LbuCas13a is a potent anti-phage effector against phage T4

Two parsimonious explanations for the phylogenetic distribution of Cas13 effectors are that either Cas13 effectors are relatively ineffective anti-phage systems, limiting their phylogenetic spread owing to evolutionary pressure, or that Cas13 effectors are potent anti-phage systems, but the fitness cost of their abortive-infection-like effects^30,34 causes selection against cas13 loci. To explore these possibilities, we tested the anti-phage activity of the most- and least-widely dispersed Cas13 effectors on the basis of our analysis of bacterial phylogeny—Cas13a and Cas13d, respectively (Fig. ​(Fig.1).1). We selected LbuCas13a from Leptotrichia buccalis and RfxCas13d from Ruminococcus flavefaciens due to their extensive biochemical characterization^29,42–45. We additionally selected an engineered variant of LbuCas13a (eLbuCas13a) that was recently reported to have lower basal trans-RNA cleavage activity and, thus, reduced toxicity when expressed in E. coli⁴⁵. Notably, none of the Cas13 orthologues here have been investigated for anti-phage activity. While a Cas13a orthologue from Listeria seeligeri has been used to restrict temperate and nucleus-forming phages^19,30,33,35, LbuCas13a comes from a phylogenetically distinct sub-clade of Cas13a effectors (Supplementary Fig. 1).

To establish an E. coli phage challenge assay for LbuCas13a and RfxCas13d, we created ‘all-in-one’ plasmids for inducible expression of cas13 using anhydrotetracycline (aTc) alongside a constitutively expressed crRNA (direct repeat-spacer) (Fig. 2a,b). During phage infection, phage RNAs are transcribed, including a crRNA-targeted transcript (orange, Fig. ​Fig.2a).2a). Upon recognition, Cas13 activates HEPN-mediated RNA cleavage, although the extent of trans-cleavage may be reduced for Cas13d relative to Cas13a⁴³. Depending on the extent of Cas13-mediated RNA cleavage, phage-encoded Cas13 resistance, protospacer mutation rate and phage-encoded function containing the protospacer, phage may overcome the resulting general transcript degradation.

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Fig. 2

Comparison of Cas13a and Cas13d in E. coli phage challenge assays with lytic phage T4.

a, Experimental architecture of Cas13 phage defence. Cas13 is expressed under aTc control alongside a crRNA. During phage infection, Cas13 unleashes toxic cis- and trans-cleavage if Cas13 detects its crRNA target. b, crRNA architecture employed in this study. c, Overview of T4 genes and transcript locations targeted by Cas13 in T4 phage challenge experiments. Approximate gene architecture is shown in forward orientation. crRNA locations are highlighted in orange. d, T4 phage infection in bacteria expressing phage-targeting crRNA and either LbuCas13a or RfxCas13d. EOP values represent the average of three biological replicates for a single crRNA. EOP data are presented as mean±s.d. e, T4 phage plaque assays comparing the efficacy of Cas13a and toxicity of Cas13d. A representative plaque assay from three biological replicates is shown. An RFP-targeting crRNA is shown as a negative control.

Source data

To test the phage-restriction capacity of LbuCas13a and RfxCas13d outside their native context, we individually targeted a small panel of genes in phage T4. Phage T4 is a classical virulent dsDNA phage with a 169kb genome and well-characterized genetic content^46,47. From the perspective of phage genome editing, T4 represents an empirical challenge, displaying considerable variability in Cas-restriction efficacy for Cas9 and Cas12a, owing in part to modified glucosyl-5-hydroxymethylcytosine nucleotides^26,27,36 and endogenous DNA-repair mechanisms²⁰. For these reasons, we hypothesized that RNA targeting could be a superior strategy to inhibit T4 and related phages.

We designed a panel of Cas13 crRNAs targeting T4 transcripts with diverse design criteria (Fig. ​(Fig.2c2c)⁴⁶. Targeted regions of T4⁴⁶ RNA sequences included essential genes (major capsid protein (mcp), transcriptional activator motA), a conditionally essential gene (deoxycytidylate hydroxymethylase gp42), a non-essential gene (accessory capsid protein soc), an early-infection gene (motA), a middle-infection gene (gp42), late-infection genes (mcp, soc), encompassing regions early in coding sequences (CDSs) (mcp, soc), middle in CDS (soc) and untranslated regions around the ribosome binding site (RBS) (gp42, motA) (Supplementary Table 1 and Fig. 2). We included a red-fluorescent protein (RFP)-targeting crRNA as a negative control. Broadly, this panel of crRNAs represents a careful exploration of Cas13 targeting the diversity of feature types present in a phage transcriptome.

Remarkably, in phage infection experiments, we observed robust phage restriction for all crRNAs tested using LbuCas13a (Fig. ​(Fig.2d).2d). Independent of gene essentiality, timing of expression or position on transcript, we found that crRNA-guided LbuCas13a could restrict phage T4 over 100,000× when targeting mcp, gp42, motA or soc (Supplementary Fig. 2). In contrast, crRNA-guided RfxCas13d exhibited highly variable and less-efficient phage restriction. Further, RfxCas13d exhibited phage-independent E. coli growth inhibition during RfxCas13d expression (Supplementary Figs. 2–4), and we also observed a high degree of phage escape for RfxCas13d relative to LbuCas13a (Fig. ​(Fig.2e2e and Supplementary Fig. 2). It is possible that RfxCas13d lacks crucial components required for full phage defence or reduced toxicity, such as the WYL domain-containing proteins that appear in its native gene neighbourhood (Supplementary Fig. 5). Our results suggest that LbuCas13a is a remarkably potent single-protein defence system of phage T4 relative to other CRISPR-Cas systems^20,26,27,36.

Cas13a confers resistance to diverse E. coli phages

To the best of our knowledge, no single Cas effector (or antiviral defence protein) has been shown to confer broad-spectrum phage resistance against diverse dsDNA phages. To uncover the phage phylogenetic limits of Cas13a anti-phage activity, we challenged E. coli expressing LbuCas13a with a phylogenetically diverse panel of dsDNA E. coli phages. To generate a representative sampling of E. coli phages, we constructed a protein-sharing network from 2,307 phage genomes visualizing the relatedness of currently known E. coli phages (Fig. ​(Fig.3a).3a). From this network, we assembled a panel of eight dsDNA and one single-stranded DNA (ssDNA) E. coli phages scattered across the E. coli phage phylogeny (Fig. ​(Fig.3a,3a, Supplementary Figs. 6 and 7 and Table 2). This panel includes both model E. coli phages (T4, T5, T7, λ and M13) and non-model E. coli phages (EdH4, MM02, N4 and SUSP1). With the sole exception of phages T4 and MM02, these phages bear minimal nucleotide sequence similarity to each other (Fig. ​(Fig.3a3a and Supplementary Fig. 7). Furthermore, these phages have diverse lifestyles and reflect a realistic model sampling of the genetic diversity found among known E. coli phages. One of these phages displays temperate (λ), another displays chronic infection (M13⁴⁸), while the remaining seven display obligately lytic life cycles. They comprise diverse lifestyles including documented plasmid-transfer-promoting (that is, ‘superspreader’)⁴⁹, DNA compartmentalization¹⁶ and pseudolysogeny⁵⁰ phenotypes. In aggregate, these phages not only represent genotypic diversity but also encompass a mixture of host-takeover strategies, modes of entry and degrees of previous characterization.

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Fig. 3

Comparison of LbuCas13a anti-phage activity across dsDNA E. coli phage phylogeny.

a, Network graph representation of E. coli phages and their relatives. Nodes represent phage genomes that are connected by edges if they share significant similarity as determined by vContact2⁷⁶ (protein similarity). Nodes are shaded red if they are classified as an E. coli phage and blue if they only share similarity. Nodes are shaded black if they were assessed for sensitivity to LbuCas13a. b, EOP experiments for Cas13a designed to target an early or late transcript. EOP values represent the average of three biological replicates for a single crRNA compared to an RFP-targeting negative control crRNA. Phages T4, EdH4, λ, T5 and T7 have additional crRNAs that were tested and are presented in Supplementary Figs. 2, 8, 10, 14 and 15, respectively.

Source data

For each phage, we designed a pair of Cas13a crRNAs targeting either a putative early gene (DNA polymerase (dnap)), RNA polymerase (rnap (T7)), a lytic regulator (cro), replication protein (II (rep) (M13)) or a putative late gene (major capsid protein (mcp VIII) (M13)). An overview of Cas13-mediated phage restriction can be found in Supplementary Table 2, diversity of crRNAs tested in Supplementary Fig. 6 and a by-phage summary of results in Supplementary Figs. 2, 8–15. In aggregate, we observed substantial anti-phage activity for all 18 guides across the nine phages tested (Fig. ​(Fig.3b3b and Supplementary Table 2). Most crRNAs reduced phage infectivity 10⁵–10⁶-fold, with the sparse observation of mature plaque-forming units (p.f.u.). Across this entire study, we observed no mature p.f.u.s above 0.01% frequency in wildtype (wt) phage lysates (Supplementary Figs. 2, 8–15). We observed a single guide (targeting T5 dnap) to yield general toxicity and growth inhibition during LbuCas13a induction (Supplementary Fig. 16). This constraint required us to perform assays in the absence of induction, achieving a mere 10²-fold restriction (Supplementary Fig. 14). However, employing the reduced-toxicity LbuCas13a mutant, eLbuCas13a⁴⁵, we observed both phage restriction at 10⁶-fold (Supplementary Fig. 14) and slightly reduced toxicity in the absence of phage (Supplementary Fig. 16). Thus, we believe that the subpar phage restriction by LbuCas13a was attributed to elevated background toxicity of the T5pol spacer rather than an inability to target this phage gene.

Interestingly, SUSP1 and M13 consistently displayed a small degree of resistance to Cas13a (Fig. ​(Fig.3b).3b). Both early- and late- transcript targeting guides only decreased phage infectivity 5,000–10,000-fold compared with all other phages showing 10⁵–10⁶-fold infectivity reduction. We further investigated the efficacy of SUSP1-targeting crRNAs in a plate-reader assay at a wide range of multiplicities of infection (MOIs) (Supplementary Fig. 17). Compared to a non-targeting crRNA control, we found that both SUSP1dnap- and SUSP1mcp-targeting guides conferred phage resistance at all MOIs tested, including MOIs >10. These results indicate that Cas13a targeting not only conferred substantial population-level protection against SUSP1 infection, but also single-cell protection³⁰. Potentially, this discordance with the abortive-infection model of Cas13 protection observed previously³⁰ reflects a feature of LbuCas13a, a feature of fitness in a non-native host for Cas13 or a feature of SUSP1 and should be investigated further. Overall, we find that LbuCas13a is capable of anti-phage activity with no identified limits across the tested coliphage phylogeny.

A generalizable markerless method for editing phage genomes

The editing of virulent phage genomes has remained a major challenge for phage engineering and reverse genetics, largely due to the lack of universally applicable genetic tools or reliance on a native CRISPR-Cas system^{25,26,28,37,51–54}. While the introduction of foreign gene content into phages is relatively straightforward to perform with homologous recombination (HR), ultimately the selection or screening for these rare recombinants is limiting even in well-characterized phages⁵³. Given that LbuCas13a phage-restriction efficacy appears to have very little variability in terms of guide (Fig. ​(Fig.2),2), target (Figs. ​(Figs.22 and ​and3)3) and phage choice (Fig. ​(Fig.3),3), we suspected that Cas13a-mediated phage restriction would be an ideal tool for counterselection during phage genome editing. The high counterselection stringency observed earlier in this study obviates the need for selection markers, creating opportunities for multi-loci editing. Furthermore, the absence of protospacer-adjacent motif (PAM) requirements for LbuCas13a targeting²⁹ suggests that virtually any position within or nearby a phage transcript could be edited and selected through LbuCas13a counterselection.

In principle, edits in the phage genome introduced through homologous recombination can escape LbuCas13a targeting, while wildtype phage cannot (Fig. ​(Fig.4a).4a). To introduce and select for edits, we performed a simple two-stage homologous recombination and enrichment process (Fig. ​(Fig.4a,4a, Supplementary Fig. 18 and Methods). Briefly, we employed two strains per edit: an editing strain containing a homologous recombination vector hosting a verification-primer binding site as well as 250bp flanking phage homology arms, and a counterselection strain containing LbuCas13a and crRNA targeting the transcript carrying the locus to be edited. A pair of locus-specific examples are shown in Fig. 4b,c. We first infected the editing strain with wildtype phage at low MOI and collected the lysate consisting of a mixture of wildtype and edited phages (‘HR’ phage lysate) (Fig. ​(Fig.4a4a and Supplementary Fig. 18a). Then we diluted this lysate, infected the counterselection strain at low MOI and collected the resultant lysate (‘HR+E’ phage lysate) (Fig. ​(Fig.4a4a and Supplementary Fig. 18b).

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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 T4soc. 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 T4soc 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 T4soc, T4GT7, T7gp1.7, EdH4gp004 and EdH4gp214. 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.

Source data

As a proof of concept that such an editing approach is immediately applicable to reverse genetics in a diversity of phages, we designed a small panel of edits across phages T4, T7 and EdH4. In particular, we designed four single deletions (for example, Fig. ​Fig.4b)4b) encoding for edited phages T4soc, T7gp1.7, EdH4gp004 and EdH4gp214. While T4soc and T7gp1.7 (a nucleotide kinase) are known non-essential genes^46,55 under standard laboratory conditions, phage EdH4 has neither been edited previously, nor is there any knowledge of its genes’ essentialities before this study. Thus, EdH4 represents a pressure test for how extensible this editing strategy is to other non-model phages. As an example of more complex edits, we also designed a large edit originally identified during forward genetic screens on T4 mutant T4GT7³⁶ (hereafter, this edit in the wildtype T4 background is referred to as ‘T4wtGT7’). This edit consists of a large 3.2kb deletion in T4, fully deleting 12 genes and truncating T4gp52.1 and T4rIIB (Fig. ​(Fig.4c4c).

We designed two crRNAs disrupted by the edited phage locus of interest as well as an additional verification guide to confirm the entire gene deletion (examples for T4soc and T4wtGT7 are shown in Fig. 4b,c, respectively). When tested against wildtype phages T4, T7 and EdH4, candidate crRNAs for T4soc, T4ndd (nucleoid disruption protein), T4denB (endonuclease IV), T7gp1.7, EdH4gp004 (hypothetical protein) and EdH4gp214 (hypothetical protein) were approximately as effective in phage restriction as crRNAs targeting definitively essential genes such as mcp (Supplementary Figs. 2, 8 and 15). However, when targeting these putatively non-essential genes, we observed that plaques emerge at 10⁻³–10⁻⁴% frequency, potentially reflecting a low rate of mutative escape permitted by the genes’ non-essentiality. In line with the model of Cas13a primarily imparting phage defence through RNA trans-cleavage activity, these results indicate that the primary counterselection pressure does not depend on the essentiality of the crRNA target. One of these crRNAs, the verification crRNA for the EdH4gp004 deletion, displayed elevated toxicity upon expression and is the only crRNA in this study we could not get to function. Ostensibly, LbuCas13a’s auto-toxicity is due to the extensive self-complementarity within the spacer of the mature crRNA (Supplementary Fig. 19) and potentially reveals a design constraint to be explored in future studies.

After each stage of editing (Fig. ​(Fig.4a),4a), lysates were collected and titred against counterselection strains expressing LbuCas13a targeting the wildtype version of the edited locus (‘enrichment crRNA’ and ‘verification crRNA’), targeting an unedited locus (‘positive control crRNA’ (mcp crRNA)) and targeting a non-existent locus (‘negative control crRNA’ (RFP crRNA)) (Supplementary Figs. 20–24). By comparing the estimated titre against the non-targeting crRNA, we obtained a phenotypic estimate of the relative prevalence of edits within the population (that is, editing penetrance). Before enrichment (‘HR’), we observed targeted Cas13a-resistant infectious centres at 0.01–1% frequency for all five edits (Fig. ​(Fig.4d).4d). Of particular note, edits for EdH4 and T4wtGT7 were generally lower in abundance, suggesting lower HR frequencies of editing for EdH4, as well as larger modifications. Importantly, after enrichment (‘HR+E’), targeted Cas13a-resistant infectious centre edited phages comprised nearly 100% of the population, while emergent general Cas13 resistance remained low (Fig. ​(Fig.4d4d and Table ​Table1).1). In addition to confirming edits using the verification crRNA, we also PCR-verified nine plaques from the ‘HR+E’ lysates spotted on the counterselection crRNA for each edit (Supplementary Figs. 25–29). Unbiased PCR-derived Sanger sequencing further confirmed that the nature of Cas13 resistance was due to the designed edit in all cases.

Table 1

Summary of Cas13a-mediated phage genome editing

Edit name	Phage	Edited locus	Edit style and scope	Survivors detected?	Plaques screened	Mutant success rate (%)
T4soc	T4	soc	Deletion	Yes	9	100
T7gp1.7	T7	gp1.7	Deletion	Yes	9	100
EdH4gp004	EdH4	gp004	Deletion	Yes	9	100
EdH4gp214	EdH4	gp214	Deletion	Yes	9	100
T4wtGT7	T4	gp52.1-rIIB	Large deletion	Yes	9	100
soc-C	T4	soc	SNP (1)	No	N/A	N/A
soc-S	T4	soc	SNP (3)	No	N/A	N/A
soc-F	T4	soc	SNP (11)	Yes	9	100
dnap-C	T4	dnap	SNP (3)	Yes	9	100
dnap-S	T4	dnap	SNP (5)	Yes	9	100
dnap-F	T4	dnap	SNP (9)	Yes	9	100

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Following specific editing experiments, no plaques were detected. Thus, screening plaques and calculating efficiency were not applicable (N/A).

Phage propagation and scaling

Phages were propagated through commonly used protocols in LB media or LB top agar overlays (0.7%)⁶⁴. Unless stated otherwise, phages were propagated on E. coli BW25113 (lacI⁺rrnB_T14 ΔlacZ_WJ16 hsdR514 ΔaraBAD_AH33 ΔrhaBAD_LD78 rph-1 Δ(araB–D)567 Δ(rhaD–B)568 ΔlacZ4787(::rrnB-3) hsdR514 rph-1). Phages N4, T4, T5 and T7 were scaled on E. coli BW25113⁶⁵. Phage SUSP1 was a gift from Dr Sankar Adhya and scaled on E. coli BW25113⁴⁹. Phages EdH4 and MM02 were obtained from DSMZ culture collection and scaled on E. coli BW25113 (DSM 103295 and DSM 29475, respectively)⁶⁶. Phage λ cI857 bor::kanR was a gift from Dr Drew Endy and scaled as described previously⁶⁷. All phages were titred through 2µl spots of 10× serial dilution of phage in SM buffer (Teknova) on E. coli BW25113 in a 0.7% top agar overlay. Phage M13 was obtained from ATCC (15669-B1) and propagated on DH5α F’Iq genotype cells (NEB).

Plasmid construction

A description of all plasmids, associated plasmid accessions and oligonucleotides to build them can be found in Supplementary Tables 3 and 4. Design of plasmids was performed manually, with visualization and sequence alignment performed in SnapGene. All plasmids used in this study were verified using whole-plasmid sequencing services offered by the UC Berkeley DNA Sequencing Facility. All plasmids were maintained as strains and maintained at −80°C in 25% glycerol (Sigma).

All-in-one LbuCas13a, eLbuCas13a and RfxCas13d plasmids were designed to include a Cas13 effector under tetR-pTet control and a crRNA placeholder under constitutive expression on a p15a-CmR backbone. The crRNA placeholder sequence consisted of a constitutive promoter followed by the corresponding CRISPR direct repeat, a BsaI dropout site (aaacAGAGACCTCGTTTACCTATCGGTCTCatgct; BsaI sites shown in bold, flanking regions in lower case, and BsaI overhangs underlined), and a terminator. LbuCas13a, eLbuCas13a and RfxCas13d entry vectors were constructed through Gibson assembly (NEB, E2611L)⁶⁸, yielding plasmids pBA559, pBA560 and pBA562, respectively. Assembly of pBA559, pBA560 and pBA562 used PCRs derived from pEJC 1.2 Lbu, pEJC 1.2 Lbu A12 and pEJC 1.5 CasRX vectors that were gifts from Drs Emeric Charles and David Savage⁴⁵. Gibson reactions were purified with DNA Clean & Concentrator-5 (Zymo Research) and electroporated into DH10b (NEB, Intact Genomics).

crRNA spacers were introduced to pBA559, pBA560 and pBA562 through BsaIHFv2 (NEB, R3733L) golden-gate assembly⁶⁹. Spacers were ordered as two complementary oligonucleotides with 4bp 5′ overhangs matching the BsaI-digested destination plasmid staggered ends, phosphorylated with T4 PNK (NEB) at 37°C for 30min and duplexed (10uM) by melting at 100°C for 5min, followed by slow cooling to room temperature over 15min. PNK-annealed spacer duplex (100fmol) were used as insert template in each golden-gate reaction. Golden-gate reactions for crRNA assembly were purified with DNA Clean & Concentrator-5 (Zymo Research), electroporated into DH10b (NEB, Intact Genomics) and plated on LB+Ch at 37°C.

HR donor vectors were assembled through BbsI (NEB, R0539L) golden-gate assembly⁶⁹. For HR vectors, pBA707 was used as an entry vector. pBA707 contains a BBR1-KanR backbone with an RFP dropout cassette. Briefly, the RFP dropout cassette consists of an RFP expression cassette flanked by BbsI restriction sites revealing 3′-ATAG-5′ and 5′-AGGA-3′ overhangs. Upon successful digestion and ligation with an appropriate insert containing 5′-TATC-3′ and 3′-TCCT-5′ overhangs, both the RFP expression cassette and BbsI sites are lost, revealing RFP colonies.

For gene deletion vector designs, gene fragments (ordered from TWIST Biosciences) consisting of BbsI cut sites compatible with pBA707 flanking sequences encoding for the corresponding gene deletion (gbBA086, gbBA089, gbBA102 and gbBA103) (Supplementary Table 5; for design details, see ‘Homologous recombination donor vector design’). For recoding vector designs, 5′-phosphorylated (with T4 PNK; NEB) and annealed oligonucleotides were used for UP-homology (oBA1761/oBA1762 or oBA1765/oBA1766), DN-homology (oBA1763/oBA1764 or oBA1767/oBA1768) and mutated protospacer (oBA1769/oBA1770, oBA1771/oBA1772, oBA1773/oBA1774, oBA1775/oBA1776, oBA1777/oBA1778 or oBA1779/oBA1780). Golden-gate reactions were purified with DNA Clean & Concentrator-5 (Zymo Research), electroporated into DH10b (NEB, Intact Genomics) and plated on LB+K at 37°C. In all cases, RFP-negative colonies were chosen for sequence verification.

crRNA design

A complete summary of the spacers used in this study can be found in Supplementary Table 1. We manually designed all Cas13a/d crRNAs as 31nt spacers complementary to target phage transcripts (that is, a 31nucleotide spacer identical to the reverse-complement of a phage gene). Spacers were chosen with no substantial bias against or towards any protospacer flanking sequence and minimal additional heuristics. Spacers were exclusively chosen to target predicted phage transcripts or a non-targeting control on the basis of published genome sequences for phage λ (J02459.1), EdH4 (MK327930.1), M13 (NC_003287.2), MM02 (MK373784.1), N4 (NC_008720.1), SUSP1 (NC_028808.2), T4 (NC_000866.4), T5 (NC_005859.1) and T7 (NC_001604.1). Because DH10b harbours λ-like prophage, 80lacZΔM15, spacers were designed to avoid similarity to the DH10b genome (NC_010473.1)⁷⁰.

When targeting the CDS of a phage gene, the transcript corresponding to the first 31 nucleotides of a CDS was targeted by default. For verification of gene deletions, an additional guide targeting 0–15 nucleotides downstream of the transcript was chosen. When targeting ‘RBS’ sequences of T4gp42 and T4motA, spacers were designed to target transcripts beginning with the -4 to -6 positions of the gene. For all negative controls, an RFP-targeting spacer (‘AACTCTTTGATAACGTCTTCGCTACTCGCCA’) was used as it represented a functional spacer targeting an RNA transcript absent within our experiments.

Homologous recombination donor vector design

Gene deletions in phages T4, T7 and EdH4 HR vectors were designed with 250bp phage homology (‘UP’), a common primer binding region for verification and 250bp phage homology (‘DN’). For T4soc, T7gp1.7 and EdH4gp0214 gene deletions, phage homology was chosen leaving both the native start and stop codons intact. For re-creation of the T4 ‘GT7-like’ large deletion³⁶, homology was inferred from alignment of T4GT7 (KJ477686.1) to wildtype T4 (NC000866.4). This design yielded a 3,254bp deletion of T4 from positions 165,257–168,510, entirely removing 12 genes and partially removing T452.1 and T4rIIB.

For minimal recoding edits, HR donor vectors were designed with 52nt of homology upstream (UP) and downstream (DN) of a targeted protospacer on the phage genome. To encode minimal edits, predicted codons were converted to silent mutations in a single codon (-C), seed region (-S) or full protospacer (-F) using a coding table for E. coli. When possible, codons were maximally altered and rare codons avoided to minimize non-Cas13 phenotypic consequence. The seed region was estimated as previously observed in vitro⁴⁴.

Efficiency of plaquing assays

Bacteriophage assays were conducted using a modified double agar overlay protocol. For each Cas13-crRNA-phage combination, a strain of DH10b (NEB, Intact Genomics) containing a Cas13-crRNA plasmid (Supplementary Table 3) was grown overnight at 37°C and 250r.p.m. To perform plaque assays, 100µl of saturated overnight culture was mixed with molten LB Lennox top agar supplemented with appropriate inducer and antibiotics and decanted onto a corresponding LB Lennox agar plate (to final overlay concentrations of 0.7% (w/v) agar, 5nM aTc and 34µgml⁻¹ chloramphenicol). For all phage experiments in this study, no supplementary CaCl₂ or MgSO₄ salts were added. For pBA675 and pBFC1053, toxicity was apparent at 5nM aTc, so lower levels of aTc were used (0 and 1nM aTc, respectively). For pBA769, assays were performed at 10nM aTc to achieve restriction against phage SUSP1. Overlays were left to dry for 15min under microbiological flame. For each Cas13-crRNA-phage combination, 10X serial dilutions of the appropriate phage were performed in SM buffer (Teknova), and 2µl of each dilution were spotted onto the top agar and allowed to dry for 10min. Plaque assays were incubated at 37°C for 12–16 hours. After overnight incubation, plaques were scanned using a standard photo scanner and plaque-forming units (p.f.u.s) enumerated. In cases where individual p.f.u.s were not enumerable but clearings were observed at high phage concentrations, we interpreted these cases as ‘lysis from without’ and indicated a lack of productive phage infection⁷¹. As an estimate of an upper bound of phage infection for these cases, the most concentrated dilution at which no individual plaques were observed was approximated as 1 p.f.u. Efficiency of plaquing (EOP) calculations for a given condition were performed by normalizing the mean p.f.u. for a condition to the mean p.f.u. of a non-targeting control: mean(p.f.u._condition)/mean(p.f.u._{negativecontrol}). All plaque assays were performed in biological triplicate. Calculations were performed using GraphPad Prism.

Liquid growth curve assays

Liquid phage experiments were performed in a Biotek plate reader at determined levels of aTc induction. Briefly, for each Cas13-crRNA combination, a strain of DH10b (NEB, Intact Genomics) containing a Cas13-crRNA plasmid (Supplementary Table 3) was grown overnight at 37°C and 250r.p.m. Strains were seeded in wells at 8×10⁶ colony-forming units (c.f.u.), and 200µl of LB+Ch media containing 0, 1nM, 10nM or 100nM aTc was added to each well. Infection was monitored in a Biotek Cytation 5 plate reader for 16hours, with 200r.p.m. shaking at 37°C, with optical density (OD)₆₀₀ readings every 5min. All growth assays were performed in biological triplicate beginning from three independent overnight bacterial cultures. Data were plotted using the seaborn package in Python.

Liquid phage infection assays

Liquid phage experiments were performed in a Biotek plate reader at determined MOIs. Briefly, for each Cas13-crRNA-phage combination, a strain of DH10b (NEB, Intact Genomics) containing a Cas13-crRNA plasmid (Supplementary Table 3) was grown overnight at 37°C and 250r.p.m. Strains were seeded in fresh media (LB+Ch+10nM aTc) to an OD₆₀₀ of 0.04 and 200µl transferred to a 96-well plate (Corning 3904), achieving a final cell count of ~8×10⁶c.f.u. per well. Appropriate phages were diluted in SM buffer (Teknova) to a maximal titre of 10¹¹p.f.u. per ml and 10X serially diluted 7 times. To begin phage infection, 1µl of phage was added to achieve MOIs of 1.25*10-6 to 12.5. Infection was monitored in a Biotek Cytation 5 plate reader for 16hours, with 200r.p.m. shaking at 37°C, with OD₆₀₀ readings every 5min. All infection assays were performed in biological triplicate beginning from three independent overnight bacterial cultures. Data were plotted using the seaborn package in Python.

Phage genome editing experiments

A graphical overview of the phage genome editing experiments is shown in Supplementary Fig. 18. All assays were performed in biological triplicate beginning from three independent overnight bacterial cultures. All editing workflows occurred in parallel processes (that is, ‘editing’ replicates).

To create genome-edited phage lysates, a phage-editing strain consisting of DH10b (NEB, Intact Genomics) containing a homologous recombination vector (pBA1015 (T7gp1.7), pBA1018 (T4wtGT7), pBA1030 (EdH4gp004), pBA1031 (EdH4gp214), pBA1032 (T4soc) or pBA787-pBA792 (recoding experiments)) (Supplementary Table 3) was grown overnight in LB+K media at 37°C and 250r.p.m. Strains were diluted into fresh media (LB+K) to an OD₆₀₀ of 0.04 and 200µl transferred to a 96-well plate (Corning 3904), achieving a final cell count of ~8×10⁶c.f.u. per well. Wildtype phage was added to each well to achieve an MOI of 0.01 (~8×10⁴p.f.u. of phage). Infection was monitored in a Biotek Cytation 5 plate reader at 200r.p.m. shaking at 37°C, with OD₆₀₀ readings every 5min. Infection was allowed to proceed until there was a visible population crash (~4.5–7hours depending on the phage). Lysates were transferred to a 96-well block (Greiner 780271-FD), and one drop of chloroform (Sigma) was added to lyse remaining bacteria. These lysates comprise a mixture of homologous recombination-edited phage and wildtype phage and comprised the ‘HR’ phage lysate. Blocks were covered with an aluminum seal (Corning 6570). ‘HR’ phage lysates were stored at 4°C until use and titred before enrichment.

To enrich genome-edited phage lysates, a phage counterselection strain consisting of DH10b (NEB, Intact Genomics) containing an ‘enrichment’ Cas13a vector (pBA1034 for T7gp1.7, pBA1038 for T4wtGT7, pBA1042 for EdH4gp004, pBA1044 for EdH4gp214, pBA691 for T4soc and T4soc recoding, or pBA778 for T4dnap recoding) (Supplementary Table 3) was grown overnight in LB+Ch media at 37°C and 250r.p.m. Strains were diluted into fresh media (LB+Ch+10nM aTc) to an OD₆₀₀ of 0.04 and 200µl transferred to a 96-well plate (Corning 3904), achieving a final cell count of ~8×10⁶c.f.u. per well. ‘HR phage lysate’ was added to each well to achieve an MOI of 0.01 (~8×10⁴p.f.u. of total phage titre). Infection was monitored in a Biotek Cytation 5 plate reader at 200r.p.m. shaking at 37°C, with OD₆₀₀ readings every 5min. Infection was allowed to proceed until there was a visible population crash (~7hours). Lysates were transferred to a 96-well block (Greiner 780271-FD), and one drop of chloroform (Sigma) was added to lyse remaining bacteria. These lysates comprise an enriched mixture of homologous recombination-edited phage and wildtype phage and comprised the ‘HR+E’ phage lysate. Blocks were covered with an aluminum seal (Corning 6570). ‘HR+E’ phage lysates were stored at 4°C until use.

Determination of phage genome editing penetrance

Phage-editing penetrance was determined by plaque assay of ‘HR’ and ‘HR+E’ lysates on non-selective and wt-phage-counterselective strains. For T7gp1.7 experiments, pBA1034 was used as the ‘enrichment crRNA’, pBA1035 as the ‘verification crRNA’, pBA678 as the ‘positive control crRNA’ and pBA620 as the ‘negative control crRNA’. For EdH4gp004 experiments, pBA1042 was used as the ‘enrichment crRNA’, pBA1043 as the ‘verification crRNA’, pBA823 as the ‘positive control crRNA’ and pBA620 as the ‘negative control crRNA’. For EdH4gp214 experiments, pBA1044 was used as the ‘enrichment crRNA’, pBA1045 as the ‘verification crRNA’, pBA823 as the ‘positive control crRNA’ and pBA620 as the ‘negative control crRNA’. For T4soc experiments, pBA673 was used as the ‘enrichment crRNA’, pBA674 as the ‘verification crRNA’, pBA647 as the ‘positive control crRNA’ and pBA620 as the ‘negative control crRNA’. For T4wtGT7 experiments, pBA1038 was used as the ‘enrichment crRNA’, pBA1039 as the ‘verification crRNA’, pBA647 as the ‘positive control crRNA’ and pBA620 as the ‘negative control crRNA’. For soc recoding edits, 10nM aTc induction was used for strains containing pBA620 as a negative control and pBA691 as an ‘enrichment crRNA’ Cas13 vector. For dnap edits, 5nM aTc induction was used for strains containing pBA620 as a negative control and pBA778 as an ‘enrichment crRNA’ Cas13 vector. For all edited phages, penetrance was defined as p.f.u._enrichment/p.f.u._negative. Average penetrance was calculated across independent editing attempts. Penetrance calculations were performed in Graphpad Prism.

To confirm the genotype of edits, we performed unbiased PCRs followed by Sanger sequencing. In addition, for gene deletions we performed N-terminal and C-terminal PCRs. Primers for unbiased PCRs were designed to amplify from the phage genome 150–300bp outside of the UP and DN homology arms supplied from the editing vectors. For N-terminal and C-terminal PCRs, reverse (oBA2074:) and forward (oBA2075) facing primers touchdown on the small primer binding site provided on HR vectors (GATAAGAGACGGCTCAACGCCCGTCTCACAGC). PCRs were performed on three individual plaques from each ‘HR+E’ lysate after plaquing on the ‘enrichment crRNA’ strain. Plaques were picked into 50µl SM buffer (Teknova) and allowed to diffuse out of the plaque plug at 4°C overnight. To prepare for PCR and denature phage virions, 10µl of these samples were transferred to PCR tubes and boiled at 100°C for 10min. PCRs were visualized using 1% agarose gels stained with SYBR-SAFE and imaged using a BioRad gel imager using auto-exposure settings. Sanger sequencing traces were visualized using Geneious.

Cas13 phylogenetic tree

Cas13-annotated protein sequences were compiled from NCBI and were identified in GTDB r95 using custom cas13 Hidden Markov Models. All sequences that did not contain two R/Q/N/K/H/****H sequence motifs were removed. CD-HIT v4.8.1⁷² was used to cluster sequences, with a length cut-off of 0.9 and sequence similarity of 0.9. Sequences were then independently aligned using MUSCLE v3.8.31 and manually trimmed in Geneious^73,74. A maximum-likelihood phylogenetic tree was built from the alignment using IQ-TREE v1.6.12⁷⁵ with the following parameters: -st AA -nt 48 -bb 1000 -m LG+G4+FO+I. Accession numbers used to construct the Cas13 phylogenetic tree are provided in Supplementary Table 6.

Phage genome comparisons network

Protein-protein phage genome comparisons were performed with VConTACT2⁷⁶ MCL clustering (rel-mode Diamond, vcs-mode ClusterONE) of the protein sequences of the Prokaryotic Viral RefSeq 201 phage database and the phages used during experiments in this study. Produced viral clusters that neither contained E. coli phage nor shared an edge with a viral cluster containing any E. coli phage were removed together with singletons to simplify the network.

Average nucleotide identity phage genome comparisons were performed with Gepard⁷⁷ using a word length of 10bp. For source genomes, we used a concatenation of the nine phage genomes used in this study: T4 (NC_000866.4), MM02 (MK373784.1), SUSP1 (NC_028808.2), EdH4 (MK327930.1), N4 (NC_008720.1), T7 (NC_001604.1), λ (J02459.1), T5 (NC_005859.1) and M13 (NC_003287.2).

We thank members of the Doudna lab, the Innovative Genomics Institute and m-CAFEs SFA for helpful discussions, encouragement and feedback. Original templates for LbuCas13a, eLbuCas13a and RfxCas13d were kindly provided by E. Charles and D. Savage. Phage SUSP1 was a generous gift from S. Adhya. We also thank H. Asahara, E. Wagner, A. Lam and R. Sachdeva for piloting custom full-plasmid sequencing and analysis pipelines that were instrumental to this study. The mature version of this pipeline is publicly accessible through the UC Berkeley Sequencing Facility. Main text figures ​figures22 and ​and44 as well as Supplementary Figure 18 were created using BioRender.com. This project, B.A.A. and T.H. were supported by m-CAFEs Microbial Community Analysis & Functional Evaluation in Soils ([email protected]), a Science Focus Area led by Lawrence Berkeley National Laboratory based upon work supported by the US Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231. J.A.D. is an Investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.