Metabolic Engineering of Escherichia Coli BL21 Strain Using Simplified CRISPR-Cas9 and Asymmetric Homolog Arms Recombineering

Abstract

BackgroundThe recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA or homolog arms for precise genome editing. However, it is often overlooked the process to generate the homolog arms which is a time-consuming, costly and inefficient step.ResultsIn this study, we first optimized CRISPR-Cas protocol in BL21 strain and successfully deleted 10 kb gene from the genome in one round of editing. To further simplify the protocol, asymmetric homolog arms as PCR fragments was used. It can be obtained by one-step PCR reaction with two primers and purified with desalting columns. Unlike conventional homolog arms that are prepared through overlapping PCR, cloning to plasmid or annealing synthetic DNA fragments, our method significantly shortened the time taken and reduced the cost to prepare the homolog arms. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across BL21 genome. Noteworthy, gRNA design is important for CRISPR-Cas system and a general heuristic gRNA design was proposed in the study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene production from ~15,000 ppm to > 40,000 ppm.ConclusionsOur work has optimized the homolog arms design for gene deletion in BL21 strains. The protocol efficiently edited BL21 strain to improve lycopene production. The same workflow is applicable to all E. coli strain which would be useful for genome rewiring to further increase metabolite production in microbial cell factories.