Collections/CRISPR-Cas for Diagnosis
Introduction
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 [4][5]. 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:
- 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.
- 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.
It is important to point out that, as parts are built upon other part of the collection, the complete documentation is only included in the most complex category part of the section (gRNA construct or Efficient gRNA construct), and not in the previous steps (gRNA spacer, gRNA and Efficient gRNA).
3. Parts
The following table contains all the initial parts embedded in the collection. When future teams use parts that fit here, they are invited to add them in this table by indicating their team name, the type and species of Cas employed, part code and name, structure in case of the composite parts, and finally their function and part collection category. The idea is to group multiple parts depending on the Cas protein that is used, so that if the nuclease type is already in the table, the parts are added in the table cells that correspond to it.
| Team | Cas protein | Part Code | Name | Structure (composite parts) | Function | Category |
|---|---|---|---|---|---|---|
| ASFAST (CCU Taiwan 2019) | LbCas12a | BBa_K2927005 | LbCas12a | - | Binds a specific DNA fragment and cuts it as well as any other DNAs present in the media. | Cas protein |
| ASFAST (CCU Taiwan 2019) | BBa_K2927006 | Repeat gRNA | - | The repeat gRNA sequence binds to LbCas12a protein, which promotes crRNA recognition of its target double-stranded DNA through sequence complementation, specified by the spacer sequence. | gRNA Spacer | |
| ARIA (UPF Barcelona 2021) | BBa_K3791000 | Spacer gRNA Ampicillin | - | Target a specific DNA fragment of a certain ampicillin-resistance gene. | gRNA Spacer | |
| ARIA (UPF Barcelona 2021) | BBa_K3791001 | Spacer gRNA Chloramphenicol | - | Target a specific DNA fragment of a certain chloramphenicol-resistance gene. | gRNA Spacer | |
| ARIA (UPF Barcelona 2021) | BBa_K3791002 | Spacer gRNA Erythromycin | - | Target a specific DNA fragment of a certain erythromycin-resistance gene. | gRNA Spacer | |
| ARIA (UPF Barcelona 2021) | BBa_K3791003 | Spacer gRNA Kanamycin | - | Target a specific DNA fragment of a certain kanamycin-resistance gene. | gRNA Spacer | |
| ARIA (UPF Barcelona 2021) | BBa_K3791004 | Spacer gRNA Spectinomycin | - | Target a specific DNA fragment of a certain spectinomycin-resistance gene. | gRNA Spacer | |
| ARIA (UPF Barcelona 2021) | BBa_K3791005 | gRNA Ampicillin | Repeat + Spacer gRNA Ampicillin | Assembly with functional LbCas12a for DNase activity performance. Specific recognition of target ampicillin-resistance gene. | gRNA | |
| ARIA (UPF Barcelona 2021) | BBa_K3791006 | gRNA Chloramphenicol | Repeat + Spacer gRNA Chloramphenicol | Assembly with functional LbCas12a for DNase activity performance. Specific recognition of target chloramphenicol-resistance gene. | gRNA | |
| ARIA (UPF Barcelona 2021) | BBa_K3791007 | gRNA Erythromycin | Repeat + Spacer gRNA Erythromycin | Assembly with functional LbCas12a for DNase activity performance. Specific recognition of target erythromycin-resistance gene. | gRNA | |
| ARIA (UPF Barcelona 2021) | BBa_K3791008 | gRNA Kanamycin | Repeat + Spacer gRNA Kanamycin | Assembly with functional LbCas12a for DNase activity performance. Specific recognition of target kanamycin-resistance gene. | gRNA | |
| ARIA (UPF Barcelona 2021) | BBa_K3791009 | gRNA Spectinomycin | Repeat + Spacer gRNA Spectinomycin | Assembly with functional LbCas12a for DNase activity performance. Specific recognition of target spectinomycin-resistance gene. | gRNA | |
| ARIA (UPF Barcelona 2021) | BBa_K3791010 | gRNA Ampicillin construct | T7 promoter + Repeat + Spacer gRNA Ampicillin | gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target ampicillin-resistance gene. | gRNA construct | |
| ARIA (UPF Barcelona 2021) | BBa_K3791011 | gRNA Chloramphenicol construct | T7 promoter + Repeat + Spacer gRNA Chloramphenicol | gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target chloramphenicol-resistance gene. | gRNA construct | |
| ARIA (UPF Barcelona 2021) | BBa_K3791012 | gRNA Erythromycin construct | T7 promoter + Repeat + Spacer gRNA Erythromycin | gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target erythromycin-resistance gene. | gRNA construct | |
| ARIA (UPF Barcelona 2021) | BBa_K3791013 | gRNA Kanamycin construct | T7 promoter + Repeat + Spacer gRNA Kanamycin | gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target kanamycin-resistance gene. | gRNA construct | |
| ARIA (UPF Barcelona 2021) | BBa_K3791014 | gRNA Spectinomycin construct | T7 promoter + Repeat + Spacer gRNA Spectinomycin | gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target spectinomycin-resistance gene. | gRNA construct | |
| ARIA (UPF Barcelona 2021) | BBa_K3791015 | Efficient gRNA Ampicillin | Repeat + Spacer gRNA Ampicillin + 4 NTs + Repeat | Efficient assembly with functional LbCas12a for DNase activity performance. Specific recognition of target ampicillin-resistance gene. | Efficient gRNA | |
| ARIA (UPF Barcelona 2021) | BBa_K3791016 | Efficient gRNA Chloramphenicol | Repeat + Spacer gRNA Chloramphenicol + 4 NTs + Repeat | Efficient assembly with functional LbCas12a for DNase activity performance. Specific recognition of target chloramphenicol-resistance gene. | Efficient gRNA | |
| ARIA (UPF Barcelona 2021) | BBa_K3791017 | Efficient gRNA Erythromycin | Repeat + Spacer gRNA Erythromycin + 4 NTs + Repeat | Efficient assembly with functional LbCas12a for DNase activity performance. Specific recognition of target erythromycin-resistance gene. | Efficient gRNA | |
| ARIA (UPF Barcelona 2021) | BBa_K3791018 | Efficient gRNA Kanamycin | Repeat + Spacer gRNA Kanamycin + 4 NTs + Repeat | Efficient assembly with functional LbCas12a for DNase activity performance. Specific recognition of target kanamycin-resistance gene. | Efficient gRNA | |
| ARIA (UPF Barcelona 2021) | BBa_K3791019 | Efficient gRNA Spectinomycin | Repeat + Spacer gRNA Spectinomycin + 4 NTs + Repeat | Efficient assembly with functional LbCas12a for DNase activity performance. Specific recognition of target spectinomycin-resistance gene. | Efficient gRNA | |
| ARIA (UPF Barcelona 2021) | BBa_K3791020 | Efficient gRNA Ampicillin construct | T7 promoter + Repeat + Spacer gRNA Ampicillin + 4 NTs + Repeat + L3S2P21 terminator | Efficient gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target ampicillin-resistance gene. | Efficient gRNA construct | |
| ARIA (UPF Barcelona 2021) | BBa_K3791021 | Efficient gRNA Chloramphenicol construct | T7 promoter + Repeat + Spacer gRNA Chloramphenicol + 4 NTs + Repeat + L3S2P21 terminator | Efficient gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target chloramphenicol-resistance gene. | Efficient gRNA construct | |
| ARIA (UPF Barcelona 2021) | BBa_K3791022 | Efficient gRNA Erythromycin construct | T7 promoter + Repeat + Spacer gRNA Erythromycin + 4 NTs + Repeat + L3S2P21 terminator | Efficient gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target erythromycin-resistance gene. | Efficient gRNA construct | |
| ARIA (UPF Barcelona 2021) | BBa_K3791023 | Efficient gRNA Kanamycin construct | T7 promoter + Repeat + Spacer gRNA Kanamycin + 4 NTs + Repeat + L3S2P21 terminator | Efficient gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target kanamycin-resistance gene. | Efficient gRNA construct | |
| ARIA (UPF Barcelona 2021) | BBa_K3791024 | Efficient gRNA Spectinomycin construct | T7 promoter + Repeat + Spacer gRNA Spectinomycin + 4 NTs + Repeat + L3S2P21 terminator | Efficient gRNA transcription for latter assembly with functional Cas12a and DNase activity performance. Specific recognition of target spectinomycin-resistance gene. | Efficient gRNA construct |
Table 1: CRISPR-Cas for Diagnosis parts.
References
[1] de Puig, H., Lee, R. A., Najjar, D., Tan, X., Soenksen, L. R., Angenent-Mari, N. M., Donghia, N. M., Weckman, N. E., Ory, A., Ng, C. F., Nguyen, P. Q., Mao, A. S., Ferrante, T. C., Lansberry, G., Sallum, H., Niemi, J., & Collins, J. J. (2021). Minimally instrumented SHERLOCK (miSHERLOCK) for CRISPR-based point-of-care diagnosis of SARS-CoV-2 and emerging variants. Science Advances, 7(32). doi: 10.1126/sciadv.abh2944
[2] Liu, T. Y., Knott, G. J., Smock, D. C. J., Desmarais, J. J., Son, S., Bhuiya, A., Jakhanwal, S., Prywes, N., Agrawal, S., Díaz De León Derby, M., Switz, N. A., Armstrong, M., Harris, A. R., Charles, E. J., Thornton, B. W., Fozouni, P., Shu, J., Stephens, S. I., Kumar, G. R., . . . Doudna, J. A. (2021). Publisher Correction: Accelerated RNA detection using tandem CRISPR nucleases. Nature Chemical Biology. doi: 10.1038/s41589-021-00882-8
[3] Agrawal, S. (2021, 1st January). Rapid, point-of-care molecular diagnostics with Cas13. MedRxiv. doi: 10.1101/2020.12.14.20247874
[4] East-Seletsky, A., O’Connell, M. R., Knight, S. C., Burstein, D., Cate, J. H. D., Tjian, R., & Doudna, J. A. (2016). Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature, 538(32)(7624), 270–273. doi: 10.1038/nature19802
[5] Chen, J. S., Ma, E., Harrington, L. B., da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360(32)(6387), 436–439. doi: 10.1126/science.aar6245