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High-efficiency genomic editing in Epstein-Barr virus-transformed lymphoblastoid B cells using a single-stranded donor oligonucleotide strategy

While human lymphoblastoid cell lines represent a valuable resource for population genetic studies, they have usually been regarded as difficult for CRISPR-mediated genomic editing because of very inefficient DNA transfection and retroviral or lentiviral transduction in these cells, which becomes a...

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Detalles Bibliográficos
Autores principales: Johnston, Andrew D., Simões-Pires, Claudia A., Suzuki, Masako, Greally, John M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694121/
https://www.ncbi.nlm.nih.gov/pubmed/31428700
http://dx.doi.org/10.1038/s42003-019-0559-3
Descripción
Sumario:While human lymphoblastoid cell lines represent a valuable resource for population genetic studies, they have usually been regarded as difficult for CRISPR-mediated genomic editing because of very inefficient DNA transfection and retroviral or lentiviral transduction in these cells, which becomes a substantial problem when multiple constructs need to be co-expressed. Here we describe a protocol using a single-stranded donor oligonucleotide strategy for ‘scarless’ editing in lymphoblastoid cells, yielding 12/60 (20%) of clones with homology-directed recombination, when rates of <5–10% are frequently typical for many other cell types. The protocol does not require the use of lentiviruses or stable transfection, permitting lymphoblastoid cell lines to be used for CRISPR-mediated genomic targeting and screening in population genetic studies.