Journal Article

Intracellularly synthesized ssDNA for continuous genome engineering

Pan, Yingjia, Lian, Jiazhang, Huang, Lei, Dong, Chang, Zhang, Yu, Liu, Wenqian

2025

Overview

Dynamic and continuous genome editing systems based on different in vivo single-stranded DNA (ssDNA) synthesis mechanisms are burgeoning ssDNA-mediated genome engineering tools.Retron-based systems are promising ssDNA-mediated tools for continuous engineering of prokaryotic and eukaryotic cells.Approaches to improve the efficiency of ssDNA-based continuous editing systems include biopart optimization, host engineering, and coupling with CRISPR systems.In vivo-synthesized ssDNA can be used for DNA-based data storage, high-throughput functional variant screening, and gene-specific continuous in vivo evolution, which are difficult to achieve with conventional chemically synthesized ssDNA. 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. 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.

• Publisher: Elsevier Ltd,Elsevier Limited
• Subject: Bacteria / Binding sites / continuous in vivo evolution / CRISPR / CRISPR-Cas Systems - genetics / Cytotoxicity / DNA, Single-Stranded - biosynthesis / DNA, Single-Stranded - genetics / E coli / Editing / Efficiency / Evolution / Gene Editing - methods / Genetic engineering / Genome editing / Genomes / Gram-positive bacteria / Internal Medicine / intracellular ssDNA production / Mutation / Plasmids / Proteins / retron / Single-stranded DNA / Transfer RNA