Sep 20, 2023
Organoid Electroporation using CRISPR RNP method
- mns1,2
- 1CRUK Cambridge Institute;
- 2Cambridge Stem Cell Institute
Protocol Citation: mns 2023. Organoid Electroporation using CRISPR RNP method. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvor74xv4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 06, 2023
Last Modified: September 20, 2023
Protocol Integer ID: 76464
Abstract
Organoid electroporation using ribonucleoprotein (RNP) CRISPR based approach for highly efficient genome editing.
Materials
Table 1: WENRAFI media composition. Modified from Fujii et al., 2018. Cell Stem Cell.
| A | B | C | D | |
| Optimised organoid media (replacement of p38i)- WENRAFI | Stock concentration | Volume | Final concentration | |
| ADF+++ | pure | 13.08 | ||
| Wnt3a conditioned medium | pure | 25 ml | 50% | |
| R-spo conditioned medium | pure | 10 ml | 20% | |
| Primocin (Invivogen #ant-pm-1) | 50mg/ml (500x) | 100μl | 500 μg/mL | |
| B-27® Supplement (Invitrogen #17504-044) | 50x | 1000 μl | 1x | |
| Nicotinamide (Sigma #N0636, in water) | 1 M (100x) | 500 μl | 10 mM | |
| N-Acetylcysteine (Sigma # A9165, in water) | 500 mM (400x) | 125 μl | 1.25 mM | |
| A3801 (Tocris #2939,in DMSO, ) | 5 mM (10,000x) | 5 μl | 500 nM | |
| mEGF (Invitrogen Biosource #PMG8043) | 100 ng/μl (2,000x) | 25 μl | 50 ng/mL | |
| mNoggin (Peprotech #250-38) | 100 ng/μl (1,000x) | 50 μl | 100 ng/mL | |
| IGF-1 (Biolegend, 590904) | 100 ng/μl | 50 μl | 100 ng/mL | |
| FGF-2 (Peprotech, #100-18B) | 100 ng/μl | 25 μl | 50 ng/mL | |
| Total | 50 mL |
Table 2: ENAFI media composition
| A | B | C | D | E | |
| ENAFI (EGF, Noggin, ADF, FGF2, IGF1) | Stock concentration | Final concentration | ENAFI+ Y+Chir (48h before) | ENAFI+ Y+Chir+DMSO (24h before and elec day) | |
| ADF+++ | pure | 24010 | 23697.5 | ||
| Primocin (Invivogen #ant-pm-1) | 50mg/ml (500x) | 500 μg/mL | 50 | 50 | |
| B-27® Supplement (Invitrogen #17504-044) | 50x | 1x | 500 | 500 | |
| Nicotinamide (Sigma #N0636, in water) | 1 M (100x) | 10 mM | 250 | 250 | |
| N-Acetylcysteine (Sigma # A9165, in water) | 500 mM (400x) | 1.25 mM | 62.5 | 62.5 | |
| A3801 (Tocris #2939,in DMSO, ) | 5 mM (10,000x) | 500 nM | 2.5 | 2.5 | |
| mEGF (Invitrogen Biosource #PMG8043) | 100 ng/μl (2,000x) | 50 ng/mL | 12.5 | 12.5 | |
| mNoggin (Peprotech #250-38) | 100 ng/μl (1,000x) | 100 ng/mL | 25 | 25 | |
| IGF-1 (Biolegend, 590904) | 100 ng/μl | 100 ng/mL | 25 | 25 | |
| FGF-2 (Peprotech, #100-18B) | 100 ng/μl | 50 ng/mL | 12.5 | 12.5 | |
| Y-27632 | 10 mM | 10 uM | 25 | 25 | |
| CHIR99021 | 10 mM | 5 uM | 25 | 25 | |
| DMSO | 1.25% | 312.5 | |||
| Total | 25mL | 25mL |
Organoid Expansion (Day -5)
Organoid Expansion (Day -5)
3h
3h
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).
Media Preparation (Day -2)
Media Preparation (Day -2)
10m
10m
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).
Media Preparation (Day -1)
Media Preparation (Day -1)
10m
10m
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).
Single Cell Dissociation (Day 0)
Single Cell Dissociation (Day 0)
1h
1h
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.
Place the tubes in a 37 °C water bath for 00:30:00 . Pipette vigorously every 5 min, 10 times with 10 mL pipette and 10 times with a 1,000-μl pipette with broken tip.
30m
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 500 µL 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.
Making RNP complex
Making RNP complex
30m
30m
Mix 1 µL of 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 00:20:00 at Room temperature .
20m
Spin and pellet correct number of cells before washing with 300 µL 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 20 µL of P3 buffer supplemented with 10 μM Y-27632 per condition.
Add 2 µL of RNP complex per condition.
Mix well and load 20 µL into electroporation chamber (16-well nucleovette strips).
Electroporation
Electroporation
30m
30m
Leave 00:10:00 at Room temperature before electroporation.
10m
Perform electroporation on Lonza Amaxa 4D Nucleofector with program DS-138.
Incubate at 37 °C for 00:10:00 .
10m
Seeding cells
Seeding cells
20m
20m
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.
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 00:10:00 to solidify the Matrigel.
10m
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.
Media change (Day +1)
Media change (Day +1)
10m
10m
Next Day: change media back to WENRAFI (Wnt and Rspo conditioned, Table 1) supplemented with 10 μM of Y-27632.
DNA extraction and screening (Day +7)
DNA extraction and screening (Day +7)
5h
5h
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 03:00:00 hours.
3h
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.