Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassigning codons to noncanonical amino acids, the generation of new-to-nature proteins offers countless opportunities for bioproduction and biocontainment in industrial chassis. A key bottleneck in genome recoding efforts, however, is the low efficiency of recombineering, which hinders large-scale applications at acceptable speed and cost. To relieve this bottleneck, we developed ReScribe, a highly optimized recombineering tool enhanced by CRISPR-Cas9-mediated counterselection built upon the minimal PAM 5′-NNG-3′ of the Streptococcus canis Cas9 (ScCas9). As a proof of concept, we used ReScribe to generate a minimally recoded strain of the industrial chassis Pseudomonas putida by replacing TAG stop codons (functioning as PAMs) of essential metabolic genes with the synonymous TAA. We showed that ReScribe enables nearly 100% engineering efficiency of multiple loci in P. putida, opening promising avenues for genome editing and applications thereof in this bacterium and beyond.

中文翻译：

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ReScribe：一种结合多重重组工程和 Minimal-PAM ScCas9 的无限制工具，用于基因组重新编码恶臭假单胞菌

基因组重新编码能够将新功能整合到微生物的 DNA 中。通过将密码子重新分配给非经典氨基酸，新天然蛋白质的产生为工业底盘中的生物生产和生物防护提供了无数机会。然而，基因组重新编码工作的一个关键瓶颈是重组的低效率，这阻碍了以可接受的速度和成本进行大规模应用。为了缓解这一瓶颈，我们开发了 ReScribe，这是一种高度优化的重组工具，由 CRISPR-Cas9 介导的反选择增强，基于犬链球菌Cas9 (ScCas9) 的最小 PAM 5'-NNG-3'。作为概念验证，我们使用 ReScribe 生成工业底盘Pseudomonas putida的最小重新编码菌株通过用同义的 TAA 替换必需代谢基因的 TAG 终止密码子（起 PAM 的作用）。我们表明，ReScribe 使恶臭假单胞菌中多个基因座的工程效率接近 100% ，为基因组编辑及其在该细菌及其他领域的应用开辟了有希望的途径。