Gene Knockout Protocol

Example: A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

In this protocol, we describe in detail every step in the generation of CRISPR-concatemer vectors, from gRNA design to the Golden Gate reaction and to confirmation of successful cloning. We also provide a highly efficient protocol for the transfection of CRISPR-concatemers into mouse small intestinal organoids by electroporation and subsequent growth factor withdrawal experiments.

1. gRNA Design for the CRISPR Vector

Design gRNAs against the genes of interest using a CRISPR gRNA design tool of choice.
Make sure the gRNAs do not contain the BsmBI recognition site by using a restriction mapping tool.
Add specific CRISPR vector overhangs to each oligo.

2. Cloning of gRNAs into the CRISPR Vector

3. Transfection of Intestinal Organoids by Electroporation

a. Pre-electroporation

1. On day 0 of the transfection procedure, split organoids in a 1:2 ratio.
When splitting organoids for electroporation, seed a minimum of 6 wells of a 48-well plate per transfection.
Seed the organoids in 20 μL-basement matrix drops and grow them in WENR + Nic medium (Wnt + EGF + Noggin + R-spondin + Nicotinamide)
at 37 °C, 5% CO2 in a humidified incubator.

2. On day 2, change medium by replacing WENR+Nic with 250 µL of EN (EGF + Noggin) + CHIR99021 (Glycogen Synthase Kinase-3 inhibitor) +
Y-27632 (ROCK inhibitor), without antibiotics.

3. On day 3, change the organoid medium to EN + CHIR99021 + Y-27632 + 1.25% v/v Dimethyl sulfoxide (DMSO), without antibiotics.

b. Preparation of the cells

4. On day 4, disrupt the basement matrix domes using a 1 mL pipette tip and transfer organoids to a 1.5 mL tube. Pool contents of four wells of a
48-well plate into a tube.

5. Mechanically break organoids into small fragments by pipetting up and down with a P200 pipette approximately 200 times. Centrifuge at room
temperature, 5 min at 600 x g.

6. Remove the medium and resuspend the pellet in 1 mL of a cell culture grade recombinant protease. Incubate at 37 °C for a maximum of 5 min
and then check a 50-µL drop of sample under an inverted light microscope with a 4x objective.

7. Transfer the cell suspension to a low-binding 15 mL tube and halt the dissociation by adding 9 mL of basal medium without antibiotics.
Centrifuge at room temperature, 5 min at 600 x g, then discard the supernatant and resuspend the pellet in 1 mL of reduced serum medium.

8. Count the number of cells with a Bürker's chamber and use a minimum of 1 x 105 cells per electroporation reaction. Add 9 mL reduced serum
medium to the 15 mL tube and centrifuge at room temperature, 3 min at 400 x g.

c. Electroporation

9. Remove all of the supernatant and resuspend the pellet in an electroporation solution. Add a total amount of 10 µg DNA to the cell suspension
and add electroporation solution to a final volume of 100 µL and keep the cell-DNA mixture on ice. Use CRISPR vectors in combination with a
Cas9 expression plasmid in a 1:1 ratio.

10. Include a separate transfection mix containing a GFP plasmid to evaluate transfection efficiency.

11. Add the cell-DNA mixture to the electroporation cuvette and place it in the electroporator chamber. Measure the impedance by pushing the
appropriate button on the electroporator and ensure that it is 0.030-0.055 Ω.

12. Add 400 µL of electroporation buffer + Y-27632 to the cuvette and then transfer all to a 1.5 mL tube. Incubate at room temperature for 30 min
to allow cells to recover and subsequently spin them at room temperature for 3 min at 400 x g.

13. Remove the supernatant and resuspend the pellet in 20 μL/well of basement matrix. Seed approximately 1 x 104 to 1 x 105 cells per well in a
48-well plate and add EN + CHIR99021 + Y-27632 + 1.25% v/v DMSO medium. Incubate at 37 °C.

14. On day 5, change the medium to EN + CHIR99021 + Y-27632, and check transfection efficiency by observing GFP expression. Keep organoids
at 37 °C and refresh EN + CHIR99021 + Y-27632 medium after 2 days.

15. On day 9, change the medium to WENR + Nic + Y-27632 and incubate at 37 °C. Note: Y-27632 can be removed after 7-10 days (on day

d. Growth Factor Withdrawal

16. 10-14 days after electroporation, split the organoids in a 1:3 ratio in a 48-well plate following the above-mentioned steps.

17. Resuspend the organoid pellet in 20 µL of basement membrane matrix and let it solidify at 37 °C for 10 min. Then, overlay 250 µL of growth
factor-deprived medium (e.g. EN) to test whether knockout of the target genes has been achieved5.

18. Split the organoids under growth factor-deprived conditions for a minimum of 2 - 3 passages to see a difference in survival between wild type
wildtype (WT) control organoids and mutant organoids.

NOTE: Wildtype organoids should not be able to survive in growth factor-deprived medium over two passages, while mutant lines should be able to