Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair

Kunwoo Lee; Michael Conboy; Hyo Min Park; Fuguo Jiang; Hyun Jin Kim; Mark A Dewitt; Vanessa A Mackley; Kevin Chang; Anirudh Rao; Colin Skinner; Tamanna Shobha; Melod Mehdipour; Hui Liu; Wen-Chin Huang; Freeman Lan; Nicolas L Bray; Song Li; Jacob E Corn; Kazunori Kataoka; Jennifer A Doudna; Irina Conboy; Niren Murthy

doi:10.1038/s41551-017-0137-2

Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair

Nat Biomed Eng. 2017:1:889-901. doi: 10.1038/s41551-017-0137-2. Epub 2017 Oct 2.

Authors

Kunwoo Lee¹, Michael Conboy², Hyo Min Park¹, Fuguo Jiang³, Hyun Jin Kim^{2

4

5}, Mark A Dewitt^{3

6}, Vanessa A Mackley^{1

2}, Kevin Chang³, Anirudh Rao³, Colin Skinner², Tamanna Shobha², Melod Mehdipour², Hui Liu¹, Wen-Chin Huang², Freeman Lan², Nicolas L Bray^{3

6}, Song Li², Jacob E Corn^{3

6}, Kazunori Kataoka^{4

5

7}, Jennifer A Doudna^{3

6

8

9

10}, Irina Conboy¹¹, Niren Murthy¹²

Affiliations

¹ GenEdit, Berkeley, CA, 94720-0001, USA.
² Department of Bioengineering, University of California, Berkeley, Berkeley, CA, 94720, USA.
³ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, 94720, USA.
⁴ Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-0033, Japan.
⁵ Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, 113-0033, Japan.
⁶ Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA, 94720, USA.
⁷ Innovation Center of NanoMedicine, Institute of Industry Promotion-KAWASAKI, Kawasaki, 210-0821, Japan.
⁸ Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, 94720, USA.
⁹ Department of Chemistry, University of California, Berkeley, Berkeley, CA, 97420-1460, USA.
¹⁰ Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
¹¹ Department of Bioengineering, University of California, Berkeley, Berkeley, CA, 94720, USA. iconboy@berkeley.edu.
¹² Department of Bioengineering, University of California, Berkeley, Berkeley, CA, 94720, USA. nmurthy@berkeley.edu.

Abstract

CRISPR/Cas9-based therapeutics, especially those that can correct gene mutations via homology directed repair (HDR), have the potential to revolutionize the treatment of genetic diseases. However, HDR-based therapeutics are challenging to develop because they require simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types, and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.

Abstract

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