A team of researchers from the University of Texas at Austin have discovered a new CRISPR-associated (Cas) protein that can degrade single- and double-stranded DNA as well as single-stranded RNA. The work, published in Nature alongside a second companion paper, also describes how a small alteration to the protein leads to the degradation of single-stranded DNA only. This finding has the potential to revolutionise testing for viruses such as COVID-19.
A new discovery
CRISPR-Cas systems are a naturally occurring component of the prokaryotic immune system. This adaptive immune mechanism consists of guide RNA that is complementary to a target and a Cas endonuclease that “cuts” genetic material. In recent years, this system has been exploited for use in gene editing.
Many different Cas proteins exist, each with their own distinct features and functions. One of these proteins is Cas12a; this endonuclease is often favoured in gene editing due to its ability to create “sticky” ends when cutting DNA, which can help to ensure the specificity of any subsequent insertions. Recently, a new Cas endonuclease that shares its most recent common ancestor with Cas12a has been discovered. To learn more about this new protein, named Cas12a2, David Taylor and his team analysed structural and biochemical properties of the protein and compared these to Cas12a.
The world is your oyster
Typically, a CRISPR-Cas system will protect an organism through destruction of foreign bodies such as a phage or virus. However, this study has uncovered a unique defence mechanism used by the newly characterised Cas12a2 protein. Cas12a2 induces abortive infection – death of the host cell to prevent further transmission. Cas12a2 does not use any secondary messengers in this process, instead inducing non-specific cleavage of single- and double-stranded nucleic acids. This sets it apart from its Cas12a relative, which does not trigger abortive infection and cannot cleave such a wide-range of genetic materials.
The team then used cryo-EM to compare the structure of Cas12a2 with that of the Cas12a protein. They discovered that the two proteins are in fact largely dissimilar, sharing only around 20% sequence identify. Cas12a2 was described as having an “oyster”-like shape, in contrast to the more typical triangular shape exhibited by Cas12a (Figure 1). Cas12a2 also contains a unique lobe, which the researchers hypothesised protects it from anti-CRISPR proteins.
Figure 1: Image comparing the structure of a.) Cas12a2 and b.) Cas12a. Cas12a2 exhibits a more “oyster”-like shape, whilst Cas12a is more triangular. Adapted from Bravo et al. 2023.
Upon binding to its target RNA, Cas12a2 was seen to undergo significant, unique conformational changes – more so than other Cas proteins. These structural alterations expose the protein’s active site and create a groove that can fit both single- or double-stranded DNA or RNA. Upon binding by Cas12a2, the helix is clamped and bent – in the case of double-stranded DNA, this causes the double helix to come apart, exposing single strands. This is followed by indiscriminate cleavage of nucleic acids and destruction of the cell. First author Jake Bravo said, “this is what makes Cas12a2 different from all the other DNA-targeting systems.”
With the use of CRISPR-Cas systems having become so prevalent in many areas, the characterisation of the unique Cas12a2 protein leads to more questions. How can the functions of this protein be exploited to advance the field and add to the technology we already have?
To combat these questions, the team conducted further experiments by inducing mutations in Cas12a2 to assess the impact this would have on its ability to degrade nucleic acids. One mutation prevented the protein from cleaving double-stranded DNA and single-stranded RNA, meaning it could only “cut up” single-stranded DNA. This is of particular interest, as identification of single-stranded DNA is a common method in viral diagnostics.
The researchers hypothesise that new efficient tests for viral detection could be developed off the back of this discovery. Saliva samples could be exposed to the mutated CRISPR-Cas12a2 system, destroying single-stranded viral target DNA. A fluorescent signal could then be used to indicate successful degradation of the target, indicating the presence of the virus in the sample. This potential technology could be ground-breaking in the detection of viruses such as COVID-19 and Ebola, with hopes that any test could be cheap and easy to carry out. This type of test would also be very adaptable. Taylor stated that “if some new virus comes out tomorrow, all you have to do is figure out its genome and then change the guide RNA in your test, and you’d have a test against it.” The findings also highlight the versatility of CRISPR systems and the importance of exploiting different Cas endonucleases.