DeTurk, E. C. | bioRxiv Science

bioRxiv2026

Stepwise DNA unwinding gates TnpB genome-editing activity

TnpB is a compact RNA-guided endonuclease and evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through a discrete, long-lived partially unwound intermediate state before reaching a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, Ymu1-WFR, stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in plants. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.

biochemistry↗

bioRxiv2025

Structure and evolution-guided design of minimal RNA-guided nucleases

The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome editing capabilities. However, generating diverse multi-domain proteins with robust enzymatic properties remains challenging. Here we use an artificial intelligence-driven strategy that couples structure-guided inverse protein folding with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease. High-throughput functional screening of AI-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant and human cells. Cryo-EM-based structure determination of the most divergent active variant revealed new stabilizing contacts in the RNA/DNA interfaces across conformational states, demonstrating the design potential of this approach. Together these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space. One-sentence abstractAn evolution- and structure-conditioned model enables design of active RNA-guided nucleases with new nucleic acid contacts resolved by cryo-EM.

synthetic biology↗