Bacterial Genome Editing Using CRISPR/Cas9 Coupled with Lambda Red Recombineering for Potential Generation of an Antigen 43 Knockout

SUMMARY To study the effect of differential Antigen 43 expression on biofilm formation, we attempted to generate a knockout mutant of the flu gene in a well-characterized Escherichia coli lab strain, MG1655. CRISPR/Cas9 technology was used, in tandem with lambda Red recombination machinery to assist with homologous recombination. A pCasRed plasmid expresses the Cas9 endonuclease, trans-activating CRISPR RNA, and lambda Red machinery. A pCRISPR-SacB-gDNA plasmid was successfully constructed to express guide RNA, allowing targeted cleavage by the Cas9 enzyme at the flu gene. Double stranded donor DNA oligonucleotides were designed as a repair template in recombineering to remove the protospacer and protospacer adjacent motif sequence, while inserting an in-frame stop codon at the target locus. Mutagenesis was attempted by heat shock transformation with pCasRed, pCRISPR-SacB-gDNA, and donor DNA. After screening by colony PCR and sequencing of the PCR products, a flu knockout mutant could not be identified. In addition, high levels of unedited background escaper colonies were observed. We propose that improved guide RNA targeting sites, the use of longer donor DNA, and transformation by electroporation may improve CRISPR/Cas9 mutagenesis efficiency.