1989/01/01 by Rasmus O. Bak, Matthew H. Porteus, Peter Sykes · 1 citation
Biochemistry, Genetics and Molecular Biology · Chemistry · Medicine · #CAR-T cell therapy research #CRISPR and Genetic Engineering #Cancer and biochemical research #Inorganic and Organometallic Chemistry #Various Chemistry Research Topics #Virus-based gene therapy research
paper · pdf · doi:10.1016/j.celrep.2017.06.064
openalex publication_date 1989/01/01 · openalex created_date 2016/06/24 · openalex updated_date 2026/08/01
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<sup>+</sup> 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.