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

Ru R, Yao Y, Yu S, Yin B, Xu W, Zhao S, Qin L, Chen X - Cell Regen (Lond) (2013)

Bottom Line: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research.Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency.This new technique may advance the clinical application of TALEN technology.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Zinc-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.

Results: In 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.

Conclusions: TAT-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.

No MeSH data available.


Related in: MedlinePlus

Disruption of endogenous genes by direct delivery of TALEN proteins. (A) Frequency of endogenous CCR5 gene disruption in HeLa cells and hiPSCs subjected to three consecutive treatments with TALEN proteins as determined by the Surveyor assay. Surveyor nuclease cleavage at mismatches produces products of 254 bp and 277 bp. (B) Representative sequence analysis of the CCR5 locus in HeLa cells after three consecutive treatments with 3 μM TAT-TALEN proteins under hypothermic conditions. Multiple deletions (dashed) and insertions (lowercase) are aligned with the cleavage site (wild type [WT]). Underlines highlight binding sites for TALEN L and TALEN R.
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Fig5: Disruption of endogenous genes by direct delivery of TALEN proteins. (A) Frequency of endogenous CCR5 gene disruption in HeLa cells and hiPSCs subjected to three consecutive treatments with TALEN proteins as determined by the Surveyor assay. Surveyor nuclease cleavage at mismatches produces products of 254 bp and 277 bp. (B) Representative sequence analysis of the CCR5 locus in HeLa cells after three consecutive treatments with 3 μM TAT-TALEN proteins under hypothermic conditions. Multiple deletions (dashed) and insertions (lowercase) are aligned with the cleavage site (wild type [WT]). Underlines highlight binding sites for TALEN L and TALEN R.

Mentions: HeLa cells were subjected to treatment with TALENs at 37°C for 1 hour and then continuously cultured at 37°C for 24 hours before the next treatment. After three treatment cycles, a Surveyor nuclease assay was performed to evaluate gene disruption. TAT-TALENs disrupted CCR5 at 37°C in a dose-dependent manner, whereas standard TALENs exerted no effects on CCR5 disruption. As shown in Figure 5A, HeLa cells subjected to three consecutive treatments with 3 μM TAT-TALEN proteins showed a modification frequency of 3% as measured by the Surveyor assay. This modification frequency increased to 16% when the cells treated with TAT-TALEN were incubated at 30°C. Standard TALENs still did not disrupt CCR5 gene under hypothermic conditions. To verify the reliability of the Surveyor assay, TAT-TALEN mediated CCR5 disruption was confirmed by sequence analysis of cloned CCR5 alleles amplified from treated HeLa cells (Figure 5B).Figure 5


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

Ru R, Yao Y, Yu S, Yin B, Xu W, Zhao S, Qin L, Chen X - Cell Regen (Lond) (2013)

Disruption of endogenous genes by direct delivery of TALEN proteins. (A) Frequency of endogenous CCR5 gene disruption in HeLa cells and hiPSCs subjected to three consecutive treatments with TALEN proteins as determined by the Surveyor assay. Surveyor nuclease cleavage at mismatches produces products of 254 bp and 277 bp. (B) Representative sequence analysis of the CCR5 locus in HeLa cells after three consecutive treatments with 3 μM TAT-TALEN proteins under hypothermic conditions. Multiple deletions (dashed) and insertions (lowercase) are aligned with the cleavage site (wild type [WT]). Underlines highlight binding sites for TALEN L and TALEN R.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230761&req=5

Fig5: Disruption of endogenous genes by direct delivery of TALEN proteins. (A) Frequency of endogenous CCR5 gene disruption in HeLa cells and hiPSCs subjected to three consecutive treatments with TALEN proteins as determined by the Surveyor assay. Surveyor nuclease cleavage at mismatches produces products of 254 bp and 277 bp. (B) Representative sequence analysis of the CCR5 locus in HeLa cells after three consecutive treatments with 3 μM TAT-TALEN proteins under hypothermic conditions. Multiple deletions (dashed) and insertions (lowercase) are aligned with the cleavage site (wild type [WT]). Underlines highlight binding sites for TALEN L and TALEN R.
Mentions: HeLa cells were subjected to treatment with TALENs at 37°C for 1 hour and then continuously cultured at 37°C for 24 hours before the next treatment. After three treatment cycles, a Surveyor nuclease assay was performed to evaluate gene disruption. TAT-TALENs disrupted CCR5 at 37°C in a dose-dependent manner, whereas standard TALENs exerted no effects on CCR5 disruption. As shown in Figure 5A, HeLa cells subjected to three consecutive treatments with 3 μM TAT-TALEN proteins showed a modification frequency of 3% as measured by the Surveyor assay. This modification frequency increased to 16% when the cells treated with TAT-TALEN were incubated at 30°C. Standard TALENs still did not disrupt CCR5 gene under hypothermic conditions. To verify the reliability of the Surveyor assay, TAT-TALEN mediated CCR5 disruption was confirmed by sequence analysis of cloned CCR5 alleles amplified from treated HeLa cells (Figure 5B).Figure 5

Bottom Line: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research.Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency.This new technique may advance the clinical application of TALEN technology.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pathogen Biology, State Key Laboratory of Respiratory Disease, Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 China.

ABSTRACT

Background: Zinc-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.

Results: In 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.

Conclusions: TAT-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.

No MeSH data available.


Related in: MedlinePlus