May 06, 2020
Footprint-Free Genome Editing of iPSC Using Alt-R CRISPR/Cas9
- 1Washington University in Saint Louis - WUSTL (MO);
- 2Washington University in St Louis
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Jacob Marsh, Rj Martinez, Celeste M M. Karch 2020. Footprint-Free Genome Editing of iPSC Using Alt-R CRISPR/Cas9. protocols.io https://dx.doi.org/10.17504/protocols.io.bfmmjk46
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: April 27, 2020
Last Modified: December 10, 2020
Protocol Integer ID: 36237
Keywords: iPSC, genome editing, Alt-R, CRISPR/Cas9,
Preparing iPSCs for Nucleofection
Preparing iPSCs for Nucleofection
Coat 1 well of a 6-well plate with 1 mL of Matrigel for 01:00:00 at 37 °C
Add 5 mL of DMEM/F12 to a 15ml conical tue
Thaw vial of cells in 37 °C water bath for approximately 00:01:00
Wipe off excess water from tube and spray with 70% Ethanol before placing vial into biosafety cabinet
Transfer thawed cells into conical tube containing 5 mL DMEM/F12
Note
If some cells remain in vial use 1 mL DMEM/F12 to rinse vial clean and transfer into the 15ml conical
Swirl to mix cells in the DMEM/F12 and 750 rpm, 00:03:00
Note
Avoid rough mixing or suspension of cells; this helps to keep the cells clustered
After centrifugation, aspirate off most supernatant, leaving a small amount in the tube. Do not try to aspirate all the way down to the pellet (may or may not visible)
Add 4 µL of Rock Inhibitor to cell pellet and resuspend in 2 mL of mTesR1 in order to achieve a 10 micromolar (µM) solution. Gently swirl to mix
Note
Avoid rough mixing or suspension of cells; this helps to keep the cells clustered
Aspirate Matrigel from coated 6-well plate, and pipet cell mixture into well.
Incuate overnight at 37 °C
After 24:00:00 change media and allow cells to recover
Once cells have reached 80-90% confluency, split culture as single cells into 3 wells of a 6 well tissue culture plate.
Coat 3 wells of a 6-well plate with 1 mL of Matrigel for 01:00:00 at 37 °C
Aspirate media from well containing cells
Wash cells with 2 mL of PBS
Add 1 mL of Accutase per well
Incubate cells and Accutase at 37 °C for 00:10:00 . Gently tap plate upon removal from incubator to help dislodge cells from Matrigel
Collect cells in 5 mL of PBS and transfer to 15ml conical tube
750 rpm, 00:03:00
Resuspend cell pellet in 6 mL mTesR1 + 5 micromolar (µM) Rock Inhibitor and plate 2 mL into each freshly coated Matrigel well
Perform the single cell passage (Step 12) at least two additional times prior to using cells for Nucleofection
Continuing iPSC Culture
Continuing iPSC Culture
Perform a single cell passage approximately 48:00:00 prior to nucleofection
Note
You will need three million cells per nucleofection and one million cells per GFP control. Therefore, three wells of a confluent of a 6-well tissue culture plate are sufficient
Coat three wells of a 6-well plate for nucleofection using Matrigel and incubate at 37 °C for 01:00:00 prior to splitting cells
Just prior to splitting cells for nucleofection, aspirate Matrigel from coated wells and add 3 mL of DMEM/F12 + 10% FBS supplemented with 10 micromolar (µM) Rock Inhibitor per well
RNP Complex
RNP Complex
Gather the following reagents for Alt-R Reactions and let them thaw on ice
| Reagent | Stability and Storage | |
| Alt-R crRNA | 6 months stability at -80°C | |
| Alt-R tracrRNA | 6 months stability at -80°C | |
| Alt-R Cas9 | 6 months stability at -80°C | |
| Electroporation Enhancer (IDT - Catalog # 1075915) | 6 months stability at -80°C | |
| sgRNA (if needed) | 6 months stability at -80°C |
Prepare Alt-R Reactions
Prepare fresh Alt-R gRNA by adding 2.5 µL of 200 micromolar (µM) Alt-R crRNA and 2.5 µL of 200 micromolar (µM) Alt-R tracrRNA in equal volumes. Heat mixture at 95 °C for 00:05:00 . Let cool to Room temperature
Note
The Alt-R gRNA should be prepared fresh before each use
Resuspend Alt-R Cas9 and Electroporation Enhancer in PBS to a final concentration of 100 micromolar (µM)
Combine Alt-R gRNA solution and Alt-R Cas9 + Electroporation Enhancer solution together and incubate at Room temperature for 00:15:00
| Component | Final Concentration | |
| PBS | - | |
| Alt-R gRNA | 120 pmol | |
| Alt-R Cas9 | 104 pmol | |
| Enhancer | 100 µM | |
| Donor ssODN or GFP* | 100 µM |
*GFP = pMax GFP control in a separate tube
Note
RNP Complex stability limited to 48:00:00 at 4 °C . Do not freeze
Split Cells for Nucleofection
Split Cells for Nucleofection
Split cells for nucleofection
Aspirate media from cells
Wash each well with 2 mL of PBS and aspirate
Add 1 mL of Accutase per well
Incubate at 37 °C for 00:10:00
Collect cells in 5 mL of PBS and transfer to a 15ml conical tube
750 rpm, 00:03:00
Count Cells for Nucleofection
Count Cells for Nucleofection
Resuspend cell pellet in 1 mL of PBS in the 15ml conical tube, then dilute cells 1:10 (10 µL of cell suspension + 90 µL of PBS) in a 1.7ml tube
Use 10 µL of diluted cells for cell counts
Using all four corners of the countess slide, calculate the average number of cells
Multiply the average by 10,000 (104)
Multiply product from step 20.2 by 3 to get the total number of cells
[Average # of Cells x 10,000 x 3] = Total Number of Cells
Take total number of cells calculated in step 20.3 and divide by three million
Take answer from step 20.4 and divide by three to get the volume of cells necessary for nucleofection
Centrifuge Cells for Nucleofection
Centrifuge Cells for Nucleofection
Transfer the desired volume of cells (calculated in step 20.5) to microcentrifuge tube
90 x g, 00:05:00
Aspirate PBS from cell pellet
Prepare Lonza Kit Reagents for Nucleofection
Prepare Lonza Kit Reagents for Nucleofection
Make reaction mix from Lonza Kit: P3 Primary Cell 4D (V4XP-3024). Each reaction requires a total of 100 µL of reaction mix.
Note
If performing more than one reaction, it is best to make a Master Mix
Combine 82 µL of P3 Solution and 18 µL of Supplement into a 1.7ml microcentrifuge tube
Combining Lonza Kit Reagents and DNA for Nucleofection
Combining Lonza Kit Reagents and DNA for Nucleofection
Combine reaction mix from step 24 (100 µL ) with previously complexed DNA from step 17
Mix reaction mix and DNA with cell pellets (step 23) by pipetting up and down with p200 pipette
Note
Try to pipette as little as possible. Pipette only until mixed
Transfer 100 µL of reaction mix + DNA + cells to a cuvette
Note
Ensure no bubbles are in the transferred mixture of cells in the cuvette. This can interfere with the nucleofection's success
Nucleofection
Nucleofection
Nucleofect with Lonza Program CA-137 in P3 Solution
Let cuvette and cells incubate for 00:10:00 at Room temperature
Transfer cells/DNA solution to approproate pre-coated well containing 2 mL of DMEM/F12 + 10% FBS + 10 micromolar (µM) Rock Inhibitor
Incubate at 37 °C overnight
Post Nucleofection
Post Nucleofection
Continue culturing the iPSC in 1 well of a 6 well plate for 5-7 days post nucleofection, changing mTesR1 daily.
24:00:00 post-nucleofection - add mTesR1 with 5 micromolar (µM) Rock Inhibitor
48:00:00 post-nucleofection - add mTesR1 with 2.5 micromolar (µM) Rock Inhibitor
72:00:00 post-nucleofection - add mTesR1 with 1 micromolar (µM) Rock Inhibitor
Continue culturing cells in mTesR1 until confluent (~5 days)