Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases.

Despite the recent discoveries of and interest in numerous structural variations (SVs)--which include duplications and inversions--in the human and other higher eukaryotic genomes, little is known about the etiology and biology of these SVs, partly due to the lack of molecular tools with which to create individual SVs in cultured cells and model organisms. Here, we present a novel method of inducing duplications and inversions in a targeted manner without pre-manipulation of the genome. We found that zinc finger nucleases (ZFNs) designed to target two different sites in a human chromosome could introduce two concurrent double-strand breaks, whose repair via non-homologous end-joining (NHEJ) gives rise to targeted duplications and inversions of the genomic segments of up to a mega base pair (bp) in length between the two sites. Furthermore, we demonstrated that a ZFN pair could induce the inversion of a 140-kbp chromosomal segment that contains a portion of the blood coagulation factor VIII gene to mimic the inversion genotype that is associated with some cases of severe hemophilia A. This same ZFN pair could be used, in theory, to revert the inverted region to restore genomic integrity in these hemophilia A patients. We propose that ZFNs can be employed as molecular tools to study mechanisms of chromosomal rearrangements and to create SVs in a predetermined manner so as to study their biological roles. In addition, our method raises the possibility of correcting genetic defects caused by chromosomal rearrangements and holds new promise in gene and cell therapy.

[1]  C. Larsson,et al.  Analysis of the cytogenetic stability of the human embryonal kidney cell line 293 by cytogenetic and STR profiling approaches , 2004, Cytogenetic and Genome Research.

[2]  Jin-Soo Kim,et al.  Genome editing with modularly assembled zinc-finger nucleases , 2010, Nature Methods.

[3]  Martin Raff,et al.  DNA Replication Mechanisms , 2002 .

[4]  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.

[5]  P. Green,et al.  Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. , 2002, Blood.

[6]  Seung Woo Cho,et al.  Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. , 2009, Genome research.

[7]  P. Visscher,et al.  Rare chromosomal deletions and duplications increase risk of schizophrenia , 2008, Nature.

[8]  J. Hoeijmakers,et al.  Chromosomal stability and the DNA double-stranded break connection , 2001, Nature Reviews Genetics.

[9]  G. Smyth,et al.  ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. , 2009, Journal of immunological methods.

[10]  Eunji Kim,et al.  Targeted chromosomal deletions in human cells using zinc finger nucleases. , 2010, Genome research.

[11]  Benjamin P. Blackburne,et al.  Mutation spectrum revealed by breakpoint sequencing of human germline CNVs , 2010, Nature Genetics.

[12]  Ryan E. Mills,et al.  Discovery of common Asian copy number variants using integrated high-resolution array CGH and massively parallel DNA sequencing , 2010, Nature Genetics.

[13]  B. Rovin,et al.  The Influence of CCL 3 L 1 Gene – Containing Segmental Duplications on HIV-1 / AIDS Susceptibility , 2009 .

[14]  Jin-Soo Kim,et al.  Analysis of targeted chromosomal deletions induced by zinc finger nucleases. , 2010, Cold Spring Harbor protocols.

[15]  J. Lupski,et al.  A DNA Replication Mechanism for Generating Nonrecurrent Rearrangements Associated with Genomic Disorders , 2007, Cell.

[16]  David R. Liu,et al.  Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection , 2011, Nature Methods.

[17]  T. Ichisaka,et al.  Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors , 2007, Cell.

[18]  Thuy D. Vo,et al.  Transient cold shock enhances zinc-finger nuclease–mediated gene disruption , 2010, Nature Methods.

[19]  J. Oldenburg,et al.  Haemophilia A: from mutation analysis to new therapies , 2005, Nature Reviews Genetics.

[20]  Marc Parmentier,et al.  A Dual-Tropic Primary HIV-1 Isolate That Uses Fusin and the β-Chemokine Receptors CKR-5, CKR-3, and CKR-2b as Fusion Cofactors , 1996, Cell.

[21]  D. Conrad,et al.  Global variation in copy number in the human genome , 2006, Nature.

[22]  Jeffrey C. Miller,et al.  An unbiased genome-wide analysis of zinc-finger nuclease specificity , 2011, Nature Biotechnology.

[23]  Stylianos E. Antonarakis,et al.  Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A , 1993, Nature Genetics.

[24]  M. Nikiforova,et al.  Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells. , 2000, Science.

[25]  M. Hurles,et al.  Large, rare chromosomal deletions associated with severe early-onset obesity , 2010, Nature.

[26]  G. Melen,et al.  Etoposide induces MLL rearrangements and other chromosomal abnormalities in human embryonic stem cells. , 2009, Carcinogenesis.

[27]  P. Stankiewicz,et al.  Structural variation in the human genome and its role in disease. , 2010, Annual review of medicine.

[28]  Vanessa Taupin,et al.  Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo , 2010, Nature Biotechnology.

[29]  L. Feuk,et al.  Detection of large-scale variation in the human genome , 2004, Nature Genetics.

[30]  Jin-Soo Kim,et al.  Preassembled zinc-finger arrays for rapid construction of ZFNs , 2011 .

[31]  J. Orange,et al.  Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases , 2008, Nature Biotechnology.

[32]  A. Bradley,et al.  Chromosome engineering in mice , 1995, Nature.

[33]  M. Jasin,et al.  Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4/ligase IV during chromosomal translocation formation , 2010, Nature Structural &Molecular Biology.

[34]  Adam James Waite,et al.  An improved zinc-finger nuclease architecture for highly specific genome editing , 2007, Nature Biotechnology.

[35]  Ying Sun,et al.  The β-Chemokine Receptors CCR3 and CCR5 Facilitate Infection by Primary HIV-1 Isolates , 1996, Cell.

[36]  M. Nikiforova,et al.  Chromosomal breakpoint positions suggest a direct role for radiation in inducing illegitimate recombination between the ELE1 and RET genes in radiation-induced thyroid carcinomas , 1999, Oncogene.

[37]  Thuy D Vo,et al.  Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures , 2011, Nature Methods.

[38]  Philip M. Kim,et al.  Paired-End Mapping Reveals Extensive Structural Variation in the Human Genome , 2007, Science.

[39]  A. Scharenberg,et al.  Zinc-finger nucleases: a powerful tool for genetic engineering of animals , 2010, Transgenic Research.

[40]  P. Jeggo,et al.  Radiation-induced genomic rearrangements formed by nonhomologous end-joining of DNA double-strand breaks. , 2001, Cancer research.

[41]  J. Lupski,et al.  Mechanisms of change in gene copy number , 2009, Nature Reviews Genetics.

[42]  Toni Cathomen,et al.  Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion , 2010, Nucleic acids research.

[43]  A. Børresen-Dale,et al.  COMPLEX LANDSCAPES OF SOMATIC REARRANGEMENT IN HUMAN BREAST CANCER GENOMES , 2009, Nature.

[44]  Gary D Bader,et al.  Functional impact of global rare copy number variation in autism spectrum disorders , 2010, Nature.

[45]  Thomas Gaj,et al.  Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. , 2010, Journal of molecular biology.

[46]  Jin-Soo Kim,et al.  Surrogate reporters for enrichment of cells with nuclease-induced mutations , 2011, Nature Methods.

[47]  Fyodor Urnov,et al.  Chromosomal translocations induced at specified loci in human stem cells , 2009, Proceedings of the National Academy of Sciences.