PDF(Pubmed)
Abstract：
Prime 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 (DETECTOR: \"detection of targeted editing of CFTR in organoids\").
摘要：
Prime编辑是最近的，CRISPR衍生的基因组编辑技术，能够引入精确的核苷酸取代，插入,和删除。这里,我们提出了纠正L227R-和N1303K-CFTR的主要编辑方法，导致囊性纤维化的两个突变,不符合目前市场批准的调节疗法的条件.我们证明,在对CFTR基因进行DNA校正后，复杂的糖基化，本地化,and,最重要的是,CFTR蛋白的功能在HEK293T和16HBE细胞系中恢复。这些发现随后在患者来源的直肠类器官和人鼻上皮细胞中得到验证。通过对原代干细胞中预测和实验鉴定的候选脱靶位点的分析，我们确认了以前关于高初素编辑(PE)特异性的报道及其在治愈性CF基因编辑治疗中的潜力.为了便于将来在翻译CF模型中筛选遗传策略，开发了一种机器学习算法,用于动态定量类器官中的CFTR功能(检测:“类器官中CFTR靶向编辑的检测”).
