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[Preprint]. 2024 Oct 11:2024.10.10.617714. [Version 1] doi: 10.1101/2024.10.10.617714

Figure 1. PhageMaP creates barcoded genome-wide knockout libraries in phages.

Figure 1.

(A) Scheme for generating barcoded phage knockout libraries. Oligos containing target-specific 95nt homology arms and sgRNAs are cloned into a high-copy number vector. 16N barcodes are subsequently added to generate a barcoded donor plasmid library. This library is infected by WT T7 and Cas9-RecA-mediated homologous recombination (HR) inserts barcodes into designated loci. (B) Screening of the PhageMaP libraries on diverse hosts. The pre- and post-selection abundances of phage variants are quantified with deep sequencing. Each variant is then scored to create context-specific gene essentiality profiles. (C) Validation of barcode insertions into four T7 genomic loci. Cas9-RecA-mediated HR was carried out using 1 of 4 sgRNAs or a mix of the 4 sgRNAs in pooled format. The four genomic loci were sequenced, and the percentage of recombined phages was determined by finding the ratio of insertions to total reads. (D) Distribution and mapping of the T7 PhageMaP library. (E) Distribution and mapping of the Bas63 PhageMaP library.