RfxCas13d (CasRx)

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RfxCas13d, also known as CasRx, is a RNA-guided endonuclease. This protein can trace its origins to the bacterium Ruminococcus flavefaciens. In its natural environment, CasRx plays a vital role in the immune system of the bacteria, where it functions as a defense mechanism against viral RNA. Guided by CRISPR RNA sequences, the protein is adept at targeting and cleaving viral RNA, thus preventing viral replication within the cell.

Usage and Biology

When compared to other Cas13 proteins, CasRx offers some advantages such as a higher RNA-targeting precision. This feature minimizes off-target effects, setting RfxCas13d apart from other Cas13 proteins, such as Cas13a and Cas13b. Consequently, it is an ideal choice for RNA manipulation and editing.

RfxCas13d's unique CRISPR-derived RNA guide sequences make it highly programmable, allowing it to target specific RNA sequences with ease. This versatility can be used for a couple of purposes, such as gene expression modulation, the study of RNA-based diseases... The precision and programmability of CasRx position it as a new tool in order to explore the RNA biology.

Protein Production

The novelty of our approach was based on using the CasRx enzyme, which we tried to produce and purify by ourselves. Multiple obstacles were on our way, for instance difficulties with production or purification processes. Here we provide you with some explanations as to what could be a reason for such a struggle.

Expression and Purification

The expression and purification of CasRX were based on this article

1. Streak LB-Agarose plate containing 100 µL of Kanamycin and 100 µL of Chloramphenicol (plates already in the fridge) with cell suspension containing the plasmid. Incubate the plates overnight at 37°C.

2. The next day inoculate 15 mL of LB media containing 15 µL of Kanamycin with one single colony and incubate overnight at 37°C on a rotary shaker at 190 RPM.

3.The next day inoculate 750 mL of TB media containing 765 µL of kanamycin and 765 µL of chloramphenicol with 15 mL of starter culture. Then shake at 37°C, 190 RPM.

4. Monitor OD every hour until cells reach an optical density of 0.4 – 0.6, then transfer flasks to 4°C for 30 min to allow flasks to cool prior to induction.

5. Critical step: For optimal expression, it is important to strictly follow the indicated OD of 0.4–0.6 at the time-point of induction.

6.Cool on ice for 15 mins

7. Take 20 µL of the flask in a tube (non-induced sample)

8. Induce expression by adding 765 µL of 0.2M IPTG (0.2 mM final concentration) and shake cultures for 20 hours on a rotary shaker at 190 RPM, in a pre-chilled 18°C incubator.

Then we proceeded to purify, however, due to availability of the machines, we purified protein at room temperature, instead of 4°C, which is one of the reasons we did not manage to purify it. Once the purification process was finished, we abandoned this idea, and made an expression test in small volumes.

1. We prepared 4 cultures of 10 ml in TB, with 10 µl of Kanamycin and Chloramphenicol. Then cultures were left to grow at 37°C, expected OD is between 0.4-0.6.

2. Of these 4 cultures, 1 was used as “non induced”, 3 others were induced at different IPTG concentrations, precisely 0.25mM, 0.5 mM and 1 mM.

3. After 20h at 18°C on 190 RPM, we took 1 ml of each culture, sonicate it up until the solution clarified. 45 µl were taken from each tube, then mixed with 15 µl of the blue.These were our TOTAL fractions, the ones called “supernatant + pellet”.

4. Secondly, we used the rest of the volume, 955 µL, we centrifuged it for 7 min, took 45 µL of the supernatant and added 15 µL of the blue. These were “supernatant only” fractions.

In total we got 8 fractions, including 2 non induced, and 6 others induced at different IPTG concentrations. However, once we’ve done SDS PAGE, we didn’t see any significant difference between non induced and induced fractions. We started by quantifying it in ImageJ, to be sure that there is no difference. However, each fraction, for example, was not sonicated in the same manner, sometimes it took more time, sometimes less. There was no lysis buffer in this experiment, so maybe the lysis was not finished. We put 10 µl of each fraction of the gel, but some columns are more intense than others, as if different conditions were applied to them. So the quantification was not very useful.

First of all, our protein can be too big for the bacteria and we did not have enough time to verify it and test it in other cells, and we also trusted the authors of the article we relied on.

Secondly, we sequenced our plasmid to see if there are any mutations, we found 1 in the ORI region of the plasmid, which could also be a reason. This pseudo mutation was, when referred in AddGene, at the position of a “F” .Then, if we had more time, we would have tested different temperatures and volumes of the cultures.

Cas13a

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For contributions, we add information in the repository : BBa_K2306012

The following figure shows the template of the guides we used to target WNV virus sequences (see Proof of concept)

Part Collection

This year, the iGEM Montpellier team designed a library of guide RNA and target RNA sequences for West Nile and Chikungunya viruses.

To address the challenge of rapidly mutating viral RNA targets, we developed a software tool capable of detecting a wide range of natural variants.

In this process, we employed the SHERLOCK system for pathogen detection within mosquitoes. Our approach involved constructing consensus guide RNA sequences and testing them on synthetic target sequences at first. Initially, we blasted a reference sequence of the target virus to gather similar sequences of viral variants. These sequences were subjected to multiple alignments, and based on the aligned nucleotide frequencies, we generated a consensus sequence. This consensus sequence significantly enhances the probability of hybridization with a broad spectrum of viral RNA variants, thereby improving the efficiency of our detection system.

WNV Sequences

We also had 1 sequence for the Chikungunya Virus: 29 nucleotides sequence (CCT GCA ACG TGC GTA CCC ATG TTT GAG GT) located on the Viral methyltransferase. We didn’t keep this sequence for further trials as it was present in a lot of other organisms, thus we didn’t test it nor keep it.

New Composite Part: BBa_K4697000

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Introducing the template

Our project aimed to tackle the issue of detecting pathogens within mosquitoes and promptly relaying this information to researchers and epidemiologists. To achieve this, we turned to the SHERLOCK-CRISPR Cas13 system, a tool for molecular biology. But we needed to devise a systematic approach for our proof of concept.

First, we designed synthetic templates, which would serve as guide RNA (gRNA) and target sequences specifically customized for our target virus, West Nile Virus (WNV) in our case. These templates were crafted to ensure high specificity and accuracy in the detection process.

Afterwards, polymerase chain reaction (PCR) technique was employed to amplify the synthetic templates using primers. The amplification allowed us to generate enough copies of the gRNA, which is essential for the detection process.

Following the amplification, a step of transcription is needed, to pass from DNA to RNA through the T7 promoter. This part is necessary as CasRx functions using RNA molecules, and our target is a RNA virus.

Usage and Biology

Using the product of the PCR, a Cas enzyme (LwCas13a in our case), and a fluorescent probe (BIOTIN-FAM for us), we can proceed with the pathogen detection.

It works when the Cas enzyme detects the gRNA, and when the latter one binds with a target sequence, initiating the cleaving process of the target, and, at the same time, of the fluorescent probe in the vicinity. This causes a fluorescent signal to be emitted that can be detected.

Fluorescence signal happens when the FAM fluorophore gets away from the quencher, making a quantifiable signal that can be detected using sensors, and defining the presence or not of pathogens.

Using this as a base, we designed a template for the detection of RNA viruses, which helps getting insight for these reasons:

1. Pathogen Detection: The primary purpose of our project is to detect the presence of pathogens, such as the West Nile virus, within mosquitoes. This can be done thanks to the T7 transcription for going from DNA to RNA, and with the complex that will be formed using the scaffold to associate with the Cas enzyme and the reverse complementary sequence of the target in order to bind and produce a reaction.

2. Specificity: Using the complementary sequence of the target, and thanks to the built-in specificity of the Cas enzyme, great specificity can be achieved, as a low amount of mismatches are allowed for the binding to work (example for Cas13a: more than 2 mismatches makes no signals, refer to part:BBa_K2306012 for more informations)

3. Real-Time Detection: To address the need for rapid results, the use of the SHERLOCK-CRISPR Cas13 system provides immediate identification of pathogens, minimizing delays in response.

Design of the template

Enhancer: needed for a better transcription process, we used GAAAT, but, according to the following reference, GAATT would have been a better choice Reference: Phage t7 promoters for boosting in vitro transcription. Thomas Conrad, Sascha Sauer, 2021 April 01, Google Patent, https://patents.google.com/patent/WO2021058145A1/en

T7 promoter: or any promoter for a transcription process, the sequence used is TAA TAC GAC TCA CTA TAG

Scaffold for the enzyme used, as we used LwCas13a, the sequence would be GAT TTA GAC TAC CCC AAA AAC GAA GGG GAC TAA AAC

another possibility for example would be to change the sequence with the scaffold for CasRx, using the following sequence CCC CTA CCA ACT GGT CGG GGT TT

, the reverse complementary sequence of the target

Primers

Once the basic parts are settled, 2 primers can be made out of them, with one never changing and the other ever changing:

forward (never changing primer) → 5’ - GAA ATT AAT ACG ACT CAC TAT AGG ATT TAG ACT ACC CCA AAA ACG AAG GGG ACT AAA AC - 3’

reverse (ever changing primer) → 5’ - target sequence + GTT TTA GTC CCC TTC GTT TTT GGG GTA GTC TAA ATC - 3' (reverse complementary sequence of the LwaCas13a scaffold)

Table with exemples of sequences

Results

Here, 6 different sequences provide fluorescence detection using our template as a guide. The detection was made using the West Nile Virus (WNV) as a target.

Kinetic can also be observed using the guides, allowing us to show that detection is possible as early as 30 minutes in the reaction.