Fig. 4: Cas13 facilitates a robust engineering strategy across diverse phages.

a, Overview of a simple two-step editing process. Wildtype phage T4 infects homology vector-containing strain at a low MOI, yielding a mixed population of wt (orange) and edited (purple) phages (‘HR’). This population is diluted and infects a LbuCas13a-expressing strain targeting the wt locus, enriching for edited phages relative to wt (‘HR+E’). b, Example gene deletion design for T4∆soc. Top: gene organization of wt T4soc locus shown with approximate locations of soc protospacers (orange) and homology arms (pink box). Bottom: gene organization of edited T4∆soc locus. The encoded deletion removes both soc protospacers, enabling enrichment of edited phages. c, Example large multi-gene deletion design from T4gp52.1 to T4rIIB (T4wtGT7). Top: gene organization of wt T4GT7 locus shown with approximate locations of T4ndd and T4denB protospacers (orange) and homology arms (pink box). Bottom: gene organization of edited T4GT7 locus. The encoded deletion removes both soc protospacers, enabling enrichment of edited phages. d, Editing penetrance (Methods) from three engineering replicates of the editing and enrichment process shown in a for T4∆soc, T4GT7, T7∆gp1.7, EdH4∆gp004 and EdH4∆gp214. In all cases, ‘negative control crRNA’ refers to an RFP-targeting crRNA, ‘positive control crRNA’ refers to the corresponding phage’s mcp-targeting crRNA, ‘enrichment crRNA’ refers to the crRNA used during the enrichment step shown in a and ‘verification crRNA’ refers to the deletion-targeting crRNA not used during enrichment. The ‘verification crRNA’ for EdH4 yielded a very toxic phenotype to establish a titre and is denoted with a red asterisk. Editing penetrance data are presented as mean ± s.d.

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