Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR.
Dis Model Mech
; 16(10)2023 10 01.
Article
en En
| MEDLINE
| ID: mdl-37772705
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.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Ribonucleoproteínas
/
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas
Límite:
Humans
Idioma:
En
Revista:
Dis Model Mech
Asunto de la revista:
MEDICINA
Año:
2023
Tipo del documento:
Article
País de afiliación:
Reino Unido