The specificity found within the mechanisms of the CRISPR system can be both a boon and a bane. Thanks to things like the PAM sequence, CRISPR-Cas9 has found itself as a useful tool to accurately target desired gene sequences at an unparalleled level. No other gene editing technology can really come close to matching the success rate of its targeting system. It even includes several copies of its gene target spacers in what is known as the seed region so that, if one of them was harmed or destroyed whether by radiation or any other method, it wouldn’t undermine the activity of the system.
But there are also cases where having such a narrowed focus can be detrimental to the activity of CRISPR as a whole. If a virus (or any sequence that is human targeted) has any sort of mutation in the identified gene, then Cas9 can find itself unable to properly cleave and deal with the offending genetic material. Since the PAM sequence and its guide RNAs require a perfect match before cleavage will be initiated.
For most human usage of the tool, this isn’t an issue. There isn’t likely to be a mutation in the targeted sequence because the sequence itself was determined and chosen ahead of time. But there are still cases and experiments, especially in more longer-lasting tests requiring CRISPR to remain active, that could lead to issues and changes in the directed nucleotide sequence. And for bacteria in the wild, a highly specific Cas9 system can leave them open to attack from mutant bacteriophages.
Looking Back On CRISPR
However, there is another form of CRISPR, a somewhat more antique version, that can deal with this problem. In our prior long-form primer article on CRISPR, one of the varieties discussed was CRISPR-Csm1, which is a part of the Type III-A grouping. It is commonly referred to today by its unique and very important Cas protein, forming the name CRISPR-Cas10. It is this particular protein that lets it avoid the pitfalls that Cas9 can run into when the target genetic sequence no longer matches its recorded sequence.
The first thing to note about the CRISPR-Cas10 system is that it lacks a PAM sequence. It just outright doesn’t have one. This was a noted curiosity in our previous article, but new research from scientists in a collaboration between The Rockefeller University and Cornell University has investigated further into the workings of this novel biological machinery.
They used the system found in Staphylococcus epidermidis and a phage sequence known by the bacteria’s CRISPR system. Then, the researchers created a phage library that had that sequence and a number of mutation variants of it. Lastly, they put all of these variants into plasmids and saw if the CRISPR-Cas10 complex could still respond to them. And indeed, for most of them it could. Even with mutations altering their coding, Cas10 remained able to recognize the sequences for the phage genes that they were.
Cas10’s Relaxed Specificity
It is believed that this is possible due to the relaxed specificity of Cas10. Since it lacks a PAM sequence and only uses a single spacer for the target gene in its seed region, the system instead looks more broadly for sequences that more or less match it. The complex doesn’t require a perfect match in order to initiate cleavage, just a good enough one. This, in turn, allows it to foment a strong and immediate immune response to invading viruses without having to have a number of guides to be made.
On average, the Cas10 system was two orders of magnitude less likely to have a phage sequence escape detection than Cas9 and other types. The disparity may be even more broad than that, as the researchers hit the limits of their detection capabilities once they went beyond 1 in 10,000,000,000 (10 billion) escapees. Additionally, the reasons for escape differed between the two.
As might be expected, the reason for escape with Cas9 is usually due to a mutation in the PAM sequence (always a G nucleotide). But for Cas10, what was required was more or less a complete deletion of the target gene. The only phages that got away are those that removed the gene of interest almost entirely so that the Cas10 complex couldn’t detect it at all. Because this is the type of mutation that would work on any CRISPR system, it can easily be said that Cas10 is one of, if not the best, at catching invading genetic material.
This is even more true if the target gene is made to be one that is indispensable to the phage genome and cannot be removed. Then they have no chance at all of escaping. If an entire species of bacteria with Cas10 did obtain and utilize such a sequence, it would have the potential of driving the virus in question to extinction. It’s possible that this has happened many times before.
Evolution and Usage
In a way, it could be said that even this older CRISPR system was the epitome of the immune system arms race between bacteria and phages. Why then did so many move to Cas9 and other systems? Perhaps they just never evolved Cas10 or the attacking phage population rarely had significant mutations to escape detection, making a more loose specificity unnecessary. Or that this more hazy focus resulted in the Cas10 system targeting the host genome by accident, making it detrimental to the survival of some bacteria that use it. There may also be other, as yet unknown, reasons for Cas10 not becoming ubiquitous throughout the bacterial community.
For now, we can certainly take advantage of the fact that Cas10 allows for a broader gene detection system and it will definitely help improve the rapidly growing scientific field around CRISPR and everything its assorted complexes can accomplish.
Photo CCs: Virus created with blender 249b 001 from Wikimedia Commons
