Collections/CRISPR-Cas for Diagnosis
This part collection aims to nest all these parts needed for the CRISPR-Cas technology to be used as a detection system with diagnostic purposes. The creators of this collection are ARIA, the 2021 UPF Barcelona iGEM team, who make use of the CRISPR-Cas technology for detecting antibiotic resistance genes in, as a proof-of-concept, laboratory samples.
1. CRISPR-Cas12/13 as detection systems
Recently (ends of 2021), especially due to the COVID-19 pandemic situation, fast diagnostic procedures based on CRISPR-Cas with the use of different Cas12 and Cas13 protein species, have gained a lot of relevance because of having proved to successfully work [1][2][3]. That is why ARIA thinks that the CRISPR-Cas for Diagnosis Part Collection might be widely useful for future iGEM teams making use of this emergent application of the technology.
The principles behind the detection of a specific target DNA/RNA fragment by these two types of Cas proteins (12/13, respectively) are simple. They are based on the emergence of a fluorescent signal due to the collateral trans cleavage activity of these Cas nucleases, which means that when they get activated by the presence of the target fragment of genetic material, they not only cut it but also cut any other DNA which is present in the media [refs]. By taking advantage of this property, any desired target sample can be detected simply by introducing with it a reporter which releases a fluorophore when being cut, producing in this way a fluorescent signal that can be measured with a fluorescence detector.
2. Part Collection
This part collection is created with the purpose of helping other future iGEM teams for the guidance in the design of their parts needed to accomplish the full detection system described above. For that, we have created and documented it in a way that it is easy-to-use and user-friendly, as it is as much structured and organized as possible, being all the part pages linked between them in a way that an external user can comfortably replicate our design.
- Categories
As for the collection categories, we propose a basic structure in which each of the gRNA design steps is considered:
- gRNA spacer: all the sequences that are explicitly the spacer (variable part) of the guide RNAs, needed for telling the CRISPR-Cas technology at play what to detect and cleave. For that, these sequences are exactly designed to target a specific gene fragment of interest, which in case of our collection, is the gene desired to be detected (for a diagnostic purpose).
- gRNA: each of them include not only the spacer sequence but also the repeat sequence, this latter one being common for all the designed guide RNAs aiming to be used with the same Cas protein species.
- gRNA construct: these include, apart from the gRNA (repeat+spacer), the promoter needed for its proper and desired transcription.
- Efficient gRNA: these are the same as the gRNAs but improved in their performance. In our case, based on the literature, we designed another set of gRNAs with a different architecture than the previous one (repeat+spacer), which was experimentally demonstrated to work better and is: repeat + spacer + 4 nucleotides + repeat.
- Efficient gRNA construct: this is the efficient gRNA with the addition of the promoter but in this case also the terminator.
- Cas protein: Cas protein (12 or 13) species used for the CRISPR-Cas detection system.
- gRNA repeat: conserved sequence of the gRNA for the same Cas protein species.
3. Parts
| Team | Cas protein | Part Code | Name | Structure (composite parts) | Function | Category |
|---|---|---|---|---|---|---|
| ARIA (UPF Barcelona 2021) | BBa_K3791000 | Spacer gRNA Ampicillin | Target a specific DNA fragment of a certain ampicillin-resistance gene. | gRNA Spacer | ||
| BBa_K3791001 | Spacer gRNA Chloramphenicol | Target a specific DNA fragment of a certain chloramphenicol-resistance gene. | gRNA Spacer | |||
| BBa_K3791002 | Spacer gRNA Erythromycin | Target a specific DNA fragment of a certain erythromycin-resistance gene. | gRNA Spacer | |||
| BBa_K3791003 | Spacer gRNA Kanamycin | Target a specific DNA fragment of a certain kanamycin-resistance gene. | gRNA Spacer | |||
| BBa_K3791004 | Spacer gRNA Spectinomycin | Target a specific DNA fragment of a certain spectinomycin-resistance gene. | gRNA Spacer |