SOP002 Human pluripotent stem cell HDR-mediated gene editing using CRISPR/Cas9 and PiggyBac technology

Day -1

Coat each well of a 6-well plate with 75µL of Laminin-521 (Biolamina 100µg/ml stock) diluted in 1mL of DPBS+Ca+Mg to make a final coating concentration of 7.5µg/ml. Incubate at 4°C . Prepare three wells of a 6 well plate per desired gene editing per PSC cell line.

Replace the medium of the PSCs with fresh 1.5mL StemFlex + 1micromolar (µM) rock inhibitor at least 1 hour before splitting, this ensures a good recovery of the PSCs following nucleofection.

Prepare 350µL P3 solution following the Lonza Nucleofector kit instructions (Cat No. V4XP-3024). Split P3 solution into three 100µL vials. Take pX458 Cas9 GFP and PiggyBac plasmid (e.g. pMV) out of the freezer and allow to reach 4Room temperature.

Add 1.5µg px458 Cas9 GFP plasmid and 1.5µg PiggyBac plasmid (e.g. pMV) per 100µL P3 reaction. Do this for two out of the three tubes and leave the last tube without plasmid or pMV plasmid only (random plasmid integration control) or px458 GFP with no gRNA (gene editing control). Mix thoroughly and leave at 4Room temperature in the hood.

Remove medium from wells. Wash once with 1X DPBS and then add 1mL Acutase per well. Incubate at 37°C for 0h 10m 0s if cells have been grown on Laminin-521.

While cells are incubating on acutase wash the Lam-521 coated plate that you prepared on step 1 with

PBS-Ca-Mg to remove all traces of the PBS+Ca+Mg. Add 2mL StemFlex media containing 1micromolar (µM) Rock inhibitor and put back in 37°C incubator.

Flush cells from the plates surface (See step 5) and then transfer to a 15mL falcon (pool all three same cell line wells into the same falcon tube). Wash wells with 2mL HES medium each and add to falcon. Pass cells through a 100micromolar (µM) cell strainer to remove large debris clumps. Count live cells using the Contessa II and add 2 million cells per 15mL falcon tube. Do this for three falcons. Spin cells down at 900rpm,0h 0m 0s. Remove medium and gently resuspend pellet in the 100µL P3 solution + Cas9 + PiggyBac plasmid (don’t forget the negative control too).

Transfer each of the three samples into three 100µL Lonza Nucleofector cuvettes (make sure you don’t insert any bubbles). Take samples to the Amaxa 4D nucleofector and nucleofect using program CL113.

Transfer the nucleofected 100µL samples to the previously prepared Lam-521 coated 6-well plates containing 2 ml StemFlex + Rock inhibitor per well, 100µL of nucleofected sample per well. Wash the cuvette from any remaining cells using StemFlex and add also to the well. Place back in the 37°C incubator and incubate 0h 10m 0s.

24 hours post nucleofection remove rock inhibitor and replace media with fresh StemFlex.

48 hours post nucleofection select cells with StemFlex + puromycin at 0.5µg/ml + rock inhibitor.

72 hours post nucleofection (24 hours post Puro selection) remove rock inhibitor and continue puro selection at 0.5µg/ml.

96 hours post nucleofection (48 hours post Puro selection), check cells and keep at 0.5µg/ml puro. At this point some clear clones will be forming in the plate, see Figure 4I. Use the objective marker to mark clonal colonies (Figure 4).

7 days post nucleofection and 5 days post puro selection at 0.5µg/ml (See Figure 3), your negative control sample (Cas9 with no gRNA or PiggyBac only controls) should be all dead. At this point you can continue at 0.5 or go ahead an increase the concentration of puromycin to 0.7µg/ml. At day 9 puro resistant PSC colonies should look like Figure 4III.

At around day 14 your puro resistant colonies should be about the same diameter as your objective marker mark. Now it is a good time to pick them and transfer them to a Lam-521 coated 12 well plate. 1 hour before picking colonies, add 1.5mL fresh StemFlex media with Rock inhibitor (this reduces cell death upon colony picking). Also add 1mL of StemFlex+rock inhibitor to each well of the Lam-521 coated plate.

Use the dissection microscope under the tissue culture hood for the picking. Using a 10µL tip scratch the PSC colony in a squared pattern, making small PSC clumps of cells out of the whole PSC colony. Then suck all the clumps and transfer them to the 12 well plate well. If clumps are too large you may pipette up and down a couple of times but do so very gently as the cells can get stressed and die at this point.

Allow the picked clones to recover 0h 10m 0s under StemFlex + Rock inhibitor. 24 hours later replace media with fresh StemFlex only.

48 hours post colony picking and, once the PSC colonies have recovered, add 0.5µg/ml-0.7µg/ml puromycin and keep under selection.

At around day 21 (see Figure 3), the 12 well plate wells should be around 30% confluent. Split them at 1:2 ratio into a new lam-521 coated 12-well plate. Keep ½ of the cells for genomic extraction using Quickexctract reagent (see manufacturer instructions).

Once you have extracted your genomic DNA (gDNA) carry out PCRs on the gDNA to amplify your gene editing region of interest and for the presence of the left and right homology arms of your PiggyBac plasmid (Figure 5A). For example, for SNCA E46K repair, use the NGS primers to PCR the region of interest and primers 11 and 22 for the left homology arm and primers 12 and 23 for the right homology arm. Use Q5 polymerase (NEB) for the NGS PCR and 2xTaq polymerase (NEB) for the homology arm PCRs. Figure 5Ai shows a diagram of where the homology arm primers are binding. Figure 5Bi shows the presence of left and right homology arm PCR products, indicating the CRISPR/Cas9 HDR mediated knock-in of the PiggyBac cassette containing your repair DNA sequence was successful.

Send the PCR products of the NGS PCR products for sanger sequencing using primer F27. If the sequencing shows correction and left and right homology are present for a particular clone this indicates that there is a good chance that that particular clone is corrected. Discard the clones that show no homology arms or correction.

At around day 25 split the gene edited puro resistant clones (Once they are about 80% confluent) and also freeze 1 vial of each. Use 500µL StemCell Banker reagent for the freezing, this reagent allows you to store the cells at -80°C for a short time and is more stable than other options. Culture positive clones under Lam-521 and StemFlex conditions and puromycin 0.5µg/ml-0.7µg/ml until they are 80% confluent.

At around day 28, nucleofect positive clones with an excision only transposase (e.g. pBX plasmid) to remove the PiggyBac selection cassette and leave only the gene edit of interest behind in a scar-less manner. (Figure 5AI-II). To do so nucleofect 3 million cells of your iPS clone with 3uf of pBX plasmid using the same directions as steps 2 to 9, in terms of plating, media and process.

48 hours post pBX transfection of each positive clone, day 30, check for GFP expression under the epifluorescence microscope and sort all GFP sorted cells into a Lam-521 coated plate containing StemFlex and CloneR (sort anything between 500 (ideally) to 4000 live GFP+ve cells per 6-well plate well).

At day 32, if PSC cells have recovered and started forming colonies, you can start Ganciclovir negative selection at 4micromolar (µM) for 120h 0m 0s.

At day 37 remove GCV from the media and allow cells to recover for 3 days. Pick up to 4 clones per each pBX transfected positive clone and transfer to a Lam-521 coated plate, follow steps 15 to 20. Carry the PCR of the gene edited region of interest using the NGS primers but also primers 14 and 15, this primer set flanks the PiggyBac cassette meaning that if the PiggyBac cassette remains present in that particular clone you will not be able to amplify it because the cassette is still integrated. If the cassette is not there any longer you should get a product that you can Sanger sequence. For Sanger sequencing of the primers 12 and 15 PCR product use F27 and R29 primers.

The resulting genotyped positive gene edited clones following pBX transfectiona and GCV selection should show no homology arm PCR products as shown in Figure 5Bii. The final Sanger sequencing result should be very clean and give the same result when sequencing the NGS of primers 14+15 PCR product, chromatogram should look something like Figure 5C.