RNA

Part:BBa_K4697000

Designed by: David Lequen   Group: iGEM23_Montpellier   (2023-10-12)


Template for Guide RNA for Cas13 detection

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

Figure 1: Scheme representing all the parts of the template and the designing of the primers for LwCas13a.
Figure 2: Scheme representing all the parts of the template and the designing of the primers for RfxCas13d.

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

Finally, 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

Table 1: Primers used for targeting sequences of West Nile Virus (WNV) using the LwCas13a.

Results

Graph 1: Fluorescence emission of the different pairs of synthetic targets and guides. Testing all the different guides
with their specific synthetic target sequence to see if there is any difference in the fluorescence level
of detection between the 2 main sequences, and the sub parts of each.

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.

Graph 3: Fluorescence detection for old RNA (ng/uL)

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

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None