Institut for Biomedicin

Rasmus Bak

CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

  • Rasmus O Bak
  • Matthew H Porteus, Department of Pediatrics, Stanford University, Stanford, CA 94305, USA. Electronic address: mporteus@stanford.edu.

The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno-associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of ∼4.5 kb limits the donor size. Here, we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for the integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing. We use the methodology in primary human T cells and CD34(+) hematopoietic stem and progenitor cells to site-specifically integrate an expression cassette that, as a single donor vector, would otherwise amount to a total of 6.5 kb. This approach now provides an efficient way to integrate large transgene cassettes into the genomes of primary human cells using HR-mediated genome editing with AAV vectors.

OriginalsprogEngelsk
TidsskriftCell Reports
Vol/bind20
Nummer3
Sider (fra-til)750-756
Antal sider7
ISSN2211-1247
DOI
StatusUdgivet - 18 jul. 2017
Eksternt udgivetJa

Se relationer på Aarhus Universitet Citationsformater

ID: 116723625