For years, scientists from all over the world have been searching for microbes in the ice of Antarctica, in the deepest trenches of the oceans, and in the most hostile volcanic environments on the planet. His goal is to find new Proteins with which to improve current gene editing techniques. This could open the door to a new era of science and medicine in which a multitude of diseases are cured by correcting the genome of patients with astonishing ease. Today a study led by Spanish scientists is published that is unique in its kind since they have not searched for these new molecules in space, but in time: they have resurrected proteins from extinct organisms that lived billions of years ago.

The researchers have focused on recreating Cas9 enzymes, the molecules that work like scissors capable of cutting the DNA of any living being at a specific point, and that are the basis of the CRISPR system of gene editing. Since it was devised in 2012, the technique has revolutionized biomedical research, as it makes it possible to rewrite the instruction book of any organism, and now it is beginning to have its first applications in the treatment of some diseases in humans. But this editing system is not perfect. It can introduce potentially dangerous errors into the genome. Hence the need to search for new genetic editing tools.

CRISPR is the immune system of many bacteria and archaea. It allows them to embed virus genetic sequences into their own genome to preserve their robot portrait. If the virus reappears, CRISPR identifies it and Cas9 kills it by cutting its genome. One of the biggest questions in this field is how this bacterial immune system originated, which is much older than that of humans.

Searching for an answer, a team made up of some of Spain's leading gene editing experts used a technique that reconstructs the genome of extinct organisms. The technique is known as ancestral sequence reconstruction. It uses powerful computers to compare the complete genomes of living things today—each made up of billions of letters of DNA—and estimates what the genome of their common ancestors would look like. In this way, researchers have made an amazing journey through time to recover Cas proteins present in extinct microbes. The oldest that they have achieved is from 2,600 million years ago. They have also made intermediate stops to rescue extinct proteins from microorganisms that lived 1,000 million, 200 million, 137 million, and 37 million years ago.

Researchers have created new CRISPR systems using these ancient proteins and injected them into human cells. The results, published in Nature Microbiology , show that despite being so primitive, all proteins are capable of genome editing.

Researchers have seen something like fast-forward evolution in the lab. The oldest protein of all can only cut single strands of DNA, perhaps the simplest and most primitive—human DNA is made up of double strands. But the rest of the more recent Cas molecules can already cut human DNA with increasing effectiveness and in fact have been able to correct two genes, TYR and OCA2, that cause albinism.

In the early 90s of the last century, the biologist Francis Mojica gave his name to CRISPR as part of his studies of microbes that lived in the hostile environment of the salt flats of Santa Pola (Alicante), a job for which he was in the pool Nobel Prize. The researcher also analyzed other sequences called PAM that is fundamental, since they allow the microbe to distinguish between the genome of a virus and its own. Without PAMs, a bacterium could kill itself. What the study shows is that the oldest Cas cut without the need for PAM. Mojica, the co-author of the current work, highlights its importance for understanding the origin and evolution of CRISPR. "Thanks to this reconstruction, we see how the immune system of microbes became less harmful to their carriers and more and more specific for each virus," he highlights. Furthermore, “this work is important because it opens up a huge toolbox for creating better CRISPR systems,” he says.

Raúl Pérez-Jiménez, a researcher at the NanoGUNE Basque cooperative research center in nanoscience and co-author of the study, details the potential of the study. “These are the oldest Cas proteins that have ever been obtained. We think they are like a diamond in the rough. Now we are going to study how we can make them as efficient as the current ones or even better”, he points out.

The fact that early proteins were more generalists may be an advantage, allowing them to do things that current CRISPRs are not capable of, such as cutting double and single-stranded DNA as well as RNA sequences at the same time. “They are like a Swiss army knife. They have scissors, corkscrew, needles, and screwdrivers. They are probably not the best tools in their class, but they have them all”, details Pérez-Jiménez.

The researcher and his partner Borja Alonso Lerma have patented these new molecules, which have been bought by Integra Therapeutics, a company co-founded by Marc Güell, a scientist at the Pompeu Fabra University in Barcelona, ​​also co-author of the study, and which is looking for new editing formulas. genetics to treat different diseases. The head of the company's scientific advisory board is the charismatic George Church, one of the world's foremost experts in this field.

Miguel Ángel Moreno Pelayo, head of genetics at the Ramón y Cajal Hospital in Madrid and co-author of the work, highlights that the reconstruction of ancient proteins opens the possibility of designing new forms of synthetic CRISPR "that do not exist in nature." Among other projects, his team develops this type of molecule to try to correct genetic defects in patients with amyotrophic lateral sclerosis. “We are facing a new paradigm”, sums up the scientist.

Another of those responsible for the study is Lluís Montoliu, a researcher at the National Center for Biotechnology in Madrid, who highlights another advantage of the primitive Cas proteins. The potential for gene editing of the CRISPR system was discovered in bacteria of the species S. pyogenes . These microbes can cause infections, so many people have antibodies that can trigger immune reactions against the CRISPR extracted from them. The primitive Cass, on the other hand, is very different from any current version, so they are not detected by the immune system, a great advantage to avoid rejection in future medical applications, Montoliu argues.

The researcher proposes a final reflection on the results of the study. Why didn't eukaryotes, the large group of multicellular organisms to which we humans belong, evolve a CRISPR-based immune system? "Because it's dangerous," reasons the scientist. “The most primitive CRISPR systems already allowed DNA to be cut, but they were very unselective, which probably ended up killing the organism they were trying to protect. In the world of bacteria, the individual is not important, what matters is the population, and this system allowed them to evolve and perfect an immune system even at the price of killing many along the way”, he concludes.

Miguel Ángel Moreno Mateos, an expert in gene editing at the Andalusian Center for Developmental Biology, celebrates the new study. “Particularly fascinating is the resurrection of ancient Cas9 [proteins] and the analysis of their activity billions of years later,” he points out. “These resurrected Cas9s present new possibilities with considerable potential in biotechnology, although further study and analysis must be carried out for this to become a reality,” he adds.