Improved cell-penetrating zinc-finger nuclease proteins for precision genome engineering

Molecular Therapy. Nucleic Acids
Jia LiuCarlos F Barbas

Abstract

Safe, efficient, and broadly applicable methods for delivering site-specific nucleases into cells are needed in order for targeted genome editing to reach its full potential for basic research and medicine. We previously reported that zinc-finger nuclease (ZFN) proteins have the innate capacity to cross cell membranes and induce genome modification via their direct application to human cells. Here, we show that incorporation of tandem nuclear localization signal (NLS) repeats into the ZFN protein backbone enhances cell permeability nearly 13-fold and that single administration of multi-NLS ZFN proteins leads to genome modification rates of up to 26% in CD4(+) T cells and 17% in CD34(+) hematopoietic stem/progenitor cells. In addition, we show that multi-NLS ZFN proteins attenuate off-target effects and that codelivery of ZFN protein pairs facilitates dual gene modification frequencies of 20-30% in CD4(+) T cells. These results illustrate the applicability of ZFN protein delivery for precision genome engineering.

References

Aug 15, 2000·Annual Review of Biophysics and Biomolecular Structure·S A WolfeC O Pabo
May 3, 2003·Nature Reviews. Genetics·Clare E ThomasMark A Kay
May 6, 2003·Science·Matthew H Porteus, David Baltimore
Feb 25, 2005·Molecular Therapy : the Journal of the American Society of Gene Therapy·Abdelatif Elouahabi, Jean-Marie Ruysschaert
Feb 20, 2007·Current Gene Therapy·Frédéric Pâques, Philippe Duchateau
Mar 16, 2007·Proceedings of the National Academy of Sciences of the United States of America·Erica A MoehleM C Holmes
May 10, 2007·Journal of Biomedicine & Biotechnology·Frank JacobsenLars Steinstraesser
Mar 25, 2008·Proceedings of the National Academy of Sciences of the United States of America·Yolanda SantiagoTrevor N Collingwood
Feb 14, 2009·PLoS Genetics·Shondra M Pruett-MillerMatthew H Porteus
Nov 26, 2009·Science·Matthew J Moscou, Adam J Bogdanove
Dec 30, 2009·Nature Methods·Louise C LaurentJeanne F Loring
May 4, 2010·Nature Methods·Yannick DoyonMichael C Holmes
Aug 19, 2010·Nature Reviews. Genetics·Fyodor D UrnovPhilip D Gregory
Dec 24, 2010·Nature Biotechnology·Jeffrey C MillerEdward J Rebar
Jul 1, 2011·Nucleic Acids Research·Araksya IzmiryanOlivier Danos
Aug 9, 2011·Nature Biotechnology·Richard GabrielChristof von Kalle
Aug 11, 2011·Genetics·Dana Carroll
Jan 26, 2012·Molecular Therapy : the Journal of the American Society of Gene Therapy·Jinyun YuanBruce E Torbett
Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Jul 4, 2012·Nature Methods·Thomas GajCarlos F Barbas
Jan 5, 2013·Science·Le CongFeng Zhang
Jan 5, 2013·Science·Prashant MaliGeorge M Church
Feb 27, 2013·Current Pharmaceutical Biotechnology·Weiwei WangGustav Steinhoff
Apr 17, 2013·Molecular Therapy : the Journal of the American Society of Gene Therapy·Lijing LiDavid L DiGiusto
May 15, 2013·Trends in Biotechnology·Thomas GajCarlos F Barbas
May 25, 2013·Annual Review of Genomics and Human Genetics·David J Segal, Joshua F Meckler
Aug 21, 2013·Nucleic Acids Research·Zhong ChenSteffen E Meiler
Aug 29, 2013·Biotechnology and Bioengineering·Thomas GajCarlos F Barbas
Mar 7, 2014·The New England Journal of Medicine·Pablo TebasCarl H June
Apr 3, 2014·Nature Reviews. Genetics·Hyongbum Kim, Jin-Soo Kim

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Citations

Mar 2, 2016·Biochemical and Biophysical Research Communications·Michela Deleidi, Cong Yu
Sep 22, 2015·Epigenetics & Chromatin·Benjamin I Laufer, Shiva M Singh
Sep 18, 2015·Frontiers in Plant Science·Andriy BilichakFrançois Eudes
Jan 10, 2019·3 Biotech·Shengwang Jiang, Qingwu W Shen
Dec 3, 2016·Cold Spring Harbor Perspectives in Biology·Thomas GajJia Liu
Sep 1, 2017·Nature Communications·H F MoffettM T Stephan
Sep 16, 2020·Advanced Drug Delivery Reviews·Jie LiNiren Murthy
Jul 9, 2020·Molecular Therapy. Methods & Clinical Development·Pedro R L PerdigãoJoao Goncalves
Jun 20, 2017·Current Opinion in Chemical Biology·Patrick Ho, Yvonne Y Chen
Feb 20, 2021·Cytokine: X·Julian J Freen-van Heeren
Apr 19, 2018·Journal of the American Chemical Society·Romain RouetJennifer A Doudna
Oct 3, 2015·Genome Research·Isaac B Hilton, Charles A Gersbach

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Methods Mentioned

BETA
gene knockout
transfection
flow cytometry
gene modification
cleavage assay

Software Mentioned

FACSDiva
FlowJo Star

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