Team:Lethbridge HS/Design
Design Overview
Our goal in this project was to increase the ease and efficiency at which pathogenic infections are detected and targeted, utilizing a CRISPR-Cas13a system. Our proposed detection system would involve the enzyme Cas13a, CRISPR RNA and collateral signal RNA Mango dehydrated on paper test strips. An RNA sequence that would correspond to the bacterial infection we want to detect will be encoded in the CRISPR RNA, allowing Cas13a to associate and form a ribonucleoprotein complex. Upon being exposed to the specific pathogenic RNA sequence, the system would be activated causing the Cas13a to cleave any RNA within its proximity including that of the RNA Mango. The cleaving of the RNA Mango should result in a color change on the paper test strip. Theoretically, the same system could be used to target a pathogenic infection. Due to the specificity of the system, the Cas13a system would only cleave the RNA found within the particular pathogenic bacterial cell, therefore killing only the bacteria and preserving the other elements within the microbiome that would normally be affected by current antibiotics. We are currently looking at several methods to administer this system, one of which is using a phagemid system. Please watch our video for an overview of our systems!
Pathogenic Bacteria
When determining which pathogenic bacteria to target, we wanted to fulfill two main criteria:
i) Is the current diagnosis of the infection time-consuming? Our system focuses on fast and efficient diagnosis, and therefore, it would be most beneficial for use with infections containing long diagnostic procedures.
ii) Are the current treatments for the pathogen broad-spectrum antibiotics? Our system is most useful for infections currently treated using general antibiotics, as it eliminates a specific bacterial strain, preventing any side effects associated with broad-spectrum antibiotics.
From our research, we decided to target Staphylococcus aureus and Clostridium difficile, with Escherichia coli as our proof of concept using Green Fluorescent Protein and Red Fluorescent Protein. Here is our alignment of the two proteins to show how we decided to select the region of the GFP mRNA to target with our crRNA that would not permit the cleavage of the RFP transcript.
Cas13a
We chose to use the CRISPR Cas13a protein for its unique property of collateral RNA cleavage, which makes it different from other Cas proteins such as Cas9 or Cas12a. Targeting RNA is more beneficial for our project as it is more abundant in the cell, allowing it to be easily targeted. When the Cas13a-crRNA complex detects the presence of the pathogenic target sequence, the Cas13a will become activated, cutting all surrounding RNA in the vicinity, of which will include our fluorescent RNA Mango.
We chose to investigate Cas13a proteins from four different bacterial species: Lsh Cas13a, Lwa Cas13a, Lba Cas13a, and Lbu Cas13a.
Each different variation of the Cas13a protein has unique strengths and weaknesses that have been previously researched (Knott et al., 2018; Abudayyeh, et al., 2017; Tambe et al., 2018; Gootenberg et al., 2017 ).
Abudayyeh et al. (2017) states that Leptotrichia wadei (LwaCas13a) was the most effective of the fifteen orthologs in E. coli. It is as effective as RNA interference (RNAi) and a more specific RNA targeting option (Abudayyeh et al 2017). This improved specificity would make it ideal for our system. Our team decided to further conduct experiments to narrow down which Cas protein would be more suitable for our system. We ended up successfully purifying LbuCas13a (Figure 1).
Figure 1. Structural overview of Lbu Cas13a (figure from Liu et al., 2017). (A)Cas13a is made up of multiple domains that are involved in RNA recognition, binding, and cleavage. The recognition (REC) lobe recognizes the hairpin loop of the crRNA. The crRNA bound to the target will locate onto the nuclease (NUC) lobe. This causes the activation of the HEPN domains (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain), which causes cleavage of the target RNA and surrounding RNAs. (B) Schematic of the interaction between the crRNA and the target RNA. (C) Complete structural complex of LbuCas13a, crRNA, and target RNA.
CRISPR RNA
The CRISPR RNA (crRNA) is a synthetic RNA component that is necessary to facilitate the recruitment of the target RNA and activation of Cas13a. These contain a direct repeat stem loop that is what Cas13a will recognize. This is followed by the target sequence that will base pair with the sequence on the actual transcript that is being targeted. Our crRNAs were designed based on Munich 2017’s designs for their crRNAs. Our discussion with Dr. Vamvaka also helped influence the length of our target sequence as this would impact the specificity of our system to prevent off-targeting effects.
Fluorescent Indicator
In choosing an indicator for our paper strips, we were looking to find a molecule that could be cleaved by the Cas13a and be easily seen by the naked eye. RNA Mango is a high-affinity RNA aptamer that binds to the fluorophore thiazole orange with nanomolar affinity, with KD ≈ 3nM ( Autor et al., 2018 ). On its own, thiazole orange (TO1), an asymmetric cyanine fluorophore, has very little fluorescence; however, when bound to the RNA Mango aptamer, the system undergoes a 1100-fold increase in fluorescence. The fluorescence of thiazole orange occurs when the monomethine bridge connecting the two heterocycles rigidifies by insertion into double-stranded helical nucleic acids, securing the thiazole orange in place within the aptamer (Dolgosheinna et al. 2015).
Figure 2. Structural overview of RNA Mango aptamers (figure from Autor et al., 2018 ). (Top) Generated variants of RNA Mango aptamers. We are working with RNA Mango II. (Middle) G Quadruplex structure for RNA Mango I. (Bottom) Top view of the core nucleotides for RNA Mango I. Green colouring represents biotinylated- thiazole orange-1 in the middle and bottom images.
Incorporating RNA Mango into our paper strip detection system provides clear identification of pathogenic presence. In comparison to other fluorescence techniques, RNA Mango is almost 2 times greater in fluorescence to the RNA Spinach aptamer, therefore demonstrating its higher fluorescence efficiency. Upon recognition of the specific pathogenic bacterial RNA of the target, the engineered Cas13a will initiate the collateral RNA cleavage effect, where surrounding RNA is randomly cut—including the RNA Mango aptamer and TO1-Biotin Complex. Its cleavage will result in the detachment of the aptamer from the fluorophore, wherein colour loss will occur. By analyzing the fluorescence changes of RNA Mango, the presence of a pathogen can be visually detected on our paper strips. This makes our system applicable to many scenarios as it does not require any laboratory analysis to detect the pathogen.
Figure 3. Overview of of our diagnostic tool. Our paper strips will have lyophilized Cas13a, crRNA, and RNA Mango on them. If the targeted pathogen is present in the sample, we should see a loss of the orange colour as the collateral effect occurs. If not, the strip should remain orange.
Phagemid
A potential method to deliver our Cas13a system is by using phagemids. Phagemids are phage-derived, DNA cloning vectors (Qi et al. 2012) that have very efficient transformation properties. Some components of phagemids include the replication origin of a plasmid, the selective marker, the intergenic region, a phage coat protein gene, restriction enzyme recognition sites, DNA encoding a single peptide, and a promoter.
Phagemids would be an efficient method of delivering our system as phagemids are basically phage plasmids (hence the name). This means that our Cas13a system would be able to be enclosed within the phagemid and injected into bacteria in our body. Upon entrance into each bacterial cell, the Cas13a will be inactive until it identifies the RNA sequence corresponding to its crRNA, where it will initiate cell death through the collateral effect.
When looking at phagemids we wanted to use for our project, we debated using a broad range phage or a more specific phage. We decided that we would go with a cocktail of phages ranging from broad range ones to more specific phages. This ensures that all of the cells we want to infect will be infected while maintaining a level of specificity.
Figure 4. Overview of therapeutic system. The phagemid encoding for the CRISPR-Cas13a complex will be encapsulated within a non-replicating bacteriophage. This can then enter bacteria within its host range and the CRISPR-Cas13a parts will be produced using the host’s machinery. Upon the presence of the target pathogenic RNA transcript, Cas13a will be activated and cause the death of only the pathogenic bacteria.
References
- Abudayyeh O., Gootenberg J., Essletzbichler P., Han S., Joung J., Belanto J., Verdine V., Cox D., Kellner M., Regev A., Lander E., Voytas D., Ting A., Zhang F. (2017). RNA targeting with CRISPR-Cas13a. Nature 550(7675): 280-284.
- Autor, A., Jeng S, C.Y., Cawte A., D., Abdolahzadeh, A., Galli, A., Panchapakesan, S.S.S., Rueda, D., Ryckelynck, M., and Unrau, P.J. (2018). Fluorogenic RNA Mango Aptamers for imaging small non-coding RNAs in mammalian cells. Nat Commun 9(1): 656.
- Elena V. Dolgosheina, Sunny C. Y. Jeng, Shanker Shyam S. Panchapakesan, Razvan Cojocaru, Patrick S. K. Chen, Peter D. Wilson, Nancy Hawkins, Paul A. Wiggins, Peter J. Unrau. RNA Mango Aptamer-Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking. 2015. Retrieved 2019 from: https://bit.ly/2AMr7Ye
- Gootenberg, J.S., Abudayyeh, O.O., Wook Lee, J., Essletzbichlet, P., Dy, A.J., Joung, J., Verdine, V., Donghia, N., Daringer, N.M., Freije, C.A., Myhrvold, C., Bhattacharyya, R.P., Livny, J., Regev, A., Koonin, E.V., Hung, D.T., Sabeti, P.C., Collines, J.J., and Zhang, F. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356(6336):438-442.
- Knott, G.J., East-Seletsky, A., Cofsky, J.C., Holton, J.M., Charles, E., O’Connell, M.R., and Doudna, J.M. (2018). Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme. Nat Struc Mol Biol 10: 825-833.
- Liu, L., Li, X., Ma, J., Li, Z., You, L., Wang, J., Wang, M., Zhang, X., and Wang, Y. (2017). The molecular architecture for RNA-guided RNA cleavage by Cas13a. Cell 170(4): 714-726.
- Tambe, A., East-Seletsky, A., Knott, G.J., Doudna, J.A., and O’Connell, M.R. (2018). RNA-binding and HEPN-nuclease activation are decoupled in CRISPR-Cas13a. Cell Rep 24(4): 1025-1036.