title: Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases.
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 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-IL2Rγnullmice, 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.
authors.affiliation: Department of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Carmen F
authors.initials: CF
authors.lastname: Bjurström
authors.affiliation: Department of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Mojadidi
authors.affiliation: Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: John
authors.initials: J
authors.lastname: Phillips
authors.affiliation: Department of Pediatrics, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Caroline
authors.initials: C
authors.lastname: Kuo
authors.affiliation: Department of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Lai
authors.affiliation: Department of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Georgia R
authors.initials: GR
authors.lastname: Lill
authors.affiliation: Department of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Cooper
authors.affiliation: Department of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Kaufman
authors.affiliation: Department of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Fabrizia
authors.initials: F
authors.lastname: Urbinati
authors.affiliation: Department of General Internal Medicine and Health Services Research, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Roger P
authors.initials: RP
authors.lastname: Hollis
authors.affiliation: Department of Microbiology, Immunology, & Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA; Department of Pediatrics, University of California, Los Angeles, Los Angeles, California, USA. Electronic address: dkohn@mednet.ucla.edu.
authors.forename: Donald B
authors.initials: DB
authors.lastname: Kohn
keywords: BCL11A; CRISPR/Cas9; TALENs; fetal hemoglobin; nonhomologous end joining; zinc finger nucleases