Protocols

(Adapted from X.Lian et. al. Nature Protocol 2013)

Media/Reagents:

· For plating iPSCs for iPSC-CM differentiation

o Autoclaved/sterile passaging tool and pipet tips.

o 0.5 mM EDTA in D-PBS

o mTeSR1 (or Essential 8 flex media)

o Geltrex-coated 12-well or 6-well plates

o Centrifuge

o Hemocytometer

o 10 mM Y27632 (Rock inhibitor) stock (Tocris #1254 or others)

o Access to room temp 15 mL tube centrifuge (use at 300 x g).

· iPSC-CM Differentiation: Days -3 Onward

o mTeSR1 (better than Essential 8 media in differentiation experiments)

o CHIR99021: 10 mM in Sterile DMSO (ordered from Cayman Chemical)

o RPMI/B27 – insulin (B27 supp – insulin: Life Technologies #0050129SA)

§ (RPMI: use the media prepared by the tissue culture facility)

§ stored at 4°C for up to 1 month

o RPMI/B27 + insulin (B27 supplement: Life Technologies #17504-44)

§ stored at 4°C for up to 1 month

o IWP2 (Tocris #3533-10 mg)

Note: Cell differentiates the best in 12-well plates. 6-well or 24-well plates could work but the efficiency (% of cTnT+ cells) is generally not as good.

Plating iPSCs for iPSC-CM Differentiation

1. Before starting:

a. Note: Make sure that cells are at 80-90% confluence.

b. Warm mTeSR1 media to RT (do not use water bath).

c. Prepare 12-well Geltrex-coated plate(s).

2. Undifferentiated iPSCs were normally maintained in Essential 8 flex media (ThermoFisher) in 6-well plates.

3. Aspirate medium from wells and add 1 mL of RT 0.5mM EDTA to each well (of a 6-well plate).

4. Incubate at 37˚C for 10 min.

5. Add 1 mL mTeSR1 (or Essential 8) to each well (of 6-well plate) and pool cells in a 15 mL conical. Mix the cells using a 5 mL pipet.

6. Aspirate supernatant and resuspend cells (generally around 150,000-200,000 depending on the strain) in mTeSR1 + 5 uM Y27632 (Rock inhibitor) at to reach a confluency of around 20-30% in each well of a 12-well plate coated with Geltrex. Add mTeSR1 + 5 uM Y27632 (Rock inhibitor) medium to each well to make a final volume of 1 mL (<1.5 mL) in each well of the 12-well plates. After seeding, be sure to shake plates to ensure that cells are evenly distributed. This time point corresponds to day -4

iPSC-CM Differentiation: Days -3 Onward

1. Day -3, day -2, and day -1: Aspirate media and replace with 2 mL/well RT mTeSR1.

2. Day 0: The cells should be around 90% confluent on this day, and this confluency is very important. Aspirate media and replace with 2 mL/well 12 uM CHIR99021 RPMI/B27-insulin media. Record the time. May need to test range of CHIR99021 (6-14 uM). In my hand, 12 µM works the best for WTC-11 strain.

3. Day 1 (24 hours later exactly): Aspirate media and replace with 3 mL/well of RT RPMI/B27-insulin.

4. Day 3 (72 hrs after CHIR99021 addition): Collect 1.5 mL/well medium from each well and mix with 1.5 mL/well fresh RPMI/B27-insulin (in 50 mL tube) – this is called combined medium.

a. Add 1 uL of 5 mM IWP2 (final concentration is 5 uM) for each 1 mL combined media.

b. Prior to aspirating, gently rock the plate back and forth to get cell debris into suspension, ensuring that the cell debris will be removed after aspiration.

c. Aspirate the remaining 1 mL/well and add 3 mL/well of combined media + IWP2.

5. Day 5: Aspirate media and replace with 3 mL/well RT RPMI/B27-insulin.

6. Day 7 and every 3 days following (Tues., Fri., Mon., etc.): Aspirate media and replace with 3 mL/well RT RPMI/B27 (+ insulin).

a. Spontaneous beating monolayers are generally observed from day 8. Robust spontaneous contraction should occur by day 12.

b. Cells can be maintained with this spontaneous beating phenotype for 6+ months.

Characterization of hiPSC-derived CMs via Flow Cytometry (from X.Lian et. al. Nature Protocol 2013)

1. Wash the differentiated cells with 1 ml of PBS per well in a 12-well plate. Aspirate the PBS.

2. Add 0.4 ml of 0.25% (wt/vol) trypsin-EDTA per well and incubate the cells in a 37°C for 15 min. Pipette the mixture 5–10 times with a P1000 tip to singularize the cells, and then add 1 mL of RPMI20 medium and transfer the cell mixture into a 1.7mL tube.

3. Centrifuge cells at 700g for 5 min at room temperature and aspirate the supernatant.

4. Add 1 ml of 1% (vol/vol) formaldehyde in 1X PBS (MAKE FRESH!!!) to resuspend the cell pellet and then incubate at room temperature for 20 min.

5. Centrifuge the cells at 2000g for 5 min at room temperature, aspirate the supernatant and then resuspend the fixed cells in 1 mL 1XPBS. Repeat this step twice to remove the formaldehyde completely.

6. At this point, the cells can be stored at 4 degree (up to two weeks).

7. Centrifuge the cells at 700g for 5 min at room temperature, aspirate the supernatant and then add 1 ml of 90% (vol/vol) cold methanol per tube. Incubate the mixture at 4 °C for 15 min.

8. Centrifuge the cells at 800g for 5 min at room temperature, aspirate the supernatant and add 1 ml of FlowBuffer-1. Repeat this wash two times to remove the methanol. At the last wash, distribute the cells into two tubes so that an isotope control can be performed for each sample.

9. Centrifuge the cells at 800g for 5 min at room temperature, and resuspend the cell pellet in 250 μl of FlowBuffer-2 with the appropriate dilution of primary antibody (1:200 dilution for Troponin T antibody from ThermoFisher MA5-12960; or 1.5uL of BD Alexa Fluor® 647 Mouse Anti-Cardiac Troponin T, cat 565744). Incubate the mixture for 1 h at room temperature or at 4 °C overnight. Always do an isotype control for each sample (e.g. BD Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, cat 557732)

[If use the alexa647 conjugated antibody, the following step should be skipped]

10. Wash the cells with 2 ml of FlowBuffer-2, centrifuge the cells at 200g for 5 min at room temperature, and resuspend the cell pellet in 250 μl of FlowBuffer-2 containing 1:1,000 dilution of secondary antibody (CellSignaling 4408S, anti-mouse Alexa488; can be combined with goat anti-rabbit Alexa647 if a second marker is checked). Incubate the mixture for 30 min at room temperature in the dark.

11. Wash the cells with 2 ml of FlowBuffer-2 (as in step 8) two times; resuspend the cell pellet in 300 μl of FlowBuffer-1 and transfer it into flow round-bottom tubes. Place the flow tubes on ice and perform the flow cytometric analysis.

Reagents:

Troponin T antibody:

BD Alexa Fluor® 647 Mouse Anti-Cardiac Troponin T, cat 565744

or

Thermo-Fisher MA5-12960, https://www.thermofisher.com/antibody/product/Cardiac-Troponin-T-Antibody-clone-13-11-Monoclonal/MA5-12960

Secondary antibody: CellSignaling 4408S, Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate), https://www.cellsignal.com/products/secondary-antibodies/anti-mouse-igg-h-l-f-ab-2-fragment-alexa-fluor-488-conjugate/4408

FlowBuffer-1 (500 ml) Add 2.5 g BSA into 500 ml PBS and filter using a 500 ml Stericup filtration system. The medium can be stored at 4 °C for up to 6 months.

FlowBuffer-2 (550 ml) Add 2.5 g BSA and 50 ml 1% Triton X-100 solution into 500 ml PBS and filter using a 500 ml Stericup filtration system. The medium can be stored at 4 °C for up to 6 months.

We present an optimized protocol for introducing mutations in human iPSCs, and this protocol could also be easily adapted for other cell types.

1 iPSC feeder-free culture

Most iPSC strains have been adapted to feeder-free culture nowadays. We routinely use a well-characterized human iPSC strain WTC-11 made by Dr. Bruce R. Conklin’s group (Gladstone Institute of Cardiovascular Disease, UCSF, cell line distributed by the Coriell Institute #GM25256). A variety of iPSC culture media and coating matrix formulations have been developed in the past two decades, and many of them are available commercially: (a) medium - Essential 8 medium (Thermo Fisher A1517001) and mTeSR1 (StemCell Technologies 85850); (b) coating matrix - Vitronectin (Thermo Fisher A14700), Matrigel (Corning 356234) and Geltrex (Thermo Fisher A1413201). These media and coating materials can be used in any combination. We regularly maintain our iPSC strains in Essential 8 Flex medium (E8 Flex, Thermo Fisher A2858501) combined with Vitronectin as the coating matrix. This combination maintains the cell pluripotency very well, and E8 Flex medium offers every-other-day medium change instead of the regular daily medium change with most other media.

For preparing the culture medium

Ø E8 Flex Medium Preparation

o For 500 ml E8 Flex Bottle

§ Thaw E8 Flex supplement (stored in –20 ºC freezer) at room temp (takes ~2-3 h on bench, or ~30 min in a water bath or metal block)

§ The remaining steps are done in a sterile hood

· Mix E8 Flex supplement by shaking the bottle several times

· Transfer entire E8 Flex supplement bottle (10 ml) to the 500 ml E8 Medium bottle

§ Add 2.5 ml of 100x Penicillin-Streptomycin (Sigma-Aldrich P4333) to the medium. Aliquot in 50-ml conical tubes and store for up to two weeks at 4 ºC

For passaging the cells:

Ø Coating plates with Vitronectin (VTN-N)

o Recombinant VTN-N (Gibco A14700) is aliquoted into 30 or 60 µl that can coat 3 or 6 wells of a 6-well plate. These aliquots are then stored in a -80 ºC freezer.

o To coat a 6-well plate, remove an aliquot of VTN-N from the -80 ºC Freezer. Thaw at room temp (takes ~1-2 min)

o Add VTN-N to sterile dPBS (Dulbecco's Phosphate-Buffered Saline)

§ 6 ml for 60 µl aliquot

§ 3 ml for 30 µl aliquot

· Note: Use a 200 µl pipette and rinse the tube with dPBS to collect all the VTN-N solution

o Mix the VTN-N solution by capping the tube and inverting several times

o Add 1 ml of VTN-N solution to each well that will be coated

o Leave plate in a sterile hood for at least 1 hour

§ Note: Take the E8 medium out of the fridge at this point and place in the hood to warm up

Ø Passaging

o Let E8 medium and VTN-N coated plates equilibrate to room temperature (if the 6-well plates were stored at 4 ºC)

o Aspirate old E8 medium from the 6-well plate

o Rinse each well with 2 ml of dPBS

o Aspirate dPBS and add 1 ml of 0.5 mM EDTA in dPBS

o Place in incubator for 5-8 min

o Aliquot E8 Flex Medium into a separate tube

§ For most passages use a 1:6 or 1:8 dilution --> for each well passaging, dilute cells into 6 ml of new medium

o After 5 min, look at cells under microscope - cells will separate and colonies will appear to round up. Colonies will also have holes in them.

o Aspirate out EDTA solution

o To recover cells, add E8 Flex medium into the well (~2 ml per well of the 6-well plate) from the aliquoted medium

§ Gently squirt medium to remove cells from the entire well. Try to avoid making bubbles and pipetting too much because mechanical stress is deleterious.

§ Use a 5 or 10 ml pipette tip to avoid creating too much mechanical stress

o Collect and add the recovered cells to the aliquoted E8 Flex medium

o Gently mix the cell solution by capping the tube and inverting several times

o Remove the VTN-N solution from the new plate by aspiration

o Add 2 ml of the cell solution to each well (recommended: add 5 µM inhibitor of ROCK (Rho-associated, coiled-coil containing protein kinase), Tocris 1254)

o Place the new 6-well plate into the incubator and shake back and forth several times to spread the cells evenly across the surface

o Change medium on the next day

o After the first medium change, E8 Flex medium can be changed every other day until confluency

For freezing of iPSCs:

o Prepare Freezing Medium

§ Combine 0.9 ml of E8 Flex Medium with 0.1 ml of DMSO for each well of a 6-well plate to be frozen down

§ Place freezing medium into the 4 ºC fridge until ready to use

o Aspirate old E8 medium from the 6-well plate

o Rinse each well with 2 ml of dPBS

o Aspirate out dPBS and add 1 ml of 0.5 mM EDTA (in dPBS)

o Place in incubator for 5-8 min. Meanwhile, take the E8 Flex medium out of fridge.

o After 4 min, look at cells under microscope - cells will separate and colonies will appear to round up. Colonies will also have holes in them.

o Aspirate out EDTA solution

o To recover cells, add freezing medium into the well (1 ml per well) from the aliquoted medium

§ Gently squirt medium to remove cells from the entire well. Try to avoid making bubbles and over-pipetting to avoid mechanical stress.

§ Use a 5-10 ml pipette again to avoid creating too much mechanical stress

o Combine the contents of all identical wells into a tube and then aliquot 1 ml of cell suspension into each cryotube

o Place into the cell cryofreezing container (Thermo Scientific 5100-0001) and move to -80 ºC overnight

o After overnight storage at -80 ºC, transfer the cells to the large liquid nitrogen tank.

For thawing iPSCs:

Ø Coat 6-well plates with Vitronectin as described above

Ø Thaw iPSCs

o Add 10 ml of room temperature E8 medium into a 15 ml tube

o Remove iPSCs from the liquid nitrogen storage tank

o Immerse vial in 37 ºC water bath and swirl it gently

o When only a small ice crystal is left, move to the hood (use caution as the label might come off)

o Collect the cells with a 5 ml pipette and add dropwise into 10 ml of E8 Flex medium

§ Drop-wise prevents osmotic damage to the cells

o Rinse the vial with 1 ml of E8 Flex medium to collect any remaining cells

o Spin down the cell suspension in a 15 ml tube at 200 g for 5 min

o Aspirate the supernatant

o Resuspend cells in 2 ml (or appropriate volume based on the cell number and dish to be used) of E8 medium (per vial thawed) by pipetting up and down several times. Add 5 µM ROCK inhibitor (important for robust cell survival after thawing).

o Aspirate the VTN-N solution from the plate

o Add 2 ml of cell solution per well

o Place in the incubator and shake plate back and forth several times to spread the cells evenly

o Change medium on the next day to E8 Flex medium without ROCK inhibitor

o After the first medium change, E8 Flex medium can be changed every other day until confluency

2 Transfection of CRISPR and donor plasmids

Two plasmids are used for the delivery of the editing machinery: a CRISPR plasmid containing the SpCas9 and sgRNA and a donor plasmid containing the homologous recombination cassette with homology arms and antibiotic resistance cassette (Fig. 2).

It is generally more difficult to transfect iPSCs than other commonly used immortalized cell lines (e.g., HEK293T), but several relatively effective methods have been developed including electroporation and lipid-mediated transfection (14,15). As electroporation-based transfection may have low cell survival rate, we routinely use Lipofectamine 3000 (Thermo Fisher L3000015) or Stem Reagent (Thermo Fisher STEM00015) to deliver the CRISPR and donor plasmids.

Ø Seeding iPSCs for transfection

o Passage the cells on the day before the transfection, aiming to obtain ~40-50% confluency at transfection in a 6-well plate.

Ø Transfection using Lipofectamine 3000

o Prepare an appropriate volume (2 ml per well for 6-well plates) of regular E8 medium (Thermo Fisher A1517001) without antibiotics, warm up to room temperature. Note: we have found that transfection works better in regular E8 medium compared to the E8 flex medium, and removal of the antibiotics is essential for cell survival after transfection.

o For each well of a 6-well plate, dilute 3 µg DNA (1.5 µg of the CRISPR plasmid and 1.5 µg of the donor plasmid) in 125 µl Opti-MEM medium (Thermo Fisher 11058021). Add 6 µl P3000 reagent (2 µl/µg DNA), mix well. It is important to include a “donor DNA only” control to differentiate stable genomic integration from transient expression. For this control, omit the CRISPR plasmid and only include 1.5 µg donor plasmid.

o For each well of a 6-well plate, dilute 7.5 µl Lipofectamine 3000 reagent in 125 µl Opti-MEM medium. Mix well.

o Combine the diluted DNA and the diluted lipofectamine reagent. Mix it well and incubate at room temperature for 15 min.

o During the 15 min, take out the cultured iPSCs (in 6-well plates) from the incubator. Aspirate the medium and replace it with the freshly prepared E8 medium without antibiotics.

o Add 250 µl of the DNA-lipid complex to each well. Gently stir the plate to mix well.

o After 6 hours or on the next day, replace the medium with E8 flex medium. mCherry fluorescence can be examined to determine transfection efficiency. mCherry intensity and transfection efficiency are expected to be similar between the experiment and the “donor DNA only” control.

As an alternative to our plasmid-based CRISPR delivery system, pre-assembled ribonucleoprotein preparations of Cas9 and sgRNA may be able to achieve high efficiency (16,17), but we have not tested this approach in iPSCs.

3 Drug selection

The purpose of the drug selection is to select not only the transfected cells, but also the successful genomic integration. In general, iPSCs lose the transient transfected plasmids gradually and completely at around two weeks post-transfection. This means that the selection needs to be performed at least for two weeks. A key to successful selection is to use Matrigel or Geltrex, rather than vitronectin, as the coating matrix.

Ø Passage cells on to Matrigel or Geltrex coated 10-cm plate

o Maintain the growth of transfected iPSCs in the 6-well plate by changing E8 flex medium every other day.

o When the wells reach 80% confluency, passage the cells in each well to a new 10-cm plate coated with Geltrex (Thermo Fisher A1413301) or Matrigel (Corning 354277).

o To coat a 10-cm plate with Geltrex or Matrigel, take out a 70 µl aliquot of Geltrex or Matrigel that was stored in -80 ºC. Place it immediately on ice and bring the ice bucket in a sterile hood.

o In the hood, add 1 ml of cold DMEM (Dulbecco′s Modified Eagle′s Medium) basal medium without any supplement (Gibco 12800-082) to the Geltrex/Matrigel. Pipette up and down to thaw and dilute the Geltrex/Matrigel.

o Add 6 ml of the cold DMEM medium to a new 10-cm plate, and then add the diluted Geltrex/Matrigel. Rock back and forth to mix the solutions.

o Place the plate in 37 ºC incubator for at least 30 min, and then the plate is ready to be used for iPSC culture. If the coated plate won’t be used on the same day, wrap it in parafilm and store it at 4 ºC.

o Cell passaging is done similar to what is described above. In detail, cells grown in each well of the 6-well plate are detached using 0.5 mM EDTA in dPBS (note: try breaking cell clusters when pelleting) and transferred to 11 ml of E8 flex medium in a 10-cm plate. Importantly, 5 µM Rock inhibitor is added to the medium to prevent cell death during matrix change. Place the 10-cm plate in 37 ºC 5% CO₂ incubator for overnight.

o On the next day, replace medium with 11 ml fresh E8 flex medium to remove the Rock inhibitor. Check cell survival and attachment under a microscope.

Ø Drug selection

o Perform medium change every other day until the confluency reaches 70-80%.

o Thaw a 1 mg/ml puromycin aliquot (diluted from Gibco A1113802). Add 5.5 µl to 11 ml fresh E8 flex medium (final concentration of puromycin is 0.5 µg/mL) and mix it well.

o Replace the old medium in the 10-cm plate with the fresh medium supplemented with 0.5 µg/ml puromycin.

o On the next day, a large fraction of cells should have lifted from the plate, while some cell clusters remain attached. Change medium to E8 flex supplemented with 0.25 µg/ml puromycin. This medium change also helps to remove the lifted cells.

§ Note: we use half concentration of puromycin starting at the second day of the selection, because we observed that a lower concentration of puromycin is required at lower cell density. In other words, on the first day of selection a higher concentration is needed when the cell density is high, while a reduced concentration is required for survival of the positive clones. We have confirmed that the optimal concentration of puromycin positively correlates with the cell density by performing drug killing curves.

o Replace medium every other day with E8 flex supplemented with 0.25 µg/ml puromycin for around two weeks until the survival colonies are almost touching each other.

o During the two-week selection process, the “donor DNA only” control gradually loses its viable colonies, and successful genomic integration is indicated by a large difference in the number of surviving colonies between the experiment and the “donor DNA only” control.

4 Colony isolation and clonal screening

How many colonies need to be screened may depend on several factors: (a) the efficiency of the homologous recombination using the transfected DNA donor, (b) the accessibility of the specific genomic locus, (c) whether homozygous or heterozygous mutants are desired. Factors 1 and 2 can be optimized in experimental design. Regarding factor 3, 48-72 colonies would be ideal to obtain a homozygous mutant, while 24 colonies are often sufficient to screen for a heterozygous mutant.

Ø Pick colonies using a stereo microscope

o Prepare Matrigel/Geltrex-coated 24-well plates similar to what was described above for 10-cm plates. The volume of DMEM-diluted Matrigel/Geltrex to be add in each well is 300 µl. Incubate the plates at 37 ºC for at least 30 min.

o Prepare the culture medium by supplementing E8 flex medium (already contains 0.5 x Penicillin-Streptomycin as described earlier) with 5 µM Rock inhibitor and 0.5 µg/ml Amphotericin B (Gibco 15290018). Amphotericin B prevents fungal contamination and can be omitted if the colony picking environment is sterile (e.g., the stereo microscope is placed in a sterile hood).

o Remove the Matrigel/Geltrex solution from the 24-well plates. Distribute 500 µl of prepared E8 flex medium in each well of the 24-well plates.

o Under a stereo microscope (Nikon SMZ1500), examine the surviving colonies and select the ones with circular shape, smooth and well-defined edge, single layer and uniformed organization of cells. Using an ethanol-wiped pipetman with a 10 µl sterile filtered tip (USA Scientific 1180-3810), scrap the selected colony in stripes carefully under the microscope without touching other colonies.

o Aspirate the stripes of the picked colony carefully under the microscope and place them in a well of the coated 24-well plates. Label the well with a check mark on the lid, to avoid cross-contamination.

o Repeat the picking process for other undifferentiated colonies until the number of desired colonies is reached (48-72 colonies for homozygous and 24 for heterozygous).

o Rock the plates back and forth several times to evenly distribute the scraped cells.

o (optional) Spin down the plates in a swinging-bucket tabletop centrifuge at 200g for 4 min.

o Place the 24-well plates in a 37ºC 5% CO₂ incubator.

Ø Maintaining the picked colonies

o Change medium on the next day with regular E8 flex medium without Rock inhibitor and Amphotericin B.

o Perform medium change every other day until the cells are confluent enough to be passaged. Please note that different wells will reach confluency at different days.

o Examine the wells that are ready to be passaged under the microscope. Mark the wells with cells that have good stem cell morphology (smooth colony edge, tight cell-to-cell contact in each colony and so on). Omit the wells that have a significant fraction of undifferentiated cells. Only proceed with the marked wells for passaging.

o Aspirate the medium, wash cells once with dPBS, and add 300 µl 0.5 mM EDTA in dPBS. Incubate at 37ºC for 7 min, aspirate the EDTA solution, and resuspend the detached cells in 1 mL E8 flex medium.

o Split the 1 mL resuspended cells into two fractions: 400 µl for cryo-preservation and 600 µl for genomic DNA extraction.

o For cryo-preservation, mix the 400 µl resuspended cells with 500 µl E8 flex medium and 100 µl DMSO. Perform slow freezing as described earlier.

o Spin down the remaining 600 µl at 800g for 5 min. Discard the supernatant and store the pellet in -20 ºC until ready for genomic DNA extraction.

5 Validation of successful integration using PCR and western blot

PCR validation is key to characterization of the guided repair at the Cas9 cleavage site, and it can help to determine the homozygous or heterozygous status of the mutation. Besides the conventional PCR approaches, droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with precision and sensitivity (18). Sanger sequencing of the PCR products is recommended to further validate the sequence alteration. We have adapted a previously developed quick genomic DNA extraction procedure(19), and here we describe a comprehensive protocol for genomic DNA PCR.

While correct repair at the DNA level is primary, correct expression of the edited gene also needs to be examined. This is important because mRNA processing, translation and protein stability may be perturbed as a result of a DNA sequence alteration.

Ø Genomic DNA PCR

o Supplement the lysis buffer (100 mM Tris-HCl pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, stored at room temperature) with 100 µg/mL Proteinase K (Invitrogen AM2544)

o Resuspend the cell pellet in 200 µl supplemented lysis buffer. Incubate at 55 ºC for 30 min.

o Add 200 µl isopropanol to each tube at room temperature. Invert several times to mix. A white precipitate may be visible.

o Spin down at 13,000g for 15 min.

o Aspirate the supernatant and air-dry the pellet for 3-5 min.

o Resuspend the pellet in 20 µl of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8). Incubate the solution at 55 ºC for 30 min to dissolve the nucleic acids.

o Set up two PCR reactions (e.g., primer pairs P1+P2 and P1+P3 in Fig. 3A) for each clone and a parental genomic DNA control: 0.02 U/µl Phusion DNA polymerase (Thermo Scientific F530), 1X Phusion GC buffer, 200 µM each dNTPs, 3% DMSO, 0.5 µM each primer, 0.5 µl genomic DNA in a 20 µl reaction.

o Proceed with the following thermal cycles: 98ºC for 30 s; 30 cycles of {98ºC for 10 seconds, X(calculated using the ThermoFisher Tm calculator)ºC for 30 s and 72ºC for 30 s per kilobase of the amplicon}c; 72ºC for 30 s; 4ºC hold.

o Add 4 µl 6X loading dye (New England Biolabs B7024S), and load 12 µl on a 1% agarose 1XTAE gel with 0.5 µg/mL ethidium bromide (Sigma-Aldrich E1510). Also load a lane with 1 µg of the 1 kb plus DNA ladder (Invitrogen 10787018). Run gel at 120 volts for 35 min (adjust the time according to amplicon sizes). Visualize the gel using an Alpha Innotech FluorChem HD2 chemiluminescent imager (ProteinSimple, Inc).

o PCR fragments for validated clones are gel-extracted using a gel extraction kit (Omega Bio-Tek D2500-02) and the DNA is sequenced by the Sanger method.

Ø Western blot to validate expression

o PCR-validated clones are thawed and grown in E8 flex medium. For well-behaved (undifferentiated and proliferating normally) clones, cells are passaged to two wells in 6-well plates. One well is used for cryo-preservation or downstream applications, while the other one is used for western blot analysis of the protein product of the edited gene.

o At cell confluency, aspirate the medium and add 20 µl lysis buffer (1X NuPAGE LDS Sample Buffer (Invitrogen NP0008) supplemented with 1.25 unit of Benzonase (Millipore E1014, to degrade DNA and RNA)) directly to the adherent cells.

o Incubate the 6-well plate in a 37ºC incubator for 10 min. Rock the plate every 3 min.

o Transfer the lysate to a 1.7 mL Eppendorf tube and resume 37ºC incubation for another 20 min.

o The lysate can be immediately used for western blot analysis or stored at -20ºC.

o For western blot analysis, load 10 µl of each lysate in a NuPage 4-12% Bis-Tris gel (Invitrogen # NP0322BOX, 12 well) and include a protein ladder (SeeBlue Plus2 Pre-stained Protein Standard, Invitrogen LC5925).

o Run gel(s) at 140-150 V for 1 h in 1X MES running buffer (Invitrogen NP000202).

o Transfer the proteins from the gel to a nitrocellulose membrane (Amersham Protran 0.45 NC, GE Healthcare 10600002) in cold 1X Western buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS). Run at 0.5 Amp constant for 1 hour.

o Disassemble and examine that the pre-stained protein ladder has been transferred to the nitrocellulose membrane.

o Place the membrane in a square petri dish, and block the membrane with 10 mL blocking buffer for 15 min (StartingBlock T20 PBS Blocking, Thermo Scientific 37539).

o Discard the blocking buffer and add 10 mL of diluted primary antibody in the blocking buffer. Incubate overnight with rotation.

o Remove the primary antibody solution and store it at 4ºC with a supplement of 0.02% sodium azide (can be used for at least 5 more times). Wash the membrane with PBST (1X PBS pH 7.4, 0.05% Tween) for three times.

o Add secondary antibody diluted in the blocking buffer. Incubate for one hour with rotation.

o Wash the membrane with PBST for three times, and 1XPBS once.

o Develop western with SuperSignal West Pico Chemiluminescent Substrate kit (Termo Scientific 34080). Mix 5 mL of SuperSignal West Pico Stable Peroxide Solution (#1856135) and 5 mL of SuperSignal West Pico Lumino/Enhancer Solution (#1856136) first, and then add the 10 mL solution to the membrane.

o Wait for one min, and image with an Alpha Innotech FluorChem HD2 chemiluminescent imager.

Ø Verified iPSC clones can be sent to WiCell (https://www.wicell.org) for karyotyping to confirm that there is no genome abnormity. Pluripotency of the clones can be examined by immunofluorescence using SOX2, OCT4 and SSEA4 antibodies as described previously (20). Moreover, p53 loss of function and other associated effects have been seen in CRISPR-manipulated stem cells (21). To help guard against genome sequence mutations acquired during the CRISPR process, which could be misleading, multiple independent clones should be obtained and tested.

Ø The validated clones are now ready for downstream analysis, which can include directed differentiation, next-generation sequencing, and proteomic analysis.

6 Optional removal of the LoxP cassette using Cre

The LoxP-flanked cassette can be removed by transfection of the Cre recombinase. This transfection can be performed after validation of the single clones from the last step, but more efficiently it can be performed immediately in the polyclonal population after the puromycin selection. The latter avoids repeating the clonal screening steps and significantly saves time and effort.

o [continued from the end of section 3] All colonies in the 10-cm petri dish are detached using 0.5 mM EDTA in dPBS. Half of cells are split into a well of a 6-well plate for Cre transfection, while the other half are cryo-preserved in E8 flex medium with 10% DMSO.

o On the next day, transfect the cells with a Cre expression plasmid with a Blasticidin S resistance gene (e.g., addgene 140284). Transfection protocols are same as described earlier.

o Blasticidin S selection is performed similar to what was described earlier for puromycin, except for the concentration: 2 μg/mL blasticidin is applied on the first day of selection, 1 μg/mL on the second and third day (drug concentration is reduced for the same reason as mentioned above in the puromycin selection), and 0 μg/mL afterwards.

o Clonal screening and western validation are performed exactly as described earlier.