Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing

CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.

CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.
CRISPR-Cas systems confer diverse RNA-guided antiviral and anti-plasmid adaptive immunity in prokaryotes 1 . CRISPR genomic loci record phage infections over time in the form of sequence arrays comprising foreign DNA sequences (spacers) flanked by direct repeats. Array transcription and processing generate CRISPR RNAs (crRNAs) that associate with one or more cognate Cas proteins to form ribonucleoprotein complexes capable of recognizing crRNA-complementary DNA or RNA 2 . Upon target binding, Cas effectors disrupt phage infection using DNA cleavage [3][4][5] , RNA cleavage 6 , secondary messenger production 7,8 or transcriptional silencing 9 . These programmable biochemical activities have been applied as genome editing tools in bacteria and eukaryotes 10 .
Our results are consistent with previous CRISPR search endeavours, suggesting that Cas13 effectors are some of the rarest Cas proteins currently identified 2 . Although RNA-targeting type-III CRISPR-Cas systems are relatively abundant in bacterial phyla 2 , we wondered whether the sparse occurrence of Cas13 effectors means that generalized resistance (for example, through RNA recycling 41 ) or specialized resistance (for example, through anti-CRISPR 33 ) to Cas13 is relatively rare as well.

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. 1). We selected LbuCas13a from Leptotrichia buccalis and RfxCas13d from Ruminococcus flavefaciens due to their extensive biochemical characterization 29,[42][43][44][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. coli 45 . 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. 2a). Upon recognition, Cas13 activates HEPN-mediated RNA cleavage, although the extent of trans-cleavage may be reduced for Cas13d relative to Cas13a 43 . 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.
To test the phage-restriction capacity of LbuCas13a and RfxC-as13d outside their native context, we individually targeted a small progression, thereby limiting infection of neighbouring cells 30 . Four Cas13 subtypes (a-d) have been identified and differ by primary sequence and size as well as auxiliary gene association and extent of cis-versus trans-RNA cleavage activity 2 . Since all known viruses produce RNA 32 , Cas13 is capable of inhibiting double-stranded DNA (dsDNA) phages, primarily shown through studies investigating temperate 30,33,34 and nucleus-forming 19,35 phages. Class 2 CRISPR effectors tested so far have limitations and guide variability in overcoming the diversity of genetic content encoded in phages 12,19,20,27,[36][37][38] . It remains unclear whether an RNA-targeting Cas13 can broadly protect bacteria from a range of dsDNA phages.
Here we characterized the ability of a single Cas13a variant to restrict wide-ranging phage infections in model bacterium Escherichia coli. Phage infection assays show that LbuCas13a is a robust inhibitor of phage infections across the E. coli phage phylogeny. Further, Cas13-mediated phage restriction is robust across a diversity of phage genome-protection strategies, lifestyles, genes and transcript features, enabling direct and specific phage interference. We demonstrate that Cas13's potent broad-spectrum antiviral activity can be applied as a sequence-specific counterselection system suitable for recovering phage variants with edits as minimal as single codon replacement. Our results highlight the vulnerability of phage RNA molecules during phage infection and provide a robust generalizable strategy for phage genome engineering.

Cas13 homologues are rare across bacterial phyla
Phages encode diverse anti-defence strategies against the bacterial defence systems they are likely to encounter 13,33,39,40 , which in turn can render these systems ineffective for either phage immunity or phage engineering. To determine whether Cas13 might be useful as both a broad-spectrum phage defence and a phage genome editing tool, we began by investigating the distribution of Cas13 effectors across bacterial phyla. We performed a bioinformatic search for Cas13 proteins across NCBI and Genome Taxonomy Database (GTDB) genomes, culminating in a non-redundant set of 224 Cas13 protein sequences ( Fig. 1 and Supplementary Fig. 1). Consistent with previous classification efforts 2 , Cas13 subtypes cluster into four clades 13a-d. We found Cas13b to be most widespread, yet predominantly found within Bacteroidota. In contrast, Cas13c and Cas13d subtypes appeared least common, primarily found in Fusobacteriota and Bacillota (formerly Firmicutes), respectively. We found Cas13a to be phylogenetically more widely dispersed, although relatively limited in total number of homologues,  20 . 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. 2c) 46 . Targeted regions of T4 46 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. 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 , and we also observed a high degree of phage escape for RfxCas13d relative to LbuCas13a ( Fig. 2e 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 Fig. 3a 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 (M13 48 ), while the remaining seven display obligately lytic life cycles. They comprise diverse lifestyles including documented plasmid-transfer-promoting (that is, 'superspreader') 49 , DNA compartmentalization 16 and pseudolysogeny 50 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. For each phage, we designed a pair of Cas13a crRNAs targeting either a putative early gene (DNA polymerase (dnap)), RNA polymerase Article https://doi.org/10.1038/s41564-022-01258-x (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 Fig. 16). This constraint required us to perform assays in the absence of induction, achieving a mere 10 2 -fold restriction ( Supplementary Fig. 14). However, employing the reduced-toxicity LbuCas13a mutant, eLbuCas13a 45 , we observed both phage restriction at 10 6 -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 Lbu-Cas13a 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. 3b). Both early-and late-transcript targeting guides only decreased phage infectivity 5,000-10,000-fold compared with all other phages showing 10 5 -10 6 -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 protection 30 . Potentially, this discordance with the abortive-infection model of Cas13 protection observed previously 30 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][52][53][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 phages 53 . Given that LbuCas13a phage-restriction efficacy appears to have very little variability in terms of guide (Fig. 2), target (Figs. 2 and 3) and phage choice (Fig. 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 targeting 29 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. 4a). To introduce and select for edits, we performed a simple two-stage homologous recombination and enrichment process (Fig. 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 250 bp 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. 4a 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. 4a and Supplementary Fig. 18b).
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. 4b) encoding for edited phages T4∆soc, T7∆gp1.7, EdH4∆gp004 and EdH4∆gp214. 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 T4GT7 36 (hereafter, this edit in the wildtype T4 background is referred to as 'T4wtGT7'). This edit consists of a large 3.2 kb deletion in T4, fully deleting 12 genes and truncating T4gp52.1 and T4rIIB (Fig. 4c).
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. 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. 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. 4d and Table 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.

PAMless Cas13a enables minimal edits in phage genomes
We aimed to further take advantage of the flexibility enabled by Cas13a's PAMless nature by creating and enriching minimal edits that only Cas13a could easily select for 20,26,27,36 , using T4 as a model virulent phage. We designed six mutants at either the non-essential soc gene or essential dnap using silent mutations, thus 'recoding' the target gene (Fig. 5). We designed these mutants to recode only a single codon (soc-C, dnap-C), recode the entire seed region (soc-S, dnap-S) 44 or recode the full target (soc-F, dnap-F) (Fig. 5a-c). To facilitate homologous recombination-mediated edits, we flanked the intended mutation with 52 bp of native phage homology (Fig. 5a) [33][34][35]. In total, these consisted of 36 plaques across 4 unique edits (soc-F, dnap-C, dnap-S, dnap-F) and 12 independent editing processes. Strikingly, we found all 36 analysed plaques to have the intended mutation (Table 1 and Supplementary Figs. 36 and 37). It should be noted that for one engineering replicate of soc-F, we found two of the three plaques to encode a single nucleotide mutation (SNP) just outside of the site of the HR arm, probably reflecting an HR-induced source of error. Nonetheless, this editing process represents a simple straightforward route for enriching phage genome edits as small as one codon, as illustrated in the case of dnap-C.

Discussion
We report that LbuCas13a transcript targeting is a broadly applicable, programmable phage counterselection pressure that can readily be converted into a phage genome editing tool. Despite belonging to one of the rarest CRISPR-Cas systems, we found LbuCas13a to be a potent RNA-guided anti-phage system. We challenged E. coli expressing Cas13a with nine diverse phages scattered across the E. coli phage phylogeny and found Cas13a to be effective at restricting all of them (>5,000-fold) (Fig. 3). While we anticipated that many phages would not harbour specific type-VI anti-CRISPR systems due to Cas13's relative scarcity across bacterial phyla (Fig. 1), these results also suggest that it is rare to encode mechanisms to broadly recover from or prevent RNA degradation in phages. Furthermore, we observed very high crRNA efficacy and consistency between these phages and designed targets. Cas13a anti-phage activity was consistent and effective across gene essentiality, gene expression timing and target location within transcribed phage loci. In addition, LbuCas13a has no PAM requirements, has limited anti-tag inhibition 56 and, as inferred from failed recoding attempts soc-C and soc-S, is tolerant to small mutations within its protospacer 44 (Fig. 5d). Given these flexible target site constraints for LbuCas13a, we found the primary constraint on crRNA design to be spacer-specific auto-toxicity independent of the phage (Supplementary Figs. 3, 4 and 19). In E. coli, we found this toxicity readily circumventable by decreasing expression levels, using a reduced toxicity variant eLbuCas13a or by designing an alternative crRNA. Based on these observations, it appears that phages are generally vulnerable to Cas13a targeting.
Leveraging the broad vulnerability of phages to Cas13a, we demonstrated how this robust counterselection could be employed to enrich markerless genome edits in multiple E. coli phages. Most Cas-based counterselection methods show extensive crRNA or phage variability 25,28,35,52 , rely on native CRISPR host biology [51][52][53][54] and/or yield a high rate of escape mutants 35,51 . Interestingly, during preparation of this manuscript, Guan et al. reported the use of LseCas13a and its cognate anti-CRISPR as a positive-selection strategy against nucleus-forming phages 35 . While they observed greater crRNA variability and a lower on-target editing penetrance, it is possible that a combination of use in a different host with different mutation rates and expression systems (Pseudomonas aeruginosa) and use of a different Cas13a orthologue (LseCas13a) is responsible for the crRNA variability. In contrast, we observed little variability in Cas13a counterselection efficacy across the 9 phages and 31 crRNAs tested in this study (with only one crRNA failing entirely; Supplementary Fig. 19). When applied to markerless genome editing, we measured a mutational penetrance of 100%-81/81 plaques across nine unique edits, three diverse phages and three independent editing attempts (Figs. 4 and 5 and Table 1). Through HR-mediated deletions or disruptions in the wildtype protospacer sequence, we proved that a wide diversity of phage genome edits are experimentally tractable, ranging from large deletions down to a single codon replacement. Due to the combination of flexibility and efficacy of phage targeting enabled by Cas13a, we anticipate that this phage selection strategy can enrich nearly any viable edit at transcribed loci in phages whose hosts can harbour and express LbuCas13a.
Possibly, the highly potent anti-phage activity observed in Cas13a is related to the relative scarcity of type-VI CRISPR-Cas systems. All known type-VI systems are thought to facilitate anti-phage activity through mechanisms similar to abortive infection 30,34 . Although the use of crRNA confers specificity for the activation of Cas13, in the absence of phage, we noticed toxicity upon LbuCas13a expression and substantial toxicity with RfxCas13d expression ( Supplementary  Figs. 3, 4 and 19). Potentially, the simultaneously increased toxicity and reduced phage restriction seen with RfxCas13d could be remedied by expression of additional proteins from its native locus, such as a pair of WYL domain-containing genes nearby ( Supplementary Fig. 5), which has augmented orthologue RspCas13d in plasmid-restriction  Article https://doi.org/10.1038/s41564-022-01258-x contexts previously described 57 . Additionally, we observed substantial auto-toxicity for LbuCas13a in conjunction with two crRNAs (targeting T5 dnap and EdH4gp004_2). The former was remedied by using a reduced trans-cleavage variant of LbuCas13a 45 (Supplementary Figs. 14 and 16). However, we could not reduce the toxicity of the second crRNA targeting EdH4gp004, ostensibly due to intense secondary structure of the mature crRNA, raising a possible constraint on spacer design and natural selection ( Supplementary Fig. 19). Nonetheless, from a phage restriction perspective, the high reliability of crRNA efficacy we observe in tandem with flexible crRNA design afforded by Cas13a means that these occasional limitations are easily circumventable. Perhaps the genetic stability and performance of this phage counterselection system would be more limited as it is applied in more diverse bacteria, phages with higher mutation rates and extends to weakly transcribed target sites. In some respects, the seemingly universal efficacy of Cas13a against phages is surprising. RNA-cleaving HEPN domains, such as those in Cas13a 6,29 , are widely found across the tree of life, including E. coli and related bacteria [58][59][60] . Although phages encode inhibitors against HEPN domains 33,61 and other endogenous RNAses, their ability to mitigate the toxic and anti-phage effects of Cas13a-mediated RNA trans-cleavage are relatively limited. Potentially, this reflects a conflicting role of RNA degradation as the infecting phage wrestles with the host for control of the transcriptome 41,62 . In contrast, phages encode a diversity of mechanisms to mitigate the effects of dsDNA cleavage, including nuclease inhibitors 11,13,14,63 , DNA modifications 15,17 , DNA-repair mechanisms 20,21 and nucleic acid compartmentalization 16,18,19 . This comparative vulnerability to degenerate RNA cleavage we observe for phages at large highlights the centrality of RNA for viral infection 32 .

Bacterial strains and growth conditions
Cultures of E. coli were grown in lysogeny broth (LB Lennox) at 37 °C and 250 r.p.m. unless stated otherwise. When appropriate, 34 µg ml −1 chloramphenicol (+Ch) or 50 µg ml −1 kanamycin (+K) sulfate was supplemented to media. All bacterial strains were stored at −80 °C for long-term storage in 25% sterile glycerol (Sigma). Cloning and assays were primarily performed in DH10b genotype cells (NEB, Intact Genomics). For constructs targeting phage M13, cloning and assays were performed in DH5α F'I q 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 Savage 45 . 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 assembly 69 . Spacers were ordered as two complementary oligonucleotides with 4 bp 5′ overhangs matching the BsaI-digested destination plasmid staggered ends, phosphorylated with T4 PNK (NEB) at 37 °C for 30 min and duplexed (10 uM) by melting at 100 °C for 5 min, followed by slow cooling to room temperature over 15 min. PNK-annealed spacer duplex (100 fmol) 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.

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 31 nt spacers complementary to target phage transcripts (that is, a 31 nucleotide 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.  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 ('AACTCTTTGA-TAACGTCTTCGCTACTCGCCA') 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 250 bp phage homology ('UP'), a common primer binding region for verification and 250 bp 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 deletion 36 , homology was inferred from alignment of T4GT7 (KJ477686.1) to wildtype T4 (NC000866.4). This design yielded a 3,254 bp 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 52 nt 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 vitro 44 .

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 250 r.p.m. To perform plaque assays, 100 µl 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, 5 nM aTc and 34 µg ml −1 chloramphenicol). For all phage experiments in this study, no supplementary CaCl 2 or MgSO 4 salts were added. For pBA675 and pBFC1053, toxicity was apparent at 5 nM aTc, so lower levels of aTc were used (0 and 1 nM aTc, respectively). For pBA769, assays were performed at 10 nM aTc to achieve restriction against phage SUSP1. Overlays were left to dry for 15 min under microbiological flame. For each Cas13-crRNA-phage combination, 10X serial dilutions of the appropriate phage were performed in SM buffer (Teknova), and 2 µl of each dilution were spotted onto the top agar and allowed to dry for 10 min. 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 infection 71 .
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 250 r.p.m. Strains were seeded in wells at 8 × 10 6 colony-forming units (c.f.u.), and 200 µl of LB+Ch media containing 0, 1 nM, 10 nM or 100 nM aTc was added to each well. Infection was monitored in a Biotek Cytation 5 plate reader for 16 hours, with 200 r.p.m. shaking at 37 °C, with optical density (OD) 600 readings every 5 min. 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 250 r.p.m. Strains were seeded in fresh media (LB+Ch+10 nM aTc) to an OD 600 of 0.04 and 200 µl transferred to a 96-well plate (Corning 3904), achieving a final cell count of ~8 × 10 6 c.f.u. per well. Appropriate phages were diluted in SM buffer (Teknova) to a maximal titre of 10 11 p.f.u. per ml and 10X serially diluted 7 times. To begin phage infection, 1 µl 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 16 hours, with 200 r.p.m. shaking at 37 °C, with OD 600 readings every 5 min. 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).

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 T7∆gp1.7 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 EdH4∆gp004 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 EdH4∆gp214 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 T4∆soc 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, 10 nM aTc induction was used for strains containing pBA620 as a negative control and pBA691 as an 'enrichment crRNA' Cas13 vector. For dnap edits, 5 nM 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 150-300 bp 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 (GATAAGAGACGGCTCAACGCC-CGTCTCACAGC). PCRs were performed on three individual plaques from each 'HR+E' lysate after plaquing on the 'enrichment crRNA' strain. Plaques were picked into 50 µl SM buffer (Teknova) and allowed to diffuse out of the plaque plug at 4 °C overnight. To prepare for PCR and denature phage virions, 10 µl of these samples were transferred to PCR tubes and boiled at 100 °C for 10 min. 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 v4.8.1 72 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 v1.6.12 75 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 VConTACT2 76 MCL clustering (rel-mode Diamond, vcs-mode Clus-terONE) 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.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.