EXPERT system Expands the Editing Scope of Prime Editing and Enhances its Efficiency

Prime editors (PEs) hold great promise in modern biotechnology. However, their editing scope is limited as PEs can only modify the downstream sequences of the pegRNA nick. Here, we report the development of the Extended Prime Editor system (EXPERT) to overcome this limitation by using an extended pegRNA (ext-pegRNA) with modified 3' extension, and an additional sgRNA (ups-sgRNA) targeting the upstream of the ext-pegRNA. We demonstrate that EXPERT can efficiently perform various types of prime editing on both sides of the ext-pegRNA nick, a task that is unattainable by canonical PEs. EXPERT exhibits prominent capacity in replacing sequences up to 88 base pairs and inserting sequences up to 100 base pairs within the upstream region of the ext-pegRNA nick. Compared to canonical PEs such as PE2, the utilization of the EXPERT strategy significantly enhances the editing efficiency of all types, up to 122 times higher. Safety wise, the use of ups-sgRNA does not increase the undesirable insertion and deletion (indel) rates at the target site; nor did we observe increased off-target editing rates genome-wide. Our work introduces EXPERT to the toolbox of gene editing, which is expected to improve the versatility and function of PEs.