Establishing a Retron-Based Cytosine Base Editor for Targeted Hypermutation in Escherichia coli

Abstract Current cytosine base editors (CBEs) are limited to unidirectional C to T conversions, restricting their applications. Retrons, bacterial genetic elements, encode a reverse transcriptase that generates multicopy single-stranded DNA (msDNA) by reverse transcribing specific non-coding RNA (ncRNA). This msDNA mimics Okazaki fragments during DNA replication, making retrons promising for gene editing. Here, we developed a retron-based cytosine base editor (RCBE) by fusing cytosine deaminase with reverse transcriptase (RT-CDA) within the retron system. RCBE first transcribes ncRNA, allowing RT-CDA to deaminate cytosine on the ncRNA. The modified ncRNA is then reverse transcribed into msDNA, where RT-CDA induces further cytosine deamination. This mutant msDNA introduces specific mutations into target gene sequences, enabling both C to T and G to A conversions. Using RCBE, we demonstrated accelerated molecular evolution of the rpoB gene in Escherichia coli . High-throughput sequencing confirmed that RCBE achieves a mutation rate of up to 0.2% in regions with high GC content. Our findings establish RCBE as a versatile tool, particularly suitable for directed evolution in GC-rich regions, with broad potential applications across various bacterial and eukaryotic hosts.