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

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Skoufou-Papoutsaki, Nefeli  ORCID logo
Adler, Sam 
D'Santos, Paula 
Mannion, Liz 
Mehmed, Shenay 


Organoids combined with genetic editing strategies, 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 been only 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 of intestinal organoid editing using a Ribonucleoprotein CRISPR-based approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different anatomical gut locations and developmental timepoints from multiple patient samples with no observed off-target editing. The method allowed us to study the effect of the 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.



Genome editing, Organoids, PTEN

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The Company of Biologists
Wellcome Trust (103805/Z/14/Z)
Wellcome Trust (102160/B/13/Z)