4.6 Article

How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes

期刊

ANALYST
卷 147, 期 23, 页码 5528-5536

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ROYAL SOC CHEMISTRY
DOI: 10.1039/d2an01318e

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资金

  1. World Anti-Doping Agency [21E06MT]
  2. Manfred-Donike Institute for Doping Analysis (Cologne, Germany)
  3. German Sport University Cologne, Germany

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The CRISPR/Cas toolkit is a frequently used gene editing technique, but it can also be used illegally for performance enhancement in sports. This study presents a promising strategy for the direct detection of illicit CRISPR/Cas methods in doping control samples using the SHERLOCK system. The method showed specificity and high sensitivity, and was able to detect the presence of CRISPR/Cas traces in serum samples. The proof-of-concept study in a mouse model also demonstrated the potential applicability of the test strategy to authentic doping control samples in the future.
The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool kit constitutes one of today's most frequently used gene editing techniques. Editing of virtually any DNA sequence can be realised, due to the quickly progressing research into different Cas effectors and their ever-expanding range of targets. Moreover, the simplicity and cost-effectiveness of those CRISPR tools can, unfortunately, also facilitate the illicit utilisation of CRISPR/Cas in order to achieve performance enhancements amongst athletes. Consequently, there is an urgent need for the direct detection of illegally applied CRISPR/Cas methods in doping control samples, for which a promising strategy is presented herein employing Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) for targeted nucleic acid detection. An analytical method was developed that enables the detection of sgRNA associated with Cas9 from Streptococcus pyogenes (SpCas9) in serum samples by means of reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection via SHERLOCK in combination with a complementary gel-based screening procedure in order to uncover doping attempts with lipid mediated CRISPR ribonucleoprotein (RNP) complexes. Initial qualitative method characterisation confirmed the specificity of both procedures and established a detection sensitivity of 10 nM uncomplexed target sequence and 100 pM sgRNA in the form of RNP complexes. Furthermore, a proof-of-concept in vivo adimistration study simulating a hypothetical gene doping scenario employing a mouse model revealed a detection window of 8 h after intravenous injection, supporting the principal applicability of the test strategy to authentic doping control samples in the future.

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