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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 sequences1. In the absence of Cas13a activation, the physical proximity of the quencher dampens fluorescence from the Fluor and there is no fluorescence activity. When Cas13a is activated, the RNA substrate is cleaved, and the Fluor and quencher are spatially separated in solution, emitting a bright green signal when excited by light of the appropriate wavelength. We did most of our experiments using the RNaseAlert system and the corresponding results are in the Cas13a and target subsections.
The fluorescence signal quality of the RNaseAlert assay was very good, however one cannot use this readout system without the fluorescence detector. Also, since the RNaseAlert is a modified RNA, it is somehow expensive in comparison to other readouts.
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Spinach Aptamer Readout
After the successful experimentation of the Cas13a with the RNaseAlert, we also tried out RNA aptamers for our readouts. For this, we used the Spinach aptamer which binds to the DFHBI changing its 3D structure2. We activated the Cas13a by the specific target, which then cleaved the Spinach aptamer bound to DFHBI and were able to show that the fluorescence activity slowly decreases (Figure 1).
Although we could see a clear decrease in the fluorescence activity as soon as the Cas13a is activated, the original level of fluorescence is lower than in case of RNaseAlert. This could be due to the fact that as soon as the spinach aptamer binds to the DFHBI, the fluorescence is already released. And regarding the time factor needed to mix all the reaction components, we lose some fluorescence before the Cas13a cleavage activity starts.
Figure 1: The fluorescence activity of the Spinach aptamer decreased with the increase in 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 by digestion of the RNA part which has the 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
- 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 teh 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 order 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 couldn’t 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 the cloning. The N-terminal part alone couldn’t 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. The marker used was the 2-log DNA ladder.
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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. The marker used was the 2-log DNA ladder.
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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.
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 RNase Alert.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.
b>Figure 4: 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.
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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 different Cas13a proteins namely Lbu, Lwa and Lsh with RNaseAlert and in parallel we also successfully tested the Spinach Aptamer with the Lbu Cas13a. 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 AuNp readout could be the potential colorimetric readout which could be further optimized, 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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