Elizabeth Kellogg

Precise insertion of large DNA cargo at defined genomic sites, without inducing double-strand breaks, remains a major limitation of current genome-editing technologies. Here we address this challenge by using de novo protein design to create highly active, new-to-nature RNA-guided transposons. The resulting system, NovoCAST, is the simplest CRISPR-associated transposon (CAST) described to date. NovoCAST exhibits sharply defined integration profiles, up to 3 orders of magnitude increase in activity relative to native CASTs, and exceptionally high on-target specificity. Structural and biochemical analyses confirm that the designed proteins fold and function as intended. Finally, we demonstrate robust programmable genomic integration in human cells, underscoring the translational potential of this approach. Together, these results establish de novo protein design as a powerful strategy for building efficient, precise genome-integration systems and provide a foundation for the development of clinically relevant, programmable gene integration tools.