ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida

[Image: see text] 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.