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Results: Readouts
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For our experimental design, we used different fluorescence and colorimetric readouts as stated below. The fluorescence read-outs worked well, and especially the RNaseAlert allowed us to characterize our Cas13a detection in a variety of contexts: native protein, lyophlized, in bulk, on paper, from virus target, bacterial target, in vitro and in vivo RNA. For our colorimetric read-outs, we could not reach a full integration with target RNA detection, but where able to assemble the necessary constructs and start characterizing them.
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RNaseAlert Readout
To characterize our Cas13a, we first turned to the standard of the field, namely the RNaseAlert detection kit, which uses the short modified RNA sequence RNaseAlert. This was used in recent work to characterize Cas13a and detect pathogen RNA sequences1,2. 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 spatial separation in solution now allows the fluorophore to emit light, leading to a 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. The corresponding results are specifically described in the Cas13a and targets subsections.
In general, the RNaseAlert assay works 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”, which is the most sensitive fluorescence detector ever built by an iGEM team to our knowledge. Since a colorimetric readout would overcome this problem by making an easy visualization possible, we also worked on different colorimetric readouts.
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Spinach Aptamer Readout
Here, we used the Spinach aptamer (a 80-nucleotides RNA) which binds the DFHBI fluorophore, changing its spatial conformation and thereby enables fluorescence3. 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 inversely proportional to the target concentration.
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ssDNA Readout
For this readout, we wanted to link the cleavage of an RNA strand (due to Cas13a activation) to an amplification scheme based on ssDNA. A dimer is formed between a ssDNA sequence and an inhibitor ssRNA sequence. This RNA is composed of three regions binding to the ssDNA separated by polyU loops (Figure 2), so that upon cleavage of the polyU loops by Cas13a, the melting temperature of the dimer is lowered and the cleaved ssRNA falls off. The ssDNA is freed and can be used into an amplification scheme: we envisioned that it would either complete a linear transcription template (known as genelet) that is single-stranded in its promoter region, and activate its transcription (reference:Timing molecular motion and production with a synthetic transcriptional clock
Elisa Francoa, Eike Friedrichsb, Jongmin Kimc, Ralf Jungmannb, Richard Murraya, Erik Winfreec,d,e, and Friedrich C. Simmelb,1 )
, or it would bind the PCR DNA template. In both cases, either transcription or PCR would lead to amplification of the signal. A transcription signal could be read with a nucleic acid binding dye, or could further linked to translation, to create a colored protein read-out such as aeBlue. Using transcription translation as an amplification of a detection into a colorimetric readout was successfully shown by Pardee et al.cite Pardee
. Similarly, DNA amplification could be signaled with nucleic acid binding dyes and fluorescence could be read with our detector.
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 10-fold 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 Gibson-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 RNAseA-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 upper left graph). 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 right 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 RNaseA 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 successfully tested Cas13a Lbu and Lwa with RNaseAlert and in parallel the Spinach Aptamer with the Cas13a Lbu. Based on our extensive experience with RNaseAlert, we can say that this fluorescence readouts can be implemented in variety of systems and is highly reproducible. However, our work on colorimetric readouts was too preliminary to discuss reproducibility of those circuits.
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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. This is why we constructed our detector, and we successfully used it to characterize Cas13a activity with RNaseAlert. Additionally, we were also able to try explore different colorimetric readouts and lay the groundwork for their success. The idea of using AuNP for a colorimetric readout is quite promising, taking into account the positive result it gave with the RNaseA. The challenges we faced with our colorimetric circuits lead 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.
- East-Seletsky, A., O’Connell, M. R., Knight, S. C., Burstein, D., Cate, J. H., Tjian, R., & Doudna, J. A. (2016). Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature, 538(7624), 270-273.
- 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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