Organoid Electroporation using CRISPR RNP method

Expand organoids as previously described. Aim for 10-20 wells of organoids for sufficient cell numbers, depending on the numbers of conditions you want to test. Feed organoids with WENRAFI media (Table 1).

48 h before electroporation, replace the medium with 250 μl of ENAFI medium supplemented with 5 μM CHIR99021 and 10 μM Y-27632 (Table 2).

24 h before electroporation, replace the medium with 250 μl of ENAFI medium supplemented with 5 μM CHIR99021, 10 μM Y-27632 and 1.25% (vol/vol) DMSO (Table 2).

Remove the medium from the organoids and add 500µL of TrypLE Express supplemented with 10 μM Y-27632 to each well. Scrape the Matrigel off the bottom of the wells with a 1,000-μl pipette. Split the organoids in 2-4 15 mL Falcons to have smaller volume for the dissociation process.

Place the tubes in a 37°C water bath for 0h 30m 0s. Pipette vigorously every 5 min, 10 times with 10 mL pipette and 10 times with a 1,000-μl pipette with broken tip.

Thaw Cas9 and guide On ice.

Add basal medium up to 10 ml and centrifuge for 4 minutes at 500g. Combine separate Falcon tubes at this stage to have a bigger pellet.

Aspirate and discard the supernatant. If pellet is loose, do a second centrifugation step in an ependorf with 500-1000 μl of media left.

Aspirate and discard supernatant. Add 500µL of Opti-MEM media and pipette well to mix.

Count number of cells with a haemocytometer (take 10 μl). Determine number of conditions (100,000 cells per condition). You will need to include negative control, no Cas9.

Mix 1µLof Cas9 and 1µL of guide (1:3.33 ratio), you will need to add 2 μl per condition.

Standard concentration: 5ug True Cut Cas9 v2 (Invitrogen, A36499- 500 μg at 5μg/μl) and 100pmol synthetic guide (Synthego- custom made, supplied 3 nmol lyophilised reconstituted with 30 μl water for 100pmol/μl).

Make complex and leave 0h 20m 0s at Room temperature.

Spin and pellet correct number of cells before washing with 300µL of PBS. Centrifuge at 500g for 4 minutes.

While washing with PBS make P3 suppl buffer (20 μl/reaction) (Lonza, V4XP-3032).

Buffer P3: 16.4 μl and Supplement 1: 3.6 μl for a total of 20 μl per condition (recommended to make at least 10% excess for pipetting error). Supplemented with 10μM Y-27632, leave at Room temperature.

Completely remove and discard the supernatant. Resuspend in 20µLof P3 buffer supplemented with 10 μM Y-27632 per condition.

Add 2µLof RNP complex per condition.

Mix well and load 20µL into electroporation chamber (16-well nucleovette strips).

Leave 0h 10m 0sat Room temperature before electroporation.

Perform electroporation on Lonza Amaxa 4D Nucleofector with program DS-138.

Incubate at 37°Cfor 0h 10m 0s.

Add 80µL of warm ENAFI media+ Y+ Chir+DMSO to each chamber. Remove 100 μl into seperate ependorfs. Wash each chamber with another 100µL of media to ensure you have taken all cells.

Centrifuge at 500g for 4 minutes.

Remove and discard the supernatant and suspend the pellet with 20-25 μl Matrigel per well. Set up 2 wells per condition.

Place the plate in a 37°C incubator for 0h 10m 0s to solidify the Matrigel.

Once matrigel has solidified, add 250 μl of ENAFI medium supplemented with 5 μM CHIR99021, 10 μM Y-27632 and 1.25% (vol/vol) DMSO (Table 2) to each well.

Next Day: change media back to WENRAFI (Wnt and Rspo conditioned, Table 1) supplemented with 10 μM of Y-27632.

7 days after electroporation, extract DNA from half or a third of the well using PicoPure DNA extraction kit (Invitrogen, KIT0103). Perform 65°C lysis step for 3h 0m 0s hours.

Perform PCR using primers that span the guide (500-800 bp) and submit for Sanger sequencing in both directions.

Analyse sanger trace using ICE Synthego. You will need to upload a control trace (No Cas9) for each edited trace.

https://ice.synthego.com/#/