Our project aims to design an alternative solution to combat the presence of cyanobacteria in Alberta waterways. Although they are a natural part of healthy aquatic ecosystems, in warm slow-moving waters with excess nutrients such as nitrogen and phosphorus, cyanobacteria multiply rapidly, forming blooms. These overgrowths block sunlight, cause oxygen depletion when they decay, and some species also produce toxins such as microcystins that are hazardous to wildlife, livestock, pets, and humans. Conventional treatments often have off-target effects, and do not take into consideration the release of toxins. Furthermore, mitigation strategies such as decreasing fertilizer use are difficult to implement due to the importance of the agriculture industry in Alberta.
Our main goals for this project were to create a target-specific system that addresses the issue of overgrown cyanobacteria blooms that also mitigates the effects of microcystins. To achieve this, our proposed treatment will incorporate two elements encapsulated together, and delivered simultaneously. These are the CRISPR Cas13a system, and microcystinase (mlrA enzyme). The CRISPR Cas13a system involves the delivery of a crRNA that will bind to a specific cyanobacteria mRNA sequence, which activates cleavage of RNA by the Cas13 enzyme. The mlrA enzyme will linearize microcystin peptides, decreasing their toxic effects.
Since these goals concern multiple aspects of cyanobacteria, we have decided to aim for a two-year project. This year we have focused our efforts on the theoretical design and modelling aspects of our system, while we plan to carry out testing for proof of concept in 2022.
The Problem
> Cyanobacteria, also known as Blue-green algae are photosynthetic bacteria that live in fresh, brackish, or marine water environments. In recent years, high phosphorus and nitrogen levels in bodies of water have resulted in an increase in cyanobacteria levels. The Albertan prairies have an especially prominent cyanobacteria problem, due to naturally nutrient-rich sedimentary bedrock and soil, exacerbated by fertilizer runoff, resulting in increased phosphorus and nitrogen levels in bodies of water. Combined with warm summers these conditions allow cyanobacteria to thrive.
The rapid growth of cyanobacteria results in the formation of blooms on the surface of stagnant water that can be thick enough to block sunlight. In addition to this, when blooms decay they consume large amounts of oxygen. This combination can deplete oxygen levels below the threshold for aquatic organism survival.
Additionally, a key defense adaptation against grazers that contributes to bloom proliferation is the production of toxins by some species, such as Microcystis aeruginosa. When blooms decay these toxins get released and can persist in the environment for up to three weeks, which increases the likelihood of being inadvertently consumed by wildlife, livestock, pets, or humans (Jones and Orr, 1994). The most abundant toxin in affected Albertan lakes produced by M. aeruginosa is microcystin-LR (MC-LR) (Zurawell, et al., 2005). Microcystins cause liver failure by inhibiting protein phosphatase and are also linked to promoting liver tumor growth (Zurawell, et al., 2005).
Current Treatment methods
Current treatment methods to eradicate algal blooms include the application of algaecides, chemicals such as copper sulfate, and mitigation strategies such as regulating the usage of fertilizers. Employing copper sulfate and algaecides harms other microorganisms that are necessary for healthy aquatic ecosystems and do not deal with the large release of microcystins when cyanobacteria die. In addition to this, regulating the use of fertilizers is impractical in Alberta, where agriculture is a prevalent industry. Current Treatment methods
The Solution
Our proposed treatment, CyaNoMore, tackles the overgrowth of cyanobacteria while addressing the problem caused by the release of microcystins. The CRISPR-Cas13a system will be designed to be cell-specific to M. aeruginosa, preventing any off-target effects. Meanwhile, the incorporation of the microcystins enzymes (mlrA) will prevent harm caused by the release of microcystin-LR following cell death.
CRISPR-Cas13a
Clustered regularly interspaced short palindromic repeats (CRISPR) encompasses nucleotide-editing systems that act as a bacterial protective responses against foreign genetic material. Our chosen CRISPR-system uses Cas13a, which we have chosen for our project originates from Leptotrichia buccalis (Lbu) and is an RNase that cleaves single-stranded RNAs (ssRNA) (O'Connell, 2019). The CRISPR RNA (crRNA) can be engineered to bind to a specific sequence on cyanobacteria mRNA. Once the Cas13a-crRNA complex binds to the mRNA, the Cas13a enzyme activates and cleaves ssRNA non-discriminately.
MlrA
Using molecular modeling we examined the binding interactions of cyanotoxin inactivation by mlrA. Sphingomonas sp. are bacteria that produce enzymes that can degrade microcystins (Bourne et al., 2001). One of the critical enzymes in this gene cluster is the mlrA, which functions to linearize microcystin-LR (Wei et al., 2021).
Fig. 1. Homology model of mlrA based on template 4cad.1.C. Constructed using the SWISS-MODEL engine (Bienert et al., 2017).
References
Bourne, D. G., Riddles, P., Jones, G. J., Smith, W. & Blakeley, R. L. Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR. Environ. Toxicol. 16, 523–534 (2001).
Jones, G. J. & Orr, P. T. Release and degradation of microcystin following algicide treatment of a Microcystis aeruginosa bloom in a recreational lake, as determined by HPLC and protein phosphatase inhibition assay. Water Research vol. 28 871–876 (1994).
O’Connell, M. R. Molecular Mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR–Cas Systems. J. Mol. Biol. 431, 66–87 (2019).
Wei, J. et al. Characterization and Mechanism of Linearized-Microcystinase Involved in Bacterial Degradation of Microcystins. Front. Microbiol. 12, 1–10 (2021).
Zurawell, R. W., Chen, H., Burke, J. M. & Prepas, E. E. Hepatotoxic cyanobacteria: A review of the biological importance of microcystins in freshwater environments. J. Toxicol. Environ. Heal. - Part B Crit. Rev. 8, 1–37 (2005).
