Crafting genetic diversity: unlocking the potential of protein evolution

Genetic diversity is the foundation of evolutionary resilience, adaptive potential, and the flourishing vitality of living organisms, serving as the cornerstone for robust ecosystems and the continuous evolution of life on Earth. The landscape of directed evolution, a powerful biotechnological tool inspired by natural evolutionary processes, has undergone a transformative shift propelled by innovative strategies for generating genetic diversity. This shift is fuelled by several factors, encompassing the utilization of advanced toolkits like CRISPR-Cas and base editors, the enhanced comprehension of biological mechanisms, cost-effective custom oligo pool synthesis, and the seamless integration of artificial intelligence and automation. This comprehensive review looks into the myriad of methodologies employed for constructing gene libraries, both in vitro and in vivo, categorized into three major classes: random mutagenesis, focused mutagenesis, and DNA recombination. The objectives of this review are threefold: firstly, to present a panoramic overview of recent advances in genetic diversity creation; secondly, to inspire novel ideas for further innovation in genetic diversity generation; and thirdly, to provide a valuable resource for individuals entering the field of directed evolution.