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Optimizing CRISPR/Cas9 System to Precisely Model Plasminogen Activator Inhibitor-1 Point Mutations in Mice

Yang Liu, Thomas L. Saunders, Thomas Sisson, Robert Blackburn, David S. Ginsberg, Duane Day
doi: https://doi.org/10.1101/254805
Yang Liu
1Molecular Innovations, Novi, Michigan, the United States of America
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  • For correspondence: yang@mol-innov.com
Thomas L. Saunders
2Transgenic Animal Model Core and Department of Internal Medicine, Medical School, University of Michigan, Ann Arbor, Michigan, the United States of America
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Thomas Sisson
3Department of Internal Medicine, Medical School, University of Michigan, Ann Arbor, Michigan, the United States of America
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Robert Blackburn
1Molecular Innovations, Novi, Michigan, the United States of America
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David S. Ginsberg
1Molecular Innovations, Novi, Michigan, the United States of America
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Duane Day
1Molecular Innovations, Novi, Michigan, the United States of America
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Abstract

CRISPR/Cas9 has become a powerful genome editing tool in recent years. CRISPR/Cas9 can be utilized to not only efficiently generate knock out models in various organisms, but also to precisely model human disease or variants to study gene function and develop therapies. However, the latter remains challenging because of low knock-in (KI) efficiency. In this study, precise gene editing modeling plasminogen activator inhibitor-1 (PAI-1) ‐tissue plasminogen activator (tPA) binding deficiency and PAI-1-vitronectin binding deficiency were generated respectively in mice. Optimization of single guide RNAs (sgRNA) and repair templates, and utilization of restriction fragment length polymorphism (RFLP) to detect KI events are described. Injection of sgRNA/Cas9/single-stranded oligodeoxynucleotide (ssODN) into mouse zygotes resulted in homozygous changes of two silent mutations and changed Arg369>Ala, which abolishes PAI-1 inhibitory activity against tPA. Targeting Arg124 and Gln146 simultaneously involved in vitronectin binding proved to be challenging. However, we successfully generated these relatively distant mutations (23 amino acids apart) seamlessly. Generation of the Arg124 mutation alone was achieved with over 60% efficiency along with the integration of a restriction site, compared to the relatively low double mutation frequency. In summary, our data indicates that the distance between desired mutations and CRISPR-induced double-stranded break (DSB) site is the most critical factor for achieving high efficiency in precise gene modification.

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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-ND 4.0 International license.
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Posted January 26, 2018.
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Optimizing CRISPR/Cas9 System to Precisely Model Plasminogen Activator Inhibitor-1 Point Mutations in Mice
Yang Liu, Thomas L. Saunders, Thomas Sisson, Robert Blackburn, David S. Ginsberg, Duane Day
bioRxiv 254805; doi: https://doi.org/10.1101/254805
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Optimizing CRISPR/Cas9 System to Precisely Model Plasminogen Activator Inhibitor-1 Point Mutations in Mice
Yang Liu, Thomas L. Saunders, Thomas Sisson, Robert Blackburn, David S. Ginsberg, Duane Day
bioRxiv 254805; doi: https://doi.org/10.1101/254805

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