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 with homology arms for precise genome editing. However, generation of homology arms is a time-consuming, costly and inefficient process that is often overlooked.ResultsIn this study, we first optimized a CRISPR-Cas genome engineering protocol in the Escherichia coli (E. coli) BL21 strain and successfully deleted 10 kb of DNA from the genome in one round of editing. To further simplify the protocol, asymmetric homology arms were produced by PCR in a single step with two primers and then purified using a desalting column. Unlike conventional homology arms that are prepared through overlapping PCR, cloning into a plasmid or annealing synthetic DNA fragments, our method significantly both shortened the time taken and reduced the cost of homology arm preparation. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across the BL21 genome. Noteworthy, gRNA design is important for the CRISPR-Cas system and a general heuristic gRNA design has been proposed in this study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene yield by ~ threefold.ConclusionsOur work has optimized the homology arms design for gene deletion in BL21. The protocol efficiently edited BL21 to improve lycopene production. The same workflow is applicable to any E. coli strain in which genome engineering would be useful to further increase metabolite production.
Highlights
Escherichia coli BL21 strain is one of the most utilized bacterial platforms for recombinant protein production and metabolic engineering [1,2,3]
Optimization of clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9 knock‐out in E. coli B strain We adapted the two-plasmid system developed by Jiang and co-workers to BL21 [6, 11], in which Cas9 and single guide RNA (sgRNA) were overexpressed on the pCas and pTarget plasmid, respectively (Fig. 1a)
Both the gRNA targeting sequence and protospacer adjacent motif (PAM) sequence were deleted after homologous recombination, and the colonies formed on agar plate were expected to have genome modifications
Summary
Escherichia coli BL21 strain is one of the most utilized bacterial platforms for recombinant protein production and metabolic engineering [1,2,3]. The complex targets the protospacer DNA which is complementary to crRNA and generates a double-strand (ds) break via the recognition of a protospacer adjacent motif (PAM) sequence, 5’-NGG-3’ [5]. A donor DNA is required for specific deletion, insertion, or mutation of the genome sequence during HR. In E.coli, λ-Red recombinases are often used for efficient recombination of the chromosome and donor DNA [6, 7]. The 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 with homology arms for precise genome editing. Generation of homology arms is a time-consuming, costly and inefficient process that is often overlooked
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