Continuous targeted hypermutation with a tunable mutation window.

Targeted in vivo hypermutation drives rapid sequence diversification within specific DNA regions of living cells, enabling continuous evolution of biomolecules for enhanced functionality. In living organisms, this is prominently illustrated by the preferential somatic hypermutation of complementarity-determining regions during antibody affinity maturation. This particular targeting strategy employs focused mutagenesis in predefined, functionally important intragenic regions and should ideally allow for target range adjustment to support broader applications. However, molecular tools incorporating these features remain limited. Here, we present RESPECTevo, a system for focused hypermutation of endogenous and heterologous regions up to ~200 bp with predefined boundaries. By leveraging specific targeting capability, spacer length flexibility, and mismatch tolerance of the Escherichia coli Cascade complex, RESPECTevo enables highly specific targeted hypermutation, target range modulation, and continuous hypermutation across dual regions. Collectively, RESPECTevo serves as an efficient platform for hypermutating user-defined target regions in living cells, facilitating continuous protein evolution for biotechnological applications.