Design and testing of CRISPRi knockdowns to assess gene essentiality in phages lambda and P1.

(a) Schematic of CRISPRi assay system. (b) Representative images of plaque assays to validate the dCas12a CRISPRi system using gene targets with known essentiality. We employed crRNAs targeting 2 essential genes of phage λ: genes encoding major capsid protein (E) or DNA packaging subunit (Nu1). For phage P1, we used crRNA targeting 3 essential genes: encoding the major capsid protein (gene 23 encoding Mcp), DNA packaging subunit (PacA), and tape measure protein (Sit); and 3 nonessential genes: ppp, upfB, or ddrB. For comparison, phage plaques appearing on an E. coli BW25113 lawn expressing a nontargeting crRNA as a control are shown for both phages (Ctrl).