Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

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Agudelo, Daniel | Carter, Sophie | Velimirovic, Minja | Duringer, Alexis | Rivest, Jean-François | Levesque, Sébastien | Loehr, Jérémy | Mouchiroud, Mathilde | Cyr, Denis | Waters, Paula | Laplante, Mathieu | Moineau, Sylvain | Goulet, Adeline | Doyon, Yannick

Edité par CCSD ; Cold Spring Harbor Laboratory Press -

International audience. 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.

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