Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing

Abstract

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.

Fig. 1. Maximum-likelihood phylogeny of Cas13 proteins and their distribution across the bacterial tree of life.

The four known subtypes, Cas13a–d, each form their clade (inner track) with a skewed distribution across bacterial taxa (outer track). A Vibrio cholerae Cas9 (UIO88932.1) was used as the outgroup. Cas13 subtypes and microbial taxa that encode Cas13 are denoted in the colour bar. Source data

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

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 vContact2 (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

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

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

Fig. 5. Minimal edits in phage T4 enabled by Cas13a counterselection.

a, Homologous recombination vector design consists of a recoded Cas13a protospacer flanked by 52 bp of homology to the phage genome. b, Recoding design for a T4 non-essential gene, soc, with introduced silent mutations shown in magenta. Three designs with differing mutations were tested (soc-C, soc-S, soc-F). Underlined nucleotides represent the edge of the Cas13a CRISPR repeat. c, Recoding design for a T4 essential gene, dnap, with introduced silent mutations shown in magenta. Three designs with differing degrees of mutations were tested (dnap-C, dnap-S, dnap-F). Underlined nucleotides represent the edge of the Cas13a CRISPR repeat. d, Editing penetrance from three biological replicates of the editing and enrichment process shown in b for soc-C, soc-S and soc-F. Edited phage lysates with no detectable plaques are noted with ND. e, Editing penetrance from three biological replicates of the editing and enrichment process shown in b for dnap-C, dnap-S and dnap-F. Editing penetrance in d and e are presented as mean ± s.d. f, Unbiased sequencing of T4soc loci from individual plaques from three independent editing attempts. Deviations from wildtype are highlighted. g, Unbiased sequencing of T4dnap loci from individual plaques after editing attempts dnap-C (top), dnap-S (middle), and dnap-F (bottom), each with three independent editing attempts. Deviations from wildtype are highlighted. Sanger sequencing traces for all verified plaques including those shown in f and g can be found in Supplementary Figs. 16 and 20. Source data