Las bacteria They arose about 3.5 billion years ago. Since then, they have perfected a mechanism by which their DNA stores bits of the virus so that if they are reinfected in the future, this defense literally cuts the infection out of the body. This system, baptized as CRISPR (acronym in English for ‘clustered and regularly spaced short palindromic repeats’) by its discoverer, the geneticist from Alicante Francis Mojicahas become a revolutionary technique that allows the creation of ‘à la carte’ genetic modifications: from wheat suitable for celiacs to sheep that give better wool, including new experimental therapies that have become the hope of many patients with genetic diseases. And all in a precise and cheap way, only using the mechanism refined over millions of years by the ‘humble’ bacteria.
The method is ‘simple’: CRISPR uses a s guides and a protein (Cas9 nuclease) to target specific areas of DNA and cut. From there, the ends are naturally glued together and the gene is inactivated. That is why it has been baptized as the ‘genetic shortstop‘. However, most bacteria for which this mechanism is known live so closely around us that we are immune to their system, and these proteins do not work with us. That is why scientists have been searching remote areas for some time, such as Antarctica, the top of Everest or the Mariana Trench, to find much more exotic bacterial species with which we have not had contact and their proteins (Cas nucleases). work on us. The approach of the Spanish group is completely new.
“We, instead of looking in space, have searched in time,” he explains to ABC Louis Montoliu, researcher and vice-director of the National Center for Biotechnology (CNB-CSIC) and the Center for Networked Biomedical Research in Rare Diseases (CIBERER-ISCIII). “We’ve kind of ‘back to the future’ looking for different nucleases.” His team, along with Raul Perez-Jimenez, a researcher at CIC nanoGUNE and that of Mojica himself at the University of Alicante, along with other collaborators are the architects of this ‘journey to the past’, which has led them to resurrect CRISPR systems for bacteria that lived up to 2,600 million years ago. His work has just been published in the journal ‘Nature Microbiology’.
The time machine: a supercomputer
The idea was born in the heads of researchers back in 2018. Instead of going to remote places, why not travel through the history of DNA and rescue ancient bacteria with different nucleases, as is done when studying extinct species at through current relatives? The first problem was how to get hold of this ancient genetic material: the oldest remains found to date date back two million years. “It may seem like a lot; but if you take into account that the Earth was created 4.5 billion years ago and that there is evidence that the first bacteria arose 3.5 billion years ago, there is a lot of scope to explore,” says Montoliu. At that moment, the team led by Pérez-Jiménez, an expert in paleoenzymology and bioinformatics, came into play.
Their job is to observe the evolution of proteins from the origin of life to the present day. They make ancestral reconstructions of proteins and genes from extinct organisms to see what qualities they had and if they can be used in biotechnological applications. In this specific case, they gathered 59 species of current bacteria of which their CRISPR Cas9 system is known and the data was put into a supercomputer that, later, was in charge of going back in time to reverse the immune system of the bacteria. Like when the DNA of different species is related to find their common ancestors, but in this case only with the CRISPR system (in no case was the entire bacterium resurrected).
The computer allowed them to make five ‘stops’ on their journey through time to observe – in a computational way, at least – what the CRISPR systems of ancient bacteria were like; specifically, they obtained results from ago 37, 137, 200, 1.000 y 2.6 billion years. The idea was then to ‘resuscitate’ these systems with the enzyme sequences provided by the computer and test them in living human cells to see if they could continue to cut DNA.
Bacteria refine their weapons
“It was pretty daring, because we had no idea what could come out of it,” Montoliu says. “But we found that these systems not only work in living cells, but are more versatile than current versions, which could have revolutionary applications in the future, such as in treatments for genetic diseases.” The Spanish team verified that these systems did indeed work and that, furthermore, they share 50% of their structure with those of today, although it is true that they work more crudely the older they are.
Image of Cas9, an endonuclease enzyme associated with the CRISPR system, acting on target DNA
“CRISPR from 37 million years ago does the job best with today’s cells, and it gets more and more ‘crude’ the further back we go. But considering that the oldest DNA we have recovered is two million years old, this work is totally revolutionary.” This also reinforces the idea that the bacterial system has been refined over time, adapting to new viruses.
Montoliu stresses that this experiment is based on data reconstructed by computer; that is, they are not directly based on recovered DNA. “Without remains we cannot know, at least with current technology, if these systems were exactly like this millions of years ago,” says the researcher. “However, the fact that, for example, the oldest ones cut only one strand of DNA is consistent with the idea that the first forms of life were based on RNA and only had a single strand.”
“This research represents an extraordinary advance in the knowledge about the origin and evolution of CRISPR-Cas systems. In how the selective pressure of viruses has been polishing over billions of years a rudimentary machinery, not very selective in its beginnings, until turning it into a sophisticated defense mechanism capable of distinguishing with great precision the genetic material of unwanted invaders that it must destroy, of its own DNA that it must preserve,” says Francis Mojica, a researcher at the University of Alicante and discoverer of the CRISPR-Cas technique, in a statement.
As for future applications, this new technique opens the door for “a more versatile tool to correct mutations that until now were not editable or were corrected in an inefficient way,” he points out. Miguel Angel Moreno, head of the Genetics service at the Ramón y Cajal Hospital and CIBERER researcher. His team has developed the Mosaic Finder tool, which has made it possible to characterize, through massive sequencing and bioinformatics analysis, the effect of genome editing produced by ancestral Cass in cultured human cells. That is, how these ancient systems worked in current living cells.
For his part, Ylenia Jabaleraa project researcher at nanoGUNE, maintains that “this scientific achievement makes it possible to have genetic editing tools with properties different from the current ones, much more flexible, which opens up new avenues in the manipulation of DNA and treatment of diseases such as ALS, cancer, diabetes, or even as a diagnostic tool for diseases.