Expanding the genome editing toolkit

In recent years, life science research has been transformed by the emergence of genome editing technologies based on the CRIS- PR-Cas system. Cas9 is a CRISPR-associated DNA endonuclease that can be programmed by short guide RNA molecules to cut genomic DNA whose sequence matches the guide RNA. This can be exploited to introduce genetic modifications near Cas9-generated DNA cuts. In addition to basic research, the Cas9-mediated genome editing technology holds great promises for industrial and clinical applications, and has sparked a major bioethical debate.

The group of Martin Jinek, Department of Biochemistry, University of Zurich, uses structural biology to study the molecular mechanism of Cas9. By using x-ray crystallography, they determined the atomic structures of the Cas9 and its complex with guide RNA and target DNA molecules and elucidated the mechanisms of action by which Cas9 binds target DNA.

RNA-guided DNA binding and cleavage by Cas9 depends on the presence of a short sequence known as protospacer adjacent motif (PAM) in the target DNA. Although the PAM requirement greatly contributes to the precision of DNA cutting, it also restricts the utility of the natural Cas9 enzyme to target genomic sequences juxtaposed to the canonical PAM sequence NGG. Thanks to a method known as directed protein evolution, a new generation of Cas9 enzymes has been devel- oped. These artificial variants, known as VQR, EQR and VRER Cas9, recognize non-canonical PAM sequences such as NGAG and NGCG.

Carolin Anders, Katja Bargsten and Martin Jinek now report the crystal structures of all three engineered Cas9 variants bound to their cognate DNAs. Their findings reveal a structural plasticity of PAM recognition whereby Cas9 variants remodel the shape of the bound DNAs in order to optimally contact their PAM sequences. Instead of altering the three dimensional structures of the Cas9 enzymes, substitutions at specific amino acid positions in the engineered variants induce and accommodate structural changes in the bound DNAs.

The observation of an induced fit by DNA distortion suggests new ways in which the specificity of the Cas9 enzyme could be engineered even further. As a first step towards structure-guided rational engineering, the authors also created a new Cas9 variant that is capable of recognizing the sequence NAAG to expand the spectrum of genomic sequences that can be targeted using Cas9. By revealing the molecular mechanisms underpinning the function of Cas9, the research of Martin Jinek and his colleagues aims at contributing to the development of of genome editing tools and technologies.

By Thomas Schnyder

Original Article in Molecular Cell