Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes

Post-mitotic cardiomyocytes have been considered to be non-permissive to precise targeted integration including homology-directed repair (HDR) after CRISPR/Cas9 genome editing. Here, we demonstrate that direct delivery of large amounts of transgene encoding guide RNA (gRNA) and repair template DNA via intra-ventricular injection of adeno-associated virus (AAV) promotes precise targeted genome replacement in adult murine cardiomyocytes expressing Cas9. Neither systemic injection of AAV nor direct injection of adenovirus promotes targeted integration, suggesting that high copy numbers of single-stranded transgenes are required in cardiomyocytes. Notably, AAV-mediated targeted integration in cardiomyocytes both in vitro and in vivo depends on the Fanconi anemia pathway, a key component of the single-strand template repair mechanism. In human cardiomyocytes differentiated from induced pluripotent stem cells, AAV-mediated targeted integration fluorescently labeled Mlc2v protein after differentiation, independently of DNA synthesis, and enabled real-time detection of sarcomere contraction in monolayered beating cardiomyocytes. Our findings provide a wide range of applications for targeted genome replacement in non-dividing cardiomyocytes.

Medienart:

E-Artikel

Erscheinungsjahr:

2020

Erschienen:

2020

Enthalten in:

Zur Gesamtaufnahme - volume:10

Enthalten in:

Scientific reports - 10(2020), 1 vom: 18. Sept., Seite 15348

Sprache:

Englisch

Beteiligte Personen:

Kohama, Yasuaki [VerfasserIn]
Higo, Shuichiro [VerfasserIn]
Masumura, Yuki [VerfasserIn]
Shiba, Mikio [VerfasserIn]
Kondo, Takumi [VerfasserIn]
Ishizu, Takamaru [VerfasserIn]
Higo, Tomoaki [VerfasserIn]
Nakamura, Satoki [VerfasserIn]
Kameda, Satoshi [VerfasserIn]
Tabata, Tomoka [VerfasserIn]
Inoue, Hiroyuki [VerfasserIn]
Motooka, Daisuke [VerfasserIn]
Okuzaki, Daisuke [VerfasserIn]
Takashima, Seiji [VerfasserIn]
Miyagawa, Shigeru [VerfasserIn]
Sawa, Yoshiki [VerfasserIn]
Hikoso, Shungo [VerfasserIn]
Sakata, Yasushi [VerfasserIn]

Links:

Volltext

Themen:

BRCA2 Protein
Cardiac Myosins
EC 3.6.1.-
Fanca protein, mouse
Fanconi Anemia Complementation Group A Protein
Journal Article
Myosin Light Chains
Myosin light chain 2
RNA, Guide, CRISPR-Cas Systems
Research Support, Non-U.S. Gov't

Anmerkungen:

Date Completed 19.01.2021

Date Revised 04.01.2024

published: Electronic

Citation Status MEDLINE

doi:

10.1038/s41598-020-72216-y

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

NLM31520169X