CRISPR-associated protein 13a (Cas13a), formerly Class 2 candidate 2 (C2c2)

Characteristics

Two HEPN domains (higher eukaryotes and prokaryotes nucleotide-binding) form a composite ribonuclease active site that cleaves bound single-stranded target RNA; a separate, HEPN-independent active site processes the pre-crRNA array into mature guides. Target recognition activates the HEPN site for indiscriminate collateral cleavage of nearby single-stranded RNA regardless of complementarity to the guide. For LshCas13a, robust cleavage additionally requires a single protospacer flanking site (PFS) nucleotide 3' of the target that is not G. Combined with isothermal amplification, this collateral activity detects viral and bacterial nucleic acids at attomolar sensitivity with single-base mismatch discrimination.

Applications: The SHERLOCK diagnostic platform pairs Cas13a collateral cleavage of a labeled reporter RNA with isothermal amplification to detect Zika and dengue virus, genotype human DNA, and identify pathogenic bacteria and tumor DNA mutations at attomolar sensitivity. The Leptotrichia wadei ortholog (LwaCas13a) knocks down reporter and endogenous transcripts in mammalian and plant cells at levels comparable to RNA interference with improved specificity, and dCas13a-GFP tracks RNA in live cells.

Limitations: Target binding activates indiscriminate collateral cleavage of bystander single-stranded RNA independent of guide complementarity; in bacterial assays this arrests cell growth, which is why SHERLOCK runs in vitro rather than in living cells. For LshCas13a, robust activity also requires a non-G nucleotide at the protospacer flanking site, restricting targetable sites near G-rich sequence.

References

1. Abudayyeh et al. (2017). RNA targeting with CRISPR-Cas13. Nature - Abudayyeh 2017 Cas13