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

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...

Detection of sgRNA via SHERLOCK as Potential CRISPR Related Gene Doping Control Strategy

“…The initial practical approach was adopted from Kellner et al and the experimental design was further elaborated from the preceding project and adjusted according to the new objective of assay improvement. 17,18 Reagents and Materials. A list of all utilized reagents and materials is available in the Supporting Information (SI).…”

“…Spacer design of the associated crRNA for targeted sgRNA identification was maintained from the previous study because of the beneficially chosen target sequence aiming for the sgRNA scaffold, allowing for the detection of many different sgRNAs associated with Streptococcus pyogenes despite their genomic target. 17 Therefore, this approach enables the identification of a whole substance class rather than only one analyte. Furthermore, Streptococcus pyogenes is a common human pathogen and there is no information whether this fact could interfere with the analytical method.…”

“…24 In addition, another preliminary proof-of-concept study provided first evidence that direct detection of sgRNA in blood post in vivo administration is principally possible via SHERLOCK in combination with RT-RPA. 17 To overcome thereby determined assay limitations such as visual readout of samples employing a transillumination device and the associated low sensitivity, the aim of the present study was method optimization for the identification of sgRNA in blood samples via SHERLOCK in terms of applicability and sensitivity for routine doping control purposes. Analyte detection was thus transferred to a fluorometric plate reader, and sample handling was adjusted accordingly.…”

Annual banned‐substance review 16th edition—Analytical approaches in human sports drug testing 2022/2023

“…An LOD of 1 aM of amplified plasmids was determined, and an overall turnaround time of 40 min was presented, suggesting an interesting methodology for future gene doping tests, but additional data on specificity and reproducibility might need to be provided. In a similar manner, Passreiter et al aimed at detecting the manipulation of the human genome by CRISPR/Cas interventions rather than proving the administration of transgenic material 113 . Exploiting the conserved region of the sgRNA found in CRISPR/Cas9 systems as xenobiotic target, a protocol consisting of isolating the CRISPR RNA from human serum, reverse transcription‐RPA, subsequent T7 transcription, and detection by fluorescence enabled by the amplicon‐triggered activation of Cas13a and cleavage of a fluorescence‐quenched ssRNA reporter.…”

Gene Doping Control Analysis of Human Erythropoietin Transgene in Equine Plasma by PCR-Liquid Chromatography High-Resolution Tandem Mass Spectrometry

“…With the growing concerns of gene doping threatening the integrity of sport, analytical methods have been developed for antigene doping using various technologies, including polymerase chain reaction (PCR), − sequencing, , clustered regularly interspaced short palindromic repeats (CRISPR), − and mass spectrometry (MS). − Specifically, real-time and digital PCR assays have been utilized to identify transgenes in different biological matrices. − The PCR-based detection method achieves its specificity by using primers and/or probe hybridized to the exon–exon junctions in the transgenes, indicating their exogenous origin. The unique sequence in the transgene is exponentially amplified by the specific pairs of primers, and detection is achieved in real time and/or post-PCR.…”