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Cas9 fusions for precision in vivo editing

View ORCID ProfileRyan R. Richardson, View ORCID ProfileMarilyn Steyert, View ORCID ProfileJeffrey Inen, View ORCID ProfileSaovleak Khim, View ORCID ProfileAndrea J. Romanowski, View ORCID ProfileBekir Altas, View ORCID ProfileAlexandros Poulopoulos
doi: https://doi.org/10.1101/2020.07.15.199620
Ryan R. Richardson
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD, USA
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Marilyn Steyert
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD, USA
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Jeffrey Inen
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD, USA
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Saovleak Khim
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD, USA
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Andrea J. Romanowski
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD, USA
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Bekir Altas
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD, USA
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Alexandros Poulopoulos
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD, USA
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  • For correspondence: apoulopoulos@som.umaryland.edu
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Abstract

Current Cas9 reagents can target genomic loci with high specificity. However, when used for knockin, on-target outcomes are inherently imprecise, often leading to unintended knockout rather than intended edits. This restricts applications of genome editing to ex vivo approaches, where clonal selection is possible. Here we describe a workflow using iterative high-throughput in vitro and high-yield in vivo assays to evaluate and compare the performance of CRISPR knockin reagents for both editing efficiency and precision. We tested combinations of Cas9 and DNA donor template variants and determined that Cas9-CtIP with in situ linearized donors display fold-increases of editing precision in cell lines and in vivo in the mouse brain. Iterating this process, we generated novel compound fusions, including eRad18-Cas9-CtIP that showed further fold-increases in performance. Continued development of precision editing reagents with this platform holds promise for direct in vivo knockin across model organisms and future targeted gene therapies.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted July 16, 2020.
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Cas9 fusions for precision in vivo editing
Ryan R. Richardson, Marilyn Steyert, Jeffrey Inen, Saovleak Khim, Andrea J. Romanowski, Bekir Altas, Alexandros Poulopoulos
bioRxiv 2020.07.15.199620; doi: https://doi.org/10.1101/2020.07.15.199620
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Cas9 fusions for precision in vivo editing
Ryan R. Richardson, Marilyn Steyert, Jeffrey Inen, Saovleak Khim, Andrea J. Romanowski, Bekir Altas, Alexandros Poulopoulos
bioRxiv 2020.07.15.199620; doi: https://doi.org/10.1101/2020.07.15.199620

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