To determine the type of Cas to be used as the detection system in POIROT, we compare the collateral activity of Cas3 and Cas12a. For reasons why these Cas proteins were selected as candidates, see Proposed Implementation_Amplification. Both cas3 and cas12a proteins are activated by dsDNA and exhibit collateral activity, indiscriminately cleaving ssDNA present in the vicinity.
Based on the study by Yoshimi et al. 1, we evaluated the collateral activity using the FQ Probe employed in their research. The FQ Probe is an ssDNA with a fluorescent molecule attached to the 5' end and a quencher attached near the center and at the 3' end. Normally, fluorescence is not observed due to the quenching effect. However, when the ssDNA is cleaved by collateral activity, fluorescence is emitted.
We added various concentrations of dsDNA that activate Cas to the FQ Probe and Cas3 or Cas12a, and observed the fluorescence intensity for each.
The fluorescence changes were plotted below.
For both Cas proteins, it was confirmed that without dsDNA, there was no change in fluorescence intensity. On the other hand, with dsDNA, the fluorescence intensity increased over time. The fluorescence intensity was found to increase roughly in proportion to time until it reached saturation. Therefore, by analyzing the slope of the fluorescence curve between 1 min and 3 min after the start of the reaction, the results were as shown below.
The slope of the fluorescence curve corresponds to the cleavage rate of Cas's collateral activity. It was confirmed that for Cas3, the cleavage rate fully depended on the target dsDNA concentration across the entire range of 0-30 nM. For Cas12a, it was dependent on the range of 0-10 nM. Within the 0-10 nM range of dsDNA, Cas12a's cleavage rate was approximately five times higher than that of Cas3. Given that Cas3 exhibits a broader concentration-dependent range and that suppressing false positives is crucial in POIROT, we decided to use Cas3 in the detection system. Further experiments will be conducted to link Cas3 with amplification in order to incorporate it into POIROT.
In the paper, Cas3 reaction is performed in CONAN Buffer, and we also used it in the above experiments. However, in POIROT, which plans to perform amplification and Cas reactions in the same solution, it is necessary to find a buffer that will work well for both reactions. From our experimental results, we knew that the TWJ reaction works in rCutSmart Buffer, the multistep-SDA reaction works in NEBuffer 2.1, and the Cas reaction works in CONAN Buffer. Therefore, we independently prepared UTokyo Buffer, which mimics the composition of CONAN Buffer but has a pH of 7.7, which is between rCutSmart Buffer (pH 7.9) 2, NEBuffer 2.1 (pH 7.9) 3, and CONAN Buffer (pH 7.5) 1. Then, we connected TWJ-SDA and 3step-SDA and attempted amplification starting from biomarker 1 using four types of buffers (rCutSmart Buffer, NEBuffer 2.1, CONAN Buffer, UTokyo Buffer).
For more information about the components of UTokyo Buffer, see Experiments Basic Operations
The fluorescence changes were plotted below.
When rCutSmart Buffer, NEBuffer 2.1, and CONAN Buffer were used, NC amplification was fast and could not be distinguished from the case with target contained. On the other hand, when UTokyo Buffer was used, the amplification rate of NC was suppressed, and it was possible to distinguish between target concentration 100 pM and NC. From this, it is thought that the amplification system will work if TWJ-SDA and multistep-SDA are connected using UTokyo Buffer.
In experiment 2., we confirmed that our amplification system works in UTokyo Buffer. In order to perform amplification and Cas reactions in the same solution at POIROT, we conducted an experiment to confirm whether Cas3 works in UTokyo Buffer.
When a low concentration of dsDNA was used, the fluorescence change shown in the lower left graph was obtained. The lower right shows the fluorescence change when the collateral activity of Cas3 was investigated in CONAN Buffer.
It was shown that Cas3 works without problems even in UTokyo Buffer. Therefore, if UTokyo Buffer is used, it is thought that both the amplification and Cas reactions will work, and the presence or absence of target can be distinguished using Cas3.
A comparison of the collateral activities of Cas3 and Cas12a showed that Cas3 has a wider region in which the cleavage rate is completely dependent on the target concentration. Since it is important to suppress false positives in POIROT, we decided to incorporate Cas3 into POIROT.
In addition, it was shown that amplification occurred without problems even when TWJ Amplification and multistep-SDA were connected in a uniquely adjusted UTokyo Buffer, and that it was possible to suppress the amplification speed of NC. Furthermore, Cas3 was shown to work in UTokyo Buffer. Since POIROT is intended to perform the amplification and Cas reactions in the same solution, we decided to use UTokyo Buffer, which is thought to work well for both reactions.
Yoshimi, K., Takeshita, K., Yamayoshi, S., Shibumura, S., Yamauchi, Y., Yamamoto, M., Yotsuyanagi, H., Kawaoka, Y., & Mashimo, T. (2022). CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus.iScience 25, 103830, 1-13. https://doi.org/10.1016/j.isci.2022.103830
New England Biolabs. (n.d.). rCutSmart™ Buffer. https://www.neb.com/ja-jp/products/b6004-rcutsmart-buffer
New England Biolabs. (n.d.). NEBuffer™ 2.1. "https://www.neb.com/ja-jp/products/b7202-nebuffer-2-1