Every technique has its limitation, how about ASFAST? In fact, the concentration of virus DNA can significantly affect the reaction time of ASFAST. Thus, we built a kinetic model using MATLAB 2019a and SimBiology tool (The MathWorks, Inc.), to simulate the core procedure including three processes: virus DNA detection, cis activity, and trans activity. With the kinetic model, we aim to find out the reaction rate of ASFAST under different virus DNA concentration and take this as a reference to set its detection limit.
SturctureIn the beginning, Cas12a protein binds with crRNA to form a complex which is capable of p72 virus DNA sequence recognition. In fact, Cas12a protein has tremendously high specificity to its target DNA.  To shorten the reaction time, we designed 6 different crRNAs, which complement different segments of the p72 capsid sequence. Hence, every p72 capsid DNA can activate 4 Cas proteins at once, which can speed up the test by approximately 4 times compared to using a single crRNA. Once Cas protein targets the p72 sequence, it will start to undergo cis activity, which is divided into two steps, the non-target strand (NTS) cleavage and target strand (TS) cleavage. When the cis activity has been completed, it will proceed to the trans activity, which carries out indiscriminate ssDNA cleavage, which separates the magnetic beads and dsDNA.
Figure 1. Sketch of modeling structure.
Obtaining and tuning parameters
To obtain the parameters that are used in our model, we first referred to journal articles which analyze the reaction times of targeting, cis activity and trans activity. However, they can only be considered as theoretical values because they were obtained under controlled experimental conditions. To come up with a modeling results that is more accurate, we tuned the parameters according to our own experimental data. In the future, the model and parameters will be continuously tuned and revised to improve precision.
Table 1. Parameters, values and references.
Table 1. Parameters, values and references.
|kon||1.1 X 108 Ms-1||Isabel Strohkendl et al (2018).|
|knts||5.2 X 10-2 s-1||Isabel Strohkendl et al (2018).|
|kts||5.1 X 10-3 s-1||Isabel Strohkendl et al (2018).|
|ktrans||1 X 106 Ms-1||Janice S. Chen et al (2017).|
Results and discussion
The general feature of the time-dependent concentrations of biological components from our simulations are shown in Figure 2. Since ktrans is much larger than kts and knts, the Cas-ts-nts and Cas-ts should be converted to Cas-RuvC immediately, which explains why the concentrations of Cas-ts-nts and Cas-ts are always low.
Figure 2. The relationship between all the substances and time under virus DNA concentration of 0.3 nM. Note that for the presentation purpose, where P72-4 is magnified for a thousand fold, and Cas12a-4, Cas-ts-nts, Cas-ts, and Cas-RuvC is magnified for a hundred fold.In reality, the concentration of virus DNA would vary from sample to sample. Hence, we also simulated with the virus DNA concentrations ranging from 0.1 nM to 5.0 nM shown in Figure 3, where the y-axis is the percentage of completion defined by a ratio of the concentration of product to the original concentration of reactant. Here, we considered that trans activity is completed when the percentage of completion reaches 80%. As our expectation, reducing the virus concentration will lengthen the required time of completion. For instance, it requires 71 minutes for 0.1 nM of virus DNA concentration to complete the reaction.
Figure 3. Comparison of trans activity rate under different virus DNA concentration.We extracted the required time of completion with various concentrations of virus DNA shown in Figure 4 and fitted the results using a power function that allow us to predict the required time within our simulation range. Based on the reaction time and the 0.1 nM detection limit of CRISPR-Cas12a protein, we set the limitation of virus DNA concentration that can be detected by ASFAST at 0.3 nM.
Figure 4. The relationship between the required time of reaction and the concentration of virus DNA.
To the best of our knowledge, there is no dissociation rate of trans activity reported in the literature, thus we only simulated the forward reaction of trans activity. Omitting the reverse reaction would significantly underestimate the required reaction time of ASFAST. To address this problem, we evaluation the worst-case scenario by reducing ktrans 50% (5×105 Ms-1). Figure 5 shows that the required time increases from 27 to 49 minutes under a virus DNA concentration of 0.3 nM. However, more researches has to be done on the dissociation rate of trans activity to determine the exact rate of its reverse reaction.
Figure 5. Worst-case scenario simulated under 0.3 nM of virus DNA concentration.
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