CRISPR Related Gene Doping

New Publication
_{first published 29th April 2024}

Paßreiter A, Naumann N, Thomas A, Grogna N, Delahaut P, Thevis M. Detection of sgRNA via SHERLOCK as Potential CRISPR Related Gene Doping Control Strategy. Anal Chem. 2024 May 14;96(19):7452-7459. 

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Abstract
Apprehensions about gene doping have grown consistently due to advancements in gene engineering techniques, particularly with the emergence of clustered regularly interspaced short palindromicrepeats/CRISPR-associated (CRISPR/Cas)-based tools. These tools not only provide unprecedented possibilities for illicit performance enhancement by athletes but also offer new avenues for the detection of gene doping through biosensing of nucleic acids. Hence, pursuing on a previous study, an analytical method based on reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection by means of Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) was optimized for the direct detection of sgRNA associated with Streptococcus pyogenes in serum. Detection device, assay parameters, and sample handling were adjusted, to overcome previously determined assay limitations. The conducted method characterization confirmed the methods' specificity and increased detection sensitivity from 100 pM to 1 fM sgRNA in 100 μL of serum. Furthermore, reanalysis of in vivo mouse administration samples collected in a previous proof-of-concept study was conducted with successful identification of sgRNA in all anticipated postadministration samples within the 24-h collection period. Those findings support the applicability of the refined analytical procedure for the detection of illegal doping attempts via ribonucleoprotein-based CRISPR/Cas application through sgRNA identification, offering a new potential doping control strategy for CRISPR related gene doping.

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What is CRISPR?

What is Sherlock?

How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes.
Paßreiter A, Naumann N, Thomas A, Grogna N, Delahaut P, Thevis M.Analyst. 2022 Nov 21;147(23):5528-5536. doi: 10.1039/d2an01318e.PMID: 36341480

First Steps toward Uncovering Gene Doping with CRISPR/Cas by Identifying SpCas9 in Plasma via HPLC-HRMS/MS.
Paßreiter A, Thomas A, Grogna N, Delahaut P, Thevis M.Anal Chem. 2020 Dec 15;92(24):16322-16328. doi: 10.1021/acs.analchem.0c04445. Epub 2020 Nov 25.PMID: 33237723
   Comment and summary of this article in analytica-world.com  (german translation)
  Comment and summary provided by Analytical Chemical Society