SummaryPrime editing is a recent, CRISPR-derived genome editing technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime editing approaches to correct L227R- and N1303K-CFTR, two mutations that cause cystic fibrosis and are not eligible for current market-approved modulator therapies. We show that, upon DNA correction of the CFTR gene, the complex glycosylation, localization, and, most importantly, function of the CFTR protein are restored in HEK293T and 16HBE cell lines. These findings were subsequently validated in patient-derived rectal organoids and human nasal epithelial cells. Through analysis of predicted and experimentally identified candidate... More
SummaryPrime editing is a recent, CRISPR-derived genome editing technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime editing approaches to correct L227R- and N1303K-CFTR, two mutations that cause cystic fibrosis and are not eligible for current market-approved modulator therapies. We show that, upon DNA correction of the CFTR gene, the complex glycosylation, localization, and, most importantly, function of the CFTR protein are restored in HEK293T and 16HBE cell lines. These findings were subsequently validated in patient-derived rectal organoids and human nasal epithelial cells. Through analysis of predicted and experimentally identified candidate off-target sites in primary stem cells, we confirm previous reports on the high prime editor (PE) specificity and its potential for a curative CF gene editing therapy. To facilitate future screening of genetic strategies in a translational CF model, a machine learning algorithm was developed for dynamic quantification of CFTR function in organoids (DETE CT OR: detection of targeted editing of CFTR in organoids ).Graphical abstractHighlights Prime editing can be used to precisely correct cystic fibrosis-causing mutations Efficient functional correction in patient-derived organoids and airway epithelium Machine learning tool DETE CT OR functionally screens genetic strategies in primary organoids Genome-wide evaluation of prime editing for L227R and N1303K confirms high fidelityBulcaen et al. develop prime editing strategies to correct L227R- and N1303K-CFTR, two drug-ineligible mutations causing cystic fibrosis. Delivery of all components to patient-derived rectal organoids and human nasal epithelial basal cells leads to genetic and functional correction without any detectable off-target editing events.
