Figure 2.

Construction of BaDAS recombinant phages. (A) Modification of the Bxb1 capsid and tail tube subunits. Locations of the sgRNA (green line) targeting either the capsid or tail tube subunit genes (gene 14 and 19, top and bottom, respectively), and the synthetic DNA substrates (blue lines) carrying RBM sequences (red lines) are shown. (B) CRISPR-mediated counter selection. Plasmids with inducible expression of Cas9 and sgRNAs targeting the 3′ junctions of phage Bxb1 genes 14 or 19 (and its derivative phiTM45) were transformed into M. smegmatis mc² 155. Cells were plated with or without ATc inducer (as indicated) and 10-fold serial dilutions of Bxb1, phiTM45, and a control phage, BuzzLyseyear, were spotted onto the lawns. sgRNA induction reduces Bxb1 (and phiTM45) plaquing by at least four orders of magnitude. Top panel, targeting capsid gene 14; Bottom panel, targeting tail tube gene 19. (C) Primary plaques (left panel) recovered from CRISPY-BRED engineering were screened by PCR using primers flanking gene 14, and a candidate recombinant (candidate 3) was plaque purified and secondary plaques re-screened by PCR; all candidates are recombinant. (D) Primary plaques (left panel) recovered from CRISPR selection against wild type gene 19 were screened by PCR using primers flanking gene 19, and a candidate recombinant (candidate 5) was plaque purified and secondary plaques re-screened by PCR (right panel); all candidates are recombinant.