Team:SDU-Denmark/Proof Of Concept

Proof of Concept

"Science never solves a problem without creating ten more" - George Bernard Shaw

PROSTATUS – Proof of concept

PROSTATUS utilizes the presence of RNA and DNA biomarkers to detect malignant prostate cancer and genetic predisposition to develop prostate cancer. Our first proof of concept experiment was set up to show that sgRNA/Cas-based detection of our chosen biomarkers is possible. The experiment utilized ribosomal RNA (rRNA), to detect collateral RNA cleavage from the activated sgRNA/Cas system upon binding to its target. rRNA cleavage was visualized using agarose gel electrophoresis, where clear rRNA bands indicate no activation of the sgRNA/Cas system and a smear indicated that the system had been activated and rRNA degraded. For this experiment the rs6983267-G target RNA was detected using the sgRNA-Cas complex made from the complementary sgRNA and Cas13a. The experiment tested the detection systems in 0,043 ng/µL and 0,0043 ng/µL of sgRNA. Five controls were included to ensure that the rRNA degradation was caused by collateral cleavage by the sgRNA/Cas system. A positive control with RNase A and four negative controls lacking target, Cas13a, sgRNA, and all the above, were included to ensure that rRNA cleavage was caused by collateral cleavage.

Cas13a POC
Figure 1. Proof that Cas13a is activated by the sgRNS and target sequence for rs6983267-G. All samples contain 1 µg/µL RNase A, 40 ng/µL Cas13a or water, 0,043 ng/µL sgRNA, 0,43 ng/µL target and 20 ng/µL rRNA as a reporter unless other is specified. Lane 1 is a positive control containing 2µL RNase A and 2 µL Tris-HCl. Lane 2 only contains Cas13a and rRNA. Lane 3 contains sgRNA, target and water instead of protein. Lane 4 contains Cas13a and sgRNA. Lane 5 contains Cas13a, 0,043 ng/µL sgRNA and target. Lane 6 contains Cas13a, 0,0043 ng/µL sgRNA and target. Lane 7 is a negative control only containing water and the rRNA. It is seen that Cas13a is only fully activated and cleaves the rRNA when target and sgRNA are also present (Lane 5-6). It is also seen that some rRNA is cleaved in the negative controls but not as much as in lane 5 and 6.

In Figure 1, it is observed that the CRISPR/Cas detection system is activated in the presence of target RNA. No rRNA bands are visible with either high or low concentrations of sgRNA, while these bands are present in the negative controls (lanes 2, 3, 4, and 7). This experiment shows that our sgRNA-Cas complex is fully activated when the target is present and that the same amount of rRNA is not degraded when the target is not present.

The final PROSTATUS tests, however, will not use agarose gels to confirm collateral cleavage. These tests will use flow strips and must be functional in real biological samples that contain RNase. To see if the test would perform in real samples, the possibilities of inhibiting RNases in urine before introducing the sgRNA-Cas complex was tested. In Figure 2, it is shown that the positive control has one line at the top and that the negative control has two lines where the bottom is the most intense. In the untreated urine sample (lane 3), we get a positive signal due to reporter cleavage by the RNases in the urine. It was observed that it is possible to inhibit the RNases with proteinase K (lane 4) as well as inactivate the proteinase K with PMSF (lane 5), before reintroducing the urine in lane 6. The reintroduction of the urine represents the introduction of the sgRNA-Cas complex in the PMT after inactivating the RNases already present in the urine, to avoid false positives. The experiment proves that our system can inhibit the RNases in urine with proteinase K but also that collateral cleavage can be activated again when proteinase K is inhibited. The result also shows that the flow strips are compatible with this test set up and that collateral cleavage can be analyzed on the strips.

Flow strips no saliva
Figure 2.  Flow strips after treatment with proteinase K and PMSF. All samples contain either nuclease-free water, urine or saliva together with 1 pmol RNA-reporter and 100 µL HybridDetect Assay Buffer. Strip 1 is a positive control containing RNase A and Tris HCl. Strip 2 is the negative control only containing water, RNA reporter and buffer. Strip 3 is untreated urine. Strip 4 is urine treated with proteinase K. 5 is urine treated with proteinase K and PMSF. 6 is urine treated with proteinase K, PMSF, and reintroduced urine to ensure inhibition of proteinase K. The same treatments are done for the four saliva strips (7-10). As seen, the RNases in urine are efficiently inhibited by proteinase K, which is again inhibited by PMSF allowing for reintroduced RNases to be active. This is not seen for the saliva as 3 hours with proteinase K did not inhibit the RNases.  

In summary, results indicate that when the targeted biomarker is present in a sample with sgRNA-Cas complexes, these are activated and begin collateral cleavage. Furthermore, we have shown that the RNase activity in urine can be deactivated with proteinase K. Subsequent PMSF-based deactivation of proteinase K activity was also shown to work. Finally, the flow strip format, was shown to be effective for detection of unspecific RNA cleavage, proving that the final PROSTATUS tests can function in biological samples.