Date: 16th April 2020
For organ transplant patients the level of immune system suppression following transplantation is key. Too little, and donor organ function is compromised and the chances of organ rejection increased; too much, and the essential defence functions of the host immune system are weakened. Now scientists have harnessed the power of CRISPRs to design a fast and inexpensive lateral flow assay to detect early signs of infection and rejection following kidney transplants in a bid to improve long-term patient outcomes.
Currently, post-kidney transplant care is centred on close monitoring of patients via blood tests and kidney biopsies. These are time-consuming, expensive, and invasive procedures. But what if this monitoring could be done using a simple urine sample? This was the goal of an international group of scientists led by James Collins at the Massachusetts Institute of Technology, US as the group turned their attention to CRISPR–Cas13.
CRISPRs have revolutionised the gene editing field and are now potentially set to do the same in the diagnostic market. So far we have seen the use of Sherlock Bioscience’s SHERLOCK™ platform in diagnostics using CRISPRs to detect DNA/RNA; Cas13 repurposed for viral detection and protection in human cells; and the development of the CRISPR-Chip biosensor and Genome Sensor™ able to identify genetic mutations of interest.
In this study, the starting point for the team was to design an assay to screen for two common opportunistic viruses infecting kidney transplant patients – cytomegalovirus (CMV) and BK polyomavirus (BKV) – and also CXCL9 mRNA, whose expression increases during acute cellular kidney transplant rejection. They decided that SHERLOCK (Specific High-sensitivity Enzymatic Reporter unlocking), a Cas13a-based CRISPR system that targets RNA, would be the ideal protocol.
In this work, the team used the SHERLOCK protocol to detect DNA or RNA from urine samples of patients. Put simply a pre-amplification stage of the nucleic acids was combined with Cas13 detection. Guide RNAs, in the work here specific to the targets CMV, BKV or CXCL9, recognised these genetic signatures in a sample, only when the signature was detected was the Cas13 activated. On activation target sequences were cut and so were reporter molecules, to release a signal on a paper strip, dipped in the prepared sample. The test, very much like a home pregnancy test kit, in which an extra line appeared on the strip if positive for any single target sequences.
Once optimised, the team were able to use the assay to detect both high and low levels of BKV or CMV infection. They were also able to correctly detect signs of transplant rejection (CXCL9) taken from over 100 samples from kidney transplant patients. However, at very low levels of target concentration they found that the test strip often had a second detection line rather than just one. To aid the reading of these tests, therefore, a photo analysis app was developed that could be used with a smartphone. This was able to analyse the test strips in an unbiased fashion, and decide results based on line intensity.
The team have designed an accurate, simple and easily read assay for the post-transplantation monitoring of common opportunistic viral infections and of graft rejection following kidney transplantations. By further development it is hoped this assay may offer critical, affordable urine-based diagnostic tests which will improve patient outcomes.
In fact, a patent application is pending, and the first author of the paper, Michael Kaminski is hoping to take the assay to clinical trials to make direct assessments against traditional monitoring methods.
Although simple, the assay still requires multiple steps, and it is here that the team would like to pursue further optimisation with the aim to generate a one-step test. This would then be more amenable in particular for the home environment, enabling self-monitoring of patients.
As for future applications, it is possible that the assay could be adapted for other transplant patients, or those immune-compromised for other reasons, such as those undergoing cancer treatments, diabetes or other genetic diseases.
With these types of CRISPR-based assays being so adaptable and able to potentially detect almost any viral infection it is not surprising in the current climate that there has been drive to develop CRISPR-based tests to detect SARS-CoV-2. With both Sherlock Bioscience and Mammoth Bioscience; companies founded by the pioneers of CRISPR, and others hot on the trail of this it is hoped that one will be available soon.
What is certain, is that CRISPR-based diagnostic assay are set to provide quick, simple, low-cost and accurate tests. The results here lend weight to their diagnostic use, and should be another step forward in the translation to approved clinical products in the not so distant future.
For more information please see the press release
Kaminski, M. M., M. A. Alcantar, I. T. Lape, R. Greensmith, A. C. Huske, J. A. Valeri, F. M. Marty, V. Klämbt, J. Azzi, E. Akalin, L. V. Riella and J. J. Collins (2020). “A CRISPR-based assay for the detection of opportunistic infections post-transplantation and for the monitoring of transplant rejection.” Nature Biomedical Engineering.