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

ABSTRACT: 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.

SUBMITTER: Johnston AD

PROVIDER: S-EPMC6694121 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Johnston Andrew D AD Simões-Pires Claudia A CA Suzuki Masako M Greally John M JM

Communications biology 20190814

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, ...[more]

PMID: 31428700

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Cellular expression profiles of Epstein-Barr virus-transformed B-lymphoblastoid cell lines.

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Expression analysis of Epstein-Barr virus–transformed human lymphoblastoid cell lines (LCLs) between male and female.

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Expression analysis of Epstein-Barr virus–transformed human lymphoblastoid cell lines (LCLs) between male and female.

Project description:Investigation of human X-linked imprinted gene. Comparing pooled RNA of lymphoblastoid cell lines from normal human male and female, identify the genes expressed with sex spesific manner. Expression array study with total RNA extracted from pooled male and female LCLs with eight 60-mer probe per gene. 47633 exemplar genes representing a total of 63780 transcripts/variants.

2012-01-20 | E-GEOD-35207 | biostudies-arrayexpress

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