Phage-based delivery of CRISPR-associated transposases for targeted bacterial editing

Abstract

Phage λ, a well-characterized temperate phage, has been recently leveraged for bacterial genome editing by selectively delivering base editors into targeted bacterial species. We extend this concept by engineering phage λ to deliver CRISPR-guided transposases, accomplishing large insertions and targeted gene disruptions. To achieve this, we engineered phage λ using homologous recombination paired with Cas13a-based counterselection for precise phage modifications. Initially, we established the utility of Cas13a in phage λ by conducting minimal recoding edits, deletions, and insertions. Subsequently, we scaled up the engineering to embed the comprehensive DNA-editing CRISPR-Cas transposase (DART) system within the phage genome, creating λ-DART phages. These modified λ-DART phages were then employed to infect Escherichia coli, generating CRISPR RNA-guided transposition events in the host genome. Applying our engineered λ-DART phages to monocultures and a mixed bacterial community comprising three genera led to efficient, precise, and specific gene knockouts and insertions in the targeted E. coli cells, achieving editing efficiencies surpassing 50% of the population. This research enhances phage-mediated genome editing by enabling efficient in situ gene integrations in bacteria, offering an avenue for further application in microbial community contexts. This scalable method enables flexible microbial genome editing in situ to manipulate the function and composition of diverse ecosystems.

Keywords: CRISPR; Cas; genome editing; phage; transposase.

Fig. 1.

Phage λ engineering and application overview. (A) Graphical overview of the homologous recombination-based editing and Cas13a-mediated counterselection for enrichment of edited phage λ. Strains containing editing plasmids with regions of homology to the λ genome facilitate homologous recombination events during phage infection. Edited phages can be enriched from these lysates through Cas13a counterselection by targeting the transcript of a locus missing from edited phages. (B) Graphical overview of the types of edits achieved in the phage λ genome; minimal nucleotide-level edits, deletions, and insertions. (C) Application of edited phage containing DART, the CAST system, for phage-mediated host genome editing through CRISPR RNA-guided transposition.

Fig. 2.

Cas13a counterselection enables diverse editing outcomes in the phage λ genome. (A) Overview of a nonessential region (lom-ea59) of the phage λ genome and deletion (Δlom-ea59) and insertion (Δlom-ea59::lacZ) editing outcomes. Homology arms for homologous recombination (HR) editing are labeled as gray boxes with dashed line borders. The Cas13a target transcript is labeled within the lom gene, and confirmatory PCR primers positions are shown positioned outside of the homology arms. (B) Edited fraction data determined after HR and subsequent enrichment (HR+E) of the intended deletion edits. The size of the deleted region is listed in parenthesis. (C) Edited fraction data determined after homologous recombination (HR) and subsequent enrichment (HR+E) of the intended lacZ insertion edits. (D) Phage titers were determined after the HR and HR+E steps of the editing and enrichment process for edits Δlom-ea59 and Δlom-ea59::lacZ. Phages were spotted on strains expressing Cas13a and a counterselection (lom) or a nontargeting (NT), negative control guide with homology to RFP. (E) Confirmatory PCR results from plaques made by phage enriched for the Δlom-ea59 or Δlom-ea59::lacZ edit. For reference, the corresponding WT amplicon is predicted to be 10.2 kb. (F) Overview of the editing strategy for replacing a nonessential region (lom-orf60a) with the DNA-editing all-in-one RNA-guided CRISPR-Cas transposase (DART) system. Homology arms for HR editing are labeled as gray boxes with dashed line borders. The two tested Cas13a target transcripts are labeled within the lom and orf60a genes. Confirmatory PCR primers positions are shown flanking the homology arms. (G) Edited fraction data determined after HR and HR+E for the Δlom-orf60a::DART edit. Two Cas13a guides (lom and orf60a) were individually tested for enrichment and plating for phage titering. (H) Confirmatory PCR results from plaques made by phage enriched for the Δlom-orf60a::DART edit. Amplicons were generated using unbiased primers and amplified across both homology arm junctions. For parts B, C, and G, shown are the mean, SD, and individual data points from n = 3 biological replicates.

Fig. 3.

Phage-delivered DART enables targeted genome editing integration events. (A) Overview of the phage-mediated host genome editing assay. Edited λ-DART phage are mixed with E. coli host cells, incubated, then the mixtures were collected and used with solid agar plates for enumerating and isolating bacterial colonies and soft agar overlay for phage plaque enumeration and isolation. (B) Overview of the host genome editing strategy following λ-DART infection. DART components are expressed and facilitate CRISPR RNA-guided transposition of the donor transposon to a lacZ or thyA target site. The transposon contains gentamicin resistance and GFP reporter genes. (C) Editing results for hosts infected at an MOI or 0.1 or 1 by λ-DART phages containing a nontargeting or thyA-targeting guide with DART components driven by a lac or J23119 promoter. (D) Confirmatory PCR results for integration of the 2.2-kb at the thyA target site. Associated Sanger sequencing trace data are shown below, highlighting the thyA knockout. Gray rectangles labeled “TSD” correspond to expected target-site duplication events. (E) Editing results for hosts infected at an MOI or 1 or 10 by λ-DART phages with a lac or J23119 DART promoter and a lacZ-targeting guide. (F) Confirmatory PCR results for integrating the 2.2-kb transposon at the lacZ target site. (G) Brightfield and fluorescence (GFP) microscopy images of cells infected at an MOI of 10 by λ-DART phages with a J23119-driven DART expressing a lacZ-targeting guide. (Scale bar represents 10 μm.) For parts C and E, shown are the mean, SD, and individual data points from n = 3 biological replicates, and statistical significance is denoted as *P ≤ 0.0332; ND = not detected.

Fig. 4.

Phage-mediated host genome editing in a community context. (A) Graphical overview of the 3-member community editing assay. (B) Editing results for host cells infected at an MOI of 10 by λ-DART phages containing a nontargeting or lacZ-targeting guide with DART components driven by a J23119 promoter. (C) Confirmatory PCR results for integrating the 2.2-kb transposon at the target site from E. coli colonies isolated on selective plates. (D) Endpoint brightfield and fluorescence (GFP) microscopy images from multiple fields of a mixed culture infected at an MOI of 10 by λ-DART phages harboring a J23119 promoter and a lacZ-targeting guide. Field 2 shows a crowded, layered view of the mixed community culture. (Scale bar represents 10 μm.) For panel B, shown are the mean, SD, and individual data points from n = 3 biological replicates; ND = not detected.

Fig. 5.

Extended incubation time increases phage-mediated host genome editing efficiency. (A) Overview of the phage-mediated host genome editing assay with an extended, 48-h incubation period. (B) Editing results for host cells infected at an MOI of 1, 10, or 100 by λ-DART phages expressing lacZ-targeting DART driven by a lac or J23119 promoter. (C) Endpoint brightfield and fluorescence (GFP) microscopy images from cells infected at an MOI of 100 by λ-DART phages harboring a J23119 promoter and a lacZ-targeting guide. (D) White epi-illumination and fluorescence (GFP) images of colonies grown on LB agar with X-gal. Plates show colonies from cells infected at an MOI of 100 by λ-DART phages harboring a lac or J23119 promoter and a lacZ-targeting guide, and some colonies appear dark due to blue-white screening. (Scale bar represents 10 μm.) For part B, shown are the mean, SD, and individual data points from n = 3 biological replicates, and statistical significance is denoted as ***P ≤ 0.0002 and ****P ≤ 0.0001.