Li-Hua Liu, Wei Lei, Zhiqian Zhang, Shijing Lai, Bo Xu, Qijun Ge, Jiawen Luo, Min Yang, Yang Zhang, Jinde Chen, Qiuru Zhong, Yi-Rui Wu, Ao Jiang
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引用次数: 0
Abstract
Targeted random mutagenesis is crucial for breeding, directed evolution, and gene function studies, yet efficient tools remain scarce. Here, we present obligate mobile element guided activity (OMEGA)-R, an innovative targeted random mutagenesis system that integrates SpyCatcher-enIscB and PolI3M-TBD-SpyTag, outperforming existing state-of-the-art technologies in key metrics, such as protein size, mutagenesis efficiency, window length, and continuity. OMEGA-R achieves a dramatic enhancement of on-target mutagenesis, reaching a rate of 1.4 × 10-5 base pairs (bp) per generation (bpg), with minimal off-target effects, in both Escherichia coli and Bacillus subtilis. The system also demonstrates exceptional compatibility with high-throughput screening (HTS) technologies, including fluorescence-activated droplet sorting (FADS) and phage-assisted continuous evolution (PACE). Utilizing OMEGA-R, we successfully identified a series of effective mutations within the T7 promoter (pT7), ribosome-binding site (RBS), superfolder GFP (sfGFP), and autocyclizing ribozyme (AR), which are invaluable for the development of high-performance biotechnology tools. These findings underscore the high efficiency and broad application potential of OMEGA-R.
期刊介绍:
Trends in Biotechnology publishes reviews and perspectives on the applied biological sciences, focusing on useful science applied to, derived from, or inspired by living systems.
The major themes that TIBTECH is interested in include:
Bioprocessing (biochemical engineering, applied enzymology, industrial biotechnology, biofuels, metabolic engineering)
Omics (genome editing, single-cell technologies, bioinformatics, synthetic biology)
Materials and devices (bionanotechnology, biomaterials, diagnostics/imaging/detection, soft robotics, biosensors/bioelectronics)
Therapeutics (biofabrication, stem cells, tissue engineering and regenerative medicine, antibodies and other protein drugs, drug delivery)
Agroenvironment (environmental engineering, bioremediation, genetically modified crops, sustainable development).