A massively multiplexed CRISPR-microfluidics platform to detect gene doping

Code: 23GD03HS

Gene doping is defined as “the non-therapeutic use of genes, genetic elements and/or modified cells that have the capacity to enhance athletic performance” and is categorized as a threat to the integrity of sport and the health of athletes by the World Anti-Doping Agency (WADA). The only direct approaches for detecting gene doping are PCR assays, so introducing a novel alternative approach for rapid and accurate identification of gene doping is vital. In this project, a novel ultrasensitive detection platform will be designed based on the combination of 2 different technologies i.e. CRISPR and Microfluidics. CRISPR/CAS systems can survey the whole genome and detect transgenic DNA (tDNA) using a single-stranded guide RNA (gRNA). In the presence of a target gene, a ternary complex (Cas12/gRNA/tDNA) will be formed and the quenched fluorescence probe will be degraded by collateral cleavage activity of the ternary complex. Microfluidics fluorescence detection can provide exquisite sensitivity. The combination of CRISPR/CAS as a biological gene doping detection system and a microfluidics system can provide multiplex gene doping detection (up to 96 samples with 96 detection assays per chip). Pre-amplification technologies such as recombinase polymerase amplification will be applied in order to reach single molecule (1 aM) sensitivity. Here, we will investigate more than 20 genes including CSH1, CSH1,CSH2, CSHL1, EPO, GDFB, GH1, GH2, IGF1, IGF2, MYOG, PPARd, VEGF, etc. as a proof of concept. However, the number of assay combinations could be 96 samples with 96 detection assays (96 different Cas/gRNAs). The gRNAs will be designed to target exon-exon junctions, specific sequences of viral vectors, such as CMVp (cytomegalovirus promoter), TkpA (thymidine kinase polyA from herpes simplex virus), hexon (major virus capsid protein from adenovirus), and CMVp (cytomegalovirus promoter) etc. The use of combinations of different gRNAs for the same gene (several gRNAs for different exon-exon junctions) with virus targeted-gRNAs will allow us to detect doping genes and different vectors simultaneously and dramatically increase the successful detection of tDNAs. Moreover, specific CRISPR-reference materials (C-RM) containing modified transgenic sequences will be designed to avoid false positive results due to cross-contamination. Based on the WADA guidelines for gene doping, appropriate no template controls (NTC) and positive template controls (PTC) will be used to determine the specificity, efficiency, sensitivity and limit of detection of the assay. The performance efficiency of the detection platforms will be tested in spiked blood samples. These multiplexed gene doping detection CRISPR-equipped microfluidic platforms could be an ideal and rapidly deployable detection approach, which is accurate, inexpensive and rapid, allowing for anti-gene doping assays in WADA-accredited laboratories.