T-PGI: an engineered STITCHR system for scarless, programmable genome integration

Scarless, programmable insertion of defined DNA remains a central goal for therapeutic genome editing. We introduce T-PGI (Transposon-mediated programmable genomic integration), an engineered implementation of STITCHR that preserves target-primed reverse transcription (TPRT) while substantially improving efficiency, specificity, and modularity. T-PGI uses R2Tocc, a low-background ortholog that we further engineered through defined deletions, rational point mutations, and modular domain insertions to enhance performance. The system employs paired nCas9 nicks flanking homology arms and bicistronic co-expression with dual NLSs, together with optimized RNA donor designs. Using combinatorial guide-template screening, T-PGI achieves robust integration across diverse cargos, including precise cassette insertion following multi-kilobase deletions. Short- and long-read sequencing confirm high-fidelity insertion with minimal local indels or structural variants. Collectively, these advances establish T-PGI as a practical and adaptable platform for accurate, scarless genome integration and provide concise design principles spanning enzyme architecture, donor configuration, and guide pairing for next-generation therapeutic editing.