Prime editing (PE) enables precise and versatile genome editing without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human cystic fibrosis (CF) transmembrane conductance regulator (CFTR) F508del, a three-nucleotide deletion that is the predominant cause of CF. By combining six efficiency optimizations for PE—engineered PE guide RNAs, the PEmax architecture, the transient expression of a dominant-negative mismatch repair protein, strategic silent edits, PE6 variants and proximal ‘dead’ single-guide RNAs—we increased correction efficiencies for CFTR F508del from less than 0.5% in HEK293T cells to 58% in immortalized bronchial epithelial cells (a 140-fold improvement) and to 25% in patient-derived airway epithelial cells. The optimizations also resulted in minimal off-target editing, in edit-to-indel ratios 3.5-fold greater than those achieved by nuclease-mediated homology-directed repair, and in the functional restoration of CFTR ion channels to over 50% of wild-type levels (similar to those achieved via combination treatment with elexacaftor, tezacaftor and ivacaftor) in primary airway cells. Our findings support the feasibility of a durable one-time treatment for CF.

Prime 编辑 （PE） 可实现精确和通用的基因组编辑，而无需双链 DNA 断裂。在这里，我们描述了 PE 系统的系统优化，以有效纠正人囊性纤维化 （CF） 跨膜传导调节因子 （CFTR） F508del，这是一种三核苷酸缺失，是 CF 的主要原因。通过结合 PE 工程化 PE 向导 RNA、PEmax 架构、显性阴性错配修复蛋白的瞬时表达、战略性沉默编辑的六种效率优化， PE6 变体和近端“死”单向导 RNA——我们将 CFTR F508del 的校正效率从HEK293T细胞中的不到 0.5% 提高到永生化支气管上皮细胞的 58%（提高 140 倍），在患者来源的气道上皮细胞中提高到 25%。优化还导致最小的脱靶编辑，编辑与插入缺失的比率比核酸酶介导的同源定向修复高 3.5 倍，并且 CFTR 离子通道的功能恢复到野生型水平的 50% 以上（类似于通过与 elexacaftor、tezacaftor 和 ivacaftor 的联合处理实现的水平）在原代气道细胞中。我们的研究结果支持对 CF 进行持久的一次性治疗的可行性。