May 23, 2023
CRISPR/Cas9 generation of knock-out iPSCs
- Dan Dou1,2,
- C. Alexander Boecker3,
- Erika L.F. Holzbaur1,2
- 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, USA;
- 3Department of Neurology, University Medical Center Goettingen, 37077 Goettingen, Germany
External link: https://doi.org/10.1016/j.celrep.2023.112448
Protocol Citation: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur 2023. CRISPR/Cas9 generation of knock-out iPSCs. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqj48yvk5/v1
Manuscript citation:
Dou D, Smith EM, Evans CS, Boecker CA, Holzbaur EL, Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes. Cell reports 42(5). doi: 10.1016/j.celrep.2023.112448
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 14, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71342
Keywords: Knock-out, iPSC, CRISPR, Cas9, FACS, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000350
Abstract
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.
Attachments
553-1149.pdf
119KB
Materials
Materials
- 10 cm cell culture dishes
- 6-well, 24-well, and 96-well plates
- Cell culture microscope for picking colonies
Reagents
- Synthetic sgRNA (for example Gene Knockout Kit v2 from Synthego = multi-guide mix for knockout).
- Alt-R® S.p. HiFi Cas9 Nuclease V3IDTCatalog #1081060
- Lipofectamine™ Stem Transfection ReagentThermo FisherCatalog #STEM00003
- Opti-MEM (Reduced Serum Medium)Thermo Fisher ScientificCatalog #31985062
- mTeSR™1 1 L Kit Stemcell TechnologiesCatalog #85857
- Growth Factor Reduced (GFR) Matrigel®CorningCatalog #354230
- DMEM/F-12Thermo FisherCatalog #11320033
- Accutase® solutionSigma AldrichCatalog #A6964
- Y-27632 ROCK InhibitorSelleckchemCatalog #S1049
- PBS buffer Thermo Fisher ScientificCatalog #10010023
- Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
- Falcon 40 µm Cell StrainerCorningCatalog #352340
- KnockOut™ Serum ReplacementThermo FisherCatalog #10828028
Transfection with sgRNA and recombinant Cas9
Transfection with sgRNA and recombinant Cas9
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 10 micromolar (µM) ROCK inhibitor (RI).
1 - 2 hours prior to transfection, change media to fresh, pre-warmed mTeSR + RI (2 mL /well).
Note
Replacing the media is important because remaining Accutase can interfere with Lipofectamine Stem transfection.
Prepare sgRNA:
Resuspend 1.5 nanomolar (nM) sgRNA in 15 µL of RNAse-free TE buffer to make a 100 micromolar (µM) stock solution.
Dilute required amount of sgRNA to 1 micromolar (µM) final concentration in TE buffer.
Flick tube of HiFi Cas9 enzyme (IDT) to mix and briefly spin.
Dilute required amount of HiFi Cas9 (62 micromolar (µM) stock solution) to 1 micromolar (µM) in Room temperature OptiMEM.
Note
e.g. 0.5 µL of 62 micromolar (µM) HiFi Cas9 in 30.5 µL OptiMEM. Leftover diluted Cas9 can be stored at -20 °C .
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 00:05:00 at Room temperature to allow RNP complexes to form.
5m
Add 0.4 µg GFP plasmid to RNP complex in Tube 1 and mix by pipetting.
Set up Lipofectamine Stem mix in Tube 2 by combining 100 µL OptiMEM and 4 µL Lipofectamine Stem.
Add Tube 2 solution to Tube 1 and flick tube to mix. Incubate mixture for 00:10:00 at Room temperature , then add mixture dropwise to iPSCs.
10m
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 ~72:00:00 ).
3d
FACS-sorting
FACS-sorting
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 .
Note
Because of the increased risk for microbiological contamination during FACS sorting, we recommend supplementing mTeSR medium with Penicillin/Streptomycin (P/S).
Collect transfected iPSCs with an Accutase split and resuspend into mTeSR (+RI +P/S).
Note
The media volume depends on size of the cell pellet; we have used 1.5 mL for resuspending iPSCs from 4 wells of a 6-well plate.
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 1.5 mL 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.
Note
Transfection efficiency with Lipofectamine Stem should be about 30%.
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.
Picking Colonies
Picking Colonies
Pick clones when individual colonies reach a size of ~ 1 mm.
Note
This should be ~ 9 days after FACS sorting for KOLF2.1 iPSCs.
For the first attempt of CRISPR/Cas9 editing, we recommend picking 24 colonies per condition.
Note
If none of the picked clones has the desired genotype, the editing protocol may need to be optimized and/or more clones should be picked during the next attempt.
In two 96-well plates, coat 12 wells for each condition with Matrigel; then add 200 µL 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 15 mL fresh media per dish.
Note
Add 15 mL instead of 10 mL mTeSR to each 10 cm dish for picking iPSC clones, because aspirating colonies will remove media.
Note
We find that leaving iPSCs at Room temperature for a prolonged time after picking clones seems to decrease chances of survival. Therefore, we recommend equilibrating 96-well plates before starting to pick colonies and using two 96-well plates per condition.
To pick individual clones, visualize colonies under a tissue culture microscope.
Note
Ideally, the microscope is placed in an open hood. We find that it’s also feasible to place the microscope in a regular cell culture hood and lift the glass front all the way up.
Use a P200 pipette to scratch each colony from the dish and aspirate the colony in 100 µL 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 100 µL 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.
Note
While picking clones, try to avoid colonies that look as if two different colonies have grown together (e.g., looking like the number 8). We recommend picking a random mix of smaller, medium-sized, and larger colonies.
Note
Optional: The remaining clones may be kept in culture for 1-2 more days and can then be used to make pooled cell lysates. These lysates can be analyzed per Western Blot to get a general sense of the knock-out efficiency.
Expanding individual clones
Expanding individual clones
1w 3d 0h 29m
1w 3d 0h 29m
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.
Note
For KOLF2.1 iPSCs, this should be ~ 168:00:00 after picking individual colonies. Some clones may have to be split earlier than other clones.
Splitting cells from 96-well plates to 24-well plates using Accutase:
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.
- 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 00:06:00 - 00:08:00 .
- 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.
Note
Optional: To obtain cellular DNA for screening successful CRISPR edits, pipette ~ ¼ of the volume into an Eppendorf tube.
14m
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 2 mL mTeSR + RI per well.
- Aspirate mTeSR from wells in 24-well plate and wash with PBS.
- Add 100 µL Accutase per well and incubate at 37 °C for 00:06:00 - 00:08:00 .
- While cells are at 37 °C , prepare one 1.5 mL Eppendorf tube with 200 µL mTeSR + RI for each clone.
14m
- 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 (00:03:00 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.
3m
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.
Note
All clones to be used in future studies should be confirmed to have a normal karyotype by cytogenetic analysis of G-banded metaphase cells.