The first successful kidney transplant took place in 1954, a feat which later won a Nobel Prize in Medicine. Now, there are 94,886 US-based individuals awaiting a kidney transplant, where the average wait for a viable kidney can vary between three years to more than a decade. Even with a successful transplant, patients must continue to monitor their kidney function as organ transplants may be lost to viral infections and organ rejection, highlighting the urgent need for prospective diagnostics. In a new study, a group of scientists have developed a novel CRISPR-based diagnostic tool to tackle this very issue.
“In [kidney] transplantation, we have this situation, where due to immunosuppression, patients are at risk for opportunistic infections, and on the other hand, are also at risk of organ rejection,” says Michael Kaminski, the lead author in this new study published in Nature Biomedical Engineering. He notes that current diagnostic tests involve using quantitative PCR to detect infections, which can take a few days, and biopsies to assess the risk of kidney rejection, which are costly and pose various health risks.
“I thought [that] an easier to perform test would [make diagnostics] more accessible,” says Kaminski.
During this study, Kaminski was a post-doctoral researcher at the Massachusetts Institute of Technology. There, Kaminski and his fellow scientists leveraged the specificity of the CRISPR-Cas gene editing system for diagnostics — specifically, they used CRISPR–Cas13 specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) technology to target cytomegaloviruses and BK polyomaviruses, which are two routinely tested viral infections in kidney transplant patients.
Developing this diagnostic tool was no easy feat, as the team had to heavily optimize this tool, including deciding which part of the viruses’ genome to target, and what protocols would be best to isolate genetic material. The team also went one step further and incorporated a smartphone app, a test strip (also known as a lateral-flow dipstick), and peeing into a cup into their diagnostic workflow.
“We optimized electrical flow such that you don’t have to use a plate reader, but you can just use a [lateral-flow] dipstick, similar to a pregnancy test, which you place in the final reaction,” says Kaminski. “Importantly, we observed that these lateral-flow dipsticks sometimes create very faint bands, even if there’s no sample present. We thought a smartphone-based software application would be really useful for the end-user — it would tell if the test was positive or negative.”
The team then tested this CRISPR-based diagnostic tool in over 100 clinical samples, finding that they could accurately detect the two viruses in blood and urine samples from kidney transplant patients. Similarly, their tool could reliably detect CXCL9 with 93% sensitivity, where increased expression of this biomarker means that a patient is undergoing kidney transplant rejection.
This diagnostic tool demonstrates that the CRISPR—Cas system is for far more than just gene editing and therapeutics. While it has a long way to go before application, this tool may become the go-to pee-in-a-cup option to monitor viral infections and early signs of transplant rejection in patients with kidney transplants.
“What’s really exciting about this [study] is that they do compare it directly to the clinical gold standards that are being used now, which a lot of academic [studies] don’t necessarily do. [This] is moving it closer and closer to clinical application,” says Dana Foss, who is a UC Berkeley-based post-doctoral researcher developing new methods of tissue-targeted Cas9 delivery for a range of diseases, including HIV. “I do think that CRISPR-based detection methods are coming soon to the clinic.”
Kaminski is now heading the Kidney Cell Engineering and CRISPR Diagnostics Lab at the Max Delbrück Center for Molecular Medicine. He notes that “although we have made [this tool] user-friendly, there is still some way to go until you can [apply] it at home,” and already has some next steps in mind to develop this tool further, including testing this in more patients with kidney transplants.
“To improve this technology, one of the important things is multiplexing. You could go for different targets. For example, in [this] assay, we went for cytomegaloviruses, BK polyomaviruses and CXCL9, but it would be really interesting for example, if you could go for ten different [targets] in one tube,” says Kaminski. “What other people are also trying to do is miniaturize these reactions in a volume that is compatible with microfluidics. This would be a cool advancement, and would enable multiplexing, but in parallel, in a microfluidic format.”
Foss also notes that while healthcare settings may be slow to implement changes like this, it may be different in low-resource settings, where there is “opportunity for a lot of innovation.”
“I’m excited to see this making waves in the clinic, [especially] being deployed in low-resource settings,” says Foss. “We’re seeing CRISPR therapeutics start up with clinical trials. I think that diagnostics will be a huge way that CRISPR makes positive outcomes for patients because of how amenable it is to a visual readout, and how much faster and easier it can be [when compared] to current methods.”