PIVOT: A hyper-compact, PAM-independent TIGR-Tas editor enabling efficient in vivo gene editing via AAV and LNP delivery

Abstract RNA-guided genome editors for therapeutic use require broad genomic access and efficient delivery, two properties that are rarely combined in existing nucleases. The compact, PAM-independent tandem interspaced guide RNA (TIGR)-Tas systems offer a promising alternative, although their editing activity remains low. Here, guided by deep mutational scanning of the TIGR-ParTasR nuclease and its cognate tigRNA, we identified that weak protein-nucleic acid contacts and intrinsic tigRNA instability are the main constraints on editing activity. Iterative engineering produced a PAM-Independent Versatile and Optimized TIGR-Tas editor, PIVOT, which achieves editing activity comparable to SpRY in mammalian cells with high specificity. Microinjection of the PIVOT system into mouse zygotes enabled efficient germline editing, and a single AAV vector drove durable hepatic PCSK9 knockout, reducing circulating PCSK9 by ~82% and serum LDL-C by ~47%. A covalently circularized tigRNA (Mobius) with enhanced RNA stability also supports efficient lipid-nanoparticle (LNP) delivery, providing a compact PIVOT/Mobius editor compatible with both AAV- and LNP-mediated in vivo editing.