Systematic and scalable genome-wide essentiality mapping to identify nonessential genes in phages

Abstract

Phages are one of the key ecological drivers of microbial community dynamics, function, and evolution. Despite their importance in bacterial ecology and evolutionary processes, phage genes are poorly characterized, hampering their usage in a variety of biotechnological applications. Methods to characterize such genes, even those critical to the phage life cycle, are labor intensive and are generally phage specific. Here, we develop a systematic gene essentiality mapping method scalable to new phage-host combinations that facilitate the identification of nonessential genes. As a proof of concept, we use an arrayed genome-wide CRISPR interference (CRISPRi) assay to map gene essentiality landscape in the canonical coliphages λ and P1. Results from a single panel of CRISPRi probes largely recapitulate the essential gene roster determined from decades of genetic analysis for lambda and provide new insights into essential and nonessential loci in P1. We present evidence of how CRISPRi polarity can lead to false positive gene essentiality assignments and recommend caution towards interpreting CRISPRi data on gene essentiality when applied to less studied phages. Finally, we show that we can engineer phages by inserting DNA barcodes into newly identified inessential regions, which will empower processes of identification, quantification, and tracking of phages in diverse applications.

Fig 1. 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).

Fig 2. Genome-wide CRISPRi design and assay format.

Schematic of steps involved in the arrayed CRISPRi knockdown experiments to assess gene essentiality in phage infectivity cycle. Created with BioRender.com

Fig 3. Gene essentiality landscape of phage λ.

The genome-wide map of gene essentiality is shown by calculating the EOP as the ratio of plaques appearing on E. coli BW25113 lawn expressing crRNA targeting respective lambda phage genes to plaques appearing on BW25113 lawn expressing a nontargeting crRNA. The EOP estimations were done by carrying out biological replicates and depicted the average EOP of every gene on the lambda phage genome map (Methods). Transcripts mentioned in the main text (with promoters) are indicated as thick horizontal arrows: orange, immediate-early transcripts; purple, early transcripts; and red, late transcripts. The underlying data for this figure can be found in Table 1 and S1 Data.

Fig 4. Gene essentiality landscape of phage P1.

The genome-wide map of gene essentiality is shown by calculating the EOP as the ratio of plaques appearing on E. coli BW25113 lawn expressing crRNA targeting respective P1 phage genes to plaques appearing on BW25113 lawn expressing a nontargeting crRNA. The EOP estimations were done by carrying out biological replicates and depicted the average EOP of every gene on the P1 phage genome map (Methods). The underlying data for this figure can be found in Table 2 and S1 Data.

Fig 5. Insertion and quantification of random DNA barcodes on a nonessential genomic location of lambda and P1vir phage.

(a) Schematic of phage engineering approach: Homologous recombination method was used to engineer phages with random barcodes at a nonessential genomic loci, and nuclease active Cas12a-based counterselection was used to enrich engineered phages. Schematic is shown for barcode insertion and counterselection for lambda phage at the red locus and P1 phage at res locus. Created with BioRender.com. (b) Barcode abundance of P1 phage against its PFU/ml estimations in triplicates. (c) Barcode abundance for both barcoded lambda and P1 phages, when mixed at different ratios. Estimations done in triplicates in a pool (Methods). The underlying data for this figure can be found in S1 Data.