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Nucleic acid detection — it's elementary with SHERLOCK!

Nature Reviews Genetics volume 18, pages 392–393 (2017)Cite this article

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SHERLOCK (specific high sensitivity enzymatic reporter unlocking) is based on two critical features of the Cas13a RNase (previously called C2c2). Firstly, because Cas13a is an RNA-guided RNase it can be reprogrammed with CRISPR RNAs (crRNAs) to recognise an RNA target of choice, enabling it to act as an RNA sensor. Secondly, Cas13a has a 'collateral effect' when it recognizes its target RNA; in addition to cleaving its specific target, it also cleaves nearby non-targeted RNAs. In SHERLOCK, this collateral effect is harnessed to cleave and activate a quenched fluorescent reporter RNA to indicate the presence of the target RNA of interest.

Two measures ensure that SHERLOCK achieves the attomolar (aM) sensitivity often required of diagnostic tools: use of a highly active Cas13a orthologue from Leptotrichia wadei (LwCas13a); and incorporation of a recombinase polymerase amplification (RPA) step coupled with T7-mediated transcription prior to target detection. Importantly, this amplification-transcription step enables SHERLOCK to detect DNA targets in addition to RNA targets. When applied to mock viruses (lentiviruses containing genomic RNA fragments of Zika virus (ZIKV) or the related Dengue virus (DENV)) or to ZIKV genomic RNA isolated from patient serum or urine samples, SHERLOCK was able to detect virus down to concentrations of ∼2 aM. Furthermore, the assay could discriminate between ZIKV and DENV, and was predictive of ZIKV RNA copy number and viral load in clinical samples.

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References

  1. Gootenberg, J. S. et al., Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017)

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  1. Dorothy Clyde
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Clyde, D. Nucleic acid detection — it's elementary with SHERLOCK!. Nat Rev Genet 18, 392–393 (2017). https://doi.org/10.1038/nrg.2017.40

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