TITLE: Footprint-Free Genome Editing of iPSC Using Alt-R CRISPR/Cas9
AUTHORS: Jacob Marsh, Rj Martinez, Celeste Karch

[STEPS]
?. [Preparing iPSCs for Nucleofection]
Coat 1 well of a 6-well plate with   of Matrigel for  at
1 ml
37 °C
?. [Preparing iPSCs for Nucleofection]
Add   of DMEM/F12 to a 15ml conical tue
5 ml
?. [Preparing iPSCs for Nucleofection]
Thaw vial of cells in  water bath for approximately
37 °C
?. [Preparing iPSCs for Nucleofection]
Wipe off excess water from tube and spray with 70% Ethanol before placing vial into biosafety cabinet
?. [Preparing iPSCs for Nucleofection]
Transfer thawed cells into conical tube containing  DMEM/F12
5 ml
If some cells remain in vial use   DMEM/F12 to rinse vial clean and transfer into the 15ml conical
?. [Preparing iPSCs for Nucleofection]
Swirl to mix cells in the DMEM/F12 and
Centrifuge: 750 33, 3 min
Avoid rough mixing or suspension of cells; this helps to keep the cells clustered
?. [Preparing iPSCs for Nucleofection]
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)
?. [Preparing iPSCs for Nucleofection]
Add  of Rock Inhibitor to cell pellet and resuspend in  of mTesR1 in order to achieve a  solution. Gently swirl to mix
4 µl
2 ml
Avoid rough mixing or suspension of cells; this helps to keep the cells clustered
?. [Preparing iPSCs for Nucleofection]
Aspirate Matrigel from coated 6-well plate, and pipet cell mixture into well.
?. [Preparing iPSCs for Nucleofection]
Incuate overnight at
37 °C
?. [Preparing iPSCs for Nucleofection]
After  change media and allow cells to recover
?. [Preparing iPSCs for Nucleofection]
Once cells have reached 80-90% confluency, split culture as single cells into 3 wells of a 6 well tissue culture plate.
?. [Preparing iPSCs for Nucleofection]
Aspirate media from well containing cells
?. [Preparing iPSCs for Nucleofection]
Wash cells with  of PBS
2 ml
?. [Preparing iPSCs for Nucleofection]
Add  of Accutase per well
1 ml
?. [Preparing iPSCs for Nucleofection]
Incubate cells and Accutase at  for . Gently tap plate upon removal from incubator to help dislodge cells from Matrigel
37 °C
?. [Preparing iPSCs for Nucleofection]
Collect cells in  of PBS and transfer to 15ml conical tube
5 ml
?. [Preparing iPSCs for Nucleofection]
Centrifuge: 750 33, 3 min
?. [Preparing iPSCs for Nucleofection]
Resuspend cell pellet in  mTesR1 +  Rock Inhibitor and plate  into each freshly coated Matrigel well
6 ml
2 ml
?. [Preparing iPSCs for Nucleofection]
Coat 3 wells of a 6-well plate with  of Matrigel for  at
1 ml
37 °C
?. [Preparing iPSCs for Nucleofection]
Perform the single cell passage (Step 12) at least two additional times prior to using cells for Nucleofection
?. [Continuing iPSC Culture]
Perform a single cell passage approximately  prior to nucleofection
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
?. [Continuing iPSC Culture]
Coat three wells of a 6-well plate for nucleofection using Matrigel and incubate at  for  prior to splitting cells
37 °C
?. [Continuing iPSC Culture]
Just prior to splitting cells for nucleofection, aspirate Matrigel from coated wells and add  of DMEM/F12 + 10% FBS supplemented with  Rock Inhibitor per well
3 ml
?. [RNP Complex]
Gather the following reagents for Alt-R Reactions and let them thaw on ice  AB1ReagentStability and Storage2Alt-R crRNA6 months stability at -80°C3Alt-R tracrRNA6 months stability at -80°C4Alt-R Cas96 months stability at -80°C5Electroporation Enhancer (IDT - Catalog # 1075915)6 months stability at -80°C6sgRNA (if needed)6 months stability at -80°C
AB1ReagentStability and Storage2Alt-R crRNA6 months stability at -80°C3Alt-R tracrRNA6 months stability at -80°C4Alt-R Cas96 months stability at -80°C5Electroporation Enhancer (IDT - Catalog # 1075915)6 months stability at -80°C6sgRNA (if needed)6 months stability at -80°C
?. [RNP Complex]
Prepare Alt-R Reactions
?. [RNP Complex]
Prepare fresh Alt-R gRNA by adding  of  Alt-R crRNA and  of  Alt-R tracrRNA in equal volumes. Heat mixture at  for . Let cool to
2.5 µl
2.5 µl
95 °C
0 Room temperature
The Alt-R gRNA should be prepared fresh before each use
?. [RNP Complex]
Resuspend Alt-R Cas9 and Electroporation Enhancer in PBS to a final concentration of
?. [RNP Complex]
Combine Alt-R gRNA solution and Alt-R Cas9 + Electroporation Enhancer solution together and incubate at  for   AB1ComponentFinal Concentration2PBS-3Alt-R gRNA120 pmol4Alt-R Cas9104 pmol5Enhancer100 µM6Donor ssODN or GFP*100 µM
0 Room temperature
AB1ComponentFinal Concentration2PBS-3Alt-R gRNA120 pmol4Alt-R Cas9104 pmol5Enhancer100 µM6Donor ssODN or GFP*100 µM
RNP Complex stability limited to   at  . Do not freeze
?. [Split Cells for Nucleofection]
Split cells for nucleofection
?. [Split Cells for Nucleofection]
Aspirate media from cells
?. [Split Cells for Nucleofection]
Wash each well with  of PBS and aspirate
2 ml
?. [Split Cells for Nucleofection]
Add  of Accutase per well
1 ml
?. [Split Cells for Nucleofection]
Incubate at  for
37 °C
?. [Split Cells for Nucleofection]
Collect cells in  of PBS and transfer to a 15ml conical tube
5 ml
?. [Split Cells for Nucleofection]
Centrifuge: 750 33, 3 min
?. [Count Cells for Nucleofection]
Resuspend cell pellet in  of PBS in the 15ml conical tube, then dilute cells 1:10 ( of cell suspension +  of PBS) in a 1.7ml tube
1 ml
10 µl
90 µl
?. [Count Cells for Nucleofection]
Use  of diluted cells for cell counts
10 µl
?. [Count Cells for Nucleofection]
Using all four corners of the countess slide, calculate the average number of cells
?. [Count Cells for Nucleofection]
Multiply the average by 10,000 (104)
?. [Count Cells for Nucleofection]
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
?. [Count Cells for Nucleofection]
Take total number of cells calculated in step 20.3 and divide by three million
?. [Count Cells for Nucleofection]
Take answer from step 20.4 and divide by three to get the volume of cells necessary for nucleofection
?. [Centrifuge Cells for Nucleofection]
Transfer the desired volume of cells (calculated in step 20.5) to microcentrifuge tube
?. [Centrifuge Cells for Nucleofection]
Centrifuge: 90 34, 5 min
?. [Centrifuge Cells for Nucleofection]
Aspirate PBS from cell pellet
?. [Prepare Lonza Kit Reagents for Nucleofection]
Make reaction mix from Lonza Kit: P3 Primary Cell 4D (V4XP-3024). Each reaction requires a total of  of reaction mix.
100 µl
If performing more than one reaction, it is best to make a Master Mix
?. [Prepare Lonza Kit Reagents for Nucleofection]
Combine  of P3 Solution and  of Supplement into a 1.7ml microcentrifuge tube
82 µl
18 µl
?. [Combining Lonza Kit Reagents and DNA for Nucleofection]
Combine reaction mix from step 24 () with previously complexed DNA from step 17
100 µl
?. [Combining Lonza Kit Reagents and DNA for Nucleofection]
Mix reaction mix and DNA with cell pellets (step 23) by pipetting up and down with p200 pipette
Try to pipette as little as possible. Pipette only until mixed
?. [Combining Lonza Kit Reagents and DNA for Nucleofection]
Transfer  of reaction mix + DNA + cells to a cuvette
100 µl
Ensure no bubbles are in the transferred mixture of cells in the cuvette. This can interfere with the nucleofection's success
?. [Nucleofection]
Nucleofect with Lonza Program CA-137 in P3 Solution
?. [Nucleofection]
Let cuvette and cells incubate for  at
0 Room temperature
?. [Nucleofection]
Transfer cells/DNA solution to approproate pre-coated well containing  of DMEM/F12 + 10% FBS +  Rock Inhibitor
2 ml
?. [Nucleofection]
Incubate at  overnight
37 °C
?. [Post Nucleofection]
Continue culturing the iPSC in 1 well of a 6 well plate for 5-7 days post nucleofection, changing mTesR1 daily.
?. [Post Nucleofection]
post-nucleofection - add mTesR1 with  Rock Inhibitor
?. [Post Nucleofection]
post-nucleofection - add mTesR1 with  Rock Inhibitor
?. [Post Nucleofection]
post-nucleofection - add mTesR1 with  Rock Inhibitor
?. [Post Nucleofection]
Continue culturing cells in mTesR1 until confluent (~5 days)