Construction and testing of engineered zinc-finger proteins for sequence-specific modification of mtDNA

Engineered zinc-finger proteins (ZFPs) are hybrid proteins developed to direct various effector domains (EDs) of choice to predetermined DNA sequences. They are used to alter gene expression and to modify DNA in a sequence-specific manner in vivo and in vitro. Until now, ZFPs have mostly been used to target DNA sites in nuclear genomes. This protocol describes how to adapt engineered ZFP technology to specifically modify the mammalian mitochondrial genome. The first step describes how to construct mitochondrially targeted ZFPs (mtZFPs) so that they are efficiently imported into mammalian mitochondria. In the second step, methods to test the basic properties of mtZFPs in vitro are described. Finally, we outline how the mtZFPs can be transiently transfected into mammalian cells and their mitochondrial import tested by both immunofluorescence and biochemical methods. The protocol can be completed within a week, although time-consuming DNA cloning steps may extend this.

[1]  G. Gaines.  In organello RNA synthesis system from HeLa cells. , 1996, Methods in enzymology.

[2]  Ignacio Anegon,et al.  Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases , 2009, Science.

[3]  M. Minczuk,et al.  Designer zinc-finger proteins and their applications. , 2006, Gene.

[4]  P. Christen,et al.  The mature form of imported mitochondrial proteins undergoes conformational changes upon binding to isolated mitochondria. , 1993, European journal of biochemistry.

[5]  J. Fernandez-Martinez,et al.  Overlap of nuclear localisation signal and specific DNA-binding residues within the zinc finger domain of PacC. , 2003, Journal of molecular biology.

[6]  Ronnie J Winfrey,et al.  Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. , 2008, Molecular cell.

[7]  Drena Dobbs,et al.  Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly , 2006, Nature Protocols.

[8]  Drena Dobbs,et al.  Zinc Finger Database (ZiFDB): a repository for information on C2H2 zinc fingers and engineered zinc-finger arrays , 2008, Nucleic Acids Res..

[9]  Aaron Klug,et al.  Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA , 2008, Nucleic acids research.

[10]  D. Leister,et al.  Mitochondria : practical protocols , 2007 .

[11]  Daniel F. Voytas,et al.  Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool , 2007, Nucleic Acids Res..

[12]  Drena Dobbs,et al.  An affinity-based scoring scheme for predicting DNA-binding activities of modularly assembled zinc-finger proteins , 2008, Nucleic acids research.

[13]  Carl O. Pabo,et al.  Drug discovery with engineered zinc-finger proteins , 2003, Nature Reviews Drug Discovery.

[14]  Fyodor D Urnov,et al.  Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases , 2007, Proceedings of the National Academy of Sciences.

[15]  Bradford W. Gibson,et al.  Characterization of the human heart mitochondrial proteome , 2003, Nature Biotechnology.

[16]  A. Klug,et al.  Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases , 2008, Proceedings of the National Academy of Sciences.

[17]  S Chandrasegaran,et al.  Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[18]  N. Venediktova,et al.  Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes. , 2008, Gene therapy.

[19]  N. Pfanner,et al.  Versatility of the mitochondrial protein import machinery , 2001, Nature Reviews Molecular Cell Biology.

[20]  M. Noyes,et al.  Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases , 2008, Nature Biotechnology.

[21]  J. Milbrandt,et al.  The nuclear localization signal of NGFI-A is located within the zinc finger DNA binding domain. , 1994, The Journal of biological chemistry.

[22]  Laura C. Greaves,et al.  Mitochondrial DNA mutations in human disease , 2006, IUBMB life.

[23]  Robert W. Taylor,et al.  Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids , 1997, Nature Genetics.

[24]  Jeffrey C. Miller,et al.  Highly efficient endogenous human gene correction using designed zinc-finger nucleases , 2005, Nature.

[25]  T. Hocking,et al.  Heritable Targeted Gene Disruption in Zebrafish Using Designed Zinc Finger Nucleases , 2008, Nature Biotechnology.

[26]  O. Capozzi,et al.  History of the Tfam gene in primates. , 2005, Gene.

[27]  Jonathan E. Foley,et al.  Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN) , 2009, PloS one.

[28]  A. Klug.  The discovery of zinc fingers and their development for practical applications in gene regulation , 2005 .

[29]  Vitali Alexeev,et al.  Targeted manipulation of mammalian genomes using designed zinc finger nucleases. , 2009, Biochemical and biophysical research communications.

[30]  Toni Cathomen,et al.  DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[31]  Y. Akao,et al.  Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria. , 2002, Journal of biomedical science.

[32]  Dana Carroll,et al.  Design, construction and in vitro testing of zinc finger nucleases , 2006, Nature Protocols.

[33]  M. Minczuk,et al.  Inhibition of herpes simplex virus 1 gene expression by designer zinc-finger transcription factors , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[34]  M. Minczuk,et al.  Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase , 2006, Proceedings of the National Academy of Sciences.