Gene editing of human embryonic stem cells via an engineered baculoviral vector carrying zinc-finger nucleases

Yuning Lei; Chi Lin Lee; Kye Il Joo; Jonathan Zarzar; Yarong Liu; Bingbing Dai; Victoria Fox; Pin Wang

doi:10.1038/mt.2011.12

Gene editing of human embryonic stem cells via an engineered baculoviral vector carrying zinc-finger nucleases

Yuning Lei, Chi Lin Lee, Kye Il Joo, Jonathan Zarzar, Yarong Liu, Bingbing Dai, Victoria Fox, Pin Wang

Research output: Contribution to journal › Article › peer-review

34 Scopus citations

Abstract

Human embryonic stem (hES) cells are renewable cell sources that have potential applications in regenerative medicine. The development of technologies to produce permanent and site-specific genome modifications is in demand to achieve future medical implementation of hES cells. We report herein that a baculoviral vector (BV) system carrying zinc-finger nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient expression of ZFNs specifically disrupted the CCR5 locus in transduced cells and the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a gene targeting vector, a DNA donor template and ZFNs were incorporated into the vector. These hybrid vectors yielded permanent site-specific gene addition in both immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both karyotypically normal and pluripotent. These results suggest that this baculoviral delivery system can be engineered for site-specific genetic manipulation in hES cells.

Original language	English
Pages (from-to)	942-950
Number of pages	9
Journal	Molecular Therapy
Volume	19
Issue number	5
DOIs	https://doi.org/10.1038/mt.2011.12
State	Published - May 2011

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title = "Gene editing of human embryonic stem cells via an engineered baculoviral vector carrying zinc-finger nucleases",

abstract = "Human embryonic stem (hES) cells are renewable cell sources that have potential applications in regenerative medicine. The development of technologies to produce permanent and site-specific genome modifications is in demand to achieve future medical implementation of hES cells. We report herein that a baculoviral vector (BV) system carrying zinc-finger nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient expression of ZFNs specifically disrupted the CCR5 locus in transduced cells and the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a gene targeting vector, a DNA donor template and ZFNs were incorporated into the vector. These hybrid vectors yielded permanent site-specific gene addition in both immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both karyotypically normal and pluripotent. These results suggest that this baculoviral delivery system can be engineered for site-specific genetic manipulation in hES cells.",

author = "Yuning Lei and Lee, {Chi Lin} and Joo, {Kye Il} and Jonathan Zarzar and Yarong Liu and Bingbing Dai and Victoria Fox and Pin Wang",

note = "Funding Information: This work was supported by grant from the California Institute for Regenerative Medicine (RT1-01028-1). We thank Steven Froelich and April Tai for critical reading of this manuscript.",

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AU - Lei, Yuning

AU - Lee, Chi Lin

AU - Joo, Kye Il

AU - Zarzar, Jonathan

AU - Liu, Yarong

AU - Dai, Bingbing

AU - Fox, Victoria

AU - Wang, Pin

N1 - Funding Information: This work was supported by grant from the California Institute for Regenerative Medicine (RT1-01028-1). We thank Steven Froelich and April Tai for critical reading of this manuscript.

PY - 2011/5

Y1 - 2011/5

N2 - Human embryonic stem (hES) cells are renewable cell sources that have potential applications in regenerative medicine. The development of technologies to produce permanent and site-specific genome modifications is in demand to achieve future medical implementation of hES cells. We report herein that a baculoviral vector (BV) system carrying zinc-finger nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient expression of ZFNs specifically disrupted the CCR5 locus in transduced cells and the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a gene targeting vector, a DNA donor template and ZFNs were incorporated into the vector. These hybrid vectors yielded permanent site-specific gene addition in both immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both karyotypically normal and pluripotent. These results suggest that this baculoviral delivery system can be engineered for site-specific genetic manipulation in hES cells.

AB - Human embryonic stem (hES) cells are renewable cell sources that have potential applications in regenerative medicine. The development of technologies to produce permanent and site-specific genome modifications is in demand to achieve future medical implementation of hES cells. We report herein that a baculoviral vector (BV) system carrying zinc-finger nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient expression of ZFNs specifically disrupted the CCR5 locus in transduced cells and the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a gene targeting vector, a DNA donor template and ZFNs were incorporated into the vector. These hybrid vectors yielded permanent site-specific gene addition in both immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both karyotypically normal and pluripotent. These results suggest that this baculoviral delivery system can be engineered for site-specific genetic manipulation in hES cells.

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Gene editing of human embryonic stem cells via an engineered baculoviral vector carrying zinc-finger nucleases

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