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Results: Readouts
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For our experimental design, we used different fluorescence and colorimetric readouts as stated below.
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RNaseAlert Readout
To characterize our Cas13a, we first turned to the standard of the field, namely the RNase Alert detection kit. This was used by Gootenberg and Doudna to characterize the Cas13a and detect pathogen RNA sequences11,2. In the absence of Cas13a activation there is no fluorescence detectable due to physical proximity of the quencher to the fluorophore. Upon binding of a matching target RNA to the crRNA, Cas13a develops a promiscuous RNase activity and cleaves off the quencher from the fluorophore. This spatially separation in solution allows now to excite the fluorophore leading to detectable fluorescent signal. We showed that the increase in signal is a measure of the Cas13a activation by target RNA and therefore used it for most of our experiments. You find the corresponding results in the Cas13a and target subsections.
In general, the RNaseAlert assay works out very well, but one has to consider that a diagnostic device relying on this system is dependent on a fluorescence detector. For this reason, we built and evaluated the fluorescence detector “Lightbringer”. Additionally, a colorimetric readout overcomes this problem by making an easy visualization possible. For this purpose, we already worked on different colorimetric readouts.
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Spinach Aptamer Readout
Here, we used the Spinach aptamer which binds the DFHBI fluorophore, changing its 3D structure and thereby enables fluorescence 3. Activated Cas13a cleaves the Spinach aptamer leading to the release of DFHBI. This process is detectable as a decreasing fluorescence intensity (Figure 1).
Figure 1: The fluorescence activity of the Spinach aptamer is indirectly proportional to the target concentration.
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ssDNA Readout
One of the first colorimetric readout that we tried out was the ssDNA oligo based readout. The idea is based around the formation of a RNA/DNA dimer and the freeing of the DNA oligo from the dimer by digestion of the RNA part which has poly-U loops. The overall idea of this readout was to utilize the Cas13a freed small activator DNA-oligo strand in various signal amplifying chains:
- Completion of an in vitro transcription target (in vitro TX
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- Primer for an isothermal PCR
- Completion of a DNA-target for a transcription/translation system (TX/TL)
We used parts of an already established synthetic circuit from our lab called Circuit 3 (C3). Circuit 3 already provided us with a dsDNA target (= in vitro transcription target ds-C3) with a single stranded overhang in the promotor region and the complementary short DNA oligo, which we used as our activator (= C3 activator, C3a) (Figure 2).
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Figure 2: Circuit 3 stucture with dsDNA target with the single stranded overhang.
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The annealing of the two strands only worked to some extent, needing around 8 to 10x excess of inhibitor RNA to fully anneal with 500 nM of DNA activator oligo at room temperature. Room temperature was chosen, because it is extremely difficult to generate sharp temperature transitions on a small chip, which would be needed for good annealing results. Reasons for a bad annealing reaction of the two strands could be the mix of different length products of the inhibitor RNA in vitro transcription initially or the steady state of RNA digested with RNA in solution, resulting in a reannealing of RNA with the DNA activator.
The RNA background brought up major problems in combination with the transcription measurement and brought up disordered results with increasing RNA background concentration not directly resulting in lower transcriptional burst differentiation. Problems for the realization of a ssDNA activator readout lie for example in the various reaction environments needed for the dimerization, the RNA digestion and the final amplification step, may it be as a transcription, PCR or in a transcription/translation system.
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Intein-Extein Readout
For the intein-extein system, we ordered two gblocks from IDT. These gblocks were then cloned into psB1C3 and psB4A5 respectively. The successful insertion was confirmed by sequencing at GATC. The plasmids were then opened by BbsI and the coding sequence of beta-galactosidase was inserted via a Gibbson-Assembly. Once again, we confirmed the sequences at GATC and got full length reads for both constructs. However, the two biobricks could neither be submitted nor characterized, as the recloning to remove illegal cuts sites within the sequence was not finished prior to the deadline. The characterization was not possible, as the C-terminal fragment could not be expressed in any of our E. coli expression strains, as it seemed to be toxic. The toxicity seemed to not affect E. coli Turbo, which was used for cloning. The N-terminal part alone could not be characterized as the parts only work together.
We plan to express the C-terminal part with an in vitro expression system and are confident, that the purified proteins will work together as planned.
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Figure 3: Colony-PCR of C-Terminal fragment. Samples 9 and 12 had the correct length and were confirmed with sequencing.
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Figure 4: Colony-PCR of N-Terminal fragment. Samples 8 and 9 had the correct length and were confirmed with sequencing.
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Gold nanoparticle readout (AuNP)
When first testing AuNP cleavage on paper, a positive result, i.e a color change mediated by spread of AuNPs, was not visible for Cas13a, but for the positive control with RNaseA. In the follow-up experiment, RNA-linked and DNA-linked AuNPs were examined. Cleavage of aggregates did occur in the RNAse A-containing positive control for the RNA-linkers. These preliminary results indicate that our AuNP system can be used for selective detection of RNases.
Figure 5: For nuclease activity for either target-activated Cas13a or RNase A, an even circular distribution of diffused, red-shifted AuNP around the spotted aggregate was expected (see left upper corner). This could be observed in the RNaseA containing positive controls for the AuNP with linkers in each lower left corner of U0, U5, U10 and U15, of varying lengths from 0, 5, 10 to 15-Uracil containing single-stranded linker segments, but not for Cas13 mixtures (upper left dots in U0,U5, U10 and U15, negative controls or DNA-linked AuNP.
However, some improvements of the assay should be conducted. First, aggregation should be optimized to avoid any unspecific aggregation while facilitating specific aggregation trough extraction of full-length in-vitro-transcribed RNA. Second, it would be useful to quantify the kinetics of AuNP-resuspension by RNase A and Cas13a in a plate-reader based assay,
like our experiments using RNaseAlert. Last, to optimize test conditions on the paper platform, a variety of paper materials, coatings and sealing materials should be tested. After all, looking at the exposed position of the Cas13a promiscuous cleavage site and our results on Cas13a and AuNPs, we are confident that an optimized version of this readout will present a functional tool for RNA detection.
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Reproducibility
We successfully integrated the fluorescence readouts RNaseAlert and Spinach Aptamer in our Cas13a detection system for different bacterial and viral targets. We also successfully tested Cas13a Lbu and Lwa with RNaseAlert and in parallel also the Spinach Aptamer with the Cas13a Lbu. Since we repeated the experiments with the RNAseAlert and Spinach Aptamer multiple times and with different parameters, we can say that the fluorescence readouts are well designed and can be implemented in variety of systems and is highly reproducible.
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Discussion and conclusion
The results we obtained using the RNAseAlert and Spinach Aptamer with our Cas13a system conclude that having a fluorescence readout is an efficient system. We also successfully used the RNaseAlert in our self-made fluorescence detector to characterize the Cas13a activity. Additionally, we were also able to try out different colorimetric readouts which were partially successful. The idea using AuNP for a colorimetric readout is quite promising, taking into account the positive result it gave with the RNaseA. The combination of all these challenges during the colorimetric readout leads us to believe, that there are more elegant ways to realize the colorimetric readout of an RNA digestion on a paper strip.
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References
- Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., ... & Myhrvold, C. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, eaam9321.
- Esfandiari, L., Wang, S., Wang, S., Banda, A., Lorenzini, M., Kocharyan, G., ... & Schmidt, J. J. (2016). PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor. Biosensors, 6(3), 37.
- Paige, J. S., Wu, K. Y., & Jaffrey, S. R. (2011). RNA mimics of green fluorescent protein. Science, 333(6042), 642-646.
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