Abstract：
The prime editor (PE) can edit genomes with almost any intended changes, including all 12 possible types of base substitutions, small insertions and deletions, and their combinations, without the requirement for double strand breaks or exogenous donor templates. PE demonstrates the possibility of correcting a variety of disease-causing mutations and might expand the therapeutic application of gene editing. In this study, PE was optimized based on a dual-adeno-associated virus (AAV) split-intein system in vitro by screening different split sites and split inteins. We found that splitting PE before amino acid 1105(Ser) of SpCas9 with Rma intein resulted in the highest on-target editing. The orientations of pegRNA and nicking sgRNA in the AAV vector were further optimized. To test the in vivo performance of the optimized dual-AAV split-PE3, it was delivered by subretinal injection in rd12 mice with inherited retinal disease Leber congenital amaurosis. The prime editors corrected the pathogenic mutation with up to 16% efficiency in a precise way, with no detectable off-target edits, restored RPE65 expression, rescued retinal and visual function, and preserved photoceptors. Our findings establish a framework for the preclinical development of PE and motivate further testing of PE for the treatment of inherited retinal diseases caused by various mutations.
摘要：
主要编辑（PE）可以编辑基因组与几乎任何预期的变化，包括所有12种可能的碱基置换类型，小的插入和删除，以及它们的组合，不需要双链断裂或外源供体模板。PE证明了纠正多种致病突变的可能性，并可能扩大基因编辑的治疗应用。在这项研究中,通过筛选不同的分裂位点和分裂内含肽，基于双重腺相关病毒（AAV）分裂内含肽系统对PE进行了优化。我们发现,在SpCas9的氨基酸1105(Ser)之前用Rma内含素分裂PE导致最高的中靶编辑。进一步优化AAV载体中pegRNA和切口sgRNA的取向。为了测试优化的双AAV分裂-PE3的体内性能,通过在患有遗传性视网膜疾病Leber先天性黑蒙的rd12小鼠中进行视网膜下注射递送。主要编辑以精确的方式纠正了致病突变，效率高达16％，没有可检测到的脱靶编辑，恢复RPE65表达，获救的视网膜和视觉功能，和保存的复印机。我们的发现为PE的临床前发展建立了框架，并激发了进一步测试PE以治疗由各种突变引起的遗传性视网膜疾病。
