Scientists have taken a step closer to developing a method to identify gene doping, according to research published by Analytical Chemistry. A gene editing method known as CRISPR-Cas9 won the 2020 Nobel Prize for Chemistry, as it allows scientists to precisely alter DNA. This is done by adding a ribonucleic acid (RNA) molecule and a Cas9 protein into cells. The RNA molecule ‘guides’ the protein to to the appropriate DNA sequence, simplifying the genome editing process.

The development of this new method raised concerns that it may be utilised for gene doping, a method prohibited by the World Anti-Doping Agency (WADA – see right). ‘Because it enables the precise modification of any desired DNA sequence and surpasses all hitherto existing alternatives for gene editing in many ways, it is one of the most frequently used tools fo genome editing’, explain scientists that researched methods of identifying whether CRISPR-Cas9 gene editing had taken place. ‘These advantages also potentially facilitate the illicit use of the CRISPR/Cas system in order to acheive performance enhancing effects in sporting competitions’.

Mario Thevis and colleagues focused on identifying the Cas9 protein most likely to be used in CRISPR-Cas9 gene editing, from the bacteria Streptococcus pyogenes (SpCas9), in human plasma samples. The scientists ‘spiked the SpCas9 protein into human plasma, then isolated the protein and cut it into pieces’, explained a statement from the American Chemistry Society (ACS). ‘When the pieces were analyzed by mass spectrometry, the researchers found that they could successfully identify unique components of the SpCas9 protein from the complex plasma matrix. 

‘In another experiment, inactivated SpCas9, which can regulate gene expression without altering DNA, was spiked into human plasma samples. With a slight modification, the method allowed the team to purify and detect the inactive form. Finally, the team injected mice with SpCas9 and showed that their concentrations peaked in circulating blood after two hours and could be detected up to eight hours after administration into muscle tissue.’

The scientists outlined that a qualitative method of validation was conducted with three specific peptides, allowing for a detection limit of 25ng/mL. As outlined by the ACS above, the detection method is also applicable to the detection of gene regulation using inactive Cas9. As mentioned, in order to prove that the concept worked, administration to a live mouse enabled a detection window of up to eight hours post administration. The team concluded that this confirms the suitability of this test strategy for analysis of doping samples in order to detect gene doping. 

It is understood that gene doping initially became a concern in 1988, when a Study was conducted involving a genetically modified mouse that over expressed insulin-like growth factor 1 (IGF-1) which was stronger than ordinary mice, even in old age. In 2002, preclinical studies into Repoxygen involved delivery of the gene encoding erythropoietin (EPO) as a potential treatment for anaemia. In 2004, a Study showed that ‘marathon mice’ which had been given gene therapy coding for a protein named PPAR gamma had double the endurance of untreated mice. It is understood that the scientists involved in all of these studies received calls from athletes and coaches, who remained interested despite being alerted about potential health risks.