Human Genome Editing Science, Ethics, and Governance (2017) / Chapter Skim
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Appendix A: The Basic Science of Genome Editing
Appendix A: The Basic Science of Genome Editing
Pages 217-260
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From page 217... ...
gene editing -- meganucleases, zinc fingers, and transcription activator-like effector nucleases (TALENs) • development of CRISPR/Cas9 • the accuracy of gene editing • enhancing the specificity of CRISPR/Cas9 • quality control and quality assurance for gene editing • use of dead Cas9 (dCas9)
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BREAKAGE AND REPAIR OF GENOMIC DNA Genomes and their constituent genes are made of double-stranded DNA; this DNA can be broken accidentally (e.g., by radiation) or purposefully, using proteins called endonucleases (often called nucleases)
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Zinc Finger Nucleases Zinc fingers are segments of protein that have evolved to recognize and bind to specific DNA sequences. Knowledge gained from natural zinc fingers led to the development of ZFNs as designer DNA-cutting enzymes (see Figure A-2)
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and a DNA-cleaving protein, FokI nuclease (Kim et al., 1996) , yielding "artificial restriction enzymes" that could be used to promote site-specific genome engineering by creating DSBs at defined locations in the genome (Bibikova et al., 2001, 2002, 2003)
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These divergent amino acids are largely responsible for determining the DNA-binding specificity for a single DNA base pair, which allows engineering of specific DNA-binding domains by choosing combinations of repeat segments containing appropriate amino acids. The biotech firm Cellectis reported the successful conduct of the first-ever TALEN-based gene-editing clinical trial in the United Kingdom on a girl with incurable acute lymphoblastic leukemia (ALL)
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From page 222... ...
. The key experimental breakthrough came from research showing that CRISPR allowed bacteria to acquire resistance to bacteriophages by integrating segments of the bacteriophage genome into the CRISPR loci, demonstrating that CRISPR was a new form of adaptive immunity (Barrangou et al., 2007)
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targeting a specific DNA sequence; DNA cut sites are indicated.
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ACCURACY OF GENE EDITING The potential impact of unintended changes to DNA is a key challenge for safe use of genome editing as a therapeutic strategy. Unintended changes to the genome could be caused by cleaving DNA at sites other than those that are being deliberately targeted.
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Although the IDLV capture method directly identifies DSBs that occur in living cells, it is relatively insensitive and has a high background. To overcome these limitations, genome-wide unbiased identification of DSBs enabled by sequencing (GUIDE-seq)
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. Genomic DNA is isolated from cells and treated with Cas9 nuclease in vitro.
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Human pluripotent stem cells (hPSCs) are primary cells with genetically intact quality control mechanisms, and it seems possible that off-target events will accumulate less frequently in hPSCs or in normal somatic cells than has been observed in cancer cells.
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, yields a Cas9 protein capable of cutting one strand of double-stranded DNA. Consequently, providing two guide RNAs that direct cutting on opposite strands in close proximity mediated by a dimer of Cas9n single cutters leads to an effective DSB and stimulation of both NHEJ and HDR to yield a DSB (see Figure A-3b)
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The "base editor" variant of Cas9 provides increased efficiency and precision when making changes to genomic DNA. SOURCE: Modified from Komor et al., 2016.
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The authors show that by controlling the location of the introduced point mutation relative to the Cas9-mediated DSB they can alter the efficiency of mutagenesis, generating either heterozygous or homozygous alterations in human-induced pluripotent stem cells (hiPSCs)
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By the same token, the transient nature of these changes limits their utility for correcting diseases caused by genetic mutations. Possible uses for such transient germline engineering, however, include the ability to expand germ cells, or the in vitro generation of desired stem cells or terminally differentiated cells.
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The embryos can be transplanted into a foster mother. The exogenous DNA sequences are randomly integrated into the genome of the resulting transgenic mice directly or after incubation any time up until the blastocyst stage (~100 cells)
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. Because chimera–competent ES cells were only available in the murine system, gene editing by homologous recombination was restricted to mice and could not readily be used in other species.
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(c) Germline transmission of the ES cell clone is verified by mating of the chimeric mouse with the albino host strain.
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GENOME EDITING IN EMBRYOS Homologous recombination in conventional gene targeting is an inefficient process and requires the selection of correctly targeted cell clones in cell culture. In a second step the targeted ES cell clone is injected into a host blastocyst to create a chimeric animal, which, in a third step, is mated to produce the desired mutant animal, a process that may take as much as 1 or 2 years (compare Figure A-7)
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If coinjected with a DNA vector, exogenous sequences will be inserted at some frequency at the double-strand break. CRISPR/Cas9-Mediated Gene Editing in the Zygote The injection of guide RNAs together with Cas9 RNA into the fertilized egg (zygote)
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This poses a complication if the goal of gene editing in embryos is the correction of a mutant allele. To correct a given mutation, a guide RNA and a DNA target construct are injected into the embryo.
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. This autocatalytic process was dubbed as a "mutagenic chain reaction." If a vector carrying sequences correcting a given mutation in addition to the Cas9 and the guide RNA was inserted into one allele, these sequences could serve as a template during meiosis and convert the other allele by homologous recombination resulting in two repaired alleles (see Figure A-9a)
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From page 239... ...
and "cargo" DNA sequences into a gene of interest. When expressed during meiosis, the guide RNA/Cas9 complex will introduce a double-strand break onto the other allele followed by homologous recombination driven repair, which will insert the Cas9/gRNA/transgene sequences into the wild-type allele.
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. While this approach has proven to be extraordinarily powerful in producing knockout mice, conditional mutations, reporter lines, and a variety of human disease models, it is still relatively inefficient and has not been readily applicable to direct targeted genome alterations in zygotes.
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More interesting is the possibility of direct genome editing in the sperm nuclei. Given that sperm are nondividing cells, currently only NHEJ-mediated gene editing would be possible, although the repair mechanism is presumably different from that in somatic
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Gene Editing in Germline Stem Cells There is considerable biological and clinical interest in generating gametes from stem cell lines that can be propagated indefinitely in vitro. Spermatogonial stem cells (SSCs)
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and no evidence for any endogenous stem cells. Gene Editing in Pluripotent Stem Cells Followed by Germ Cell Differentiation Pluripotent embryonic stem cells or induced pluripotent stem cells can be generated from both males and females, are readily amenable to CRISPR editing, and can be differentiated down the pathway toward meiotically competent germ cells.
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From page 244... ...
This suggests that more knowledge of how germ cells actually develop in the human, or perhaps the nonhuman primate, embryo versus the mouse embryo is needed to move this research forward. Gene Editing in Haploid ES Cells Most animals are diploids, and natural haploid cells are typically limited to mature germ cells.
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EDITING THE MITOCHONDRIAL GENOME Mitochondrial diseases are a group of maladies caused by the dysfunction of mitochondria due to mutations in mitochondrial DNA (mtDNA)
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Moreover, due to the very low activity of repair mechanisms in mitochondria, the frequency of re-ligation of target mtDNA and introduction of new mutations would be very rare. In addition, similar mitochondrial editing tools in the future could also be used to eliminate mutated mtDNA in gametes derived from stem cells.
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TABLE A-1 General Approaches to Delivering Genome-Editing Components Preferred Method Delivered Component Explanation Advantages Disadvantages Applications Nonviral Transfection Nuclease(s) as plasmid DNA, All components are Relatively simple; Cellular uptake and Cell lines and RNA, or protein assembled with a can deliver all nuclear access can be some primary glyco/lipo-polymer editing machinery rate-limiting; vehicle cells ex vivo guideRNA as plasmid DNA or vehicle that favors cell together; transient can cause cytotoxicity oligonucleotide is mixed with entry; the complex expression limiting and inflammation the nuclease is applied to cells ex cytotoxicity and in vivo; for in vivo RNA (can be complexed with vivo or injected into immunogenicity of delivery RNA needs nuclease as Ribonucleoprotein, a tissue or blood in editing machinery.
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From page 248... ...
immunogenicity of editing machinery. Electroporation A brief electric pulse Very effective Can cause cytotoxicity Cell lines and is passed across a method of delivery (increasingly from primary cells population of cells to a wide variety of protein to mRNA ex vivo in a solution that cell types ex vivo; to DNA; alleviated contains the editing transient expression by using modified nuclease reagents with limiting cytotoxicity nucleic acids)
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Microinjection Nuclease(s) as plasmid DNA, The components can Efficiencies up to Technically more Zygotes and RNA, or protein; gRNA as be introduced in a 100% challenging than other early embryos plasmid DNA or RNA.
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RNP, or for the combined the cell progeny; transduction of delivery of template and a modified version human cells well guideRNA and/or nuclease, on made with mutant tolerated; IDLV the same or separate vectors) integrase-defective provides for lentiviral vector transient nuclease (IDLV)
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From page 251... ...
expression in dividing cells; well suitable for nuclease and template delivery. Adenoviral Replication- Able to package Has shown severe acute Primary cells Vector defective virus that large fragments of toxicity in some clinical ex vivo can package >20 DNA; can be used trials; many people have kilobases of DNA for in vivo and pre-existing immunity; able to transduce a ex vivo delivery; no longer commonly wide variety of cell transient expression used gene therapy types both ex vivo in dividing cells and vector.
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2003. Enhancing gene targeting with designed zinc finger nucleases.
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1987. Targeted correction of a mutant HPRT gene in mouse embryonic stem cells.
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2011. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.
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2012. Androgenetic haploid embryonic stem cells produce live transgenic mice.
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2013. Max is a repressor of germ cell-related gene expression in mouse embryonic stem cells.
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2015. Robust in vitro induction of human germ cell fate from pluripotent stem cells.
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2013. Parthenogenetic haploid embryonic stem cells produce fertile mice.
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1989. Germ line transmission of a disrupted b2-microglobulin gene produced by homologous recombination in embryonic stem cells.
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