TY - JOUR
T1 - Targeted manipulation of mammalian genomes using designed zinc finger nucleases
AU - Kandavelou, Karthikeyan
AU - Ramalingam, Sivaprakash
AU - London, Viktoriya
AU - Mani, Mala
AU - Wu, Joy
AU - Alexeev, Vitali
AU - Civin, Curt I.
AU - Chandrasegaran, Srinivasan
N1 - Funding Information:
This work was supported partly by grants from National Institute of General Medical Sciences (GM077291) and Maryland Stem Cell Research Fund to S.C. (JHU) and partly by a grant from The Bill & Melinda Gates Foundation through the Grand Challenge Explorations Initiative to K.K. (Pondicherry Biotech Private Limited, PBPL) and a National Foundation for Cancer Research fellow Award to C.I.C. We thank Dr. Joseph Margolick’s Lab at JHU for assistance with the flow cytometry and FACS studies.
PY - 2009/10/9
Y1 - 2009/10/9
N2 - Targeted introduction of a double-stranded break (DSB) using designer zinc finger nucleases (ZFNs) in mammalian cells greatly enhances gene targeting - homologous recombination (HR) at a chosen endogenous target gene, which otherwise is limited by low spontaneous rate of HR. Here, we report that efficient ZFN-mediated gene correction occurs at a transduced, transcriptionally active, mutant GFP locus by homology-directed repair, and that efficient mutagenesis by non-homologous end joining (NHEJ) occurs at the endogenous, transcriptionally silent, CCR5 locus in HEK293 Flp-In cells, using designed 3- and 4-finger ZFNs. No mutagenesis by NHEJ was observed at the CCR2 locus, which has ZFN sites that are distantly related to the targeted CCR5 sites. We also observed efficient ZFN-mediated correction of a point mutation at the endogenous mutant tyrosinase chromosomal locus in albino mouse melanocytes, using designed 3-finger ZFNs. Furthermore, re-engineered obligate heterodimer FokI nuclease domain variants appear to completely eliminate or greatly reduce the toxicity of ZFNs to mammalian cells, including human cells.
AB - Targeted introduction of a double-stranded break (DSB) using designer zinc finger nucleases (ZFNs) in mammalian cells greatly enhances gene targeting - homologous recombination (HR) at a chosen endogenous target gene, which otherwise is limited by low spontaneous rate of HR. Here, we report that efficient ZFN-mediated gene correction occurs at a transduced, transcriptionally active, mutant GFP locus by homology-directed repair, and that efficient mutagenesis by non-homologous end joining (NHEJ) occurs at the endogenous, transcriptionally silent, CCR5 locus in HEK293 Flp-In cells, using designed 3- and 4-finger ZFNs. No mutagenesis by NHEJ was observed at the CCR2 locus, which has ZFN sites that are distantly related to the targeted CCR5 sites. We also observed efficient ZFN-mediated correction of a point mutation at the endogenous mutant tyrosinase chromosomal locus in albino mouse melanocytes, using designed 3-finger ZFNs. Furthermore, re-engineered obligate heterodimer FokI nuclease domain variants appear to completely eliminate or greatly reduce the toxicity of ZFNs to mammalian cells, including human cells.
KW - Custom-designed zinc finger nucleases
KW - Homologous recombination
KW - Homology-directed repair
KW - Non-homologous end joining
KW - ZFN-mediated gene targeting
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U2 - 10.1016/j.bbrc.2009.07.112
DO - 10.1016/j.bbrc.2009.07.112
M3 - Article
C2 - 19635463
AN - SCOPUS:69049104003
SN - 0006-291X
VL - 388
SP - 56
EP - 61
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 1
ER -