2022 article

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

Adler, B. A., Hessler, T., Cress, B. F., Mutalik, V. K., Barrangou, R., Banfield, J., & Doudna, J. A. (2022, March 26). (Vol. 3). Vol. 3.

co-author countries: Australia 🇦🇺 United States of America 🇺🇸
Source: ORCID
Added: March 31, 2022

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.