TY - JOUR
T1 - Creating designed zinc-finger nucleases with minimal cytotoxicity
AU - Ramalingam, Sivaprakash
AU - Kandavelou, Karthikeyan
AU - Rajenderan, Raja
AU - Chandrasegaran, Srinivasan
N1 - Funding Information:
This work was supported by a grant ( 52067 to K.K.) from the Bill and Melinda Gates Foundation through the Grand Challenge Explorations Initiative . S.C. was supported by a grant from the National Institute of General Medical Sciences ( GM077291 ). We thank Mr. S. Gunasegaran (President and Chief Executive Officer of Pondicherry Biotech Private Limited) for his constant support and encouragement, Dr. Jithesh Narayanan (Pondicherry Biotech Private Limited) for helpful suggestions, and Dr. Margolick's laboratory at Johns Hopkins University for assistance with the flow cytometry and FACS studies.
PY - 2011/1/21
Y1 - 2011/1/21
N2 - Zinc-finger nucleases (ZFNs) have emerged as powerful tools for delivering a targeted genomic double-strand break (DSB) to either stimulate local homologous recombination with investigator-provided donor DNA or induce gene mutations at the site of cleavage in the absence of a donor by nonhomologous end joining both in plant cells and in mammalian cells, including human cells. ZFNs are formed by fusing zinc-finger proteins to the nonspecific cleavage domain of the FokI restriction enzyme. ZFN-mediated gene targeting yields high gene modification efficiencies (> 10%) in a variety of cells and cell types by delivering a recombinogenic DSB to the targeted chromosomal locus, using two designed ZFNs. The mechanism of DSB by ZFNs requires (1) two ZFN monomers to bind to their adjacent cognate sites on DNA and (2) the FokI nuclease domains to dimerize to form the active catalytic center for the induction of the DSB. In the case of ZFNs fused to wild-type FokI cleavage domains, homodimers may also form; this could limit the efficacy and safety of ZFNs by inducing off-target cleavage. In this article, we report further refinements to obligate heterodimer variants of the FokI cleavage domain for the creation of custom ZFNs with minimal cellular toxicity. The efficacy and efficiency of the reengineered obligate heterodimer variants of the FokI cleavage domain were tested using the green fluorescent protein gene targeting reporter system. The three-finger and four-finger zinc-finger protein fusions to the REL-DKK pair among the newly generated FokI nuclease domain variants appear to eliminate or greatly reduce the toxicity of designer ZFNs to human cells.
AB - Zinc-finger nucleases (ZFNs) have emerged as powerful tools for delivering a targeted genomic double-strand break (DSB) to either stimulate local homologous recombination with investigator-provided donor DNA or induce gene mutations at the site of cleavage in the absence of a donor by nonhomologous end joining both in plant cells and in mammalian cells, including human cells. ZFNs are formed by fusing zinc-finger proteins to the nonspecific cleavage domain of the FokI restriction enzyme. ZFN-mediated gene targeting yields high gene modification efficiencies (> 10%) in a variety of cells and cell types by delivering a recombinogenic DSB to the targeted chromosomal locus, using two designed ZFNs. The mechanism of DSB by ZFNs requires (1) two ZFN monomers to bind to their adjacent cognate sites on DNA and (2) the FokI nuclease domains to dimerize to form the active catalytic center for the induction of the DSB. In the case of ZFNs fused to wild-type FokI cleavage domains, homodimers may also form; this could limit the efficacy and safety of ZFNs by inducing off-target cleavage. In this article, we report further refinements to obligate heterodimer variants of the FokI cleavage domain for the creation of custom ZFNs with minimal cellular toxicity. The efficacy and efficiency of the reengineered obligate heterodimer variants of the FokI cleavage domain were tested using the green fluorescent protein gene targeting reporter system. The three-finger and four-finger zinc-finger protein fusions to the REL-DKK pair among the newly generated FokI nuclease domain variants appear to eliminate or greatly reduce the toxicity of designer ZFNs to human cells.
KW - Genome engineering
KW - Homologous recombination
KW - Non-homologous end-joining
KW - Site-Specific modification
KW - Targeted cleavage
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U2 - 10.1016/j.jmb.2010.10.043
DO - 10.1016/j.jmb.2010.10.043
M3 - Article
C2 - 21094162
AN - SCOPUS:78650905952
SN - 0022-2836
VL - 405
SP - 630
EP - 641
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 3
ER -