Synth Syst Biotechnol. 2026 Sep 18;18:142-149. doi: 10.1016/j.synbio.2026.09.002. eCollection 2027 Apr.

ABSTRACT

Targeted mutagenesis is a crucial tool for in vivo continuous evolution. However, existing methods for long-range targeted mutagenesis in vivo often face limitations such as low efficiency, poor host compatibility, operational complexity, and uneven distribution of mutations across these windows. In this study, we developed a novel in vivo Targeted Assisted Mutagenesis via Endogenous RNAP tool (TAMER) by functionally fusing the endogenous ω subunit of RNAP with a deaminase and dCas9. This tool achieved a mutagenesis efficiency of 1.28 × 10^-3 substitutions per base (s.p.b.), which represents a 4.2 × 10⁵-fold increase over the natural mutation rate. Additionally, this method enables uniform mutation distribution, an even mutation rate, a broad mutagenesis window, low off-target effects, and cross-host portability. Overall, TAMER offers a simple, efficient, and broadly applicable strategy for in vivo targeted long-range mutagenesis.

PMID:42787729 | PMC:PMC13602049 | DOI:10.1016/j.synbio.2026.09.002