Generation of gene-of-interest knockouts in murine organoids using CRISPR-Cas9

Abstract

Gene-of-interest knockout organoids present a powerful and versatile research tool to study a gene's effects on many biological and pathological processes. Here, we present a straightforward and broadly applicable protocol to generate gene knockouts in mouse organoids using CRISPR-Cas9 technology. We describe the processes of transient transfecting organoids with pre-assembled CRISPR-Cas9 ribonucleoprotein complexes, organoid cell sorting, and establishing clonal organoid culture pairs. We then detail how to confirm the knockout via Western blot analysis.

Keywords: CRISPR; Cancer; Organoids.

Graphical abstract

Figure 1

Examples of murine gastric cancer organoid cultures (A and B) Show healthy murine gastric cancer organoid cultures in growth phase. Optimal phase to passage organoids or employ organoids in transfection. (C and D) Show murine gastric cancer organoid cultures in death phase. Do not use organoid in death phase to passage or transfect. Scalebar represent 200 μm.

Figure 2

Preparing single cells from organoids using TrypLE solution (A) Shows healthy murine gastric cancer organoids in growth phase, the optimal phase to start preparation of organoids for transfection. (B) Shows partially broken up organoids in PBS after transferring to 15 mL tube (step 10). (C) Shows partially broken up organoids after resuspension in pre-warmed TrypLE solution (step 13). (D) Shows dissociating organoids after incubation in TrypLE solution at 37°C for 5 min (step 15). (E) Shows dissociating organoids after incubation in TrypLE solution at 37°C for a total of 10 min (step 17). (F) Shows small clumps of cells or single cells from dissociated organoids after incubation in TrypLE solution for a total of 15 min (step 17). Scalebar represent 100 μm.

Figure 3

Gating strategy for single cell sorting of ATTO 500+ cells (A) Gating strategy used to sort viable ATTO 550^high cells from organoid cells transfected with RNP complexes. (B) Overlay histogram of cells transfected with RNP complexes (red) and mock transfected cells (RNPs without gRNA) (light blue) to demonstrate high ATTO 550 positivity.

Figure 4

Manual “picking” of organoids (A) Shows an organoid grown in RGF BME dome surrounded by other organoids. (B) The image shows a 1,000 μL pipette tip placed over (dark ring surrounding the organoid) an organoid immediately prior “picking” of that organoid by pipetting it into the pipette tip. (C) A micrograph showing the now empty place in the RGF BME dome where the picked organoid was. (D) Presents an image of the manual picked organoid in the new well confirming that only one organoid was picked. Scalebars represent 200 μm.

Figure 5

CRISPR knockout confirmation via Western blot analysis Western blot analysis for both a CRISPR target protein and control GAPDH. P = parental organoids; C = clonal organoids. C1, C3 and C4 represent successful complete knockouts, whereas C6 represents an incomplete knockout meaning strongly reduced protein level suggesting heterozygous loss of target gene.