr. Frosty with 2-propanol.
SECTION: Harvesting Cells for Freezing and Genotyping:
17.2. Transfer cells into a cryovial.
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18. Genotyping pool (go to the Genotyping section for more information)
 
SECTION: Harvesting Cells for Freezing and Genotyping:
18.1. Store the remaining pellet in the conical tube at -20 °C until ready to extract DNA.

SECTION: Harvesting Cells for Freezing and Genotyping:
18.2. Perform an excision PCR with an unedited line as a negative control.
SECTION: Harvesting Cells for Freezing and Genotyping:
18.3. 
 

SECTION: Single Cell Sorting by FACS
19. Preparing a 96 well-plate
 
SECTION: Single Cell Sorting by FACS
19.1. Coat a 96 well-plate with 100 µL/well of Matrigel. 

SECTION: Single Cell Sorting by FACS
19.2. Allow the plate to incubate wi for at least 30 min at 37 °C .

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19.3. Prepare 20 mL of Media (mTeSR Plus + ROCK Inhibitor (1,000X) + Clone R (10X)+ Anti-Anti (100X)).
 

 

SECTION: Single Cell Sorting by FACS
19.4. Aspirate the Matrigel from the 96 well-plate and add 100 µL/well of media to each well.

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19.5. Set the plate back into the incubator.
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20. Preparing Cells

 
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20.1. Wash the well with PBS.
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20.2. Add 0.5 mL Accutase.

SECTION: Single Cell Sorting by FACS
20.3. Incubate the plate for ~10-15 mins at 37 °C. 

SECTION: Single Cell Sorting by FACS
20.4. Quench the Accutase with 2.5 mL of PBS.

SECTION: Single Cell Sorting by FACS
20.5. Transport cells into a 15 mL conical tube.
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20.6. Spin down the cells for 3 min at 800 rpm.

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21. Transport cells to FACS machine.



SECTION: Single Cell Sorting by FACS
20.9. Count cells.
SECTION: Single Cell Sorting by FACS
20.8. Resuspend the cells in1-2 mLof warm media.



SECTION: Single Cell Sorting by FACS
20.7. Remove supernatant.
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20.10. Dilute cells to a 1.5 million cells/500 µL.
 



SECTION: Single Cell Sorting by FACS
20.11. Pass cells through a filter mesh (strainer cap) using a P1000.

Place a filter mesh on top of a FACS collection tube.
Replace filter mesh with cap for collection tube.
 

SECTION: Single Cell Sorting by FACS
21.1. Seal the 96 well-plates with media with parafilm.
SECTION: Single Cell Sorting by FACS
21.2. Seal the collection tube with parafilm.
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21.3. Clean a large container with ethanol.
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21.4. Place the 96 well-plate and collection tube into the container.
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22. FAC Sorting
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22.1. Perform single cell sorting with aBD FACS Aria Fusion (Beckton Dickinson), equipped with 355, 405, 488, 561 and 640 nm lasers. 

 

SECTION: Single Cell Sorting by FACS
22.5. Use forward scatter area versus height and side scatter area versus height gates to make the single cell determination. The specifications of the sort layout include single cell precision, 96 well collection device and target event of 1.
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22.4. Drop delay determination and 96 well plate set-up setup using Accudrop beads (Becton Dickinson). 
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22.3. In certain experiments mCherry fluorescence (excitation 561 nm, emission 610 nm) can be used to define sorting parameters. 
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22.2. Set forward a scatter threshold of 15,000 to eliminate debris from list mode data, and fix the number of events to be collected.
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23. Quarantining Cells (Day 0)
SECTION: Single Cell Sorting by FACS
23.1. Move cells to the quarantine incubator (or a separate incubator from other cell lines) during the duration of 7-day Anti-Anti treatment and before confirming the cells are mycoplasma negative

Perform a mycoplasma test between Day 3 and Day 7.
SECTION: Single Cell Sorting by FACS
24. Days 1-3

 
SECTION: Single Cell Sorting by FACS
24.1. Do not change media.
SECTION: Single Cell Sorting by FACS
24.2. 
Ensure there is 1 cell/well and there is no contamination. 
 

SECTION: Single Cell Sorting by FACS
25. Day 4
 
SECTION: Single Cell Sorting by FACS
25.1. Prepare fresh media without ROCK Inhibitor. For 12 mL

 

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25.2. Aspirate spent media using a multichannel aspirator.
SECTION: Single Cell Sorting by FACS
25.3. Pipette 100 µL/ well of the new media into each well of a 96 well-plate.
 


SECTION: Single Cell Sorting by FACS
25.4. Check there is 1 colony/well. Ensure colonies are growing.
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26. Day 6
SECTION: Single Cell Sorting by FACS
26.1. Prepare fresh media without ROCK Inhibitor and Clone R. For 12 mL.


 

SECTION: Single Cell Sorting by FACS
26.2. Aspirate spent media using a multichannel aspirator.
SECTION: Single Cell Sorting by FACS
26.3. Pipette 100 µL/ well of the new media into each well of a 96 well-plate.
 


SECTION: Single Cell Sorting by FACS
27. Day 8 and later
SECTION: Single Cell Sorting by FACS
26.4. Check there is 1 colony/well. Ensure colonies are growing.
SECTION: Single Cell Sorting by FACS
26.5. Perform a mycoplasma test during this stage or earlier.
SECTION: Single Cell Sorting by FACS
27.1. If the cells are 70-80% confluent or begin to grow on top of each other, move onto the next step. If not, continue with this step.
SECTION: Single Cell Sorting by FACS
27.2. If the cells are not ready, continuing feeding but with just mTeSR Plus

Aspirate spent media using a multichannel aspirator.
Pipette 100 µL/ well of mTeSR Plus into each well of a 96 well-plate.
 

Check there is 1 colony/well. Ensure colonies are growing.

SECTION: Single Cell Sorting by FACS
27.3. Keep cells in 96 well-plate for 1-7 more days or until the clones can be passaged to a smaller plate format.
SECTION: Passaging and Maintaining the Clones
28. Identify the surviving clones.
 
SECTION: Passaging and Maintaining the Clones
28.1. Using a microscope and marker, count and label the wells of the 96 well-plate where there are surviving clones.
SECTION: Passaging and Maintaining the Clones
28.2. If there are between 24 and 48 clones, then you will passage the cells in 2 x 48 well-plates
 

SECTION: Passaging and Maintaining the Clones
28.3. If there are less than 24 clones, then you will passage the cells into 2 x 24- well-plates.
 

SECTION: Passaging and Maintaining the Clones
29. Prepare the 24 or 48 well-plates.
SECTION: Passaging and Maintaining the Clones
29.1. Matrigel coat the 24 or 48 well-plates.


SECTION: Passaging and Maintaining the Clones
29.2. Incubate for at least 30 min at 37 °C. 

SECTION: Passaging and Maintaining the Clones
29.3. 
 Prepare media. For 25 mL
 
 

 
 
SECTION: Passaging and Maintaining the Clones
29.4. Aspirate the Matrigel from plates.
SECTION: Passaging and Maintaining the Clones
29.5. Add media to plates

For 48 well-plates: add 250 µL/well 
For 24 well-plates: add 500 µL/well 

SECTION: Passaging and Maintaining the Clones
30. Clump passage the clones (row-by-row)

 
SECTION: Passaging and Maintaining the Clones
30.1. Aspirate spent media from one row of the 96 well-plates.
SECTION: Passaging and Maintaining the Clones
30.2. Pipette 50 µL/well of ReLeSR.

SECTION: Passaging and Maintaining the Clones
30.3. Incubate at Room temperature for 45 s.

SECTION: Passaging and Maintaining the Clones
30.4. Aspirate ReLeSR from cells.
SECTION: Passaging and Maintaining the Clones
30.5. Incubate the 96 well-plate at 37 °C for 3 min.

SECTION: Passaging and Maintaining the Clones
30.6. Pipette 100 µL/well of mTeSR Plus w/ ROCK inhibitor to each well in the row.

SECTION: Passaging and Maintaining the Clones
30.7. Resuspend cells by pipetting up and down.
SECTION: Passaging and Maintaining the Clones
30.8. Move 35 µL into one well of the new plates and 65 µL into another well of the other new plate.

 

SECTION: Passaging and Maintaining the Clones
30.9. Once a row is passaged, disperse the cells by rocking in all four directions.
SECTION: Passaging and Maintaining the Clones
30.10. Continue row-by-row until the entire plate is passaged.
SECTION: Passaging and Maintaining the Clones
31. Day 1 post passaging
SECTION: Passaging and Maintaining the Clones
31.1. 
 

SECTION: Passaging and Maintaining the Clones
31.2. 
 

SECTION: Passaging and Maintaining the Clones
32. Harvesting one plate to genotype the clones.
SECTION: Passaging and Maintaining the Clones
31.3. If one plate is at least 70-80% confluent, move to the next step. Otherwise, maintain the plates.
SECTION: Passaging and Maintaining the Clones
32.1. Aspirate the spent media.
 
SECTION: Passaging and Maintaining the Clones
32.2. Wash the cells with 200 µL/well of PBS. 

SECTION: Passaging and Maintaining the Clones
32.3. Aspirate the PBS.
SECTION: Passaging and Maintaining the Clones
32.4. Seal the sides of the plate with parafilm to avoid evaporation
SECTION: Passaging and Maintaining the Clones
32.5. Store the plate at -20 °C and harvest DNA later or extract DNA immediately using Quick Extract.

SECTION: Passaging and Maintaining the Clones
32.6. Quick Extract Protocol.

i. Add 50 µL/well of Quick Extract. 
ii. Scrape the bottom of the well to detach cells.
 
iii. Mix by vortexing for 15 s.
iv. Transfer cells in Quick Extract to labeled PCR tubes. 
 

v. Incubate the samples at 65 °C for 6 min using a Thermocycler.
vi. Mix by vortexing.
vii. Incubate the samples at 98 °C for 2 min using a Thermocycler.




SECTION: Passaging and Maintaining the Clones
33. (Optional) Freezing the clones.



SECTION: Passaging and Maintaining the Clones
33.1. 
 

SECTION: Passaging and Maintaining the Clones
33.7. Incubate at Room temperature for  45 s.

SECTION: Passaging and Maintaining the Clones
33.6. Add 100 µL/well of ReLeSR. 

SECTION: Passaging and Maintaining the Clones
33.5. Wash cells with 200 µL/well of PBS. Aspirate

SECTION: Passaging and Maintaining the Clones
33.4. Aspirate spent media.
SECTION: Passaging and Maintaining the Clones
33.3. Grow the plate until clones are 70-80% confluent.
SECTION: Passaging and Maintaining the Clones
33.2. Obtain a Styrofoam box and clean it with ethanol.
SECTION: Passaging and Maintaining the Clones
33.8. Aspirate and incubate plate at 37 °C for 3 min.
 
SECTION: Passaging and Maintaining the Clones
33.9. Add CryoStor

For 96 well-plates: 100 µL/well.
For 48 well-plates: 200 µL/well.

SECTION: Passaging and Maintaining the Clones
33.10. Add Mineral Oil

For 96 well-plates: 100 µL/well
For 48 well-plates: 200 µL/well

SECTION: Passaging and Maintaining the Clones
33.11. Seal the plate with parafilm.
SECTION: Passaging and Maintaining the Clones
34. (Optional) Thawing clones from frozen 96/48 well-plate.
 
SECTION: Passaging and Maintaining the Clones
33.12. Place the plate in the Styrofoam box.
SECTION: Passaging and Maintaining the Clones
34.1. Coat 12 well plates with 0.5 mL/well of Matrigel.

Incubate the plate at 37 °C for at least 30 min.

SECTION: Passaging and Maintaining the Clones
33.13. Carefully transfer the box to a -80 °C freezer.

SECTION: Passaging and Maintaining the Clones
33.14. The cells are stable for up to 1 month at -80 °C.

SECTION: Passaging and Maintaining the Clones
34.2. Label Eppendorf tubes with the clone number of the clones that you will move forward with
SECTION: Passaging and Maintaining the Clones
34.3. Prepare Media. For 12 mL


 

SECTION: Passaging and Maintaining the Clones
34.4. Warm PBS to 37 °C.

SECTION: Passaging and Maintaining the Clones
34.5. Place the frozen plate on paper towels and place it in the 37 °C incubator for 10 min or until edges of the plate are thawed.

SECTION: Passaging and Maintaining the Clones
34.6. Pipette warm PBS onto wells that you want to thaw.

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34.7. Pipette the cells into their respective Eppendorf tube.

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34.8. Centrifuge at 800 rpm for 3 min.

SECTION: Passaging and Maintaining the Clones
34.9. Aspirate the supernatant
 


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34.10. Resuspend the pellet in 1 mL of media.

SECTION: Passaging and Maintaining the Clones
34.11. Pipette the cells into 1 well of a 12 well-plate.
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34.12. Place plate in incubator.
SECTION: Genotyping
35. 
 

SECTION: Genotyping
36. 
 

SECTION: Genotyping
36.1. Obtaining gene sequences: National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) 
SECTION: Genotyping
36.2. Storing and aligning sequences: SnapGene
 
SECTION: Genotyping
36.3. Designing primers: Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/)
SECTION: Genotyping
36.4. Ordering primers: Integrated DNA Technologies (www.idtdna.com/) 

Products and Services → Custom DNA Oligos → DNA Oligos (order now)
SECTION: Genotyping
36.5. Check for off targets: Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) 
SECTION: Genotyping
37. Perform an excision PCR (perform on all clones).
 
SECTION: Genotyping
37.1. Designing excision primers

These set of primers should bind outside of both the cut sites.
Length: After the excision, the expected length of the band should be between 100 bp and 1000 bp.

SECTION: Genotyping
37.2. Expectations

Unedited line: A large band or no band because of the size of the excision.
Homozygous excision: Expected band size.
Heterozygous excision: Expected band size and the unedited band (unless there is no band).
SECTION: Genotyping
37.3. Sanger Sequence

Save some PCR product to sanger sequence at a later time.
SECTION: Genotyping
38. Perform 5’ cut site excision PCR (perform only on clones that have the correct excision band).
 
SECTION: Genotyping
38.1. Design 5’ cut site primers.

One of the primers should bind on the 5’ outside of the cut site and the other primer should bind within the excision region.
Length: If the excision did not occur, the expected length of the band should be between 100 bp and 1000 bp. With the excision, we expect no band
SECTION: Genotyping
38.2. Expectations

Unedited line: Expected band size
Homozygous excision: No band
Heterozygous excision: Expected band size
SECTION: Genotyping
39. Perform 3’ cut site excision PCR (perform only on clones that have the correct excision band).
 
SECTION: Genotyping
39.1. Design 3’ cut site primers.

One of the primers should bind 3’ outside of the cut site and the other primer should bind within the excision region.
Length: If the excision did not occur, the expected length of the band should be between 100 bp and 1000 bp. With the excision, we expect no band.
SECTION: Genotyping
39.2. Expectations

Unedited line: Expected band size.
Homozygous excision: No band.
Heterozygous excision: Expected band size.
SECTION: Karyotyping and Freezing
40. Send one or two clones with correct excisions to karyotype.


SECTION: Karyotyping and Freezing
40.1. Freeze a few vials (3-4) of all clones you are karyotyping.
SECTION: Karyotyping and Freezing
41. Expand and freeze 10+ vials the clone with the correct excision and normal karyotype.