Resurrecting ancient CRISPR-Cas proteins from millions of years ago

It is possible to resurrect Cas9 proteins that could have existed in bacteria many millions of years ago, and function as gene editing tools in human cells. We have just demonstrated this in a collaborative article published in the journal Nature Microbiology.

The discovery that transformed biology: CRISPR-Cas
There is no doubt that CRISPR-Cas tools have changed the lives of many researchers. These CRISPR-Cas systems were originally described by Francis Mojica as part of an adaptive defense system that bacteria use to defend themselves against viruses (bacteriophages) that attack them. Years later, two researchers, Emmanuelle Charpentier and Jennifer Doudna, turned them into genetic editing tools, capable of inactivating or inducing targeted editing of any gene in any organism. These researchers ended up receiving a well-deserved Nobel Prize in Chemistry in 2020. The rest is the history of biology.

The vast majority of laboratories in the world continue to use the CRISPR-Cas9 system from the Streptococcus pyogenes bacteria (SpCas9), which was the first complete system described by Charpentier and Doudna in their article in the journal Science published in June 2012. Some years Feng Zhang then described a somewhat smaller Cas9 nuclease, derived from a CRISPR-Cas9 system from Staphylococcus aureus (SaCas9). This SaCas9 nuclease was smaller and therefore the entire corresponding CRISPR-Cas system could be included in a single adeno-associated virus (AAV) viral particle, instead of having to use two, as is the case with SpCas9, since it is necessary to use another virus to include the guide RNA that directs the cut and the template DNA that is to be used to recombine the desired gene after the cut generated by the nuclease.

Both Streptococcus pyogenes and Staphylococcus aureus are pathogenic bacteria for humans, commonly causing infections throughout the body (S. aureus) or otitis and laryngitis (S. pyogenes), and, therefore, our immune system already has antibodies against them and against all their components, including the SpCas9 and SaCas9 proteins. Researcher Matthew Porteus (Stanford University, CA, USA) realized this, demonstrating that most people have antibodies against these Cas9 proteins from these two bacteria. This unexpected setback was published in the journal Nature Medicine. Logically, we do not want to use as a therapeutic protein something against which the immune system will react by attacking it. That is why attempts have been made to find many other Cas9 nucleases and other Cas derived from other bacteria, far from any interaction with humans.

The number of CRISPR-Cas9 systems described has increased rapidly, as has the description of Cas proteins from metagenomes, from bioinformatic predictions, or from bacteria and archaea from the most remote and extreme places on the planet, such as the Atacama Desert. .

In search of Cas9 proteins in the distant past
An alternative to searching for new Cas9 in bacteria that inhabit the most inhospitable corners of the Earth is to search for them not in space, but in time, trying to resurrect ancestral Cas proteins that must have existed many millions of years ago.

The main problem with this approach is the lack of fossil record and organic remains from which to obtain DNA.

A few weeks ago, the oldest DNA recovered from Greenland minerals that was about two million years old was published. This may seem like a lot, but on a geological level, considering that our planet is estimated to be about 4 billion years old, it is a trifle.

We need to go beyond two million years, much more. How do we do it? This is what paleoenzymology achieves: reconstructing sequences of ancestral proteins that must have existed in already extinct organisms.

Paleoenzymology works from the sequences of these proteins in a certain group of current organisms and their phylogenetic relationships, applying maximum likelihood statistical techniques. With sufficient computational power, the most probable ancestral sequences can be inferred, which could have existed, which are compatible and which allow explaining the current diversity of sequences.

By calibrating the analysis with a timeline it is possible to deduce the age at which the ancestral protein nodes that gave rise to different groups of current Cas9 proteins are located.

The phylogenetic tree reconstructs, using a maximum likelihood algorithm, the evolutionary relationships between the Cas9 protein sequences of up to 59 species of current bacteria, more or less related, indicating the nodes from which the results have been obtained. ancestral Cas protein sequences.

Ancestors identified from up to 2 billion years ago
With the described strategy, Raúl Pérez-Jiménez's team obtained the Cas9 protein sequences of a set of 59 current, evolutionarily related bacteria, to a greater extent from the genus Streptococcus and to a lesser extent from the genus Enterococcus, Clostridium and Bifidobacterium, among others. Pérez-Jiménez put a powerful computer to work with a maximum likelihood algorithm to infer the most likely ancestral Cas9 proteins that could have existed and given rise to the diversity of current Cas9 proteins.

By calibrating the location of the nodes with a timeline, five ancestral Cas9 proteins were established, called PDCA, PCA, SCA, BCA and FCA, which could have existed 37, 137, 200, 1 000 and 2 600 million years ago respectively. These ages far exceed the possibility of finding remains of any ancient DNA, currently limited to 2 million years. The resulting ancestral Cas9 proteins resemble current SpCas9 between 56% (the oldest) and 93% (the most modern) molecular identity.

Between dinosaurs and the first eukaryotic cells
To mentally locate the estimated ages of these computationally resurrected ancestral Cas nucleases, let us remember that the Earth is ~4.500 billion years old; The oldest evidence of prokaryotic cells is from ~3.500 billion years ago; eukaryotic cells (with a nucleus, a product of the fusion of several bacteria according to the endosymbiont theory proposed by Lynn Margulis) appeared ~1.850 billion years ago; the Cambrian explosion that gave rise to most animal groups that exist today took place ~580 million years ago; The Brachiosaurus (one of the largest dinosaurs that ever existed, the first to appear in the movie Jurassic Park) became extinct ~150 million years ago and the rest of the dinosaurs became extinct 65 million years ago, except for those that gave rise to current birds.

Once the sequences of the ancestral Cas proteins were obtained, the corresponding DNA sequences that encoded them were synthesized, using codons optimized for the Escherichia coli bacteria, to obtain and purify proteins from which to carry out the in vitro experiments.

The properties of Cas as genetic scissors
The functional characterization of the five resurrected ancestral Cas proteins was done at two levels: in vitro, in the laboratory, and in vivo, in human cells.

Firstly, it was found that, as we move away from the current moment, the oldest ancestral Cas have a greater tendency to have a cutting activity of one of the two DNA strands (nickase activity) compared to a cutting activity of the two strands of DNA, as the current Cas9 has.

Secondly, single-stranded DNA and RNA (which is also single-stranded) cutting activity was also detected in the oldest ancestral Cas, while the most "modern" ancestral Cas preferentially showed a DNA cutting activity of double chain.

All of them apparently recognize, with greater or lesser success, an RNA molecule that guides the process, derived from the structure we know for the current SpCas9, which is probably not optimized for all these ancestral Cas.

The original genetic world
These observations fit with an original world essentially based on single-stranded DNA and RNA molecules, an RNA world that is one of the most accepted hypotheses of the origin of genetic material in living beings. Hence, it is logical that ancestral Cas prefer to cut single-chain molecules.

The specificity of the different ancestral Cas was evaluated by investigating their requirements for the PAM (Protospacer Adjacent Motif) sequence, which is another of the findings and names proposed by Francis Mojica. PAM allows bacteria to differentiate between the sequence to be cut present in the virus genome from the same sequence, but inserted in the bacterial genome. In this way they avoid committing suicide, degrading their own genome.

Current SpCas9 needs to have the three-nucleotide series “NGG” next to the sequence complementary to the guide RNA molecule. This requirement was maintained for the intermediate ancestral Cas, 37, 137 and 200 million years old. The oldest SpCas9, BCA and FCA, do not appear to require a specific PAM sequence for cleavage, and accept virtually any sequence adjacent to that recognized by the guide RNA.

All this can be interpreted as an evolution from Cas proteins without the need for PAM to the current ones, which need a PAM (different for each Cas9 of each bacteria). It makes sense if we think that the original diversity of virus sequences must have been much lower than the diversity of current viruses, so the need to add the PAM verification step must have appeared later.

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Source: The Conversation. Authorship:

Lluís Montoliu
Scientific researcher at the CSIC, National Center for Biotechnology (CNB – CSIC)

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