Deep sequencing of prime editor target sequences

Prime editing is a recent precision genome editing modality whose versatility offers the prospect for a wide range of applications, including the development of targeted genetic therapies. Yet, an outstanding bottleneck for its optimization and use concerns the difficulty in delivering large prime editing complexes into cells. Here, we demonstrate that packaging prime editing constructs in adenoviral capsids overcomes this constrain resulting in robust genome editing in both transformed and non-transformed human cells (up to 90% efficiencies). Using this cell cycle-independent delivery platform, we found a direct correlation between prime editing activity and cellular replication and disclose that the proportions between accurate prime editing events and unwanted byproducts can be influenced by the target-cell context. Hence, adenovector particles permit the efficacious delivery and testing of prime editing reagents in human cells independently of their transformation and replication statuses. The herein integrated gene delivery and gene editing technologies are expected to aid investigating the potential and limitations of prime editing in numerous experimental settings and, eventually, in ex vivo or in vivo therapeutic contexts.

先导编辑（prime editing）是一类新兴的高精度基因组编辑技术，其多功能性可为包括靶向基因疗法开发在内的诸多应用带来广阔前景。然而，该技术在优化与应用过程中面临的突出瓶颈，在于难以将大型先导编辑复合物递送入细胞内。本研究证实，将先导编辑构建体包装至腺病毒衣壳（adenoviral capsids）中可克服这一限制，在转化（transformed）细胞与非转化（non-transformed）人类细胞中均实现高效稳健的基因组编辑，最高编辑效率可达90%。借助这一不依赖细胞周期的递送平台，我们发现先导编辑活性与细胞增殖存在直接关联，并揭示出准确先导编辑事件与非预期副产物的比例可受靶细胞环境的影响。综上，腺病毒载体颗粒可独立于细胞的转化与增殖状态，实现先导编辑试剂在人类细胞中的高效递送与测试。本研究整合的基因递送与基因编辑技术，有望助力在多种实验场景中探究先导编辑的潜力与局限，并最终应用于体外（ex vivo）或体内（in vivo）治疗场景。