In Vivo Continuous Evolution Systems: Overview, Principles and Applications

Traditional in vitro directed evolution is constrained by discrete workflows, high labor demands, and separation from native cellular contexts. In vivo continuous evolution systems overcome these limitations by enabling autonomous and targeted mutagenesis coupled with cellular proliferation. This review outlines the key parts of these systems, including elevated mutation rates (10−5−10−4 substitutions per base), orthogonality, and autonomy. We classify current platforms into some categories: phage-assisted continuous evolution, error-prone polymerase-based DNA replication systems, reverse transcription-driven mutagenesis systems, and deaminase-fusion transcription-coupled mutagenesis systems, detailing their mechanisms, optimization strategies, and applications. Finally, we address technical bottlenecks and emerging directions, including AI-driven component design, automated platforms, expanded host compatibility, and broadened industrial applications, providing a roadmap for advancing continuous evolution technology.