Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR

Authors:
Skoufou-Papoutsaki N, Adler S, D'Santos P, Mannion L, Mehmed S, Kemp R, Smith A, Perrone F, Nayak K, Russell A, Zilbauer M, Winton DJ
In:
Source: Dis Model Mech
Publication Date: (2023)
Issue: 16(10): dmm050279
Research Area:
Cancer Research/Cell Biology
Stem Cells
Basic Research
Molecular Biology
Cells used in publication:
Epithelial, intestinal, human (InEpC)
Species: human
Tissue Origin: intestine
Platform:
4D-Nucleofector® X-Unit
Experiment

Cells were centrifuged, the supernatant was aspirated, and 300 µl PBS was added. Cells were centrifuged, the supernatant was aspirated, and the pellet was resuspended in the appropriate volume of P3 supplement buffer (20 µl per condition by mixing 16.4 µl P3 buffer and 3.6 µl supplement 1; Lonza, V4XP-3032). The cells were transferred into 16-well nucleovette strips (Lonza). After 10 min at room temperature, electroporation was performed on an Amaxa 4D Nucleofector (Lonza) with the DS-138 custom programme unless otherwise stated. The cells were then incubated for 10 min at 37°C and then 80 µl pre-warmed medium was added to the electroporation chambers. Cells were centrifuged, supernatant was discarded, and the pellet was resuspended in 20 µl Matrigel per condition and seeded in 48-well plates (100,000 cells per well).

Abstract

Organoids, combined with genetic editing strategies, have the potential to offer rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has only been up to 30%, with implications for the subsequent need for selection, including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method for intestinal organoid editing using a ribonucleoprotein-based CRISPR approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different gut anatomical locations and developmental timepoints from multiple patient samples with no observed off-target editing. The method allowed us to study the effect of loss of the tumour suppressor gene PTEN in normal human intestinal cells. Analysis of PTEN-deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss, including mTORC1 activation.