Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Daniel Agudelo (1) , Sophie Carter (1) , Minja Velimirovic (1) , Alexis Duringer (1) , Jean-François Rivest (1) , Sébastien Levesque (1) , Jérémy Loehr (1) , Mathilde Mouchiroud (2) , Denis Cyr (3) , Paula Waters (3) , Mathieu Laplante (2, 4) , Sylvain Moineau (5, 6) , Adeline Goulet (7, 8, 9, 10) , Yannick Doyon (1, 4)

1 CRCHUQ - Centre de recherche du CHU de Québec-Université Laval
2 Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Faculty of Medicine, Université Laval, Québec, QC, Canada.
3 CHUS – Centre Hospitalier Universitaire de Sherbrooke [Sherbrooke, QC, Canada]
4 Université Laval, centre de recherche sur le cancer, Québec, Canada
5 Département de Biochimie, Microbiologie et Bioinformatique [Québec, Canada]
6 Groupe de Recherche en Écologie Buccale (GREB) and Félix d’Hérelle Reference Center for Bacterial Viruses, Faculté de Médecine Dentaire
7 AFMB - Architecture et fonction des macromolécules biologiques
8 CNRS - Centre National de la Recherche Scientifique
9 AMU - Aix Marseille Université
10 LISM - Laboratoire d'ingénierie des systèmes macromoléculaires

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

Domaines: Sciences du Vivant [q-bio]

Daniel Agudelo, Sophie Carter, Minja Velimirovic, Alexis Duringer, Jean-François Rivest, et al.. Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9. Genome Research, 2020, 30 (1), pp.107-117. ⟨10.1101/gr.255414.119⟩. ⟨hal-03050601⟩