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Precise genomic deletions using paired prime editing

View ORCID ProfileJunhong Choi, View ORCID ProfileWei Chen, View ORCID ProfileChase C. Suiter, View ORCID ProfileCholi Lee, Florence M. Chardon, View ORCID ProfileWei Yang, Anh Leith, View ORCID ProfileRiza M. Daza, Beth Martin, View ORCID ProfileJay Shendure
doi: https://doi.org/10.1101/2020.12.30.424891
Junhong Choi
1Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
2Howard Hughes Medical Institute, Seattle, WA 98195, USA
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  • For correspondence: junhongc@uw.edu shendure@uw.edu
Wei Chen
1Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
3Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA 98195, USA
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Chase C. Suiter
1Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
4Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA
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Choli Lee
1Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
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Florence M. Chardon
1Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
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Wei Yang
1Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
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Anh Leith
1Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
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Riza M. Daza
1Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
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Beth Martin
1Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
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Jay Shendure
1Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
2Howard Hughes Medical Institute, Seattle, WA 98195, USA
5Brotman Baty Institute for Precision Medicine, University of Washington, Seattle, WA 98195, USA
6Allen Discovery Center for Cell Lineage Tracing, University of Washington, Seattle, WA 98195, USA
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  • For correspondence: junhongc@uw.edu shendure@uw.edu
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Abstract

Technologies that precisely delete genomic sequences in a programmed fashion can be used to study function as well as potentially for gene therapy. The leading contemporary method for programmed deletion uses CRISPR/Cas9 and pairs of guide RNAs (gRNAs) to generate two nearby double-strand breaks, which is often followed by deletion of the intervening sequence during DNA repair. However, this approach can be inefficient and imprecise, with errors including small indels at the two target sites as well as unintended large deletions and more complex rearrangements. Here we describe a prime editing-based method that we term PRIME-Del, which induces a deletion using a pair of prime editing gRNAs (pegRNAs) that target opposite DNA strands, effectively programming not only the sites that are nicked but also the outcome of the repair. We demonstrate that PRIME-Del achieves markedly higher precision in programming deletions than CRISPR/Cas9 and gRNA pairs. We also show that PRIME-Del can be used to couple genomic deletions with short insertions, enabling deletions whose junctions do not fall at protospacer-adjacent motif (PAM) sites. Finally, we demonstrate that lengthening the time window of expression of prime editing components can substantially enhance efficiency without compromising precision. We anticipate that PRIME-Del will be broadly useful in enabling precise, flexible programming of genomic deletions, including in-frame deletions, as well as for epitope tagging and potentially for programming rearrangements.

Competing Interest Statement

The University of Washington has filed a patent application based on this work, in which J.C., W.C., and J.S. are listed as inventors.

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-NC-ND 4.0 International license.
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Posted January 02, 2021.
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Precise genomic deletions using paired prime editing
Junhong Choi, Wei Chen, Chase C. Suiter, Choli Lee, Florence M. Chardon, Wei Yang, Anh Leith, Riza M. Daza, Beth Martin, Jay Shendure
bioRxiv 2020.12.30.424891; doi: https://doi.org/10.1101/2020.12.30.424891
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Precise genomic deletions using paired prime editing
Junhong Choi, Wei Chen, Chase C. Suiter, Choli Lee, Florence M. Chardon, Wei Yang, Anh Leith, Riza M. Daza, Beth Martin, Jay Shendure
bioRxiv 2020.12.30.424891; doi: https://doi.org/10.1101/2020.12.30.424891

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