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Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases

Flores Bjurström, Carmen LU ; Mojadidi, Michelle; Phillips, John; Kuo, Caroline Y; Lai, Stephen; Lill, Georgia R; Cooper, Aaron R; Kaufman, Michael; Urbinati, Fabrizia and Wang, Xiaoyan, et al. (2016) In Mol Ther Nucleic Acids 5.
Abstract
We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems
designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+progenitor cells. ZFNs and TALENs were delivered as
in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or
in vitro transcribed gRNA (gR.1).
Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the... (More)
We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems
designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+progenitor cells. ZFNs and TALENs were delivered as
in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or
in vitro transcribed gRNA (gR.1).
Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs. (Less)
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Contribution to journal
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published
in
Mol Ther Nucleic Acids
volume
5
publisher
Nature Publishing Group
external identifiers
  • scopus:85015241817
ISSN
2162-2531
DOI
10.1038/mtna.2016.52
language
English
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no
id
8f8adb85-b1c6-45e3-a61b-04a75bfafe05
date added to LUP
2016-10-27 19:16:28
date last changed
2017-11-12 04:25:31
@article{8f8adb85-b1c6-45e3-a61b-04a75bfafe05,
  abstract     = {We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems <br/>designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+progenitor cells. ZFNs and TALENs were delivered as <br/>in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or <br/>in vitro transcribed gRNA (gR.1). <br/>Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs.},
  articleno    = {e351},
  author       = {Flores Bjurström, Carmen and Mojadidi, Michelle and Phillips, John and Kuo, Caroline Y and Lai, Stephen and Lill, Georgia R and Cooper, Aaron R and Kaufman, Michael and Urbinati, Fabrizia and Wang, Xiaoyan and Hollis, Roger P and Kohn, Donald B},
  issn         = {2162-2531},
  language     = {eng},
  month        = {08},
  publisher    = {Nature Publishing Group},
  series       = {Mol Ther Nucleic Acids},
  title        = {Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases},
  url          = {http://dx.doi.org/10.1038/mtna.2016.52},
  volume       = {5},
  year         = {2016},
}