TITLE: Organoid Electroporation using CRISPR RNP method
AUTHORS: mns

[DESCRIPTION]
Organoid electroporation using ribonucleoprotein (RNP) CRISPR based approach for highly efficient genome editing.

[STEPS]
SECTION: Single Cell Dissociation (Day 0)
4. Remove the medium from the organoids and add 500 µL 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.

SECTION: Single Cell Dissociation (Day 0)
5. Place the tubes in a 37 °C water bath for 30 min. Pipette vigorously every 5 min, 10 times with 10 mL pipette and 10 times with a 1,000-μl pipette with broken tip.

SECTION: Single Cell Dissociation (Day 0)
6. Thaw Cas9 and guide on ice.

SECTION: Single Cell Dissociation (Day 0)
7. 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.
SECTION: Single Cell Dissociation (Day 0)
8. Aspirate and discard the supernatant. If pellet is loose, do a second centrifugation step in an ependorf with 500-1000 μl of media left.
SECTION: Single Cell Dissociation (Day 0)
9. Aspirate and discard supernatant. Add 500 µL of Opti-MEM media and pipette well to mix.

SECTION: Single Cell Dissociation (Day 0)
10. 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.
SECTION: Making RNP complex
11. Mix 1 µLof Cas9 and 1 µL 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 20 min at Room temperature.

SECTION: Making RNP complex
12. Spin and pellet correct number of cells before washing with 300 µL of PBS. Centrifuge at 500g for 4 minutes.

SECTION: Making RNP complex
13. 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.

SECTION: Making RNP complex
14.  Completely remove and discard the supernatant. Resuspend in 20 µLof P3 buffer supplemented with 10 μM Y-27632 per condition.

SECTION: Making RNP complex
15. Add 2 µLof RNP complex per condition.

SECTION: Making RNP complex
16. Mix well and load 20 µL into electroporation chamber (16-well nucleovette strips).

SECTION: Electroporation
17. Leave 10 minat Room temperature before electroporation.

SECTION: Electroporation
18. Perform electroporation on Lonza Amaxa 4D Nucleofector with program DS-138.  

SECTION: Electroporation
19. Incubate at 37 °Cfor 10 min.

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

SECTION: Seeding cells
21. Centrifuge at 500g for 4 minutes.
SECTION: Seeding cells
22. Remove and discard the supernatant and suspend the pellet with 20-25 μl Matrigel per well. Set up 2 wells per condition.
SECTION: Seeding cells
23. Place the plate in a 37 °C incubator for 10 min to solidify the Matrigel.

SECTION: Seeding cells
24. 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.
SECTION: Media change (Day +1)
25. Next Day: change media back to WENRAFI (Wnt and Rspo conditioned, Table 1) supplemented with 10 μM of Y-27632.
SECTION: Media Preparation (Day -2)
2. 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).

SECTION: Media Preparation (Day -1)
3. 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).

SECTION: DNA extraction and screening (Day +7)
26. 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 180 min hours.

SECTION: Organoid Expansion (Day -5)
1. 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).
SECTION: DNA extraction and screening (Day +7)
27. Perform PCR using primers that span the guide (500-800 bp) and submit for Sanger sequencing in both directions.
SECTION: DNA extraction and screening (Day +7)
28. Analyse sanger trace using ICE Synthego. You will need to upload a control trace (No Cas9) for each edited trace.
https://ice.synthego.com/#/