Universität zu Köln

Passreiter, Alina, Naumann, Nana, Thomas, Andreas, Grogna, Nicolas, Delahaut, Philippe and Thevis, Mario (2022). How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes. Analyst, 147 (23). S. 5528 - 5537. CAMBRIDGE: ROYAL SOC CHEMISTRY. ISSN 1364-5528

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Abstract

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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Passreiter, Alina UNSPECIFIED UNSPECIFIED UNSPECIFIED Naumann, Nana UNSPECIFIED UNSPECIFIED UNSPECIFIED Thomas, Andreas UNSPECIFIED UNSPECIFIED UNSPECIFIED Grogna, Nicolas UNSPECIFIED UNSPECIFIED UNSPECIFIED Delahaut, Philippe UNSPECIFIED UNSPECIFIED UNSPECIFIED Thevis, Mario UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-671296
DOI:	10.1039/d2an01318e
Journal or Publication Title:	Analyst
Volume:	147
Number:	23
Page Range:	S. 5528 - 5537
Date:	2022
Publisher:	ROYAL SOC CHEMISTRY
Place of Publication:	CAMBRIDGE
ISSN:	1364-5528
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language GENE; DELIVERY; RIBONUCLEOPROTEIN Multiple languages Chemistry, Analytical Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/67129