Going back to the beginning of CRISPR feels like traveling through a time portal. The gene editing technology has become so ingrained in our lives, in scientific literature and the news, that it feels like it’s been a decade or two already since we started using it. It comes as a bit of a shock to remember that it’s only been just a few short years since that fateful paper was published. The advancements since has come so rapidly that it’s hard to keep up and it makes one wonder what the next big uncovered secret of biology will be.

But something we forget is that CRISPR, while an important system, is still just a basic bacterial defense mechanism. Heck, we’ve found bacteriophages since that have developed “anti-CRISPR” systems to counter the bacteria that use it. The evolutionary arms race continues whether we’re paying attention or not. And that’s why some scientists have been thinking.

We already know about several other defense systems that some bacteria use, classified under names like BREX or DISARM or even just called the prokaryotic Argonaute system. They haven’t received all that much investigation in comparison, but we do know about them. So the real question becomes, how many other defense systems are there in microorganisms that we don’t know about? Are any of them similar to CRISPR or otherwise have useful effects we could utilize for gene manipulation? The only answer was just a big question mark.

A Bacterial Analysis

Researchers at the Weizmann Institute in Israel chose to do the needed investigation to find out the truth. The first step was to go through the genome databases of bacteria and isolate the regions where other defense genes were known to reside. These so-called “defense islands” had a high likelihood of housing other defense systems as well. Over 45,000 bacteria and archaea genomes were analyzed, along with the resulting proteins from 14,083 families and they were grouped based on their proximity to the islands.

This helped to narrow things down to only 277 protein families along with an additional 35 gene sets that were already previously suspected to be involved with defense systems. Lastly, 23 other protein families that were suspected as well, but didn’t meet the proximity criteria were added, resulting in a total of 335 candidate gene families.

These systems require examination in families because already known systems like CRISPR have their proteins spread across multiple genes, the Cas protein family. Since the order of these genes are highly conserved, the same can be assumed for other systems, so the best options are those where the families appear in the same format across many species. The neighboring genes to each family, out to 10 on each side, were clustered together in this multi-species scan.

Then, a look at the annotations previously added describing genes was able to cross many off the list. 39% were found to be defense-affiliated genes like transposons, but were not defense genes themselves. Another 30% were found to be defense genes that correspond to the systems we already knew about, so could be discarded. And a final 17% connected to operons likely controlling metabolic functions and not defense systems.

Transforming Those That Remain

That left 14%, or 41, candidates left. Some more filtering took out those that only correspond to a small or certain group of species, as only widespread systems were to be looked at here, leaving just 28 gene families to look into that were spread across the entire bacterial phylogeny.

Now they just had to individually test each candidate within a bacterial test subject. They chose to use the model organisms of Escherichia Coli (E. Coli) and Bacillus subtilis (B. subtilis) to see how each gene family conferred resistance to phages, if they did at all in the first place. However, one issue was that, while the use of the candidates is widespread in the bacterial kingdom, none of them were present in the model organisms being used. So they had to be inserted. The scientists went with the simpler option when dealing with bacteria and had them uptake recombinant DNA with the target gene families through a plasmid vector placed into the bacterial environment.

The promoters and other regulatory sequences from the source bacteria were also transferred, in order to ensure proper expression of the genes. They also doubled up when possible, taking the target systems from two different bacteria, just in case one of their choices had an inactive version of the proposed defense system. They also repeated this procedure with five already known defense systems in order to act as a control group. In total, from the 28 candidate systems, they were able to clone 61 separate lines in the model organisms for testing. They also tried to ensure afterwards that at least one case with each system was showing proper expression, but only 26 out of the 28 were successful.

Resisting The Phage Attack

Then the testing began with bombardment by bacteriophage, 10 types for B. subtilis and 6 for E. coli. Phage resistance was evident in 9 of the 26 systems, indicating entirely new defense systems. However, since the rest of the 26 were so associated with defense islands in the bacterial genome, the scientists decided to test one out independently to more accurately determine its function.

Dubbing it Wadjet, their analysis initially suggested that it was just a housekeeping gene family involved in chromosome maintenance. But that result made no sense for it being in the genome location it is that is often horizontally transferred to other bacteria and thus is found randomly throughout species. A critical maintenance system would be more broadly kept and in a more conserved, unchanging location.

Therefore, they hypothesized that it was a descendent system from a maintenance set of genes, yes, but that it has been repurposed as a defensive system. Based on its structure, it seems like it might prevent overdone amounts of gene transfer between species or, due to its single-strandedness in transfer, it may protect against single-stranded DNA phages. However, there are no currently known single-stranded DNA phages of B. subtilis, so they couldn’t test that hypothesis.

Breaking Down Functionality

Of the 9 actual positive resulting systems, they were able to look into three in more depth. The first they named Zorya. The mode of action for this system may use membrane depolarization when under phage attack. It may first try to sense and inactivate the phage DNA and, upon failing, may open up its own cell membrane and cause its own death as a method of preventing the spread of the phage infection to other bacterial colonies. This is shown by 80% of the experimental groups with Zorya not producing phage offspring after death.

The next system titled Thoeris had a fascinating surprise for the researchers. It appears to use a set of Toll-Interleukin Receptor (TIR) domains that are not commonly found in defensive bacterial systems. They are more common in the immune systems of mammals, plants, and invertebrates, not microorganisms. With this finding, the TIR domains have now been confirmed to be defensive structures found in all six of the kingdoms of life. Based on its use elsewhere, these domains may be used to identify particular patterns of phage, with each duplicated copy focusing on specific components.

The last system they looked at was called Druantia and it features a huge sized genomic DNA sequence and a correspondingly large protein. Some of the genes controlling it appear to be related to the already known DISARM system, but the multitude of genes and sequences in Druantia implies a higher complexity. The researchers were not able to recognize any of the other domains in it, implying it may be an entirely new mode of defensive function in prokaryotes of which there is no data on yet. More experiments will be required.

From Discovery To Knowledge

As can be seen, it will take many more studies to unearth the true capabilities of these discovered systems. The three described above need more research, the rest of the 9 positive for defensive systems will need more research, and even the rest of the 26 will need to be considered as they might have defensive functions and they just weren’t tested against the right phage or situation.

Either way, any one of these bacterial defense systems may turn out to have the potential for use in medicine or biological research. But we are only at the discovery stage right now. So much more needs to be done and will be done over the coming months and years. Who knows, another CRISPR level announcement may be right around the corner.

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Photo CCs: Scanning electron micrograph of Methicillin-resistant Staphylococcus aureus (MRSA) and a dead Human neutrophil – NIAID from Wikimedia Commons