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SHERLOCK: The case of the disruptive diagnostic

"SHERLOCK is exciting because it represents a sensitive approach to detect mutations, much like PCR, but it does not require a thermocycler and could work as a bedside test." - Dr Tilmann Buerckstuemmer, Global Head of Innovation at Horizon Discovery


Brief summary of the discovery:

Adaptation of CRISPR into a Diagnotic Platform

An exciting paper published in the latest issue of Science by the lab of Feng Zhang at the Broad Institute (Gootenberg) describes the adaptation of CRISPR-based technology into a diagnostic platform (CRISPR-Dx) that allows rapid, inexpensive, highly sensitive and specific detection of nucleic acids. At the heart of their method, dubbed SHERLOCK (for Specific High-Sensitivity Enzymatic Reporter unLOCKing), lies CRISPR associated family member Cas13a (an RNA-guided RNase formerly known as C2c2, an enzyme characterized by Zhang and colleagues in 2016 (Abudayyeh)). Cas13a is naturally expressed in bacteria and is deployed to target/counter invading phage RNA. Similar to Cas9, Cas13a displays on-target cleavage upon recognition, however Zhang and colleagues additionally found the enzyme to cleave nearby RNA molecules in a non-specific manner. The researchers effectively harnessed this "collateral cleavage" characteristic of Cas13a to generate a visual indicator of nucleic acid detection (first described in East-Seletsky).

Simluating Patient Cell-free DNA

Specifically the work describes the ability of the tool to detect both RNA and DNA at the single molecule level by coupling loop-mediated isothermal amplification to T7 RNA polymerase-based in vitro transcription. Subsequently, Cas13a protein together with a designed target-specific CRISPR RNA then bind and cleave the intended target. Crucially, Cas13a-driven collateral cleavage of a fluorescence-quenched non-specific RNA also present in the reaction mix releases the reporter signal, thereby providing a readable output. All this is performed as a single reaction at constant temperature, 37C). As proof of concept/principle, Zhang and colleagues provide evidence of the system's high sensitivity, enabling them to detect specific strain variants of Zika virus, while demonstrating high specificity by its ability to distinguish between pathogenic bacteria. Remarkably, SHERLOCK demonstrates its use as a genotyping tool through the specific identification of single nucleotide polymorphisms as well as human cancer-specific mutations in cell-free DNA samples. Patient cell-free DNA was simulated using BRAF V600E and EGFR L858R cfDNA reference standards created by Horizon Discovery.

Taken together, the promising ability of this platform to selectively detect very low copy number of virtually any DNA or RNA target, such as those present in blood, saliva, urine, and when spotted on paper holds great value for basic research, diagnostics and therapeutics. - By Dr Carlos le Sage, Senior Scientist at Horizon Discovery

  1. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F. Science. 2017 Apr 28;356(6336):438-442. doi: 10.1126/science.aam9321. Epub 2017 Apr 13. PMID: 28408723
  2. Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F. Science. 2016 Aug 5;353(6299):aaf5573. doi: 10.1126/science.aaf5573. Epub 2016 Jun 2. PMID: 27256883
  3. East-Seletsky A, O'Connell MR, Knight SC, Burstein D, Cate JH, Tjian R, Doudna JA. Nature. 2016 Oct 13;538(7624):270-273. doi: 10.1038/nature19802. Epub 2016 Sep 26. PMID: 27669025