In a special blog post, Molly-Ann Williams(@WilliamsMolly_9) and Anne Parle-McDermott (@anne_parle) from the School of Biotechnology and DCU Water Institute, Dublin City University provide an overview of how CRISPR-Cas works and how it can be applied to ecology and monitoring in particular. Read their recently published Molecular Ecology Resources paper here.
The field of CRISPR-Cas for genome editing has simply exploded since its introduction in 2012. The discovery of many different Cas enzymes with additional natural or genetically engineered functionalities, is resulting in an increase in CRISPR-Cas applications across all fields from food security to medicine.
So how can we join the revolution and apply CRISPR-Cas to the field of Ecology?
CRISPR-Cas systems consist of two main elements: a guide and a nuclease. Guides (made of RNA) direct the nuclease (Cas enzyme) to specific nucleic acid sequences (DNA or RNA). Upon target recognition the nuclease carries out the desired response, most commonly cleavage of the target sequence. The initially discovered CRISPR-Cas system relied on a nuclease called Cas9. This enzyme is involved in highly specific cleavage of target sequences that allow genome editing to occur by activating the natural repair system of the cell. More recently the applications of this system have been expanded beyond genome editing by the discovery of several new Cas enzymes with a secondary function i.e., the indiscriminate cleavage of single stranded nucleic acids upon target recognition. The discovery of these Cas enzymes has revolutionised nucleic acid diagnostics due to two main features:
- Protein-guide and cleavage molecules (Cas): able to specifically recognise target nucleic acids, cleave the target sequence and subsequently cleave other non-specific nucleic acids.
- Nucleic acids as reporters: the non-specific nucleic acids can be designed as a reporter molecule that releases measurable signal when cleaved. This allows us to visualise when the initial target sequence has been detected and apply it to diagnostics and species monitoring.
Two main elements of a CRISPR-Cas diagnostic system: Cas enzyme and guide RNA effector complex and single stranded (ss) nucleic acid reporter molecule. In this example, the nuclease is Cas12a specific to DNA detection downstream from a TTTV PAM site.
The three main Cas enzymes of interest for diagnostics are Cas12, Cas13 and Cas14 each with unique functions applicable to different types of tests (for a more detailed discussion of these enzymes visit this blog).
The Cas enzyme most relevant for single species detection from environmental DNA is the enzyme Cas12a. This nuclease can detect both ssDNA and dsDNA but can only recognise DNA sequences downstream from a TTTV protospacer adjacent motif (PAM). Importantly, Cas12a cannot detect DNA sequences missing this PAM site. This is vital when designing single species detection assays.
Do you have two closely related species that you want to distinguish? Searching your target species sequence for a site downstream of a PAM site found ONLY in your target, and not in sympatric species, will ensure highly specific recognition and prevent detection of non-target species.
What if you work with environmental RNA? Well there is a CRISPR-Cas system for you too! The Cas enzyme Cas13 differs from Cas12a in that it recognises single stranded RNA molecules with non-specific cleavage of ssRNA following target cleavage i.e., it works the same as Cas12a but targets RNA rather than DNA.
The world of CRISPR diagnostics is still in its early stages but with the discovery of new CRISPR-Cas systems with unique functions, there is no reason ecologists cannot utilise these diagnostic tools to enhance environmental monitoring using molecular techniques. For more information on using CRISPR-Cas diagnostics for single species detection from environmental DNA read our paper here.