Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs

BackgroundZinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However, the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed.ResultsIn this study, we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN, respectively. However, TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5, a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.ConclusionsTAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology.

[1]  E. Thiel,et al.  Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. , 2009, The New England journal of medicine.

[2]  E. Thiel,et al.  Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation. , 2011, Blood.

[3]  G. Ciliberto,et al.  Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation , 2004, Gene Therapy.

[4]  R. Jiao,et al.  Efficient and specific modifications of the Drosophila genome by means of an easy TALEN strategy. , 2012, Journal of genetics and genomics = Yi chuan xue bao.

[5]  Erin L. Doyle,et al.  Targeting DNA Double-Strand Breaks with TAL Effector Nucleases , 2010, Genetics.

[6]  P. Vyas,et al.  TAT opens the door. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[7]  Elo Leung,et al.  A TALE nuclease architecture for efficient genome editing , 2011, Nature Biotechnology.

[8]  Igor Antoshechkin,et al.  A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly , 2013, Nucleic acids research.

[9]  Jianyong Xu,et al.  Generation of CD34+ cells from CCR5-disrupted human embryonic and induced pluripotent stem cells. , 2012, Human gene therapy.

[10]  J. Orange,et al.  Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases , 2008, Nature Biotechnology.

[11]  M. Morris,et al.  Cell‐penetrating peptides: from molecular mechanisms to therapeutics , 2008, Biology of the cell.

[12]  Erin L. Doyle,et al.  Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting , 2011, Nucleic acids research.

[13]  B. Tuch,et al.  β cell regeneration , 2008 .

[14]  M. Lako,et al.  Concise Review: Putting a Finger on Stem Cell Biology: Zinc Finger Nuclease‐Driven Targeted Genetic Editing in Human Pluripotent Stem Cells , 2011, Stem cells.

[15]  Kevin Kim,et al.  A TALEN genome-editing system for generating human stem cell-based disease models. , 2013, Cell stem cell.

[16]  L. Lopalco.  CCR5: From Natural Resistance to a New Anti-HIV Strategy , 2010, Viruses.

[17]  Yoshio Kato,et al.  targeted gene knockout by direct delivery of zinc-finger nuclease proteins , 2012 .

[18]  Prashant Mali,et al.  Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. , 2009, Cell stem cell.

[19]  Jeffrey G. Mandell,et al.  Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases , 2006, Nucleic Acids Res..

[20]  R. Brasseur,et al.  Happy birthday cell penetrating peptides: already 20 years. , 2010, Biochimica et biophysica acta.

[21]  Thuy D. Vo,et al.  Transient cold shock enhances zinc-finger nuclease–mediated gene disruption , 2010, Nature Methods.

[22]  Samira Kiani,et al.  Genetic engineering of human ES and iPS cells using TALE nucleases , 2011, Nature Biotechnology.

[23]  Jeffrey C. Miller,et al.  A rapid and general assay for monitoring endogenous gene modification. , 2010, Methods in molecular biology.

[24]  Mark A. Kay,et al.  Progress and problems with the use of viral vectors for gene therapy , 2003, Nature Reviews Genetics.

[25]  G. Symonds,et al.  The use of cell-delivered gene therapy for the treatment of HIV/AIDS , 2010, Immunologic research.

[26]  Li Wang,et al.  Erratum: Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting (Nucleic Acids Research (2011) 39 (e82) DOI: 10.1093/nar/gkr218) , 2011 .

[27]  Joanna E. Huddleston.  HIV: HIV hides in haematopoietic stem cells , 2011, Nature Reviews Microbiology.

[28]  Vanessa Taupin,et al.  Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo , 2010, Nature Biotechnology.

[29]  T. Tzfira,et al.  A toolbox and procedural notes for characterizing novel zinc finger nucleases for genome editing in plant cells. , 2009, The Plant journal : for cell and molecular biology.

[30]  N. Cherradi,et al.  A novel concept in antiangiogenic and antitumoral therapy: multitarget destabilization of short-lived mRNAs by the zinc finger protein ZFP36L1 , 2010, Oncogene.

[31]  V. Nain,et al.  CPP-ZFN: A potential DNA-targeting anti-malarial drug , 2010, Malaria Journal.

[32]  Bing Yang,et al.  TAL effector nuclease (TALEN) engineering. , 2013, Methods in molecular biology.

[33]  Jeffry D Sander,et al.  FLAsH assembly of TALeNs for high-throughput genome editing , 2022 .