An international research group has reconstructed for the first time ancestors of the well-known CRISPR-Cas system from 2.6 billion years ago and has studied its evolution over time. The results suggest that the revitalized systems not only work, but are more versatile than current versions and could have revolutionary applications. The prestigious scientific journal Nature Microbiology has published the results of this research which, in the opinion of the research team, "opens new avenues for gene editing".
The project, directed by the Ikerbasque researcher from CIC nanoGUNE Rául Pérez-Jiménez, includes teams from the Higher Council for Scientific Research, the University of Alicante, the Center for Biomedical Research in the Network for Rare Diseases (CIBERER), the Ramón y Cajal Hospital -IRYCIS and other state and international institutions.
The acronym CRISPR is the name of repetitive sequences present in the DNA of bacteria and archaea (prokaryotic organisms). Between repeats, these microorganisms harbor fragments of genetic material from viruses that have infected their ancestors, which allow them to recognize if the infection is repeated and defend themselves by cutting the DNA of the invaders using Cas proteins associated with these repeats. It is a mechanism (CRISPR-Cas system) of antiviral defense. This ability to recognize DNA sequences is the basis of its usefulness, as if it were a pair of molecular scissors. CRISPR-Cas technology now makes it possible to cut and paste pieces of genetic material into any cell, making it possible to use it to edit DNA.
Current research efforts are focused on finding new versions of CRISPR-Cas systems with distinct properties in different places of the planet. For this, systems of different species that inhabit extreme environments are explored or molecular design techniques are applied to modify them. A radically different way to find new systems is to search for them in the past, which is precisely the basis of this research.
The nanoGUNE Nanobiotechnology group, led by Raúl Pérez-Jiménez, has spent years studying the evolution of proteins from the origin of life to the present day. They carry out ancestral reconstructions of proteins and genes from extinct organisms to observe what qualities they have and if they are usable in biotechnological applications. It is a time travel carried out by means of bioinformatics techniques. In this work that has just been published in the journal Nature Microbiology, they have reconstructed for the first time the evolutionary history of CRISPR-Cas systems, from ancestors of 2.6 billion years ago to the present day.
The research team has carried out the computational reconstruction of the ancestral CRISPR sequences, synthesized them, and studied and confirmed their functionality. "It is surprising that we can revitalize Cas proteins that should have existed billions of years ago and verify that they already had the ability to operate as gene editing tools, something that we have currently confirmed by successfully editing genes in human cells" explains Lluís Montoliu, researcher at the National Center for Biotechnology of the CSIC (CNB-CSIC) and CIBERER, and head of the team that has functionally validated these ancestral Cass in human cells in culture.
Another interesting conclusion of the study is that the CRISPR-Cas system has become more complex over time, which is a sign of its adaptive nature, which has been adapting to the new virus threats that bacteria have suffered throughout evolution. “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 must preserve”, adds the researcher at the University of Alicante and discoverer of the CRISPR-Cas technique, Francis Mojica. On the applied side, "the work represents an original way of approaching the development of CRISPR tools to generate new instruments and improve those derived from existing ones in current organisms," adds Mojica.
“Current systems are very complex and are adapted to function within a bacterium. When the system is used outside that environment, for example, in human cells, the immune system causes a rejection and there are also certain molecular restrictions that limit its use. Curiously, in ancestral systems some of these restrictions disappear, which gives them greater versatility for new applications”, emphasizes Pérez-Jiménez.
Miguel Angel Moreno, head of the Genetics service at HRYC-IRYCIS-CIBERER points out that "the ingenuity that an ancestral nuclease could have, in that it does not recognize certain regions of the genome so specifically, makes them more versatile tools for correcting mutations than until now were not editable or were corrected in an inefficient way”. His team has developed the Mosaic Finder tool, which has made it possible to characterize, through massive sequencing and bioinformatic analysis, the effect of genome editing produced by these ancestral Cass in human cells in culture.
Ylenia Jabalera, 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”.
The work is the result of international research carried out by various centers and laboratories led by nanoGUNE in collaboration with the groups led by Francis Mojica, from the University of Alicante, who coined the acronym CRISPR; Lluís Montoliu, researcher at the CNB-CSIC and CIBERER and one of the references on CRISPR in Spain; Marc Güell from Pompeu Fabra University and National Award for Research and Technology Transfer in the field of genome editing for therapeutic purposes; Miguel Ángel Moreno-Pelayo, head of the Genetics service at Hospital Ramón y Cajal-IRYCIS, and member of CIBERER, and Benjamin Kleinstiver from Massachusetts General Hospital and Harvard Medical School, a world leader in the design of CRISPR-systems. Cas.
Bibliographic reference
Evolution of CRISPR-associated Endonucleases as Inferred from Resurrected Proteins; Nature Microbiology (DOI: 10.1038/s41564-022-01265-y).
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