2018
DOI: 10.1038/s41598-017-18568-4
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Site-specific randomization of the endogenous genome by a regulatable CRISPR-Cas9 piggyBac system in human cells

Abstract: Randomized mutagenesis at an endogenous chromosomal locus is a promising approach for protein engineering, functional assessment of regulatory elements, and modeling genetic variations. In mammalian cells, however, it is challenging to perform site-specific single-nucleotide substitution with single-stranded oligodeoxynucleotide (ssODN) donor templates due to insufficient homologous recombination and the infeasibility of positive selection. Here, we developed a DNA transposon based CRISPR-Cas9 regulated transc… Show more

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Cited by 23 publications
(22 citation statements)
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References 36 publications
(39 reference statements)
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“…B2M KO in human iPSCs. The human iPSC line 1383D2 was reprogrammed from the peripheral blood cells of a healthy male donor (CTL-CP1 LP_53, Donor #40, Cellular Technology Ltd) by episomal vectors and was cultured under feeder-free, xeno-free StemFit AK02N media (Ajinomoto) with Laminin-511 E8 (Nippi) coating 72 . Before inoculating the cells, the plates were coated with 0.25-0.35 µg cm −2 of Laminin and incubated for 2 h to overnight in a 37°C CO 2 incubator.…”
Section: Methodsmentioning
confidence: 99%
“…B2M KO in human iPSCs. The human iPSC line 1383D2 was reprogrammed from the peripheral blood cells of a healthy male donor (CTL-CP1 LP_53, Donor #40, Cellular Technology Ltd) by episomal vectors and was cultured under feeder-free, xeno-free StemFit AK02N media (Ajinomoto) with Laminin-511 E8 (Nippi) coating 72 . Before inoculating the cells, the plates were coated with 0.25-0.35 µg cm −2 of Laminin and incubated for 2 h to overnight in a 37°C CO 2 incubator.…”
Section: Methodsmentioning
confidence: 99%
“…All the iPS cell lines (404C2, 585A1, 604B1, 1383D2 and Ff-XT28s05) used in this study were generated under written consent with the approval by the Ethics Committee at Faculty of Medicine or CiRA, Kyoto University. Human iPS cell lines were cultured with StemFit AK03N media and iMatrix-511 as previously reported (Ishida et al, 2018). For cytokine treatment, iPSCs were treated by 100 ng/mL LPS (Invitrogen, Cat.…”
Section: Contact For Reagent and Resource Sharingmentioning
confidence: 99%
“…Establishing EGxxFP SSA reporter cells. piggyBac-based SSA EGFP reporter vector, pPV-EF1a-EGxxFP-DMD-all-iP-A, was transfected into C2C12, Hu5 or HEK293T cells with piggyBac transposase expressing vector pHL-EF1a-hcPBase-A 44 and then selected by puromycin for 2 weeks. The bulk reporter cells were used for NanoMEDIC inoculation experiments.…”
Section: Primer Name Sequencementioning
confidence: 99%