Harnessing the central dogma: Enzyme-driven gene diversification strategies for in cellulo continuous directed evolution.

In cellulo continuous directed evolution offers a powerful route to optimize biomolecules with enhanced or novel functions by enabling rapid, iterative exploration of vast fitness landscapes. At the heart of this approach lie intracellular gene diversification strategies, which have undergone notable innovations in recent years. Although a growing list of techniques has emerged-often tailored to distinct host organisms-they share overarching design principles rooted in the central dogma. By examining how genetic mutations are introduced and propagated through nucleic acid-processing and mutating enzymes, we gain a unifying framework for comparing and adapting these strategies. Here, we review the latest intracellular gene diversification methods within this conceptual lens, highlighting their commonalities, their mechanistic underpinnings, and their adaptability. We then discuss key opportunities for future development, aiming to guide the design of next-generation in cellulo continuous directed evolution systems with greater efficiency, broader host applicability, and finer control over mutagenesis.