Affiliation:
1. CRUK Cambridge Institute, Cambridge CB2 0RE, UK 1
2. Wellcome-MRC Cambridge Stem Cell Institute, Cambridge CB2 0RE, UK 2
Abstract
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.
Funder
Wellcome Trust
Cancer Research UK Cambridge Institute, University of Cambridge
Publisher
The Company of Biologists
Subject
General Biochemistry, Genetics and Molecular Biology,Immunology and Microbiology (miscellaneous),Medicine (miscellaneous),Neuroscience (miscellaneous)
Cited by
2 articles.
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