New study reveals 2.6-billion-year-old resurrected enzymes can still edit cells

Molecular design techniques to modify

Finding unique CRISPR-Cas systems with distinctive traits in the world's most remote regions is the aim of current research. This is accomplished by employing molecular design techniques to study or modify various organisms that endure extreme environments. This research's premise—looking for new systems in the past—actually represents a fundamentally different strategy.

Ral Pérez-Jiménez is the leader of the nanoGUNE's nanobiotechnology group, which has spent years researching the development of proteins from the beginning of life to the present. Proteins and DNA from extinct species have undergone ancestral reconstructions to determine their characteristics and whether they may be applied in biotechnological applications.

”What is surprising is that we can revitalize Cas proteins that must have existed billions of years ago and find that they already had the capacity then to operate as gene editing tools; we have now confirmed that by successfully editing genes in human cells,” explained Lluís Montoliu, a researcher at the National Biotechnology Center of the CSIC (CNB-CSIC) and CIBERER, and head of the team that has functionally validated these ancestral Cas proteins in human cells in culture.

It became more complex over time

Another intriguing result in the study is that the CRISPR-Cas system has steadily become more complicated over time, indicating its adaptive nature; it has been gradually responding to new virus threats that have hung over bacteria throughout evolution.

”This research signifies an extraordinary advance in knowledge about the origin and evolution of CRISPR-Cas systems. About how the selective pressure of viruses has, over billions of years, been fine-tuning rudimentary, initially not very selective machinery; this had been taking place until a sophisticated defense mechanism was produced," added the University of Alicante researcher who discovered the CRISPR-Cas technique, Francis Mojica.

"It is a mechanism capable of distinguishing with great precision between its own DNA, which it must preserve, and the genetic material of unwanted invaders, which it must destroy. The work represents an original approach to the development of CRISPR tools to generate new tools and improve those derived from existing ones in current organisms," added Mojica.

The research was conducted internationally by numerous institutes and laboratories under the direction of nanoGUNE in partnership with Francis Mojica's teams at the University of Alicante, who popularized the term CRISPR.

The study was published in Nature Microbiology on January 2.

Study abstract:

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR–Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.