TRU-PE: A Universal, Trackable Prime Editor Toolkit for Robust Single-and Multi-Locus Genome Engineering

Abstract Prime editing (PE) holds immense promise for precise genome editing, but its wider adoption is constrained by large payloads and inefficient delivery in recalcitrant cell types. Here, we present TRU-PE (Trackable, Robust, and Universal Prime Editor), a toolkit that overcomes these barriers via a split-vector architecture coupled with fluorescence-guided enrichment. This design ensures stoichiometric component expression, yielding marked improvements in editing efficiency and accelerated workflows compatible with both plasmid and viral delivery. By integrating a polycistronic hCtRNA-sgRNA/pegRNA array, the derivative TRU-MPE system enables simultaneous editing of up to ten genomic loci. Furthermore, it enhances the performance of evolved PE editors, allows flexible vector stoichiometry and inspires novel editing reporters. Leveraging these capabilities, we successfully generated a combinatorial human iPSC-based platform for GATA2 deficiency, which accurately recapitulates genotype-specific hematopoietic defects. TRU-PE thus provides a user-friendly, scalable framework for robust single- and multi-locus genome engineering, accelerating functional genomics and therapeutic development.