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Design and specificity of long ssDNA donors for CRISPR-based knock-in

Han Li, Kyle A. Beckman, Veronica Pessino, Bo Huang, Jonathan S. Weissman, Manuel D. Leonetti
doi: https://doi.org/10.1101/178905
Han Li
1Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
6Howard Hughes Medical Institute, University of California, San Francisco, CA 94158
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Kyle A. Beckman
2Department of Pediatrics, University of California, San Francisco, CA 94158
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Veronica Pessino
3Graduate Program in Biophysics, University of California, San Francisco, CA 94158
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Bo Huang
4Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158
5Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158
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Jonathan S. Weissman
1Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
6Howard Hughes Medical Institute, University of California, San Francisco, CA 94158
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  • For correspondence: jonathan.weissman@ucsf.edu manuel.leonetti@ucsf.edu
Manuel D. Leonetti
1Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
6Howard Hughes Medical Institute, University of California, San Francisco, CA 94158
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  • For correspondence: jonathan.weissman@ucsf.edu manuel.leonetti@ucsf.edu
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Abstract

CRISPR/Cas technologies have transformed our ability to manipulate genomes for research and gene-based therapy. In particular, homology-directed repair after genomic cleavage allows for precise modification of genes using exogenous donor sequences as templates. While both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) forms of donors have been used as repair templates, a systematic comparison of the performance and specificity of repair using ssDNA versus dsDNA donors is still lacking. Here, we describe an optimized method for the synthesis of long ssDNA templates and demonstrate that ssDNA donors can drive efficient integration of gene-sized reporters in human cell lines. We next define a set of rules to maximize the efficiency of ssDNA-mediated knock-in by optimizing donor design. Finally, by comparing ssDNA donors with equivalent dsDNA sequences (PCR products or plasmids), we demonstrate that ssDNA templates have a unique advantage in terms of repair specificity while dsDNA donors can lead to a high rate of off-target integration. Our results provide a framework for designing high-fidelity CRISPR-based knock-in experiments, in both research and therapeutic settings.

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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. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted August 21, 2017.
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Design and specificity of long ssDNA donors for CRISPR-based knock-in
Han Li, Kyle A. Beckman, Veronica Pessino, Bo Huang, Jonathan S. Weissman, Manuel D. Leonetti
bioRxiv 178905; doi: https://doi.org/10.1101/178905
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Design and specificity of long ssDNA donors for CRISPR-based knock-in
Han Li, Kyle A. Beckman, Veronica Pessino, Bo Huang, Jonathan S. Weissman, Manuel D. Leonetti
bioRxiv 178905; doi: https://doi.org/10.1101/178905

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