Intracellularly synthesized ssDNA for continuous genome engineering.

Despite the prevalence of genome editing tools, there are still some limitations in dynamic and continuous genome editing. In vivo single-stranded DNA (ssDNA)-mediated genome mutation has emerged as a valuable and promising approach for continuous genome editing. In this review, we summarize the various types of intracellular ssDNA production systems and notable achievements in genome engineering in both prokaryotic and eukaryotic cells. We also review progress in the development of applications based on retron-based systems, which have demonstrated significant potential in molecular recording, multiplex genome editing, high-throughput functional variant screening, and gene-specific continuous in vivo evolution. Furthermore, we discuss the major challenges of ssDNA-mediated continuous genome editing and its prospects for future applications.