Overview
The first design made for the project was in favor of the greater stability of DNA over RNA. Despite working with the latter one, the choice leaned in favor of the longer-lasting stock and virtually limitless quantity of materials offered by amplification through PCR, for as long as enough template was present. For this purpose, the base design that has to be tested was a set of primers fixing one to another in order to reconstruct a complete sequence.
The first sequences were manually crafted since no software was available at the time. Data was acquired from NCBI after conducting a BLAST on the West Nile Virus (WNV). Additional details can be found in the Proof of Concept section In the Software section, further information can be found about the software developed by our team during the project, that facilitates and automatizes guide design. The functionality can be found there, as well as the operational mechanics and the results.
Two sequences needed to be re-ordered for the reverse primers of LwCas13a (target sequence 2.1 and 2.2) as they proved to be unsuitable for the binding with the forward primer (incorrect orientation of the 28 nucleotides sequence to target). Other than this, all processes ran smoothly, from PCR to transcription.
As the initial cycle got completed, the next step can start, involving the preparation of everything concerning CasRx (transformation, expression, purification). However, it is imperative to acknowledge what was learned and make adjustments in the future. For instance, the improvement of the gel electrophoresis, mostly because no ladder was suitable to confirm the weight of the bands except that it is below 100 bp, but also optimizing the conditions by modifying the agarose concentration and type, the buffer composition, the equipment...(see cycle 5)
The CasRx plasmid was acquired through Addgene.Upon receipt, the plasmid was initially in a strain of E.Coli not suited for expression (DH5 alpha strain). Consequently, we needed to transform it according to the reference material.The effective expression strain for the plasmid seemed to be Rosetta2 (DE3), which was already present in the lab.
To transform our plasmid into the new strain, the initial step consists of rendering the cells chemically competent for transformation.
We succeeded in making competent cells and transforming the casRX plasmid into Rosetta cells. See the Results section for more information.
Understanding that the protocol varies a lot depending on the strain, the starting point was the preparation and competency of cells, ending with the transformation itself, leading to the next cycle.
Production and purification preparation and discussion : CasRX protein was new to us, so we started by doing expression tests in LB medium to see how it grows. We saw that protein was not well produced, but still decided to try the high scale production in TB medium as it was done in the article we relied on. In order to avoid losing more time, we started preparing cultures and protocols for our purification. Some small details were slightly changed, for example buffer composition. As cold temperature equipments are heavily used in our host laboratory, we were not able to secure a time slot to perform purification at 4C and decided to try the procedure at room temperature instead. .
Refer to the CasRX expression and purification result section for more information .
First of all, we concluded that our protein is not well produced in E.coli cells and there is little to no difference between before and after IPTG induction. Also, the problem might be related to IPTG concentrations, which is why we made after purification expression tests to see the influence of 0.25 mM, 0.5 mM and 1 mM of IPTG. After staining our gel we once again observed no difference between non induced and induced fractions. We therefore tried to quantify it with ImageJ, but it did not give any significant results. We then have been suggested that we should have tried different bacteria strains or even using eukaryotes cells like Saccharomyces. To dig further, we sent our plasmid to sequencing and discovered that there were a few mutations in the ORI region which possibly could have an effect on protein production and result in the experiment failure.
In order to validate our system, it is imperative to generate quantifiable data to back-up our claims. Initially, employing an agarose gel electrophoresis was considered, but after reviewing alternative results, no conclusion could have been drawn from it. Consequently, the usage of plate reader detection sounded like a good compromise, considering that it can quantify fluorescence and provide kinetic information.
First, the Cas will detect the target sequence, thanks to the synthetic guide designed, and it will attract the protein thanks to the stem loop. Once the binding between the target and the guide is done and the Cas detects the complex, it starts cutting everything, firstly the target sequence. After that, every RNA strand in close proximity will be cut, including the string between the fluorescent molecule and the quencher, allowing the separation between those two parts and getting them away from each other, making the fluorescence not quenched, and thus detectable.
The detection results can be found in the graph 1 of the Result section.
The initial detection didn’t yield discernible kinetic reactions. In the future, it was considered placing the plate on ice prior to the sample loading to delay the reaction process as much as possible and potentially observe initial reactions kinetics on the plate reader. Additionally, discrepancies were observed in the outcomes of seemingly similar samples, such as those between 1.1 and 1.4, despite only having 2 different nucleotides between each successive sequence. Investigating this interesting difference could offer us valuable insights to help designing new target sequences or even improve our design software tool with new parameters.
For this cycle, the purpose of the experiments will be to get better gels through variations of parameters, in order to get an easier time analyzing.
Better visualization of the agarose gel is important to verify the quality of our materials all along the protocol, from initial amplifications to transcription, even more considering the length of our sample (below 100 bp). Small size samples give rise to new challenges to be visualized on gels.
In pursuit of optimisation, adjustments were made on all the parameters, here is an exhaustive list:
See final gel in the Result section. The gel was prepared using a 2% (m/V ratio) agarose composition with TBE buffer. Electrophoresis was conducted for 45 minutes at a constant voltage of 130V, and the gel was stained with Diamond Nucleic Acid Dye. The sample configuration (from left to right): 25 bp ladder, 100 bp ladder, Negative control, Positive control, Synthetic target sequences 1.1 to 1.4, Synthetic target sequences 2.1 to 2.2, 100 bp ladder, 25 bp ladder.
Obtaining a high-quality gel extends beyond parameter optimization. Depending on the choice of materials, a totally different outcome can be seen. For instance, selecting a certain fluorescent nucleic acid dye can affect both the quality of the bands and the noise during the visualization. Likewise, changing the machine necessitates modification of the combs and gel length, which influence the sample behavior under the same voltage. Agarose concentration influences the sample passage through the gel, making a lower concentration more permeable. Additionally, the quality of the agarose powder significantly impact the result consistency during the revelation.
Following the first successful detection, different conditions were tested. The first one was comparing the first and second batch of prepared RNA, while the second part contrasts the difference between raw mass per microliter (ng / µL) and normalized quantity (molecular count) per microliter (µM / µL), with also a change in either the target or the guide concentration (doubling, halving).
For the differences between old and new RNA batch as well as between ng/µL and µM/µL, check graph 2 and 3 in the Result section. For the differences between guide and target concentration, check graph 4.
This phase of the experiment investigated the difference in concentration, giving more insights about the influence on normalizing the experimental settings. It proved that normalizing gives overall better results compared to the concentration analysis, as the molarity acts as a huge factor.
As the detection worked well from a purely in vitro context, the next step aimed to transition into an in vivo setting, using infected mosquitoes. With the support of MIVEGEC, the team acquired mosquitoes infected with deactivated viruses, ensuring a safe workplace and not preventing disease development. The goal of the experiment would be to assess if the detection works in an in vivo environment.
The initial steps involved purifying viral RNA out of the mosquitoes using Macherey-Nagel’s kit, in order to facilitate the detection process. In order to verify if detection could take place in a noisy sample, water was removed from the detection reactions and was fully replaced by mosquito juice. The most stable guide observed in the first rounds of detections, which was selected as “reference sequence”, was used in a detection reaction with purified viral mosquito RNA to see if we could detect RNA coming from a real mosquito. Lastly, we tested a detection reaction with a mix of different guides to explore the feasibility of multiplexing detection possibilities and offer more flexibility to our future automatized system.
The results of this cycle can be found in graph 5 of the Result section.
Thanks to this cycle, the viability of the detection was proved to be possible in an in vivo context, allowing future perspectives. As the detection in the purified viral RNA of the mosquitoes didn’t work, some questions rose up, including the fact that the virus included the sequence or not, as well as the adequacy of the viral charge for effective detection. In the results section, we brainstormed some improvement ideas to understand experiments that did not succeed, but also improve what already works and expand the system, to push its limits further in hopes of achieving the automatized detection system, which could help many.