Design

Although Cas13 recognizes RNA, to lower the cost of our experiments, we decided to design DNA template sequences and then perform transcription to obtain target and guide RNAs. The template for all the target sequences is described in figure 1.

Procedure to obtain RNA target sequences:

The guide RNA is complementary to the 28 nucleotides of the coding strand of the target sequence. We also decided to design the guides as DNA to have enough material for a large number of experiments. The template used for all the guides is described in figure 2.

To filter the guide RNA and to choose the most specific one, we developed a program. The goal is to have an automatic pipeline that Blast a sequence against NCBI databases. We used it to blast our potential guide RNA candidates to check if there are alignments with other sequences. The analysis is run via python through the NCBI server. The principle is shown in figure 3:

We analyzed our results and decided to remove sequences that have a high probability to match with sequences in other organisms than Vibrio Aestuarianus. We end up with 9 guide RNAs that can target the sequences of interest: two guides targeting 16s, two guides targeting dnaJ, oneguide targeting flaA, one targeting vam, one targeting toxR and the last one targeting vspR.
All the DNA sequences used are reported in this table.

The test can be decomposed in two main steps:

• A. Main reaction
• B. Paper based revealing

The first step of our assay is the Cas13 activation and probe cleavage. This can be preceded by an optional target amplification step. This step will be performed in a first test tube. A second test tube contains the enzyme (Cas13a), the guide RNA (crRNA) and the probe. The probe is a15 uridine RNA molecule conjugated to biotin in 5’ and FAM (6-fluorescein amidite) in 3’. In the reaction tube one can insert the product of the RPA or directly the tested sample (see results for detailed protocol).

Our project was split in two different tests as explained in other sections:

• An in vitro test used for the characterisation of the reaction.
• A field test directly usable by oyster farmers. This test relies on the use of antibodies and RNA probes linked to molecules recognized by antibodies in a lateral flow assay.

Our diagnostic is intended to be easy to use and rapid, making it qualitative. In order to access more quantitative outputs and perform more in-depth analysis we developed an in vitro test.
The test relies on the use of a specific RNA probe linked to a fluorophore and a quencher. Thus using fluorescence output we developed a plate-reader-based Shell’lock detection. In this section we are going to detail every step of the experimental design of this approach.

The probe is the core of the fluorescent detection. We designed the cleavage reporter according to Gootemberg et al. (3) . This reporter is composed of the fluorophore 6-FAM (IDT), a poly U sequence and the quencher BHQ_1 (IDT).

Quenchers are a class of molecules that can, through different mechanisms suppress the fluorescence of a fluorophore. The most common form of quenching is through Förster resonance energy transfer (FRET). This mechanism is based on the overlapping of the excitation spectra of the quencher with the emission spectra of the fluorophore. The energy of the fluorescent emission by the fluorophore is transmitted to the quencher and can then be emitted at another wavelength or dissipated. In the case of Dark quencher the energy is dissipated which creates a decrease in the fluorescence detection.

Many mechanisms of quenching have been described in the litterature, involving energy transfer, dimerization of the fluorophore, chemical modification of the fluorophore… We decided to use Black Hole Quencher®-1 quencher from IDT. The mechanism of quenching of this particular fluorophore is the property of IDT.

The choice of the fluorophore was adapted from the quencher. We chose a fluorophore that corresponded to the characteristics imposed by the quencher i.e with an overlap between the emission spectra of the fluorophore and the absorbance of the quencher. We decided to choose FAM (6-Carboxyfluorescein ).

For our experiments we followed the protocol published in the SHERLOCK publication by Kellner et al. (1) . For this we used the LwCas13a (Leptotrichia wadei). Multiple Cas13a have been tested and used for the SHERLOCK reaction (4) . The plasmid of expression of the Cas used can be found on Addgene.

The reaction starts by the recognition of the guide RNA by the Cas protein. This step depends on the presence of a scaffold sequence in the guide in 5’ that is recognized by LwCas13a. This sequence was obtained by aligning varius guide RNA found in the literature using the same Cas (LwCas13a). After assembly of the Cas-guide complex, there is specific recognition of the target. This specificity comes from the “spacer” sequence in the guide (20nt) that is complementary to the target RNA. Upon recognition the Cas protein will become activated and will perform it’s nuclease activities:

• The unspecific “collateral” nuclease activity, which will cut unspecifically RNA present in physical proximity with the complex. In our test this will lead to the cutting of the RNA probes and the beginning of fluorescence measurement
• The specific nuclease activity, which will cleave the target RNA. Upon cleave of the target the Cas-guide will unbind from the target and be recycled for further use. This step will also stop the collateral activity.

The components of the SHERLOCK mix can be found in our protocol. The concentration of the components were adjusted for our experimental setup after characterization experiments. We performed kinetic experiments to follow the evolution of the concentration of the cleaved probes. The experiments ran over 8h by measuring at 5 minutes intervals. The complete protocols for the different experiments we carried out can be found in our protocol.