Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9
- Daniel Agudelo1,
- Sophie Carter1,
- Minja Velimirovic1,
- Alexis Duringer1,
- Jean-François Rivest1,
- Sébastien Levesque1,
- Jeremy Loehr1,
- Mathilde Mouchiroud2,
- Denis Cyr3,
- Paula J. Waters3,
- Mathieu Laplante2,4,
- Sylvain Moineau5,6,7,
- Adeline Goulet8,9 and
- Yannick Doyon1,4
- 1Centre Hospitalier Universitaire de Québec Research Center–Université Laval, Québec, Québec G1V 4G2, Canada;
- 2Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec (CRIUCPQ)–Université Laval, Québec, Québec G1V 4G5, Canada;
- 3Service de Génétique médicale, Département de Pédiatrie, Centre Hospitalier Universitaire de Sherbrooke (CHUS), et CRCHUS, Sherbrooke, Québec J1H 5N4, Canada;
- 4Université Laval Cancer Research Centre, Québec, Québec G1V 0A6, Canada;
- 5Département de biochimie, de microbiologie, et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec, Québec G1V 0A6, Canada;
- 6Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Québec, Québec G1V 0A6, Canada;
- 7Félix d'Hérelle Reference Center for Bacterial Viruses, Faculté de médecine dentaire, Université Laval, Québec, Québec G1V 0A6, Canada;
- 8Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, 13288 Marseille Cedex 09, France;
- 9Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Campus de Luminy, 13288 Marseille Cedex 09, France
Abstract
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.
Footnotes
-
[Supplemental material is available for this article.]
-
Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.255414.119.
- Received August 1, 2019.
- Accepted December 17, 2019.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
