Fanconi anemia gene editing by the CRISPR/Cas9 system.

Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder.

[1]  L. Peltonen,et al.  The rearranged L-myc fusion gene (RLF) encodes a Zn-15 related zinc finger protein. , 1995, Oncogene.

[2]  T. Leung,et al.  Phosphorylation of a Novel Myosin Binding Subunit of Protein Phosphatase 1 Reveals a Conserved Mechanism in the Regulation of Actin Cytoskeleton* , 2001, The Journal of Biological Chemistry.

[3]  J. Wagner,et al.  Haematopoeitic cell transplantation for Fanconi anaemia – when and how? , 2010, British journal of haematology.

[4]  Jennifer Doudna,et al.  RNA-programmed genome editing in human cells , 2013, eLife.

[5]  Y. Mori,et al.  Physical and functional interaction of the active zone protein CAST/ERC2 and the β-subunit of the voltage-dependent Ca(2+) channel. , 2012, Journal of biochemistry.

[6]  Christof von Kalle,et al.  Bioinformatic clonality analysis of next-generation sequencing-derived viral vector integration sites. , 2012, Human gene therapy methods.

[7]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[8]  Y. Mori,et al.  Physical and functional interaction of the active zone protein CAST/ERC2 and the β-subunit of the voltage-dependent Ca(2+) channel. , 2012, Journal of biochemistry.

[9]  Cameron S. Osborne,et al.  LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1 , 2003, Science.

[10]  F. Bushman,et al.  Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. , 2008, The Journal of clinical investigation.

[11]  J. Wagner,et al.  Unrelated donor bone marrow transplantation for Fanconi anemia. , 1996, Bone marrow transplantation.

[12]  Jin-Soo Kim,et al.  Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases , 2014, Genome research.

[13]  J. Tolar,et al.  Sleeping Beauty‐mediated correction of Fanconi anemia type C , 2011, The journal of gene medicine.

[14]  M. Buchwald,et al.  Suppression of apoptosis in hematopoietic factor-dependent progenitor cell lines by expression of the FAC gene. , 1996, Blood.

[15]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[16]  H. Cross,et al.  Mutation of HERC2 causes developmental delay with Angelman-like features , 2012, Journal of Medical Genetics.

[17]  R. Gibson,et al.  A common mutation in the FACC gene causes Fanconi anaemia in Ashkenazi Jews , 1993, Nature Genetics.

[18]  James M. Harris,et al.  Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long-term safety in preclinical nonhuman primate transplant models. , 2011, Cell stem cell.

[19]  R. Barrangou,et al.  Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria , 2012, Proceedings of the National Academy of Sciences.

[20]  M. Bohlooly-y,et al.  Phenotypic screening of hepatocyte nuclear factor (HNF) 4-γ receptor knockout mice , 2006 .

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

[22]  A. D’Andrea,et al.  Fanconi anemia and DNA repair. , 2001, Human molecular genetics.

[23]  S. Ekker,et al.  Dynamic gene expression after systemic delivery of plasmid DNA as determined by in vivo bioluminescence imaging. , 2005, Human gene therapy.

[24]  Prashant Mali,et al.  Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. , 2009, Cell stem cell.

[25]  J. Wagner,et al.  bone marrow : results of a matched-pair analysis matched unrelated donor − comparable to that of human leukocyte antigen Survival after transplantation of unrelated donor umbilical cord blood , 2001 .

[26]  B. Stoddard,et al.  Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template , 2010, Nucleic acids research.

[27]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

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

[29]  Erin L. Doyle,et al.  Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting , 2011, Nucleic acids research.

[30]  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.

[31]  P. Gregory,et al.  Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery , 2007, Nature Biotechnology.

[32]  Yolanda Santiago,et al.  Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology , 2010, Nucleic acids research.

[33]  Anthony E. Boitano,et al.  Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells , 2010, Science.

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

[35]  R. Kotin,et al.  Characterization of a preferred site on human chromosome 19q for integration of adeno‐associated virus DNA by non‐homologous recombination. , 1992, The EMBO journal.

[36]  Erin L. Doyle,et al.  Targeting DNA Double-Strand Breaks with TAL Effector Nucleases , 2010, Genetics.

[37]  D. Schindler,et al.  Fanconi anemia proteins interact with CtBP1 and modulate the expression of the Wnt antagonist Dickkopf-1. , 2013, Blood.

[38]  Michel Sadelain,et al.  Safe harbours for the integration of new DNA in the human genome , 2011, Nature Reviews Cancer.

[39]  S. Olson,et al.  The Fanconi anemia complementation group C gene product: structural evidence of multifunctionality. , 2001, Blood.

[40]  Stephan Wolf,et al.  Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing , 2010, Nature Protocols.

[41]  Christof von Kalle,et al.  TALEN-based gene correction for epidermolysis bullosa. , 2013, Molecular therapy : the journal of the American Society of Gene Therapy.

[42]  Luca Biasco,et al.  Comprehensive genomic access to vector integration in clinical gene therapy , 2009, Nature Medicine.

[43]  B. Kaina,et al.  Cisplatin sensitivity is related to late DNA damage processing and checkpoint control rather than to the early DNA damage response. , 2009, Mutation research.

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

[45]  Lei Zhang,et al.  Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. , 2010, Genome research.

[46]  Claudio Mussolino,et al.  Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects , 2012, Nucleic acids research.

[47]  S. Kaufmann.  Immunity to bacteria. , 1990, Current opinion in immunology.

[48]  P. Fei,et al.  Advances in the understanding of the Fanconi anemia tumor suppressor pathway , 2013, Cancer biology & therapy.

[49]  Minoru Takata,et al.  Functional Interplay between BRCA2/FancD1 and FancC in DNA Repair* , 2006, Journal of Biological Chemistry.

[50]  Jeffrey C. Miller,et al.  A rapid and general assay for monitoring endogenous gene modification. , 2010, Methods in molecular biology.

[51]  R. Jaenisch,et al.  Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases , 2009, Nature Biotechnology.

[52]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[53]  M. van der Burg,et al.  Targeted Genome Editing in Human Repopulating Hematopoietic Stem Cells , 2014, Nature.

[54]  Gang Bao,et al.  CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity , 2013, Nucleic acids research.

[55]  David J. Rawlings,et al.  Tracking genome engineering outcome at individual DNA breakpoints , 2011, Nature Methods.

[56]  S. Fagerlie,et al.  Functional correction of FA-C cells with FANCC suppresses the expression of interferon gamma-inducible genes. , 2001, Blood.

[57]  Margherita Neri,et al.  Site-specific integration and tailoring of cassette design for sustainable gene transfer , 2011, Nature Methods.

[58]  N. Maizels,et al.  Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair , 2014, Proceedings of the National Academy of Sciences.

[59]  M. Bohlooly-y,et al.  Phenotypic screening of hepatocyte nuclear factor (HNF) 4-gamma receptor knockout mice. , 2006, Biochemical and biophysical research communications.

[60]  C. von Kalle,et al.  Hepatocyte-Targeted Expression by Integrase-Defective Lentiviral Vectors Induces Antigen-Specific Tolerance in Mice with Low Genotoxic Risk , 2011, Hepatology.

[61]  Kun Zhang,et al.  Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. , 2014, Cell stem cell.

[62]  R. Moses,et al.  The Ashkenazi Jewish Fanconi anemia mutation: Incidence among patients and carrier frequency in the at‐risk population , 1994, Human mutation.