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.

Phages are important unrecognized players in the ecology of the oral pathogenPorphyromonas gingivalis

“…In characterizing Pg CRISPR-Cas systems we found a striking near-ubiquity of Class 2 Type VI systems, generally rare among bacteria 42 . Although Type VI-B are the most common system in Pg , the majority of spacers were encoded by Type I-B rather than Type VI-B arrays (Type I-B: 51%, I-C:17%, II-C: 11%, III-B: 4%, VI-B2: 17%, Supplementary Data 4).…”

“…Despite their relatively lower abundance, we found Type VI array spacers targeting all 24 candidate species of Pg phages at 100% identity. Recent work has demonstrated that Type VI systems offer broad spectrum activity against phages 42 , likely in part due to their lack of a requirement for protospacer associated motifs, this suggests the possibility that fewer spacers are needed by these systems to achieve coverage of diverse phages. Of note, whereas Type VI effector genes were commonly identified (Cas13b, and the Cas13b-activated membrane pore-forming Csx28 43 , identified as tm_HEPN by CCTyper 36 ), genes associated with the adaptation module (e.g.…”

“…To also allow evaluation of hits to non-phage sequences, we additionally mapped coverage on a per gene basis in bacterial genomes using the BEDTools 95 annotate function with default settings. Exploratory analyses of differences in strand level mapping of spacers from Class 1 systems [where crRNAs bind DNA (Types I-B, I-C, and III-B)], versus Class 2 systems [where crRNAs bind mRNA (Types II-C and VI-B2)] 42 , 101 , showed no consistent patterns. Both direct and reverse complement mappings were therefore counted for all spacers.…”

Continuous Multiplexed Phage Genome Editing Using Recombitrons

“…That screening effort can be reduced by imposing a counterselection on the unedited phage, such as CRISPR-based depletion of the wild-type phage [9][10][11][12] . However, this negative selection is not universally applicable to all edit types because it requires functional disruption of a protospacer sequence 13 . Phages also frequently escape CRISPR targeting 14 , which can result in most selected phages containing escape mutations outside of the intended edit 9 .…”

An efficient, scarless, selection-free technology for phage engineering

“…In recent years, several methods have been developed to engineer phage genomes using retroelements 2 and heterologous supplement of recombination enzymes 3 . We were the first group to harness the CRISPR-Cas system for the genetic engineering of phages 4 , followed by many others [5][6][7][8][9][10] . Using CRISPR-Cas for genetic engineering is straightforward, can be designed for virtually any gene, and is cost-effective.…”