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Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

View ORCID ProfileAlexandre Paix, Andrew Folkmann, Daniel H. Goldman, Heather Kulaga, Michael Grzelak, View ORCID ProfileDominique Rasoloson, View ORCID ProfileSupriya Paidemarry, Rachel Green, Randall Reed, Geraldine Seydoux
doi: https://doi.org/10.1101/161109
Alexandre Paix
Dept. of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine
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Andrew Folkmann
Dept. of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine
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Daniel H. Goldman
Dept. of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine
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Heather Kulaga
Dept. of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine
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Michael Grzelak
Dept. of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine
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Dominique Rasoloson
Dept. of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine
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  • ORCID record for Dominique Rasoloson
Supriya Paidemarry
Dept. of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine
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  • ORCID record for Supriya Paidemarry
Rachel Green
Dept. of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine
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Randall Reed
Dept. of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine
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Geraldine Seydoux
Dept. of Molecular Biology and Genetics, HHMI, Johns Hopkins University, School of Medicine
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  • For correspondence: gseydoux@jhmi.edu
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Abstract

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double-stranded) engage in a high-efficiency HDR mechanism that requires only ~35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity-sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand-annealing (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted July 09, 2017.
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Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks
Alexandre Paix, Andrew Folkmann, Daniel H. Goldman, Heather Kulaga, Michael Grzelak, Dominique Rasoloson, Supriya Paidemarry, Rachel Green, Randall Reed, Geraldine Seydoux
bioRxiv 161109; doi: https://doi.org/10.1101/161109
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Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks
Alexandre Paix, Andrew Folkmann, Daniel H. Goldman, Heather Kulaga, Michael Grzelak, Dominique Rasoloson, Supriya Paidemarry, Rachel Green, Randall Reed, Geraldine Seydoux
bioRxiv 161109; doi: https://doi.org/10.1101/161109

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