Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins

Borja Alonso-Lerma, Ylenia Jabalera, Sara Samperio, Matias Morin, Almudena Fernandez, Logan T Hille, Rachel A Silverstein, Ane Quesada-Ganuza, Antonio Reifs, Sergio Fernández-Peñalver, Yolanda Benitez, Lucia Soletto, Jose A Gavira, Adrian Diaz, Wim Vranken, Avencia Sanchez-Mejias, Marc Güell, Francisco J M Mojica, Benjamin P Kleinstiver, Miguel A Moreno-PelayoLluis Montoliu, Raul Perez-Jimenez

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)77-90
Number of pages14
JournalNature Microbiology
Issue number1
Early online date2 Jan 2023
Publication statusPublished - Jan 2023


  • CRISPR-Associated Protein 9/genetics
  • CRISPR-Cas Systems
  • Endonucleases/genetics
  • Gene Editing
  • Firmicutes/enzymology
  • RNA, Guide, CRISPR-Cas Systems


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