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| Status |
Public on Aug 31, 2023 |
| Title |
Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Organoids are currently one of the most widely used ex vivo models in epithelial biology. Combined with genetic editing strategies, organoids offer a promise of 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.
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| Overall design |
We generated PTEN KO human intestinal organoids using CRISPR and performed RNA-seq to identify the trasncriptional effect of the loss of a colorectal cancer tumour suppressor gene and functionally validate the KO.
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| Citation(s) |
37772705 |
| Submission date |
Aug 24, 2023 |
| Last update date |
Oct 23, 2023 |
| Contact name |
Maria-Nefeli Skoufou-Papoutsaki |
| E-mail(s) |
Maria-Nefeli.Skoufou-Papoutsaki@cruk.cam.ac.uk
|
| Organization name |
University of Cambridge
|
| Department |
CRUK Cambridge Institute
|
| Street address |
Robinson Way
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0RE |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (7)
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| Relations |
| BioProject |
PRJNA1009059 |
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