CRISPR/Cas9-mediated knockins in human induced pluripotent stem cells
Introduction
This protocol provides detailed information on how to achieve successful point mutation knockins with CRISPR/Cas9 in human induced pluripotent stem cells for disease modelling purposes. Make sure to read this protocol in its entirity first and prepare for each step in advance (during multiple incubation periods etc.).
Materials
Nucleofector (e.g. Amaxa IIb or 4D) and associated components (cuvettes , nucleofection solution)
Cell counter (manual haemocytometer or automated)
Geltrex LDEV Free hESC Quality 5 ml (A1413302, Thermo Fischer Scientific)
DMEM/F12 with L-Glut, HEPES 500 ml (11330-032, Thermo Fischer Scientific; can also use Sigma's without L-Glut/GlutaMAX and buffered by HCO3-)
Essential 8 Flex Medium Kit (E8/F, A2858501, Thermo Fischer Scientific; new media formulations are availabe for improved survival under low-density conditions as single cells, e.g. StemFlex)
RevitaCell Suppplement 5 ml (A2644501, Thermo Fischer Scientific; or other ROCK inhibitor supplied diluted to 10 uM final concentration)
ReLeSR passaging reagent 100 ml (#5872, Stem Cell Technologies; also available as 500 ml)
StemPro Accutase (A110501, Thermo Fischer Scientific; do not use TrypLe Express due to low viability!)
Freezing medium (either PSC Cryopreservation Kit #2644601 from ThermoFischer; mFreSR from Stem Cell Technologies is an option as well - #5855; or make your own: 90 % FBS + 10 % DMSO + 1X RevitaCell or 10 uM ROCKi)
96-well culture plates (flat bottom)
Sterile reservoirs 25 ml, 4x50 (#4312, Integra)
Multichannel pipette and aspiration adapter for 96-well plates
Fluorescence microscope (e.g. EVOS FL AMF4300, Thermo Fischer Scientific)
QuickExtract DNA Extraction Solution (#QE09050, Cambridge Bioscience - originally a product of Epicentre)
Gene editing reagents: Cas9 plasmid (e.g. pX459) and gBlocks-encoded sgRNAs (link to previous post)
Genotyping reagents (depending on method)
Procedure
Thaw the 5 ml Geltrex vial on ice in the fridge overnight.
Mix 1:1 with cold DMEM/F12 and aliquot into 500 ul aliquots; store at -20C (this is 50X).
For plate coating, thaw the 50X Geltrex stock in the fridge or on the bench if ready to use immediately.
Dilute 1:50 in cold DMEM/F12 (500 ul to 24.5 ml) - mix by pipetting up-and-down with stripette.
Coat plates (1 ml per well for 6-well plate; 6 ml per 10-cm dish).
Place coated dishes in the incubator for at least 20-30 min. Subsequently, either store in the fridge or equilibrate to room temperature prior to use.
You can keep coated plates in the fridge for up to 2 weeks (good to parafilm to avoid evaporation).
Prepare E8/F (Essential 8/Flex) according to manufacturer's instructions (essentially, remove 10 ml of the baselin medium - you can keep this for plate coating; add 10 ml of supplement that has been thawed in the fridge overnight or at RT for 1-2h; mix well).
When using the medium, only take the amount you need by aliquotting into 50 ml tubes and storing the main bottle in the fridge to avoid excessive exposure to room temperature. This is critical for successful culturing although E8/Flex has the advantage of incorporating a heat-stable FGF2.
Prepare the required number of Geltrex-coated 10-cm dishes, equilibrated to room temperature. You need one dish per planned nucleofection.
Pre-treat the cells to be passaged with RevitaCell: dilute RevitaCell 1:100 in E8/F and add 1.5 ml to each well in a 6-well plate after removing the old medium (1.5 ml is fine for a short period of time or if cells are sparse, otherwise use 2 ml for 6-wells). Make sure to prepare enough E8/F+Revita as you will need it for seeding (calculate the amount required depending on the number of 10-cm dishes that you are setting up).
Incubate at 37C for min. 30min; up to 3h.
Take cells out and transfer the 1.5 ml conditioned medium to a labelled 2 ml tube (I find 5 ml Eppendorf tubes more handy for this)
Wash the cells with 2 ml DPSB per well.
Aspirate the DPBS and treat the cells with 1 ml ReLeSR for 1 minute.
Transfer the cells to the incubator for 4-7 minutes (cell line-dependent so important to optimise; also, the original protocol says to leave them at room temperature, but I find that it works better when they are kept in incubator during this time).
In the meantime, aspirate the excess Geltrex from the plates that will be used for seeding and add 10 ml of E8/F+Revita to each plate.
Take the cells out of the incubator and add 1 ml of the conditioned medium: add this gently to the side of the well (another essential steps as stem cells are very fragile at this stage!).
Place the plate towards your body to stabilise it and tap on the side with your free hand: you should see the colonies float off and break-up into smaller pieces if you have allowed sufficient incubation time beforehand. If you have difficulties dissociating the colonies, use a P1000 pipette (not smaller!) to dislodge the cells by pipetting medium over them (not scraping), but try to be as gentle as possible when pipetting the medium up-and-down. This is really criticial and excessive pipetting will result in culture deterioration!
Gently transfer the dissociated cells to the tube with the remaining conditioned medium.
Take the required amount of cells that corresponds to your usual split ratio, taking into account that you are scaling up to a 10-cm dish. My cells are fine when split 1:15 and 1:20, but it is very cell line-depending. 1:10 is a good start if in doubt. If too dense, the stem cells will start differentiating. If too sparse, a lot will struggle to survive.
Swirl the plate to achieve equal seeding distribution across the plate.
Return the cells to the incubator and leave undisturbed for 24h. NB: try to process your cells at roughly the same time of day; this regularity keeps them healthy!
On the following day, refeed the cells with E8/F without Revita.
Skip a day of feeding as the cells are quite sparse at this stage. It is normal to see the cells struggling immediately after RevitaCell removal, but they will recover if healthy. Following one day of skipped feeding, feed daily until ready to passage again (usually after 4 days for my cells; usually when colonies become bigger than 1000 um in diameter and the medium is orange/yellow despite feeding 24h before).
Accutase StemPro should be equilibrated to room temperature (I usually aliquot this reagent into 15 ml Falcons that I keep at -20 and thaw as required).
Equilibrate your nucleofection solution to room temperature (I use a home-made nucleofection solution that works well with my stem cells; for a recipe, see the following paper: doi: 10.1016/j.ymeth.2014.05.003. You can buy your own cuvettes as well, but make sure that they correspond to your nucleofector specs and the cuvettes you would have used from the Nucleofector manufacturer (for instance, I use the following Ingenio cuvettes bought from Cambridge Bioscience: #MIR50121). This will save you a lot of money!
Prepare sufficient amount of E8/F supplemented with RevitaCell (see above).
Prepare Geltrex-coated 96-well plates (1-2 per nucleofection depending on how efficient you expect your targeting to be).
You need 100 ul of DMEM/12+Geltrex (1:50 dilution of 50X Geltrex stock, see above) per well of a 96-well plate. You will also need a multichannel and 25 ml sterile reservoirs (see Materials).
Refer to "iPSC culturing in preparation for targeting" for instructions on how to pretreat cells in preparation for passaging.
Make sure that you have your CRISPR reagents ready: sgRNA-gBlocks (3 ug) + PX459 plasmid (5 ug) + HDR template (200 pmol to 400 pmol).
When ready to passage the cells, proceed as described in "iPSC culturing in preparation for targeting" until the ReLeSR step. Instead of ReLeSR, add 2 ml of StemPro Accutase to the cells in a 10-cm dish and ensure that it covers the surface.
If you don't forget, keep the conditioned medium from the 10-cm dish.
Incubate at 37C for 7-8 minutes (cell line-dependent).
In the meantime, prepare for the next steps: label tubes and plates, aspirate excess Geltrex from 96-well plate
When the cells are ready, collect into a 15 ml Falcon with 5 ml E8/F w/o RevitaCell (not required becauase you will be spinning down soon), and pipette up-and-down GENTLY (how gently is often difficult to get right if you are not used to working with these cells; I hope to post a video soon to demonstrate this).
You have to work carefully but quickly in the next 2 steps as the Accutase continues to work until the cells are spun down and the supernatant removed.
Count the cells and calculte cells/ml.
Calculate the required volume to have 2.2x10^6 cells/nucleofection (10 % excess).
Prepare a separate 15 ml Falcon for each nucleofection and transfer the required volume of cell (I would advise to perform only 1 nucleofection at a time!).
Spin the cells down at 200g for 3 minutes.
Aspirate the supernatant and flick the tube (gently) to dislodge the pellet.
The following steps for nucleofection are dependent on the nucleofection you are using. Mine apply to the Amaxa Nucleofector IIb.
Resuspend the cells in 110 ul nucleofection solution (10 % excess) using P1000! (this is important to reduce the shear stress on the cells)
Transfer 100 ul of the suspension to a cuvette and nucleofect the cells.
In my hands, the Amaxa Nucleofector IIb B-16 programme works well.
Immediately add 500 ul of the conditioned medium to the cuvette.
Using a Pasteur pipette, transfer the cell suspension to the 15 ml Falcon containing the conditioned medium.
With a stripette (10 ml), pipette gently up-and-down to mix the cells.
Transfer to a 25 ml sterile reservoir and seed 50-100 ul into the prepared 96-well plate(s). You can use 50 ul if you want to promote enrichment even further, in which case you can seed 2 plates.
Return the plates to the incubator and leave undisturbed for 24h.
I usually repeat the procedure, but only for GFP nucleofection to assess the nucleofection efficiency.
Assess cell health on the following day. If you did set up a GFP control nucleofection, check the efficiency of nucleofection (I usually obtain 50-60 % efficiency).
If the cells look good in the 96-well plate, you can refeed them E8/F without RevitaCell. Otherwise, skip feeding and leave them in RevitaCell as before, unless there is too much debris - then refeed.
Monitor the cells and remove RevitaCell no later than 4 days post-targeting.
When the cells are 80-90 % confluent, prepare 96-well plates coated with Geltrex.
Preatreat the cells with 100 ul E8/F supplemented with RevitaCell 30min-3h prior to processing.
1X wash in 200 ul DPBS per well
60 ul ReLeSR per well for 1 minute
Remove ReLeSR and leave cells for 6-7 minutes at room temperature
In the meantime, remove excess Geltrex
Add 200 ul E8/F + RevitaCell and pipette up-and-down while scraping gently with pipette tip
Take 100 ul of cell suspension to a new Geltrex-coated 96-well plate, leaving 100 ul behind on old plate as a back-up for DNA extraction; alternatively, take the the whole 200 ul volume to the new Geltrex plate and replenish whatever is remaining on the old plate with 100 ul medium. There are always cells left, so you will be able to extract DNA from these plates on the following day.
Assess cell health the following day and determine whether to remove RevitaCell after 24h or to wait till the following day.
On the following day, aspirate the medium from the "old" plate, wash 1X with DPBS and add 30 ul ReLeSR to all wells for 5-6 minutes (do not aspirate in between).
Add 70 ul QuickExtract (I usually keep aliquots at -20C and thaw as required) and proceed with DNA extraction as follows:
Remembering to change tips (crucial!), pipette up and down in each well to ensure cell dissociation and transfer the content of each 96-well culture plate to a 96-well PCR plate.
On a thermocycler, set-up the following programme: 65C for 5 minutes, 98C for 10 minutes
Let plate cool to room temperature. 1:10 dilution of the extracted DNA can used directly in PCR reaction.
Proceed with genotyping. We usually perform digital RFLP (happy to share the protocol if interested) for which you will need access to a capillary sequencer and an analysis programme such as GeneMapper.
Once you have identified wells positive for the mutation of interest (> 1% mutation burden), expand these into a 12-well plate using ReLeSR for expansion (Geltrex coated; 500 ul per well).
Once the cells in the 12-well plate have reached the desired confluence and are ready to passage, prepare Geltrex-coated 96-well plates and pretreat the cells with E8/F supplemented with RevitaCell.
Proceed with passaging using Accutase (3-500 ul per 12-well) - see above for protocol.
Before counting, spin the cells down at 200g for 3 minutes.
In the meantime, remove excess Geltrex from the coated 96-well plate for seeding.
Aspirate the supernatant and flick the tube gently to dislodge the pellet.
Resuspend the cells in 1 ml E8/F supplemented with RevitaCell.
Determine the number of cells required to have 10,000 cells/ml and transfer the required volume to a separate tube containing 1-2 ml E8/F+RevitaCell (i.e. you will need 10,000 to 20,000 cells, respectively).
From the 10,000 cells/ml suspension, take 380 ul (~ 3750 cells) to a 15 ml Falcon containing 14.6 ml E8/F+RevitaCell.
This is enough to seed one 96-well plate; prepare more if you need to seed more than 1 (how many you seed depends on the starting mutation burden).
Transfer the cell suspension to a 25 ml sterile reservoir and seed 100 ul into a 96-well plate (i.e. 25 cells/well).
This seeding density will be cell line dependent; I usually use 10-25 cells/well depending on the cell line as I have experienced that this results in survival of only 2-3 cells.
Return the cells to the incubator and feed every 3rd day in the beginnning as they will be very sparse.
Note that any surviving cells will tend to cluster at the well edges, so be careful not to score a well as empty!
Remove RevitaCell on the 6th day when any surviving colonies should be growing; again, this is cell line-dependent, and you might be able to do it 4 days post-seeding. However, do not keep the cells in RevitaCell for more than 6 days as it inhibits proliferation.
The cells will usually be ready for genotyping 10-14 days post-seeding at clonal density. Proceed with replica-plating and genotyping as described above. If further enrichment is required, repeat the described process until a genetically homogenous population is obtained.
When you have wells with a mutation burden of minimum 30-40 %, you should either perform Sanger sequencing or targeted high-throughput sequenicng such as MiSeq to identify wells with cells that have indels; these should not be used for further expansion.