Recombineering-assisted linear CRISPR/Cas9-mediated multiplex genome editing (ReaL-MGE) for bacterial metabolic engineering v1

Recombineering-assisted Linear CRISPR/Cas9-mediated Multiplex Genome Editing (ReaL-MGE) constitutes a significant advancement in bacterial genetic engineering. This technology synergizes the RNA-guided programmability of CRISPR/Cas9 with the 5’-3’ exonuclease and single-strand DNA annealing protein activities of phage recombinases, enabling precise kilobase-scale DNA manipulation at multiple genomic loci simultaneously. ReaL-MGE mitigates off-target effects, removes substrate restrictions, and circumvents the complexities associated with assembling multiple gRNAs on circular vectors. The development of successive ReaL-MGE iterations addresses bacterial intolerance to simultaneous multi-site genomic editing. ReaL-MGE enables the precise simultaneous integration of 22 kilobase-scale sequences (>1 kb each) into distinct genomic loci of non-model bacteria, thereby expanding the scope of bacterial genome engineering. Demonstrating cross-class applicability, ReaL-MGE facilitates multiplex genome editing in Gammaproteobacteria (Escherichia coli and Pseudomonas putida) and Betaproteobacteria (Schlegelella brevitalea), highlighting its potential for diverse synthetic biology applications in biotechnology, agriculture, and environmental science. Compared to alternative bacterial multiplex genome editing technologies, ReaL-MGE offers significant advantages, including unrestricted editing sites and positions, no substrate length limitation, and enhanced convenience and safety. This study provides a comprehensive protocol detailing ReaL-MGE’s capabilities, demonstrating its superiority over prior multi-site editing techniques and its potential to transform multiplex genome engineering. The entire procedure entails approximately 9 days.