RNA-targeting CRISPR-Cas13 Provides Broad-spectrum Phage Immunity

Abstract

CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against invasive phages through general, non-specific RNA degradation upon complementary target transcript binding. Despite being RNA nucleases, Cas13 effectors are capable of inhibiting the infection of dsDNA phages but have only been investigated across a relatively small sampling of phage diversity. Here, we employ a systematic, phage-centric approach to determine the anti-phage capacity of Cas13 and find LbuCas13a to be a remarkably potent phage inhibitor. LbuCas13a confers robust, consistent antiviral activity regardless of gene essentiality, gene expression timing or target sequence location. Furthermore, after challenging LbuCas13a with eight diverse E. coli phages distributed across E. coli phage phylogenetic groups, we find no apparent phage-encoded limits to its potent antiviral activity. In contrast to other Class 2 CRISPR-Cas proteins, these results suggest that DNA phages are generally vulnerable to Cas13a targeting. Leveraging this effective anti-phage activity, LbuCas13a can be used seamlessly as a counter-selection agent for broad-spectrum phage editing. Using a two-step phage editing and enrichment approach, we show that LbuCas13a enables markerless genome edits in phages with exceptionally high efficiency and precision, including edits as small as a single codon. By taking advantage of the broad vulnerability of RNA during viral infection, Cas13a enables a generalizable strategy for editing the most abundant and diverse biological entities on Earth.

Approaches for bacteriophage genome engineering

“…Replication of Staphylococcus epidermidis phages on a host strain encoding an endogenous type III-A system, a plasmid-encoded repeat-spacer unit, and a recombination template resulted in very efficient selection for the desired phage mutants using a one-step approach (Figure 2B) [36]. The heterologously expressed RNAtargeting type VI system was also successfully applied to counter-select for mutant phages in a two-step approach [43,44].…”

“…Nevertheless, T4 could be successfully engineered by screening for effective type II spacers [49], or by combining Cas9-based genome editing with insertion of a luciferase gene for facilitated screening [48]. The type V CRISPR-Cas system, which is less affected by DNA modifications [50], and the RNA-targeting type VI system [44] were also efficiently applied to generate and select for mutant T4 phages. Both the one-step approaches with the DNA-targeting type II or V systems and the two-step approach with the type VI system eliminated the wild type T4 phages without resulting in enrichment of escape mutants [44,[48][49][50].…”

“…The type V CRISPR-Cas system, which is less affected by DNA modifications [50], and the RNA-targeting type VI system [44] were also efficiently applied to generate and select for mutant T4 phages. Both the one-step approaches with the DNA-targeting type II or V systems and the two-step approach with the type VI system eliminated the wild type T4 phages without resulting in enrichment of escape mutants [44,[48][49][50]. Spacers resulting in a high enough targeting efficiency for counter-selection were readily found for type VI [44], while for type V [50] and type II [48,49] screening for an efficient spacer was required.…”

Phage genome cleavage enables resuscitation from Cas13-induced bacterial dormancy

“…Type VI CRISPR-Cas systems can provide robust anti-viral immunity to a wide range of viruses through this abortive infection-like mechanism [13][14][15][16][17] .…”

“…Two reports have demonstrated that DNA-targeting CRISPR systems and RM can exert anti-phage immunity at the same time 21,22 . Previous investigations of type VI CRISPR immunity have been performed with methylated or otherwise restriction-resistant phages [13][14][15][16][17] . Here we find that RM systems frequently cooccur with type VI-A CRISPR systems in strains of L. seeligeri and other Listeria, and the two defenses operate simultaneously during phage infection.…”