Detecting CRISPR/Cas gene doping

(Press-News.org) All athletes want to be at the top of their game when they compete, but some resort to nefarious approaches to achieve peak muscle growth, speed and agility. Recent developments in gene editing technology could tempt athletes to change their DNA to get an edge. Now, researchers reporting in ACS' Analytical Chemistry demonstrate first steps toward detecting this type of doping both in human plasma and in live mice.

The gene editing method called CRISPR/Cas is a popular way for scientists to precisely change the DNA in many organisms, and it recently gained even more attention when key developers of the method were awarded the 2020 Nobel Prize in Chemistry. With this method, researchers add an RNA molecule and a protein into cells. The RNA molecule guides the protein to the appropriate DNA sequence, and then the protein cuts DNA, like a pair of scissors, to allow alterations. Despite the ethical concerns that have been raised about the method's potential application in humans, some athletes could ignore the risks and misuse it to alter their genes. Because CRISPR/Cas changes DNA, it is considered "gene doping" and is banned by the World Anti-Doping Agency, an independent international organization. A sufficient method to detect CRISPR/Cas gene editing needs to be developed, however. So, Mario Thevis and colleagues wanted to see whether they could identify the protein most likely to be used in this type of doping, Cas9 from the bacteria Streptococcus pyogenes (SpCas9), in human plasma samples and in mouse models.

The team spiked the SpCas9 protein into human plasma, then isolated the protein and cut it into pieces. 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 2 hours and could be detected up to 8 hours after administration into muscle tissue. The researchers say that although much work still needs to be done, this is an initial step toward a test to pinpoint athletes trying to gain an unfair advantage.

INFORMATION:

The authors acknowledge funding from the Manfred-Donike-Institute and the German Sport University.

The abstract that accompanies this paper can be viewed here.

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