WO2017173004A1 - Procédé d'édition précise de génome in vivo - Google Patents

Procédé d'édition précise de génome in vivo Download PDF

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WO2017173004A1
WO2017173004A1 PCT/US2017/024869 US2017024869W WO2017173004A1 WO 2017173004 A1 WO2017173004 A1 WO 2017173004A1 US 2017024869 W US2017024869 W US 2017024869W WO 2017173004 A1 WO2017173004 A1 WO 2017173004A1
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genome editing
cells
target sequence
sequence
cell
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Takayasu MIKUNI
Jun Nishiyama
Ryohei Yasuda
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Mikuni Takayasu
Jun Nishiyama
Ryohei Yasuda
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Humanized animals, e.g. knockin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag

Definitions

  • the present invention relates to a method for in vivo genome editing, comprising: (a) delivering a genome editing machinery (e.g. plasmid vectors encoding a Cas9 nuclease and a single-guide RNA; as well as a donor template polynucleotide), to mitotic progenitor cells of a non-human embryo through in utero electroporation; and (b) modifying at least one target sequence (i.e. a DNA sequence within the genome of the progenitor cells) by using homology-directed repair (HDR).
  • the genome editing machinery may further comprise a control of transfection, comprising a transposon, and a polynucleotide encoding a transposase.
  • Another aspect of the invention relates to a non-human embryo or a non-human animal carrying a modified target sequence in its genome, wherein said non-human embryo or non-human animal is produced by the in vivo genome editing method of the invention.
  • a further aspect of the in vivo genome editing method of the invention relates to a modified cell obtained from said non-human embryo or from said non-human animal.
  • knock-in mice in which a specific protein is tagged with an epitope tag or fluorescent protein can be used (Yang, 2009, Nat Neurosci 12 113-5).
  • dense tissue such as mammalian brain
  • several methods have been recently developed for single-cell labelling of endogenous proteins by using recombinant antibody-like proteins or a conditional tag knock-in strategy (Fortin, 2014, J Neurosci 34 16698-712; Gross, 2013, Neuron 78 971 -85).
  • none of these techniques provides rapid, scalable and high-throughput readouts for the localization of endogenous proteins.
  • Genome editing based on the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 enables rapid and efficient modification of the genome (Cong, 2013, Science 339 819- 23; Doudna, 2014, Science 346 1258096; Hsu, 2014, Cell 157 1262-78; Jinek, 2012, Science 337 816-21 ; Sander, 2014, Nat
  • CRISPR-Cas9 induces targeted DNA single- or double-strand breaks in the genome, which are then repaired through either nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways (Cox, 2015, Nat Med 21 121 -31 ; Doudna, 2014, Science 346 1258096; Hsu, 2014, Cell 157 1262-78; Sander, 2014, Nat Biotechnol 32 347-55; Yang, 2013, Cell 154 1370-9).
  • NHEJ nonhomologous end-joining
  • HDR homology-directed repair
  • CRISPR-Cas9 mediated gene knockout and knock-in rely on the completely different mechanisms (NHEJ vs HDR) and can be used for different purposes.
  • NHEJ-mediated gene knockout is based on error-prone DNA repair of Cas9-mediated DNA double strand break (imprecise and uncontrollable process) and can be used to explore the effects of disrupting a particular gene.
  • HDR-mediated gene knock-in enables precise genome editing including sequence insertion, deletion and replacement, which can be applied for many purposes such as visualization of endogenous gene products, modeling or correction of disease-related mutations etc.
  • CRISPR-Cas9 mediated gene knockout via the NHEJ pathway can be applied in post-mitotic cells of the mammalian brain (Straub, 2014, PLOS one, 9(8), e105584; Incontro, 2014, Neuron 83 1051-7, Swiech,2015, Nat Biotechnol 33 102-6; Takeo, 2015, J Neurosci 35:12518-34; Shinmyo, 2016, Sci Rep 6: 2061 1 ).
  • homologous recombination proteins are mainly expressed in the G2 phase of the cell cycle, making HDR-based gene editing difficult in post-mitotic cells such as neurons or cardiac myocytes (Hsu, 2014, Cell, 157: 1262-1278).
  • methods for stimulating HDR- based repair or alternative strategies for efficient gene insertion are urgently needed (Hsu, 2014, Cell, 157: 1262-1278).
  • the technical problem underlying the present invention is the provision of means and methods to obtain post-mitotic cells, such as neurons, with a HDR-mediated genome modification, or animals having a HDR-mediated genome modification within its post-mitotic cells.
  • the present invention relates to a method for in vivo genome editing, comprising:
  • step (b) of the in vivo genome editing method of the invention at least one target sequence (i.e. at least one genomic DNA sequence) in the mitotic progenitor cells is modified by using HDR.
  • the present invention circumvented the problem that post-mitotic cells cannot directly modified by using HDR by targeting dividing (i.e. mitotic) progenitor cells, which retain HDR activities, by in utero electroporation. Therefore, the present invention provides a novel strategy to enable sequence insertion based on CRISPR-Cas9 mediated HDR in vivo.
  • the genome editing machinery may comprise at least one polynucleotide encoding a site-specific DNA nuclease, which introduces a double or single strand break within the target sequence.
  • double strand breaks are introduced by the site- specific nuclease.
  • ZEN zinc finger nucleases
  • TALEN transcription activator-like effector nucleases
  • ZEN zinc finger nucleases
  • TALEN transcription activator-like effector nucleases
  • Cas clustered regularly interspaced short palindromic repeat
  • Cas9 clustered regularly interspaced short palindromic repeat
  • TALEN transcription activator-like effector nuclease
  • a ZFN comprises a zinc-finger DNA binding domain, which should be designed for each target gene, and a Fokl nuclease.
  • a TALEN comprises a DNA binding domain, which should be designed for each target gene, and a Fokl nuclease.
  • the nucleotide and amino acid sequences of a Fokl nuclease are commonly known in the art.
  • the Fokl nuclease may have a nucleotide sequence that has at least 80%, preferably, at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99%, and most preferably 100% identity to the nucleotide sequence of SEQ ID NO: 1 .
  • the Fokl nuclease may have an amino acid sequence that has at least 80%, preferably, at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99%, and most preferably 100% identity to the amino acid sequence of SEQ ID NO: 2.
  • the Fokl nuclease when combined with a zinc-finger DNA binding domain or a DNA binding domain, has the activity to introduce a single or double strand break into the DNA at a defined target site.
  • ZFNs and TALENs can introduce a single strand break into the DNA at a defined target side, they are often referred as zinc finger nickase (ZFNickase) and Transcription- activator-like effector nickases(TALE nickase), respectivily.
  • Cas9 as nuclease has the advantage that it solely requires the expression of the Cas9 nuclease protein in combination with one short, synthetic chimeric tracr/crRNA (a "single-guide RNA") or two short, synthetic tracr/crRNAs (a "dual-guide RNA”) that define the target specificity.
  • Cpfl as nuclease has the advantage that it solely requires the expression of the Cpfl nuclease protein in combination with one short, synthetic crRNA that defines the target specificity. Therefore, using Cas9 or Cpfl as nuclease represents a considerable simplification in the generation of target-specific single- or double-strand breaks (Cas9) or double strand breaks (Cpfl ).
  • Cas9 or Cpfl is used as site-specific DNA nuclease.
  • the nucleotide and amino acid sequences of Cfpl nucleases are commonly known in the art.
  • AsCpfl or LbCpfl may be used.
  • the Cpfl nuclease that is used in context of the invention may have a nucleotide sequence that has at least 80%, preferably, at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99%, and most preferably 100% identity to the nucleotide sequence of SEQ ID NO: 7 or 9.
  • the Cpfl nuclease may have an amino acid sequence that has at least 80%, preferably, at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99%, and most preferably 100% identity to the amino acid sequence of SEQ ID NO: 8 or 10.
  • the Cpfl nuclease when combined with a guide RNA comprising a target sequence specific crRNA molecule, has the activity to introduce double strand breaks into the DNA at a defined target site.
  • a Cas9 nuclease is used as site-specific DNA nuclease in the in vivo genome editing method of the invention.
  • the Cas9 nuclease is Streptococcus pyogenes Cas9 (SpCas9) or Staphylococcus aureus Cas9 (SaCas9).
  • Cas9 orthologs include those derived from Streptococcus thermophilus, Neisseria meningitides and Francisella novicida.
  • the Cas9 nuclease may also be a SpCas9 mutant such as eSpCas9 (Ian, 2016, Science, 351 : 84-88) or SpCas9-HF1 (Kleinstiver, 2016, Nature, 529: 490- 495), which can induce more specific cleavages than the original SpCas9.
  • the Cas9 nuclease is SpCas9 or SaCas9.
  • the nucleotide and amino acid sequences of Cas9 nucleases such as SpCas9 or SaCas9 are commonly known in the art.
  • the Cas9 nuclease that is used in context of the invention may have a nucleotide sequence that has at least 80%, preferably, at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99%, and most preferably 100% identity to the nucleotide sequence of SEQ ID NO: 3 or 5.
  • the Cas9 nuclease may have an amino acid sequence that has at least 80%, preferably, at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99%, and most preferably 100% identity to the amino acid sequence of SEQ ID NO: 4 or 6.
  • the Cas9 nuclease when combined with a guide RNA comprising a target sequence specific crRNA molecule and a trans-activating crRNA (tracrRNA) molecule, has the activity to introduce single or double strand breaks, preferably double strand breaks, into the DNA at a defined target site.
  • the specificity of the herein provided in vivo genome editing method may be further enhanced by using Cas9 variants with minimum or no off-target effects while retaining comparable on-target cleavage activity (Kleinstiver, 2016, Nature, 529: 490-495; Slaymaker, 2016, Science 351 , 84-88).
  • Cas9 is guided by a specificity-determining guide-RNA sequence (CRISPR RNA (crRNA)) that is associated with a trans-activating crRNA (tracrRNA) and forms Watson-Crick base pairs with the complementary DNA target sequence, resulting in site-specific double strand breaks (Heidenreich, 2016, Nature Reviews Neurosciences, 17: 36-44).
  • CRISPR RNA crRNA
  • tracrRNA trans-activating crRNA
  • a simple two-component system consisting of Cas9 and a fusion of the tracrRNA-crRNA duplex to a "single-guide RNA", "sgRNA"
  • a simple three-component system consisting of Cas9, a tracrRNA molecule and a crRNA molecule, wherein the two RNA molecules are forming a "dual-guided RNA”
  • Cpf 1 a single-RNA-guided nuclease, which only uses a crRNA and does not use a tracrRNA, can also be used for genome editing.
  • different Cas proteins can be targeted to specific DNA sequences simply by changing the short specificity-determining part of the guide RNA, which can be easily achieved in one cloning step.
  • the genome editing machinery may further comprise:
  • At least one guide RNA consisting of at least one target sequence specific CRISPR RNA (crRNA) molecule and at least one trans-activating crRNA (tracrRNA) molecule;
  • crRNA target sequence specific CRISPR RNA
  • tracrRNA trans-activating crRNA
  • At least one guide RNA which is a chimeric RNA molecule comprising at least one target sequence specific crRNA and at least one tracrRNA; or
  • target sequence specific CRISPR RNA crRNA
  • Makarova, 2011 Nat Rev Microbiol, 9: 467-477
  • Makarova, 2011 Biol Direct, 6: 38
  • Bhaya, 2011 Annu Rev Genet, 45: 273-297
  • Barrangou, 2012 Annu Rev Food Sei Technol, 3: 143-162
  • Jinek, 2012 Science, 337: 816-821 , Cong, 2013, Science, 339: 819-823; Mali, 2013, Science 339: 823826 or Hwang, 2013, Nature Biotechnology, 31 : 227-229.
  • crRNAs differ depending on the Cas9 system but typically contain a sequence complementary to the target sequences (or complementary to a part of the target sequence) of between 10 and 30, preferably between 15 and 25 (e.g. about 20) nucleotides length, flanked by two direct repeats (DR) of a length of between 21 to 46 nucleotides.
  • the 3' located DR of the crRNA is complementary to and hybridizes with the corresponding tracrRNA, which in turn binds to the Cas9 protein.
  • the preferred DR sequence for use with the SpCas9 or SaCas9 nuclease is the sequence as shown in SEQ ID NO: 71 (i.e. GTTTT AG AG CTA) .
  • DR sequences functioning together with Cas9 nucleases of other bacterial species may be identified by bioinformatic analysis of sequence repeats occurring in the respective Crispr/Cas operons and by experimental binding studies of the Cas9 nuclease and tracrRNA together with putative DR sequence flanked target sequences, as shown by Deltcheva, 201 1 , Nature, 471 : 602-607.
  • tracrRNA trans-activating crRNA
  • the term "tracrRNA” refers to a small RNA, that is complementary to and base pairs with a crRNA, thereby forming an RNA duplex.
  • the tracrRNA may also be complementary to and base pair with a pre-crRNA, wherein this pre-crRNA is then cleaved by an RNA- specific ribonuclease, to form a crRNA/tracrRNA hybrid.
  • the "tracrRNA” contains a sequence complementary to the palindromic repeat of the crRNA or of the pre-crRNA. Therefore it can hybridize to a crRNA or pre-crRNA with direct repeat.
  • the crRNA/tracerRNA hybrid is the so-called "guide RNA", which acts as a guide for the Cas9 nuclease, which cleaves the invading nucleic acid.
  • the preferred tracrRNA sequence for use with the SpCas9 or SaCas9 nuclease is shown herein in SEQ ID NO: 72 (i.e. TAGCAAGTTAAAATAAGGCTAGTCCGTTTTT).
  • a dual-guide RNA i.e. a guide RNA consisting of at least one target sequence specific CRISPR RNA (crRNA) molecule and at least one tracrRNA molecule
  • a desired target sequence e.g. a desired protein encoding gene
  • a dual-guide RNA may be designed by designing a crRNA and tracrRNA separately, a crRNA may be designed by a sequence that is complementary to the target sequence with a part or the entire DR sequence, a tracrRNA may be synthesized under the optimal promoter (e.g. U6 promoter) as shown by Jinek, Science, 337: 816-821 .
  • Chimeric RNA molecules comprising at least one target sequence specific crRNA and at least one tracrRNA (i.e. single-guide RNAs, sgRNAs) that target a desired target sequence (e.g. a desired protein encoding gene) can easily be designed by using routine techniques.
  • a chimeric RNA may be designed by the fusion of a sequence that is complementary to the target sequence (or complementary to a part of the target sequence) of 10-30, preferably 15-25 (e.g. about 20) nucleotides in length with a part or the entire DR sequence and with a part or the entire of a tracrRNA, e.g. as shown by Jinek, Science, 337: 816-821.
  • RNAs may be designed by unbiased genome-wide analysis to minimize the potential off-target cleavages by Cas9 (Ran, 2013, Nat Protoc 8 2281 -2308). Therefore, an online tool may be used (the CRISPR design tool, http://crispr.mit.edu/).
  • the genome editing machinery comprises a polynucleotide encoding at least one chimeric RNA molecule comprising at least one target sequence specific crRNA and at least one tracrRNA ("single-guide RNA", "sgRNA").
  • This polynucleotide may comprise a sequence encoding a sequence that is complementary to the target sequence (or complementary to a part of the target sequence) of about 20 nucleotides in length followed by a guide RNA scaffold sequence of about 76 nucleotides in length. This scaffold sequence encodes the DR sequence and the tracrRNA.
  • the site-specific nuclease is Cpfl.
  • the genome editing machinery may further comprise:
  • RNAs for a Cpfl nuclease are commonly known in the art.
  • a guide RNA may be designed by designing a sequence complementary to the target sequence of -20 nucleotides which follows a T-rich PAM as shown in SEQ ID NO: 73 (i.e. TTTN) on the 5' side (Zetsche, 2015 Cell 163 759-71 ).
  • the crRNA contains sequence that is complementary to the target sequence (or complementary to a part of the target sequence) of 10-30, preferably 15-25 (e.g. about 20) nucleotides in length.
  • the crRNA for Cpfl comprises a sequence that is complementary to the target sequence (or complementary to a part of the target sequence) of about 20 nucleotides in length followed by a 19 nucleotide sequence.
  • This 19 nucleotide sequence is a short stem-loop structure in the direct repeat.
  • Cpfl does not require an additional tracrRNA.
  • the polynucleotide encoding the Cas9 or Cpfl nuclease and the polynucleotide encoding the guide RNA may be comprised in one single nucleic acid sequence, for example in one plasmid vector.
  • separate nucleic acid sequences, e.g. separate plasmid vectors, encoding either the Cas9/Cpfl nuclease or the guide RNA may be delivered to mitotic progenitor cells.
  • multiple guide RNAs may be used to target several genes at once. This method may allow editing of multiple genes, e.g., for studying genetic interactions or modeling multigenic disorders.
  • the genome editing machinery comprises two or more different guide RNAs or two or more different polynucleotides encoding different guide RNAs.
  • the genome editing machinery comprises two or more different guide RNAs or two or more different polynucleotides encoding different guide RNAs.
  • 2-10 preferably 2-3, different guide RNAs (i.e. single- or dual-guide RNAs) or 2-10, preferably 2-3, different polynucleotides encoding different guide RNAs (i.e. single- or dual-guide RNAs) may be used in context of the present invention.
  • RNAs i.e. single- or dual-guide RNAs
  • polynucleotides encoding different guide RNAs i.e. single- or dual-guide RNAs
  • the HDR efficiency may be increased by the direct delivery of pre- assembled Cas9 protein-guide RNA ribonucleoprotein complexes (RNPs), rather than expressing these components from plasmids.
  • RNPs Cas9 protein-guide RNA ribonucleoprotein complexes
  • one embodiment of the present invention relates to the herein provided in vivo genome editing method, wherein the editing machinery comprises at least one pre-assembled Cas9 protein-guide RNA ribonucleoprotein complex (RNP), which introduces a double or single strand break within the target sequence.
  • RNP Cas9 protein-guide RNA ribonucleoprotein complex
  • the skilled person readily knows how to introduce such a complex into progenitor cells via in utero electroporation, since this complex has been efficiently introduced by the electroporation method in vitro (Lin, 2014, eLife, 3: e04766).
  • the HDR efficiency may be increased by genetic or pharmacological inhibition of the NHEJ pathway may (see, e.g., Chu, 2015, Nature biotechnology, 33: 543-548; Maruyama, 2015, Nature biotechnology, 33: 538-542).
  • HDR can be induced if a donor template is present; see, e.g., Heidenreich, 2016, Nature Reviews Neurosciences, 17: 36-44; Cong, 2013, Science, 339: 819-23; Doudna, 2014, Science, 346: 1258096; Hsu, 2014,Cell 157: 1262-78.
  • one aspect of the invention relates to the herein provided in vivo genome editing method, wherein in step (b) after introducing a single or double strand break within the target sequence, the target sequence is modified by HDR, and wherein the genome editing machinery further comprises at least one donor template polynucleotide comprising a donor nucleic acid sequence and regions homologous to the target sequence.
  • the donor template polynucleotide is preferably a single-stranded oligodeoxynucleotide (ssODN).
  • ssODN single-stranded oligodeoxynucleotide
  • the regions homologous to the target sequence are preferably localized at the 5' and 3' end of the donor nucleic acid sequence.
  • donor template polynucleotide also called “DNA donor template”, see, e.g. Heidenreich, 2016, Nature Reviews Neurosciences, 17: 36-44 refers to a nucleic acid sequence that serves as a template in the process of homologous recombination and that carries the modification that is to be introduced into the target sequence.
  • the genetic information including the modification(s), is copied into the target sequence within the genome of the embryo.
  • the donor template polynucleotide can be identical to the part of the target sequence to be replaced, with the exception of one nucleotide that differs and results in the introduction of a point mutation upon homologous recombination or it can comprise an additional gene previously not present in the target sequence.
  • the template polynucleotide may be a single-stranded nucleic acid molecule (i.e. a ssODN).
  • the donor template polynucleotide comprises regions that are homologous to the target sequence, or to parts of the target sequence.
  • plasmid vectors can be used as donor template polynucleotide, i.e. a (plasmid based) double-stranded DNA may be used as donor template polynucleotide.
  • regions homologous to the target sequence also called “homology arms” or “regions homologous to parts of the target sequence” refer to regions having sufficient sequence identity to ensure specific binding to the target sequence or to parts of the target sequence, respectively.
  • the regions homologous to the target sequence flank the donor nucleic acid sequence that carries the modification to be inserted. Therefore, it is preferred that at least two regions homologous to the target sequence or homologous to a part of the target sequence are present in the donor template polynucleotide.
  • Methods to evaluate the identity level between two nucleic acid sequences are well known in the art. For example, the sequences can be aligned electronically using suitable computer programs known in the art.
  • Such programs comprise BLAST (Altschul, 1990, J Mol Biol, 215: 403), variants thereof such as WU- BLAST, which is called AB-BLAST now (Altschul, 1996, Methods Enzymol, 266: 460), FASTA (Pearson, 1988, Proc Natl Acad Sei USA, 85: 2444) or implementations of the Smith-Waterman algorithm (SSEARCH, Smith, 1981 , J Mol Biol, 147: 195).
  • BLAST Altschul, 1990, J Mol Biol, 215: 403
  • WU-BLAST which is called AB-BLAST now (Altschul, 1996, Methods Enzymol, 266: 460)
  • FASTA Pearson, 1988, Proc Natl Acad Sei USA, 85: 2444
  • SSEARCH Smith-Waterman algorithm
  • the "regions homologous to the target sequence” or the “regions homologous to parts of the target sequence” have a sequence identity with the corresponding target sequence or with a part of the corresponding target sequence of at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, even more preferably at least 99.9% and most preferably 100%.
  • sequence identities are defined only with respect to those target sequences or parts of target the sequence that serve as binding sites for the homology arms.
  • the overall sequence identity between the entire donor template polynucleotide and the target sequence or a part of the target sequence can differ from the above defined sequence identities, due to the presence of the part of the donor template polynucleotide that is to be inserted into the target sequence (i.e. the donor nucleic acid sequence).
  • the donor template polynucleotide is a chimeric polynucleotide comprising the donor nucleic acid sequence and the regions homologous to the target sequence or to parts of the target sequence. It is preferred that in the donor template polynucleotide the regions homologous to parts of the target sequence are localized at the 5' and 3' ends of the donor nucleic acid sequence. Accordingly, in this preferred embodiment, the donor nucleic acid sequence is flanked by the two regions homologous to parts of the target sequence.
  • the donor template polynucleotide used in the in vivo genome editing method of the present invention comprises of a first region homologous to a part of the target sequence, followed by the donor nucleic acid sequence, which is followed by a second region homologous to a part of the target sequence.
  • the donor template polynucleotide may be a single-stranded oligodeoxynucleotide (ssODN).
  • oligodeoxynucleotide ODN
  • ODN oligodeoxynucleotide
  • An ODN is a single-strand ODN (ssODN) if it does not hybridized with a second, different (i.e. complementary or partially complementary) oligonucleotide strand.
  • the ssODN may fold back onto itself, thus forming a partial or complete double stranded molecule consisting of one oligodeoxynucleotide strand. However, it is preferred that the ssODN does not fold back to form a partial or complete double stranded molecule but instead is single-stranded over its entire length.
  • An ODN in accordance with the present invention refers to both oligodeoxynucleotide and polydesoxynucleotides and is between 30 and 600 nucleotides in length, preferably between 70 and 500 nucleotides in length, even more preferably between 100 and 350 nucleotides in length, and most preferably between 150 and 250 nucleotides (e.g. about 200 nucleotides) in length.
  • an epitope tag such as a HA-tag, which is about 27 nucleotides long or a FLAG-tag, which is about 24 nucleotides long
  • an about 150-250 (e.g. about 200) nucleotides long ssODN may be used.
  • the ssODN preferably comprises regions homologous to parts of the target sequence that are >40 nucleotides, preferably 40-150 nucleotides, more preferably about 80 nucleotides, flanking the donor nucleic acid sequence (i.e. insertion sequence) at both sides.
  • a double strand DNA fragment which comprises an insertion sequence franked by regions homologous to parts of the target sequence of about 0.5-2 kb, preferably of about 0.6-1.2 kb (e.g. 0.7-0.9 kb), at both sides is preferably used as donor template polynucleotide.
  • Donor template polynucleotides that induce HDR in a desired target sequence can easily be designed by using routine techniques, e.g. as described in Ran, 2013, Nat Protoc 8 2281 -2308.
  • the herein provided in vivo genome editing method in order to test the specificity of the used construct, incorrect ssODNs-sgRNA pairs may be used as a control.
  • NHEJ inhibitor Scr7; Maruyama, 2015, Nat Biotechnol, 33(5): 538-542; Chu, 2015, Nat Biotechnol, 33(5): 543-548
  • HDR enhancer Rad51 mRNA and RS-1 ; Song, 2016, Nat Commun, 7: 10548, doi: 10.1038/ncomms10548
  • the herein provided in vivo genome editing method may comprise adding a NHEJ inhibitor and/or a HDR enhancer to the progenitor cells.
  • constructs that induce HDR may be introduced into the mitotic progenitor cells together with a plasmid vector comprising a polynucleotide sequence encoding a transposon and a separate plasmid vector comprising a polynucleotide sequence encoding a transposase.
  • the transposase may comprise a transgene (e.g.
  • a fluorescent protein such as monomeric EGFP (mEGFP)
  • terminal repeats flanking the transgene of interest for transposition into the genome to occur Cadinanos, 2007, Nucleic Acids Res, 35: e87; Chen, 2012, J Neurosci Methods, 207(2): 172-180.
  • mEGFP monomeric EGFP
  • any transgene with flanking terminal repeats may be used as transposon.
  • the transposon system used in context of the present invention may be Sleeping Beauty (SB) or piggybac (PB) (VandenDriessche, 2009, Blood, 1 14:1461 -8).
  • Piggybac Compared to Sleeping Beauty, piggybac has a more precise "cut and paste" mechanism, higher transposition efficiency and larger cargo capacity (Chen, 2012, J Neurosci Methods, 207(2): 172- 180). Therefore, in context of the present invention it is preferred that the Piggybac transposon system is used.
  • hyperactive piggybac transposase and piggybac transposon vectors expressing a fluorescent protein (e.g. mEGFP) may be used as a marker of transfection (Chen, 2012, J Neurosci Methods, 207(2): 172-180; Loulier, 2014, Neuron, 81 : 505-520, Yusa, 2011 , Proc Nat Ac Sc USA, 108: 1531 -1536).
  • a fluorescent protein e.g. mEGFP
  • Such a transposon system induces genomic integration of the transgene, preventing the dilution of the transgene (e.g. a fluorescent protein) during cell divisions.
  • the transposon does not contribute to HDR-mediated genome editing.
  • the knock-in efficiency may be quantified as the ratio of the amount of introduced tag (e.g. HA-tag)/mEGFP double- positive cells to that of mEGFP positive cells.
  • Such a transposon system has the additional advantage that potential effects of the in utero electroporation in transfected cells that did not undergo HDR can be evaluated.
  • transposon and the transposase may be encoded by one chimeric or by two separate polynucleotides.
  • the genome editing machinery comprises:
  • the polynucleotides encoding the transposon and/or the transposase may include vector sequence.
  • the polynucleotides encoding the transposon and/or the transposase may be plasmid vector(s).
  • the vector endocing the transposon and/or the transposase is not restricted to be a circular vector; also linearized vectors may be used to encode the transposon and/or the transposase.
  • the transposon and the transposase are introduced with separate vectors.
  • a transposon-based gene expression vector contains terminal repeats both at 5'- and 3'-sides of a transgene; the other components in the vector are the same as in other expression vectors (e.g., promoter, polyA etc.).
  • the transposon may comprise an epitope tag and/or a fluorescent protein.
  • fluorescent proteins that can be used include CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein) and many other derivatives.
  • the fluorescent protein is mEGFP, tdTomato or DsRed2.
  • mEGNF, DsRed2 or tdTomato may be inserted in the piggybac transposon system.
  • pPB-CAG-mEGFP, pPB- CAGDsRed2 or pPB-CAG-tdTomato may be constructed by subcloning mEGFP, DsRed2 or tdTomato into pPBCAG.
  • Introducing a fluorescent protein as a marker of transfection has the advantage that positive transfected pups can be selected just after birth, e.g. by using epifluorescence.
  • the target sequence comprises at least one of the following sequences:
  • the target sequence is a genomic DNA sequence. It is preferred that the target sequence is a protein encoding gene. Said protein encoding gene is modified in step (b) of the herein provided in vivo genome editing method in the coding or non-coding region.
  • the target sequence may be selected so that the cleavage sites of the site-specific nuclease (e.g. of Cas9) are located either in the non-coding region within about 10 bp (preferably within about 2 bp) upstream of the start codon or within 10 bp downstream from the stop codon.
  • the protein encoding gene may be modified in the 3'-untranslated region and/or in the 5'- untranslated region of the gene.
  • the target sequences and donor template polynucleotides e.g. the ssODNs
  • the site-specific nuclease e.g. Cas9
  • the donor temple polynucleotide e.g. the ssODN
  • the Cas9 recognition sequences are changed after HDR is completed. If the Cas9 recognition sequence exists after HDR is completed, a mutation may be introduced in the donor template polynucleotide to disrupt PAM sequence.
  • the cleavage site of the site-specific nuclease e.g. of Cas9
  • the modification of the target sequence e.g. integration of an HA-tag
  • the modification of the target sequence may be located just downstream (i.e. 0-100bp for a tag insertion; 1-1000bp for a point mutation) of the start codon.
  • potential effects of NHEJ may be evaluated for each gene depending on the purpose of experiments.
  • the target is a secreted or a type I membrane protein with a signal sequence
  • one may select a target sequence for CRISPR-Cas9 mediated cleavage to minimize the possibility of deletion or mislocalization of the gene products.
  • one may select the target sequences so that the estimated cleavage site (3 bp upstream of the PAM) can be located in the untranslated region.
  • the genome modification e.g. insertion of an epitope tag or of a fluorescent protein into an endogenous protein
  • the genome modification is performed at the 5'-untranslated region or near the stop codon (i.e. within 10 bp downstream from the stop codon).
  • This strategy enables N- or C-terminal tagging and minimizes the effect of NHEJ.
  • NHEJ was not observed in most of the neurons.
  • NHEJ mediated gene knockout it is necessary to induce frameshift mutations, which leads to premature stop codons, within coding exons of the target gene. Therefore, target sequences and strategy that is used for inducing NHEJ considerably differs from those useful for the herein provided in vivo genome editing method.
  • the protein encoding gene may also be modified in the coding region, which may result in a loss of function of the encoded protein.
  • HDR enables versatile modification of the genome such as insertion, deletion or replacement of a sequence.
  • one aspect of the invention relates to the herein provided in vivo genome editing method, wherein the modification of the target sequence is substitution, insertion, or deletion of at least one nucleotide of the target sequence.
  • the modification of the target sequence includes introduction of specific mutations or transgenes.
  • CRISPR-Cas9 enables multiplex gene editing and in utero electroporation allows co-transfection of multiple plasmids into the same cells with high efficiency. Therefore, by using the herein provided in vivo method it is possible to target many genes in a single.
  • one aspect of the invention relates to the herein provided in vivo method, wherein at least two target sequences are modified.
  • 2-10 preferably 1 -3 (e.g. 1 or 2) different target sequences (preferably protein encoding genes) may be modified by using the herein provided in vivo genome editing method.
  • one aspect of the invention relates to the herein provided in vivo genome editing method, wherein the modification of the at least one target sequence is any one of the following modifications:
  • disease model animals e.g. mice
  • DMD Duchenne muscular dystrophy
  • nonsyndromic mental retardation to nonsyndromic mental retardation
  • Fragile X syndrome may be introduced by using the in vivo genome editing method of the invention.
  • one aspect of the present invention relates to a disease model animal (e.g. mouse) or the offspring of said disease model animal (e.g.
  • the disease model animal may be a disease model for the Rett syndrome, muscular dystrophies, such as Duchenne muscular dystrophy (DMD), nonsyndromic mental retardation, nonsyndromic mental retardation, or Fragile X syndrome.
  • DMD Duchenne muscular dystrophy
  • nonsyndromic mental retardation nonsyndromic mental retardation
  • Fragile X syndrome Fragile X syndrome
  • the herein provided in vivo genome editing method is used to develop in vivo models of brain tumors. Mutations in isocitrate dehydrogenase (IDH) 1 and 2 occur in the vast majority of low grade gliomas and secondary high grade gliomas. All mutations identified to date have been a single amino acid missense mutation in IDH1 at arginine 132 (R132) or the analogous residue in IDH2 (R172) (Cohen, 2013, Curr Neurol Neurosci Rep, 13(5): 345).
  • IDH isocitrate dehydrogenase
  • gliomas are astrocytic (70%) and the herein provided in vivo genome editing method allows precise genome editing in the astrocyte (see, e.g., Fig2.D and H), gliomas would be a good target to introduce mutations by the herein provided method to generate an in vivo model of de novo brain tumors.
  • the number of knock-in cells would be increasing due to the proliferation of cancer cells and knock-in cells/regions might be identified by the mass of cancer tissue. Thus, identification of knock-in cells might be easy in this case.
  • brain tumor model may be produced by the herein provided in vivo genome editing method by mutating the BRAF gene.
  • brain tumor models such as models for pleomorphic xanthoastrocytoma, ganglioglioma, or pilocytic astrocytoma, may be produced by introducing a V600E mutation in the BRAF gene (Dias-Santagata, 2011 , Plos One 29;6(3): e17948).
  • Rett syndrome there are eight common mutations, which arise at CpG hotspots in MECP2 and result in loss of function due to truncated, unstable or abnormally folded proteins.
  • R168X 11 .5%
  • R270X 9%
  • R255X 8.7%
  • T158 M 8.3%
  • R306C 6.8%
  • one or more of these mutations may be introduced into a non-human animal (e.g. a mouse) by using the herein provided in vivo genome editing method for preparing an animal model for the Rett syndrome.
  • DMD Duchenne muscular dystrophies
  • R54L missense human mutation
  • the R54L mutation may be introduced into a non- human animal (e.g. a mouse) by using the herein provided in vivo genome editing method for preparing an animal model for DMD.
  • the SYNGAP1 gene may be mutated.
  • the mutations K138X and/or R579X may be introduced into the SYNGAP1 gene by using the herein provided in vivo genome editing method (Hamdan, 2009, NEJM 360:599-605).
  • the IQSEQ2 gene may be mutated by the herein provided in vivo genome editing method.
  • one or more (e.g. all) of the mutations R359C, R758Q, Q801 P, R863W and E849K may be introduced in the IQSEQ2 gene (Shoubridge, 2010, Nature Genetics 42:486-488).
  • an animal model for Fragile X syndrome may be prepared by using the in vivo genome editing method of the present invention. Therefore, the mutation I367N may be introduced in the FMR1 gene (De Boulle, 1993, Nature Genetics 3:31-35).
  • the modification of the at least one target sequence may be insertion of an epitope tag and/or a fluorescent protein.
  • the epitope tag may be a HA-tag and/or the fluorescent protein may be monomeric EGFP (mEGFP).
  • the epitope tag may also be a FLAG-tag. If the herein provided in vivo genome editing method is combined with a transposon system (e.g. with the piggyback transposon system) then the epitope tag/fluorescent protein that is introduced via HDR is preferably different from the epitope tag/fluorescent protein that is introduced via the transposon system.
  • HA-tag The nucleotide and amino acid sequences of the HA-tag are shown herein as SEQ ID NOs: 11 and 12, respectively.
  • the nucleotide and amino acid sequences of the FLAG-tag are shown herein as SEQ ID NOs: 15 and 16, respectively.
  • the nucleotide and amino acid sequences of mEGFP are shown herein as SEQ ID NOs: 13 and 14, respectively.
  • a HA-tag is inserted into the at least one target sequence.
  • multiple copies of epitope tags e.g. multiple HA-tags
  • This has the advantage that an increased sensitivity can be gained.
  • the method of the invention advantageously allows labeling of two different species of proteins with different tags in single cells, providing a valuable tool for co-localization assays of a pair of endogenous proteins.
  • a HA-tag may be introduced into one target gene and a FLAG-tag may be introduced into another target gene.
  • endogenous subcellular protein localization may be examined in the context of a knockout of a different protein.
  • a method advantageously provides functional insight into the interactions between the visualized and deleted protein.
  • one protein may be labeled with a tag and/or fluorescent protein by using HDR and another gene may be knocked out through NHEJ or HDR.
  • HDR a tag and/or fluorescent protein
  • Such a method allows for the study of cell autonomous gene function and allows comparing normal and knockout cells in the same tissue. This method may be useful for analyzing the molecular mechanisms genetic diseases (such as the Rett syndrome).
  • mitotic cells can be targeted by using the herein provided in vivo genome editing method, such as mitotic progenitors in the developing brain, mitotic progenitors in the developing spinal cord, or mitotic progenitors of muscle cells.
  • the mitotic progenitor cells are progenitor cells of the developing brain, progenitor cells of the developing spinal cord, or progenitor cells of muscle cells.
  • the progenitor cells of the developing brain or of the developing spinal cord may be progenitor cells of neurons.
  • the mitotic progenitor cells are progenitor cells of the developing brain (e.g. progenitor cells of neurons).
  • HA-tag By fusing a HA-tag to either the N- or C-terminus of a variety of proteins including nuclear, cytoskeletal, vesicular, cytosolic and membrane proteins, the appended Examples demonstrate that the method of the invention can be used to visualize the subcellular localization of a broad spectrum of endogenous proteins in brain tissue.
  • the progenitor cells of the developing brain are progenitor cells of cortical pyramidal neurons, CA1 pyramidal neurons, CA3 pyramidal neurons in the hippocampus, dentate granule cells in the hippocampus, spiny stellate cells in the subiculum, granule cells in the olfactory bulb, medium spiny neurons in the striatum, basolateral amygdala neurons, granule cells in the cerebellum, Purkinje cells in the cerebellum, and/or glial cells.
  • the mitotic progenitor cells may also be progenitor cells of muscle cells.
  • progenitor cells of muscle cells may be progenitor cells of cardiomyocytes or progenitor cells of skeletal muscle cells.
  • the mitotic progenitor cells are progenitor cells of the developing brain (such as progenitor cells of neurons).
  • the in utero electroporation may be performed at any development stage of the embryo, provided that mitotic progenitor cells are targeted.
  • embryonic day (E) is commonly known in the art and refers to the day of development of the embryo. Regarding rodents such as mice, the day on which the vaginal plug is detected can be designated as embryonic day 0 (EO).
  • the non-human embryo is at any stage from embryonic day 10 (E12) to embryonic day 18 (E18), preferably from E10 to E13, most preferably at E12. Accordingly, in the herein provided method in utero electroporation may be performed from E10 to E18, preferably from E10 to E13, most preferably at E12. It is indicated that from E10 to E13, and particularly at E12, cortical progenitors for layer 2/3 neurons retain much higher HDR activity than neuro-progenitor cells at E15.
  • layer 2/3 neurons comprise the external granular/pyramidal layers, containing small to medium size pyramidal neurons. As can be seen in Fig. 1 , layer 2/3 neurons can be identified with DAPI staining by labeling the nucleus in these cells.
  • in utero electroporation may be performed at different time points depending on the progenitor cells to be targeted. More preferably, the progenitor cells in the cortex of the developing brain are targeted. Therefore, in utero electroporation is more preferably performed from E12 to 13. For example, for targeting progenitor cells in layer 2/3 pyramidal neurons in the cerebral cortex in utero electroporation may be performed from E12 to 13. For targeting progenitor cells of CA1 pyramidal neurons, or progenitor cells of dentate granule cells in the hippocampus in utero electroporation may be performed at E13.
  • Also for targeting progenitor cells of spiny stellate cells in the subiculum in utero electroporation may be performed at E13.
  • For targeting progenitor cells of granule cells in the olfactory bulb in utero electroporation may be performed at E12.
  • For targeting progenitor cells of medium spiny neurons in the striatum in utero electroporation may be performed from E1 1 to 12.
  • For targeting progenitor cells of basolateral amygdala neurons in utero electroporation may be performed from E1 1 to 12.
  • For targeting progenitor cells of granule cells in the cerebellum in utero electroporation may be performed at E13.
  • the non-human embryo may be the embryo of a mammal, of an avian or of a fish.
  • the non-human embryo is a non-human mammalian embryo.
  • the non-human mammalian embryo may be an embryo of a rodent, an embryo of a dog, an embryo of a felid, an embryo of a primate, an embryo of a rabbit, an embryo of a pig, or an embryo of a ruminant.
  • Non-limiting examples of "rodents” are mice, rats, squirrels, chipmunks, gophers, porcupines, beavers, hamsters, gerbils, guinea pigs, degus, chinchillas, prairie dogs, and groundhogs.
  • Non-limiting examples of "dogs” include members of the subspecies canis lupus familiaris as well as wolves, foxes, jackals, and coyotes.
  • Non-limiting examples of "felides” include members of the two subfamilies: the pantherinae, including lions, tigers, jaguars and leopards; and the felinae, including cougars, cheetahs, servals, lynxes, caracals, ocelots and domestic cats.
  • the term "primates”, as used herein, refers to all monkeys including for example cercopithecoid (old world monkey) or platyrrhine (new world monkey) as well as lemurs, tarsiers, apes and marmosets (Callithrix jacchus).
  • the non- human mammalian embryo is an embryo of a mouse, an embryo of a rat, or an embryo of a ferret. It is most preferred that the non-human embryo is an embryo of a mouse.
  • the mother animal of the non-human embryo i.e. the animal who is carrying the uterus wherein the embryo is developing
  • the mother animal of the non-human embryo is of the same species as the non-human embryo.
  • the in vivo genome editing method of the invention may comprise to grow the embryo into an offspring.
  • the offspring obtained from the embryo is an individual in which a region in genomic DNA has been modified. That is, an individual in which a target sequence (e.g. a protein encoding gene) has been modified can be obtained by the herein provided method.
  • a target sequence e.g. a protein encoding gene
  • one aspect of the present invention related to the herein provided in vivo method further comprising to let the embryo develop to a non-human animal. Accordingly, in one aspect of the invention the embryo will develop to a non-human animal and will be born. After birth of the non-human animal a cell that has been developed from the progenitor cell that has been modified during the herein provided in vivo genome editing method may be obtained from the non-human animal.
  • a cell that has been developed from the progenitor cell that has been modified during the herein provided in vivo genome editing method may be obtained from the non-human embryo itself.
  • one aspect of the invention relates to the herein provided in vivo genome editing method, comprising obtaining a modified cell from the non-human embryo or from the non-human animal (i.e. from the non-human animal that has been developed from the non-human embryo).
  • one aspect of the present invention relates to a non- human embryo carrying a modified target sequence in its genome, wherein said non-human embryo is produced by the herein provided in vivo genome editing method.
  • the non-human embryo of the invention carries a HDR-mediated modification in its genome in a progenitor cell or in a post-mitotic cell that has been developed from a progenitor cell (e.g. a neuron).
  • one aspect of the invention relates to a non- human embryo, which is produced by the in vivo genome editing method of the invention, wherein the non- human embryo carries a HDR-mediated genome modification in a progenitor cell or in a post-mitotic cell.
  • the non-human embryo of the invention may comprise a HA-tag sequence at the N-terminus of Doublecortin, which is expressed in postmitotic migrating and differentiating neurons in the developing brain.
  • the progenitor cell that carries a modification may be a progenitor cell of the developing brain, e.g. a progenitor cell of a neuron.
  • the post-mitotic cell that carries a modification may be a post-mitotic cell of the brain, e.g. a neuron.
  • a post-mitotic cell of the brain e.g. a neuron.
  • the in vivo genome editing method of the invention may comprise to grow the non- human embryo into an offspring resulting in a non-human embryo with a modified target sequence in its genome.
  • one aspect of the present invention relates to a non-human animal carrying a modified target sequence in its genome, wherein the non-human animal is produced by the herein provided in vivo genome editing method.
  • the non-human animal of the invention carries a HDR-mediated modification in its genome, preferably in a post-mitotic cell (e.g. in a neuron).
  • one aspect of the invention relates to a non-human animal, which is produced by the in vivo genome editing method of the invention, wherein the non-human animal carries a HDR-mediated genome modification in a post-mitotic cell.
  • the non-human animal of the invention may comprise a HA-tag sequence at the N-terminus of CamKlla, CamKI ⁇ , MeCP2, ⁇ -Adin, Rab11a, 14-3-3 ⁇ , FMRP, Arc and/or PKCct and/or at the C-terminus of CamKlla, CamKlfi, MeCP2 and/or Cav1.2.
  • the non-human animal of the invention may comprise a mEGFP sequence at the C-terminus of CamKlla and/or at the N-terminus of CamK//jS.
  • the non-human animal may comprise a FLAG-tag sequence at the N- terminus of CamK///3.
  • the post-mitotic cell that carries a modification may be a post-mitotic cell of the brain, e.g. a neuron. In the non-human animal of the invention not all cells (but only selected progenitor or post-mitotic cells) carry a genome modification.
  • a modified tissue or cells that has been developed from the progenitor cells that have been modified by using the in vivo genome editing method of the invention may be obtained from the non-human embryo of the invention (which carries a modified target sequence in its genome) or from the non-human animal of the invention (which carries a modified target sequence in its genome).
  • one aspect of the invention relates to a modified cell obtained from the non-human embryo of the invention or from the non-human animal of the invention.
  • This modified cell comprises the modification in its genome that has been introduced via HDR in the non-human embryo during the in vivo genome editing method of the invention.
  • the modified cell of the invention carries a HDR-mediated modification in its genome.
  • the modified cell of the invention is preferably a post-mitotic cell, such as differentiated neuron or a differentiated muscle cell. Accordingly, one aspect of the invention relates to a modified post-mitotic cell that has been obtained from the non-human embryo of the invention or from the non-human animal of the invention, wherein the modified post-mitotic cell carries a HDR-mediated modification in its genome. More preferably, the modified cell of the invention is a differentiated neuron.
  • the modified cell of the invention may comprise a HA-tag sequence at the N-terminus of Doublecortin.
  • the modified cell of the invention may comprise a HA-tag sequence at the N-terminus of CamKlla, CamKltf, MeCP2, ⁇ -Adin, Rab11a, 14-3-3 ⁇ , FMRP, Arc and/or PKCct and/or at the C-terminus of CamKlla, CamKlfi, MeCP2 and/or Ca v 1.2.
  • the modified cell of the invention may comprise a mEGFP sequence at the C-terminus of CamKlla and/or at the N-terminus of CamK///3.
  • the modified cell may comprise a FLAG-tag sequence at the N-terminus of CamK//jS.
  • a non-human animal e.g. a mouse comprising the modified cell of the invention.
  • the in vivo genome editing method of the invention may be applied to inducing various kinds of mutations in the genome, providing a variety of pathological cellular models in vivo.
  • this method may be used to introduce a pathological mutation and a tag sequence (to detect mutant cells) simultaneously to a gene of interest.
  • Such models have the advantage that the pathological cells can easily be compared with internal controls in the same tissue, which facilitates not only basic research but enables reliable assessment of drugs for therapeutic and side effects.
  • one aspect of the invention relates to the use of the non-human embryo of the invention, the non-human animal of the invention, or the modified cell of the invention, for selecting and/or evaluating a pharmaceutical agent or for identifying the expression, subcellular localization, distribution and/or dynamics of at least one endogenous protein.
  • the subcellular localization, distribution and/or dynamics of at least one endogenous protein may be analyzed by light or electron microscopy, e.g. by immunoelectron microscopy.
  • the PFA-fixed tissue slices or cells may be permeabilized with 0.3-0.4% Triton X-100 in PBS, blocked with 5% normal goat serum and 2% BSA in PBS or 5% normal donkey serum in PBS, and incubated overnight with a primary antibody against a tag protein such as HA or FLAG (or with primary antibodies against multiple tag proteins such as HA and FLAG) and with a primary antibody against an transfection marker such as GFP.
  • the slices or cells may then be incubated with Alexa Fluor-conjugated secondary antibodies for 1-3 hours followed by DAPI staining.
  • the stained slices or cells may be mounted on glass slides in Fluoromount-G.
  • live cells or tissue slices or the targeted organ may be directly visualized with light microscopy such as two-photon or confocal microscopy, or by electron microscopy.
  • immunostaining of brain slices may be performed at postnatal days 14-48 (P14-48). Therefore, an antibody against the tag that has been introduced into the target sequence (e.g. HA-tag) may be used.
  • the appended Examples demonstrate that HDR could be detected as early as 60 h after in utero electroporation, suggesting that HDR occurred rapidly, possibly within one or two days after in utero electroporation.
  • the in vivo genome editing method of the invention may be used for studying the protein localization (e.g. in the brain) from embryonic to adult stages.
  • Immunoelectron microscopy allows nanoscale visualization of endogenous proteins with defined ultrastructures in cells.
  • the lack of reliable antibodies compatible with electron microscopy imaging limits its application to a variety of proteins.
  • the appended Examples demonstrate that the herein provided in vivo genome editing method circumvents this problem by introducing epitope-tags into selected target genes and is thus useful for nanometer scale localization of endogenous proteins by using immunoelectron microscopy.
  • the method of the invention can also be used for labeling endogenous proteins for live Imaging. Labeling endogenous proteins with fluorescent proteins (such as mEGFP) by using the inventive method has the advantage that it allows for imaging of protein dynamics in live cells without overexpression artifacts.
  • One of the merits of the method of the invention is that high-quality antibodies can be used for the detection of inserted tags.
  • the method of the invention can be applied to image various tagged proteins without extensive optimization.
  • the herein provided method allows protein labeling in a sparse subset of cells in the tissue, the specificity of immunostaining can be easily validated by examining surrounding negative control cells in the same specimen.
  • drugs may be administrated into the herein provided modified non-human embryo or non- human animal in vivo or to the herein provided modified cell or tissue in vitro.
  • the modified non-human embryo, animal, tissue or cell may carry one or more disease specific mutations.
  • the therapeutic and side effects of the pharmaceutical agent may be analyzed by using visual inspection as well as light or electron microscopy. For example, the subcellular localization, expression and/or dynamics of endogenous proteins may be compared with those prior to administration of the pharmaceutical agent.
  • the herein provided in vivo genome editing method may also be used for visualization of endogenous proteins in human disease model animals (e.g. mice).
  • the in vivo genome editing method of the invention can be easily combined with the abundant existing resources of genetically modified animals.
  • human disease model animals e.g. mice
  • These should provide deep insights into the pathology of diseases. For example, one can image excitatory and inhibitory synapses in autism model mice by visualizing PSD-95 and gephyrin, excitatory and inhibitory synapse markers, respectively, with the herein provided in vivo genome editing method.
  • PSD-95 and gephyrin may be modified in the 3'-untranslated region and/or in the 5'-untranslated region by introducing an epitope tag or a fluorescent protein by using the herein provided method.
  • the epitope tag or the fluorescent protein may then be used to visualize PSD-95 and gephyrin.
  • Candidate drugs can be applied in disease model animals (e.g. mice) modified with the herein provided in vivo genome editing method and the disease- related protein(s) and/or molecular marker(s) of a disease can be visualized to determine effects of the drugs at molecular level in the course of diseases.
  • disease model animals e.g. mice
  • the disease- related protein(s) and/or molecular marker(s) of a disease can be visualized to determine effects of the drugs at molecular level in the course of diseases.
  • one aspect of the invention relates to a method for screening and/or evaluating drugs, comprising the steps:
  • step (c) effectively and/or side-effects of the candidate drug may be evaluated based on the effect of the candidate drug on the disease-related protein(s) and/or molecular marker(s).
  • the non-human animal is preferably a mouse and more preferably a disease model mouse.
  • mice There are many human disease model mice available that can be used in the above-described method for screening and/or evaluating drugs, such as the following:
  • Alzheimer's disease Double transgenic mice expressing a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1 -dE9) (Borchelt, 1996, Neuron 17: 1005-13)
  • Parkinson's disease A53T a-synuclein transgenic mice (Giasson, 2002, Neuron, 34:521 -533)
  • Rett syndrome MeCP2 knockout/conditional knockout mice (Guy, 2001 , Nature Genet. 27:322;
  • Fragile X syndrome Fmr1 knockout mice (Bakker, 1994, Cell, 78: 23-33)
  • One aspect of the present invention relates to the non-human animal that has been modified in step (a) of the above-described method for screening and/or evaluating drugs.
  • the invention also relates to the offspring of the non-human animal that has been modified in step (a) of the above-described method for screening and/or evaluating drugs.
  • the inventive method advantageously enables specific single-cell labeling of endogenous proteins by either N- or C-terminal epitope tagging.
  • the inventive method enables the precise evaluation of the localization of endogenous proteins and of the morphology of cellular structures without the potential morphological phenotypes caused by overexpression of fluorophore tagged-proteins.
  • the in vivo genome editing method of the invention is a highly generalizable technique that can be applied to various species of proteins from embryonic to adult stages.
  • the appended Examples demonstrate that the inventive method enables comparison of subcellular localization of endogenous proteins in various neuron subtypes and brain regions across developmental stages, providing regional and developmental specific information about protein localization.
  • the herein provided in vivo genome editing method is a scalable and high-throughput method that can, e.g., be used to identify the precise subcellular localization of endogenous proteins, which is essential for integrative understanding of a cell at the molecular level.
  • endogenous proteins can be imaged with high specificity, resolution and contrast in single cells.
  • the herein provided in vivo genome editing method may also be used for biochemistry based methods in order to determine protein-protein interactions.
  • Immunoprecipitation (IP) followed by mass spectrometry has recently emerged as a preferred method for the comprehensive determination of protein-protein interaction networks (interactome).
  • IP Immunoprecipitation
  • mass spectrometry has recently emerged as a preferred method for the comprehensive determination of protein-protein interaction networks (interactome).
  • targeting endogenous protein complexes has been challenging due to the lack of a specific antibody to the antigen.
  • the herein provided in vivo genome editing method can probe the endogenous protein with specific tag, high quality antibody against the tag can be used for IP.
  • the obtained high-quality interactome maps could be used as a powerful resource to elucidate individual protein function as well as interrogate developmental and disease mechanisms at a system level.
  • One remarkable benefit from detangling the interactome using the herein provided in vivo genome editing method is to identify the ligand for an "orphan" receptor. Determining unknown ligands for many receptors such as G protein-coupled receptors (GPCRs) is of particular interest, since a number of drugs target GPCRs while most of the relevant ligands still remain unknown. Identifying endogenous ligands is informative to design selective drugs targeting the receptors. Furthermore, by using the herein provided method also the specificity of candidate drugs (synthesized peptides etc.) that target given receptors can be tested in vivo.
  • GPCRs G protein-coupled receptors
  • the binding of tag-conjugated drugs and receptors that have been labeled by using the herein provided in vivo genome editing method may be observed by detecting the coupling of distinct tags (e.g. splitted fluorophore).
  • distinct tags e.g. splitted fluorophore
  • the in vivo genome editing method of the present invention can be used to modify mitotic progenitor cells of a non-human embryo.
  • said method is used to modify mitotic progenitor cells of a human embryo.
  • one embodiment of the present invention relates to a method for in vivo genome editing, comprising:
  • Modifying progenitor cells of a human embryo may be used for curing genetic diseases within this embryo.
  • one aspect of the invention relates to a genome editing machinery for use in treating a genetic disease within an embryo, wherein the genome editing machinery is delivered to mitotic progenitor cells of a human embryo through in utero electroporation; and at least one target sequence within the genome of these mitotic progenitor cells is modified by using HDR.
  • Genetic diseases that may be cured by using the in vivo genome editing method provided herein are, e.g. Rett syndrome, Fragile-X syndrome, Angelman syndrome, a spinocerebellar ataxia, nonsyndromic mental retardation, nonsyndromic mental retardation, or brain tumors.
  • a method for in vivo genome editing comprising:
  • the genome editing machinery comprises at least one polynucleotide encoding a site-specific DNA nuclease, which introduces a double or single strand break within the target sequence.
  • TALEN transcription activator-like effector nuclease
  • At least one guide RNA consisting of at least one target sequence specific CRISPR RNA (crRNA) molecule and at least one trans-activating crRNA (tracrRNA) molecule;
  • At least one guide RNA which is a chimeric RNA molecule comprising at least one target sequence specific crRNA and at least one tracrRNA;
  • the genome editing machinery comprises two or more different guide RNAs or two or more different polynucleotides encoding different guide RNAs.
  • the editing machinery comprises at least one pre-assembled Cas9 protein-guide RNA ribonucleoprotein complex (RNP), which introduces a double or single strand break within the target sequence.
  • RNP pre-assembled Cas9 protein-guide RNA ribonucleoprotein complex
  • step (b) after introducing a single or double strand break within the target sequence, the target sequence is modified by HDR, and wherein the genome editing machinery further comprises at least one donor template polynucleotide comprising a donor nucleic acid sequence and regions homologous to the target sequence.
  • the donor template polynucleotide is a single-stranded oligodeoxynucleotide (ssODN).
  • ssODN single-stranded oligodeoxynucleotide
  • the method of item 9 or 10 wherein in the donor template polynucleotide the regions homologous to the target sequence are localized at the 5' and 3' end of the donor nucleic acid sequence.
  • the genome editing machinery comprises:
  • a chimeric polynucleotide encoding a transposon and a transposase The method of item 12, wherein the polynucleotide(s) include(s) a vector sequence.
  • the transposon comprises an epitope tag and/or a fluorescent protein.
  • the target sequence comprises at least one of the following sequences:
  • the epitope tag is a HA-tag and/or the fluorescent protein is monomeric EGFP (mEGFP).
  • mEGFP monomeric EGFP
  • the mitotic progenitor cells are progenitor cells of the developing brain, progenitor cells of the developing spinal cord, or progenitor cells of muscle cells.
  • the progenitor cells in the developing brain or the progenitor cells of the developing spinal cord are progenitor cells of neurons.
  • progenitor cells of the developing brain are progenitor cells of cortical pyramidal neurons, CA1 pyramidal neurons, CA3 pyramidal neurons in the hippocampus, dentate granule cells in the hippocampus, spiny stellate cells in the subiculum, granule cells in the olfactory bulb, medium spiny neurons in the striatum, basolateral amygdala neurons, granule cells in the cerebellum, Purkinje cells in the cerebellum, and/or glial cells.
  • progenitor cells of muscle cells are progenitor cells of cardiomyocytes or progenitor cells of skeletal muscle cells.
  • the method of item 28 wherein the non-human mammalian embryo is an embryo of a mouse, an embryo of a rat, or an embryo of a ferret.
  • the method of any one of items 1-29 further comprising to let the embryo develop to a non- human animal.
  • the method of any one of items 1 -30 comprising obtaining a modified cell from the non-human embryo or from the non-human animal.
  • a non-human embryo which is produced by the method of any one of items 1-29, wherein the non-human embryo carries a HDR-mediated genome modification in a progenitor cell or in a postmitotic cell.
  • a modified post-mitotic cell that has been obtained from the non-human embryo of item 32 or from the non-human animal of item 33, wherein the modified post-mitotic cell carries a HDR-mediated modification in its genome.
  • the modified cell of item 34 wherein said cell is a differentiated neuron or a differentiated muscle cell.
  • a non-human animal comprising the modified cell of any one of items 34-36.
  • SLENDR single-cell labeling of endogenous proteins by CRISPRCas9-mediated homology-directed repair
  • CRISPR-Cas9-based HDR machinery was introduced into progenitor cells in the embryonic mouse brain several days before their final neurogenic divisions using in utero electroporation (IUE) (Nishiyama, 2012, Eur J Neurosci 36 2867-76; Tabata, 2001 , Neuroscience 103 865-72). It is demonstrated herein that a tag sequence for a short epitope or a longer fluorescent protein can be rapidly and precisely inserted into an endogenous gene of interest in vivo. This method is scalable to many species of proteins in diverse cell types, and permits high resolution imaging with light and electron microscopy both in fixed and live tissue.
  • SLENDR allows researchers to rapidly and precisely determine the localization and dynamics of endogenous proteins with the resolution of micro- to nanometers in various cell types, regions and ages of the brain, providing a powerful tool suitable for large-scale analysis on a broad spectrum of proteins.
  • Electroporation also called “electropermeabilization”
  • electropermeabilization refers to a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing proteins, DNA and/or RNA to be introduced into the cell.
  • Electroporation can be used to transfect eukaryotic cells (such as mammalian cells) by introducing new coding DNA. For example, it is used in the process of producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy. Electroporation has proven efficient for use on tissues in vivo, e.g. in utero.
  • In utero electroporation is an electroporation of embryos that are developing in the uterus.
  • In utero electroporation utilizes electrical pulses that create transient pores in cell membranes, allowing DNA to enter the cell. Since the negatively charged DNA will move towards the positive electrode, different cell populations can be targeted depending on the positioning of the electric field; see, e.g., Figure 4A..
  • In utero electroporation unlike cell culture or ex vivo techniques, has the advantage that transfected cells will continue to be exposed to all the physiological cues that guide normal development. This is particularly important in the brain, where structures such as axons require guidance cues from other cell types in order to develop properly. Brain development in particular involves a programmed series of proliferation and migration events, meaning that different tissue layers can be targeted based on the timing of electroporation.
  • the typical in utero electroporation procedure is performed as follows. First, all instruments have to be sterilized and the surgical area has to be wiped down with 70% ethanol. Next, an injection solution is made, e.g., by dissolving the genome editing machinery, e.g. plasmid DNA, in sterile PBS containing 0.1 mg/ml Fast Green. The Fast Green may be added to allow for visualization of the injection solution in the embryo.
  • the genome editing machinery e.g. plasmid DNA
  • a pregnant animal e.g. rodent
  • an analgesic like buprenorphine may be delivered subcutaneously.
  • the abdomen may be shaved and sterilized the incision site.
  • a vertical incision along the midline in the skin may be made; and, using scissors, it may be cut through the peritoneum.
  • the opening may be covered with sterile saline soaked gauze.
  • ritodrine hydrochloride may be applied to the exposed uterine horns.
  • the embryonic chain may be gently pulled out of the abdominal cavity, wherein the embryos are kept wet by covering with sterile pre-warmed saline.
  • the DNA solution may be injected through the uterine wall into the tissue to be transfected (e.g. the developing brain or a developing skeletal muscle).
  • the DNA solution may be injected into the lateral ventricle (for the cerebral cortex, olfactory bulb, amygdala, striatum and hippocampus) or the fourth ventricle (for the cerebellum) of each pup.
  • an electrode is placed on each side of the tissue to be transfected, with the positive electrode in the direction to which the DNA should be electroporated.
  • an electrode is placed on each side of the head, with the positive electrode in the direction to which the DNA should be electroporated.
  • Electroporation may be performed at E10 (e.g. for Purkinje cells in the cerebellum), at E1 1-12 (e.g. for the olfactory bulb, amygdala, striatum and cerebral cortex), E13 (e.g. for the cerebral cortex, hippocampus, subiculum and granule cells in the cerebellum) or E15 (e.g. for the cerebral cortex at the later stage).
  • Electric pulses e.g.
  • E10 33 V for 30 ms, 4 times with 970 ms intervals; at E11 -12, 40 V for 30 ms, 4 times with 970 ms intervals; at E13, 40 V for 50 ms, 4 times with 950 ms intervals; and at E15, 45 V for 50 ms, 4 times with 950 ms intervals
  • forceps-shaped electrodes e.g. at E10-13, CUY650P3; at E13-15, CUY650P5; Nepa Gene
  • the position and angle of the electrode may be set as described in Figure 4A.
  • the position and angle of the electrode as well as the timing of the in utero electroporation for the transfection of particular progenitor target cells is described below in Table 1.
  • Table 1 Position and angle of the electrode as well as timing of the in utero electroporation for the transfection of particular progenitor target cells.
  • the uterine horn may be placed back into the abdominal cavity.
  • 2-3 ml of warm saline may be added to the cavity.
  • the peritoneum may be sewed together with absorbable sutures, and the skin may be closed using staples.
  • Electroporated constructs can guide overexpression of wildtype or mutant proteins or block protein expression completely. Neurological phenotypes can then be assessed at either the microscopic or organismal level. In utero electroporation can also be useful for visualizing specific cell populations and the connections they make by delivering sequences encoding fluorescent proteins or epitope tags into the tissue tissue to be analyzed (e.g. into the neuronal tissue).
  • an in vivo method refers to a method, wherein a whole, living organism, i.e. a living embryo in the uterus of its living mother animal is used.
  • in utero electroporation is performed on a living mother animal in order to deliver a genome editing machinery to mitotic progenitor cells of a living embryo.
  • the embryo is preferably a non- human embryo.
  • the mother is of the same species as the embryo.
  • the term “genome editing” refers to a type of genetic engineering in which DNA is inserted, deleted or substituted (replaced) in the genome of an organism using nucleases. These nucleases create site-specific single or double (preferably double) strand breaks at desired locations in the genome. In general, the introduced single- or double-strand breaks are repaired through non-homologous end-joining (NHEJ) or homology-directed repair (HDR), resulting in a targeted substitution, insertion, or deletion.
  • NHEJ non-homologous end-joining
  • HDR homology-directed repair
  • homology-directed repair (HDR) of the single or double strand breads is induced by providing the cells to be modified with a donor template polynucleotide.
  • the nucleases that can be used for genome editing include Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector-based Nucleases (TALEN), and the CRISPR-Cas system.
  • a “genome editing machinery” comprises all molecules (i.e. polynucleotide(s) and/or protein(s)) that allow genome editing.
  • the genome editing machinery comprises a site-specific nuclease (e.g. a ZFN, a TALEN, a Cas9 nuclease, or a Cpfl nuclease) or a polynucleotide (such a plasmid vector) encoding the site-specific nuclease.
  • the site-specific nuclease is a Cas9 nuclease
  • the genome editing machinery further comprises a dual-guide RNA (i.e.
  • the genome editing machinery further comprises a crRNA, or a polynucleotide encoding this RNA.
  • the genome editing machinery further comprises a donor template polynucleotide comprising a donor nucleic acid sequence and regions homologous to the target sequence or to parts of the target sequence.
  • delivering or grammatical variations thereof mean “providing” or “introducing”.
  • delivering a genome editing machinery to mitotic progenitor cells means “providing mitotic progenitor cells with a genome editing machinery” .
  • delivering a genome editing machinery to mitotic progenitor cells means “introducing a genome editing machinery into mitotic progenitor cells”.
  • delivering a genome editing machinery to mitotic progenitor cells of a (non-human) embryo through in utero electroporation means that in utero electroporation is used for the transfection of mitotic progenitor cells of a (non-human) embryo, and that during this transfection the genome editing machinery is introduced into the mitotic progenitor cells.
  • HDR hyperchrometic recombination
  • the HDR repair mechanism can only be used by the cell when there is a homologue piece of DNA (i.e. a donor template polynucleotide) present in the nucleus. HDR mostly occurs in the G2 and S phase of the cell cycle.
  • HDR-based gene editing is difficult in post-mitotic cells such as neurons, cardiomyocytes or skeletal muscle cells.
  • NHEJ non-homologous end joining
  • the highly error-prone NHEJ pathway induces insertions and deletions (indels) of various lengths that can result in frameshift mutations and, consequently, gene knockout.
  • the HDR pathway directs a precise recombination event between a homologous DNA donor template (i.e. a donor template polynucleotide) and the damaged DNA site, resulting in accurate correction of the single or double strand break. Therefore, HDR can be used to introduce specific mutations or transgenes into the genome.
  • the donor template polynucleotide usually a ssODN
  • homologous recombination refers to a mechanism of genetic recombination in which two DNA strands comprising similar nucleotide sequences exchange genetic material. Cells use homologous recombination for the repair of damaged DNA, in particular for the repair of single and double strand breaks. The mechanism of homologous recombination is well known to the skilled person and has been described, for example by Paques, 1999, Microbiol Mol Biol Rev, 63: 349404.
  • mitotic cells are cells that are capable of proliferation. Accordingly, herein “mitotic cells” are cells that are capable of cell division. Mitotic cells generally include the epithelial, stromal (fibroblastic) and vascular (endothelial) cells that comprise the major renewable tissues and organs such as the skin, intestines, liver, kidney and so on. Mitotic cells also comprise major components of the haematopoietic system, and cells such as the glia, which support the survival and function of non-dividing neurons.
  • the mitotic cells are preferably the undifferentiated stem and progenitor cells that provide many of these tissues with the differentiated cells that are required for their function. More preferably, the mitotic cells are progenitor cell, e.g.
  • progenitor cells of the developing brain such as progenitor cells of neurons, which are capable of cell division
  • progenitor cells of muscle cells such as progenitor cells of cardiomyocytes or progenitor cells of skeletal muscle cells, which are capable of cell division.
  • post-mitotic cells are cells that are incapable of proliferation. Accordingly, herein “post-mitotic cells” are cells that are incapable of cell division. Post-mitotic cells include the differentiated neurons and muscle cells that comprise the brain, heart and skeletal muscle.
  • progenitor cells is commonly known in the art and is described, e.g., in Weissman, 2000, Cell 100 157-168.
  • progenitor cells are cells that undergo at least one additional round of division. Accordingly, herein the term “progenitor cells” also includes stem cells.
  • progenitor cells also refers to biological cells that, like stem cells, have a tendency to differentiate into a specific type of cell, but are already more specific than stem cells and are pushed to differentiate into its "target” cells. The most important difference between stem cells and progenitor cells that are no stem cells is that stem cells can replicate indefinitely, whereas progenitor cells that are no stem cells can divide only a limited number of times.
  • progenitor cells are terminally differentiated cells that are induced to undergo (a) cell division(s) and then develop to post-mitotic cells. It is indicated that progenitor cells (that are no stem cells) as well as stem cells can be targeted by the herein provided in vivo genome editing method, because the appended Examples show that neurons as well as glial cells can be targeted by the herein provided method.
  • an "embryo” is a multicellular diploid eukaryote in an early stage of embryogenesis, or development.
  • an embryo develops from a zygote, the single cell resulting from the fertilization of the female egg cell by the male sperm cell.
  • the zygote possesses half the DNA of each of its two parents.
  • the zygote will begin to divide by mitosis to produce a multicellular organism.
  • the result of this process is an embryo.
  • all stages from the zygote until birth are considered to be embryonic stages.
  • embryonic stage of development between the fifth and the eleventh weeks after fertilization, and is considered a fetus from the twelfth week on.
  • embryo comprises the human embryo as well as the human fetus.
  • electroporation may be performed when the embryo is from E10 to E18, preferably from E10 to E13, most preferably at E12.
  • Step (b) of the in vivo genome editing method provided herein comprises modifying at least one target sequence by using HDR.
  • step (b) of the inventive method results in a modified target sequence.
  • a "modified target sequence” is a genomic nucleotide (i.e. DNA) sequence which is modified during step (b) of the herein provided in vivo genome editing method.
  • the modification that is introduced into the target sequence may be at least one substitution, insertion, or deletion.
  • the "target sequence” refers to the genomic location that is to be modified by the method of the invention. Accordingly, the "target sequence” comprises but is not restricted to the nucleotide(s) subject to the particular modification, i.e.
  • the "target sequence” also comprises the sequence surrounding the relevant nucleotide(s) to be modified.
  • the “target sequence” also comprises at least 10, at least 100, at least 200, at least 500, or at least 1000 nucleotide(s) upstream and/or downstream of the relevant nucleotide(s) to be modified. More preferably, the term “target sequence” refers to the entire protein encoding gene to be modified.
  • modified includes, but is not limited to, one or more nucleotides that are substituted, inserted and deleted within the target sequence.
  • substitution refers to the replacement of nucleotides with other nucleotides.
  • the term includes for example the replacement of single nucleotides resulting in at least one point mutation. For example, 1 to 10, (e.g. 2 or 3) point mutations may be generated through substitution of nucleotides. Said point mutations can lead to an amino acid exchange in the resulting protein product but may also not be reflected on the amino acid level (i.e. silent mutations).
  • substitution are mutations resulting in the replacement of multiple nucleotides, such as for example parts of genes, such as parts of exons or introns as well as the replacement of entire genes.
  • the number of nucleotides that replace the originally present nucleotides may be the same or different (i.e. more or less) as compared to the number of nucleotides removed.
  • the number of replacement nucleotides corresponds to the number of originally present nucleotides that are substituted.
  • insertion refers to the incorporation of one or more nucleotides into the target sequence. Insertion of parts of genes, such as parts of exons or introns as well as insertion of entire genes is also encompassed by the term "insertion".
  • the insertion can result in a frameshift mutation within a coding sequence of a gene. Such frameshift mutations will alter the amino acids encoded by a gene following the mutation. In some cases, such a mutation will cause the active translation of the gene to encounter a premature stop codon, resulting in an end to translation and the production of a truncated protein.
  • insertion relates to an insertion of an exogenous sequence, such as a cDNA.
  • This cDNA is preferably a cDNA of a reporter gene, such as a fluorescent protein.
  • the fluorescent protein may be mEGFP.
  • insertion relates to an insertion of a nucleotide sequence encoding an epitope tag, such as a HA-tag or a FLAG-tag. Also loxP sites may be inserted into the target sequence.
  • deletion refers to the loss of nucleotides or larger parts of genes, such as exons or introns as well as entire genes.
  • insertion the deletion of a number of nucleotides that is not evenly dividable by three will lead to a frameshift mutation, causing all of the codons occurring after the deletion to be read incorrectly during translation, potentially producing a severely altered and most likely nonfunctional protein. If a deletion does not result in a frameshift mutation, i.e. because the number of nucleotides deleted is dividable by three, the resulting protein may be nonetheless altered as the finished protein will lack, depending on the size of the deletion, one or several amino acids that may affect the function of the protein.
  • modifications are not restricted to coding regions of protein encoding genes, but can also be introduced into non-coding regions, for example in regulatory regions such as promoter or enhancer elements or in introns.
  • modifications of the target sequence include introduction of mutations into a wildtype gene in order to analyze its effect on gene function; the replacement of an entire gene with a mutated gene or, alternatively, if the target sequence comprises mutation(s), the alteration of these mutations to identify which one is causative of a particular effect; the removal of entire genes or proteins or the removal of regulatory elements from genes or proteins as well as the introduction of fusion-partners.
  • a fusion partner may be an epitope tag (such as a HA-tag, a FLAG-tag, a his-tag, or a tap-tag) or a fluorescent protein (such as mEGFP).
  • Cas9 nuclease also called “Cas9 protein” or “Cas9 endonuclease” refers to the "clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9". This term is well known in the art and has been described, e.g. in Heidenreich, 2016, Nature Reviews Neurosciences, 17: 36-44; Makarova, 2011 , Nat Rev Microbiol, 9: 467-477 and in Makarova, 2011 , Biol Direct, 6 :38. Cas9 proteins constitute a family of enzymes that require a base-paired structure formed between an activating tracrRNA and a targeting crRNA to cleave target single or double strand DNA.
  • the Cas9 nuclease may be a modified Cas9 protein, wherein the nuclease function of the protein is altered into a nicking endonuclease function.
  • the naturally occurring Cas9 endonucleases function of cleaving both strands of a double-stranded target DNA, is altered into an endonuclease that cleaves (i.e. nicks) only one of the strands.
  • Means and methods of modifying a Cas9 protein accordingly are well known in the art, and include for example the introduction of amino acid replacements into Cas9 that render one of the nuclease domains inactive.
  • transposase and/or the site-specific nuclease may be introduced as a protein, but alternatively the transposase and/or the site-specific nuclease (e.g.
  • the Cas9 nuclease may be introduced in form of a polynucleotide encoding said protein.
  • the transposon and/or the guide RNA(s) may be introduced in form of a polynucleotide.
  • the polynucleotide encodes said site-specific nuclease (e.g. Cas9 nuclease), said transposase, said transposon, and/or said guide RNA(s) in expressible form such that expression in the embryo results in a functional site-specific nuclease (e.g. Cas9 nuclease), functional transposase, functional transposon, and/or functional guide RNA(s).
  • the coding sequences may be comprised in a vector, such as for example a plasmid, cosmid, virus, bacteriophage or another vector used conventionally e.g. in genetic engineering.
  • the vector is a plasmid vector.
  • the coding sequences inserted in the vector can e.g. be synthesized by standard methods, or isolated from natural sources.
  • the coding sequences may further be ligated to transcriptional regulatory elements and/or to other amino acid encoding sequences.
  • regulatory sequences are well known to those skilled in the art and include, without being limiting, regulatory sequences ensuring the initiation of transcription, internal ribosomal entry sites (IRES) (Owens, 2001 , Proc Natl Acad Sei, USA, 98: 1471-1476) and optionally regulatory elements ensuring termination of transcription and stabilization of the transcript.
  • IRS internal ribosomal entry sites
  • Non-limiting examples for regulatory elements ensuring the initiation of transcription comprise a translation initiation codon, transcriptional enhancers such as e.g.
  • the SV40-enhancer, insulators and/or promoters such as for example the cytomegalovirus (CMV) promoter, SV40-promoter, RSV-promoter (Rous sarcome virus), the lacZ promoter, chicken beta-actin promoter, CAG-promoter (a combination of chicken beta actin promoter and cytomegalovirus immediate-early enhancer), the gai10 promoter, human elongation factor l a- promoter, AOX1 promoter, GAL1 promoter CaM-kinase promoter, the lac, trp or tac promoter, the lacUV5 promoter, or the autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter.
  • CMV cytomegalovirus
  • SV40-promoter RSV-promoter
  • RSV-promoter Rousarcome virus
  • the lacZ promoter the lacZ promoter
  • Non-limiting examples for regulatory elements ensuring transcription termination include the V40-poly-A site, the tk-poly-A site or the SV40, lacZ or AcMNPV polyhedral polyadenylation signals. Additional regulatory elements may include translational enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Moreover, elements such as origin of replication, drug resistance gene or regulators (as part of an inducible promoter) may also be included.
  • polynucleotide refers to DNA, such as cDNA or genomic DNA, and RNA.
  • the polynucleotides used in accordance with the present invention may be of natural as well as of (semi) synthetic origin.
  • the polynucleotides may, for example, be nucleic acid molecules that have been synthesized according to conventional protocols of organic chemistry.
  • the person skilled in the art is familiar with the preparation and the use of polynucleotides (see, e.g., Sambrook and Russel “Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001)).
  • the polynucleotides used in accordance with the invention may comprise or consist of nucleic acid mimicking molecules known in the art. They may contain additional non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include, without being limiting, phosphorothioate nucleic acid, phosphoramidate nucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA).
  • the Cas9/guide RNA complex, the Cpfl/guide RNA complex, the ZEN, or the TALEN introduced in step (a) to mitotic progenitor cells specifically binds to the target sequence and introduces a single or double strand break (in the case of the Cas9/guide RNA complex, ZEN, or TALEN) or a double strand break (in the case of the Cpfl/guide RNA complex) within the target sequence.
  • ZENs and TALENs can introduce single strand breaks by using mutated (D450A) Fokl; see Ramirez, 2012, Nucleic Acids Res 40 5560-8; Wu, 2015, Proc Nat Acad Sci 1 12 1530-9.
  • the term "specifically binds to the target sequence” means that the Cas9/guide RNA complex, the Cpf1 /guide RNA complex, the ZEN nuclease, or the TALEN nuclease is designed such that the complex or ZEN/TALEN nuclease, respectively, statistically only binds to a particular sequence and does not bind to an unrelated sequence elsewhere in the genome.
  • Methods for testing the DNA- binding specificity of a Cas9/guide RNA complex, a Cpfl/guide RNA complex, a ZEN nuclease, or a TALEN nuclease are known to the skilled person and include, without being limiting, transcriptional reporter gene assays and electrophoretic mobility shift assays (EMSA).
  • the term "introduces a single or double strand break within the target sequence” relates to the interruption of the DNA strand(s) of a DNA double helix, wherein either one of the two strands (single strand break) or both strands (double strand break) in the double helix are severed.
  • the presence of such a single or double strand break within the genomic DNA triggers intracellular repair mechanisms.
  • such breaks are repaired by homologous recombination, while double strand breaks are typically repaired by either non-homologous end joining (NHEJ) or homologous recombination.
  • NHEJ non-homologous end joining
  • said single or double strand breaks are repaired by homologous recombination, i.e. by homology-directed repair (HDR).
  • HDR can be induced by providing a donor template polynucleotide comprising a donor nucleic acid sequence and regions homologous to the target sequence.
  • a donor template polynucleotide comprising a donor nucleic acid sequence and regions homologous to the target sequence.
  • targeted modification of a genome can be achieved with high specificity.
  • the genome of an embryo is modified.
  • said embryo or the animal developed from the embryo or a modified cell obtained from said embryo or from said animal
  • Methods for analyzing for the presence or absence of a modification include, without being limiting, assays based on physical separation of nucleic acid molecules, sequencing assays as well as cleavage and digestion assays and DNA analysis by the polymerase chain reaction (PCR).
  • assays based on physical separation of nucleic acid molecules include without limitation MALDI-TOF, denaturating gradient gel electrophoresis and other such methods known in the art, see for example Petersen, 2002, Hum Mutat 20: 253-259; Hsia, 2005, Theor Appl Genet 111 : 218-225; Tost, 2005, Clin Biochem 35: 335-350; Palais, 2005, Anal Biochem 346: 167-175.
  • sequencing assays comprise, without limitation, approaches of sequence analysis by direct sequencing, fluorescent SSCP in an automated DNA sequencer and Pyrosequencing. These procedures are common in the art, see e.g. Adams (Ed.), “Automated DNA Sequencing and Analysis", Academic Press, 1994; Alphey, “DNA Sequencing; From Experimental Methods to Bioinformatics", Springer Verlag Publishing, 1997; Ramon, 2003, J Transl Med 1 : 9; Meng, 2005, J Clin Endocrinol Metab, 90: 3419-3422.
  • cleavage and digestion assays include without limitation restriction digestion assays such as restriction fragments length polymorphism assays (RFLP assays), Rnase protection assays, assays based on chemical cleavage methods and enzyme mismatch cleavage assays, see e.g. Youil, 1995, Proc Natl Acad Sei USA 92: 87- 91 ; Todd, 2001 , J Oral Maxil Surg, 59: 660-667; Amar, 2002, J Clin Microbiol, 40: 446-452.
  • restriction digestion assays such as restriction fragments length polymorphism assays (RFLP assays), Rnase protection assays, assays based on chemical cleavage methods and enzyme mismatch cleavage assays, see e.g. Youil, 1995, Proc Natl Acad Sei USA 92: 87- 91 ; Todd, 2001 , J Oral Maxil Surg, 59: 660-667;
  • a successfully modified embryo may be selected by incorporation of an epitope tag and/or a fluorescent protein and analyzing whether this epitope tag or fluorescent protein is present in the embryo (or the animal developed from the embryo or the modified cell obtained from said embryo or animal); e.g. by fluorescence microscopy, immunohistochemistry or immunoelectron microscopy, as it is described in the appended Examples.
  • C showing the target sites for Cas9, sgRNA and ssODNs.
  • the sgRNA targeting sequences are underlined.
  • the protospacer-adjacent motif (PAM) sequences are indicated.
  • Recombination primer sets top; F, CaMKIIa-F1 and HA-R1 ; G, HA-F1 and CaMKIIp-R1
  • control primer sets bottom; F, CaMKIIa-F1 and CaMKIIa-R1 ; G, CaMKIIp-F1 and CaMKIIp-R1
  • HA tag sequence is marked.
  • A ⁇ -Actin
  • C DCX
  • E Rab11a
  • G CaV1.2
  • I 14-3-3 ⁇
  • K Fmrp
  • M Arc
  • Q PKCa
  • the sgRNA targeting sequences are underlined.
  • the PAM sequences are indicated.
  • the stop (A and I) and start (C, E, G, K, M, 0 and Q) codons are marked.
  • the Cas9 cleavage sites are indicated by the arrowheads.
  • A-D Electron microscopic images of dendritic spines (A) and shafts (C) in the cerebral cortex showing immunogold labeling for the HA tag (arrowheads) fused to the N-terminus of CaMKIIp in HA positive (top) and surrounding control (bottom) cells. Three-dimensional reconstructions of corresponding spines (B) and dendrites (D).
  • SLENDR is Scalable to Various Cell Types in Various Brain Regions
  • FIG. 1 Schematic illustration of IUE for targeting distinct brain regions.
  • the relative position of the electrodes (+, positive pole; -, negative pole) are shown to target different brain areas.
  • the position of electrode paddles and the injected DNA are shown in the coronal (top) and transverse (bottom) section of the brain to target the cerebral cortex (a), hippocampus and subiculum (b), olfactory bulb (c), striatum and amygdala (d) and cerebellum (e).
  • LV lateral ventricle; IV, fourth ventricle; PC, Purkinje cell; GC, granule cell.
  • DG dentate gyrus.
  • C-F Images of the subiculum (C), olfactory bulb (D), striatum (E) and amygdala (F) showing the DAPI signal (C-E) and immunoreactivities for NeuN (E, F) and the HA tag fused to ⁇ -Actin
  • C CaMKIIa
  • D to F MeCP2
  • E MeCP2
  • GL glomerular layer
  • EPL external plexiform layer
  • MCL mitral cell layer
  • GCL granule cell layer
  • BLA basolateral amygdala
  • Ctx cerebral cortex.
  • G Images of the cerebellum showing the DAPI signal and immunoreactivities for calbindin D-28k (a Purkinje cell marker) and the HA tag fused to CaMKII .
  • ML molecular layer
  • PCL Purkinje cell layer
  • GCL granule cell layer.
  • FIG. 1 Schematics of combining SLENDR with NHEJ-mediated gene knockout.
  • the HA sequence is inserted to ⁇ -Adin through HDR-mediated genome editing and a frame-shift mutation is induced in MeCP2 through NHEJ-mediated genome editing in the same cell (left).
  • some cells in the same tissue undergo only HDR-mediated genome editing (right), allowing comparison of the expression and localization of endogenous proteins within the same brain slice.
  • C showing the targeting sites for Cas9, sgRNA and HDR donor plasmid.
  • the sgRNA targeting sequences are underlined.
  • the PAM sequences are indicated.
  • the stop and start codons of CaMKIIa (A) and CaMKIfi (C) are marked.
  • FIG. 1 Graphical representation of the mouse genomic loci of CaMKIIa (A, C) and CaMKIfi (E) showing targeting sites different from Figure 1 for Cas9, sgRNA and ssODNs.
  • the sgRNA coding sequences are underlined.
  • the PAM sequences are indicated.
  • the start codon of CaMKIIa (C) and stop codons of CaMKIIa (A) and CaMKIfi (E) are marked.
  • A-H Confocal microscopic images of the cerebral cortex electroporated at E12 showing the DAPI signal (A-D and F-H) and immunoreactivities for NeuN (E) and the HA tag.
  • the HA tag sequence is marked.
  • FIG. 2 Graphical representation of the mouse genomic loci of MeCP2 showing the targeting sites for Cas9, sgRNA and ssODNs.
  • the sgRNA targeting sequences are underlined.
  • the PAM sequences are indicated.
  • the start codon is marked.
  • the Cas9 cleavage sites are indicated by arrowheads.
  • C Confocal microscopic images of the cerebral cortex electroporated at E12 showing the DAPI signal and immunoreactivities for mEGFP and the HA tag fused to the N-terminus of MeCP2 in the cortex.
  • a DCX-negative cell (non-neuronal cell) is indicated as arrowhead.
  • B-E Confocal microscopic images of the cerebral cortex showing the DAPI signal and immunoreactivities for mEGFP and the HA tag fused to the N-terminus of DCX (B), Rab11 a (C), FMRP (D) and PKCa (E).
  • F and G Images of the layer 2/3 pyramidal neurons at P9 (F) and P27 (G) showing the immunoreactivities for mEGFP and the HA tag fused to PKCa. Signal intensities for HA-PKCa and mEGFP along the yellow dashed lines are shown.
  • C Graphical representation of the mouse genomic loci of wild (upper) or recombined (lower) CaMKIIa showing the PCR primer set (CaMKIIa-F2 and mEGFP-R1 ) for PCR genotyping.
  • Example 1 Materials and Methods Animals
  • the human codon-optimized SpCas9 and sgRNA expression plasmid was a gift from F. Zhang (pX330, Addgene plasmid # 42230) (Cong, 2013, Science 339 819-23).
  • the FLAG tag sequence in SpCas9 in pX330 was removed (pX330N) for experiments in which the FLAG tag sequence is inserted into endogenous ⁇ ( Figures 5A and B).
  • the 20-base sequences which precede a 5'-NGG protospacer- adjacent motif sequence were selected to induce DNA double strand breaks within 10 bp from the tag insertion sites.
  • sgRNA scaffold of pX330 was ligated into the sgRNA scaffold of pX330 (Ran et al., 2013).
  • Single-stranded oligodeoxynucleotides (ssODNs) for HDR which contained the 27-base HA or 24-base FLAG tag sequence flanked by sequences of -80 bases on each side that were homologous to the target region, were purchased from IDT.
  • oligonucleotides Details of the oligonucleotides are described herein below.
  • mEGFP monomeric EGFP, A206K
  • plasmid-based donor templates for HDR were prepared.
  • target CaMKIIa a donor template containing the mEGFP sequence flanked by -0.9 kbp homology arms was generated and subcloned into Sacl and Sail sites of the pCAGGS vector. The -2.5 kbp fragment for HDR was cut out by the flanking restriction enzymes.
  • a donor template containing the mEGFP sequence flanked by -0.7-0.9 kbp homology arms was prepared and subcloned into EcoRI and Kpnl sites of the pUC57 vector.
  • the vector was linearized by cutting once at the EcoRI site.
  • the resulting fragment, or linearized vector were purified by QiaQuick gel extraction kit (Qiagen) and concentrated by ethanol precipitation.
  • pPB-CAG-mEGFP ,pPB-CAGDsRed2 and pPB-CAG-tdTomato were constructed by subcloning mEGFP, DsRed2 (a gift from Edward Callaway, Addgene plasmid # 15777) (Wickersham, 2007, Neuron 53 639-47) and tdTomato into pPBCAG.
  • EBNXN a gift from Sanger Institute
  • pCAG-hyPBase by subcloning hyPBase (pCMV- hyPBase, a gift from Sanger Institute) into the pCAGGS vector.
  • pCAGCaMKIIa-mEGFP and pCAG- mEGFP-CaMKII were constructed by subcloning CaMKIIa and Ca / ⁇ cDNA from Camuia (Lee, 2009, Nature 458 299-304; Takao, 2005, J Neurosci 25 3107-12) and pCMV-CaMKII (Kim, 2015, Neuron 87 813-26), respectively, into pCAG-mEGFP.
  • MeCP2 knockout previously reported sgRNA sequence (Swiech, 2015, Nat Biotechnol 33 102-6) was incorporated into pX330. All the plasmid constructs were verified by DNA sequencing.
  • mice were deeply anesthetized with 2% isoflurane (Piramal Healthcare).
  • Buprenorphine-SR 0.1 mg/mouse, ZooPharm
  • ritodorin hydrochloride 0.7-1.4 g/g of body weight; Sigma-Aldrich
  • each plasmid (pX330- derivatives, pPB-CAG-mEGFP, pPB-CAG-DsRed2, pPB-CAG-tdTomato and pCAG-hyPBase), the ssODNs for HDR and the double-stranded DNA template for mEGFP insertion were 1 ⁇ / ⁇ , 20 ⁇ and 1 ⁇ / ⁇ , respectively (Table 5).
  • the concentration of DNA was chosen based on previous IUE studies (Borrell, 2005, J Neurosci Methods 143 151-8; Chen, 2012, J Neurosci Methods 207 172-80; Imamura,
  • DNA solution was mixed with Fast Green (0.1 mg/ml, Sigma-Aldrich) and 1-2 ⁇ of the solution was injected into the lateral ventricle (for the cerebral cortex, olfactory bulb, amygdala, striatum and hippocampus) or the fourth ventricle (for the cerebellum) of each pup.
  • Fast Green 0.1 mg/ml, Sigma-Aldrich
  • Electroporation was performed at E10 (for Purkinje cells in the cerebellum), at E11 -12 (for the olfactory bulb, amygdala, striatum and cerebral cortex), E13 (for the cerebral cortex, hippocampus, subiculum and granule cells in the cerebellum) or E15 (for the cerebral cortex at the later stage).
  • Electric pulses (at E10, 33 V for 30 ms, 4 times with 970 ms intervals; at E1 1-12, 40 V for 30 ms, 4 times with 970 ms intervals; at E13, 40 V for 50 ms, 4 times with 950 ms intervals; and at E15, 45 V for 50 ms, 4 times with 950 ms intervals) were delivered with forceps-shaped electrodes (at E10-13, CUY650P3; at E13-15, CUY650P5; Nepa Gene) connected to an electroporator (NEPA21 , Nepa Gene).
  • mice Under deep ketamine-xylazine anesthesia (100 ⁇ g of ketamine -10 ⁇ g of xylazine per g of body weight, i.p.), mice were fixed by cardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brain was then removed and soaked in the fixative for 4-12 h. After rinsing with PBS, coronal vibratome sections (50 ⁇ in thickness and 100 ⁇ in thickness for embryonic brain) were prepared (VT1200, Leica).
  • the acquired images were processed using the Zen 2012 software (Zeiss) or Adobe Photoshop CS6 software (Adobe Systems) and analyzed with the ImageJ software (http://rsbweb.nih.gov/ij/).
  • Zen 2012 software Zeiss
  • Adobe Photoshop CS6 software Adobe Systems
  • ImageJ software http://rsbweb.nih.gov/ij/.
  • mEGFP knock-in cells coronal vibratome sections were imaged without immunostaining using LSM780.
  • secondary branches with similar width of apical dendrites of layer 2/3 pyramidal neurons in the somatosensory cortex were examined.
  • Z-stack images with 0.5 ⁇ intervals covering all spines in a dendrite were used to measure the fluorescence intensity of mEGFP-labeled CaMKIIa or CaMKII .
  • the intensity profile along a line crossing the center of a spine and its adjacent dendritic shaft was obtained to measure the ratio of intensity peaks corresponding to the spine and the shaft. Only isolated spines which displayed clear peaks in the intensity profile were examined. The analysis was performed using ImageJ.
  • HA-CaMKII knock-in mouse was anesthetized, and transcardially perfused with 4% paraformaldehyde and 0.1 % glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 20 min after flushing with 0.9% NaCI.
  • the brain was then post-fixed with 4% paraformaldehyde in 0.1 M PB for 3 h and sectioned into 50 ⁇ thick slices with a vibratome (VT 1200S, Leica). Brain sections with positive DsRed2 labeling were chosen for further processing.
  • the brain sections were incubated in 50 mM glycine in 0.1 M PB, cryo-protected with 15% then 30% sucrose, and subjected for two cycles of freeze-and-thaw with liquid nitrogen (Parajuli, 2012, J Neurosci 32 13555-67).
  • the sections were blocked with 10% normal goat serum (NGS) and 1 % fish skin gelatin (FSG) in Tris-buffered saline (TBS, pH 7.6), and then incubated with the rabbit anti-HA primary antibody (1 :1000, Cell Signaling Technology) in TBS with 1 % NGS and 0.1 % FSG for 48 h at 4 °C, followed by the incubation with 1 .4 nm gold-conjugated secondary antibody (1 :100, Nanoprobes) in TBS with 1 % NGS and 0.1 % FSG for 16 h at 4 °C.
  • Silver enhancement was performed with HQ silver enhancement kit (Nanoprobes) until HA positive neurons became visible under a light microscope.
  • the sections were then post-fixed in 1 % glutaraldehyde in 0.1 M PB for 10 min, fixed with 0.5% aqueous osmium tetroxide for 40 min at 4 °C, stained in 1 % aqueous uranyl acetate for 35 min at room temperature, dehydrated by sequentially replacing the solution with ethanol, acetone and then propylene oxide, and embedded in Durcupan ACE (Sigma). The resin was polymerized at 60 °C for 48 h.
  • a region containing HA positive neurons was trimmed out, and 50 nm thick sections were cut and collected onto a kapton tape by ATUMtome (Schalek, 2011 , Microscopy and Microanalysis 17 966-7) (RMC/Boeckeler).
  • the kapton tape was placed on a silicon wafer, and a layer of 5 nm thick carbon was coated on the wafer surface by the high vacuum sputter coater (ACE600, Leica).
  • the sections on the wafer were imaged under a scanning electron microscope (SEM, Merlin VP Compact, Zeiss) assisted with Atlas 5 AT software (Zeiss). We zoomed into dendrites of immunogold labeled HA positive neurons and imaged at 4 nm/pixel resolution.
  • genomic DNA was isolated from the lUE-transduced (mEGFP or DsRed2-positive) brain area.
  • DNeasy Blood & Tissue Kit Qiagen
  • genomic PCR was performed with the primers indicated in Figures 1 , 9 and 12.
  • the recombination product was further amplified with a semi-nested PCR using the first PCR product as a template.
  • the PCR product was purified by QiaQuick gel extraction kit (Qiagen) and then proceeded to DNA sequencing using one of the primers used in the semi-nested PCR.
  • Glutamate uncaging and imaging of live neurons were performed under a custom-built twophoton microscope with two ThSapphire lasers (Chameleon, Coherent) as previously described (Lee, 2009, Nature 458 299-304).
  • the lasers were tuned at the wavelength of 920 nm and 720 nm for imaging and uncaging, respectively.
  • the intensity of each laser was independently controlled with electro-optical modulators (Conoptics).
  • the fluorescence was collected with an objective (60x, 1 .0 numerical aperture, Olympus), divided with a dichroic mirror, and detected with photoelectron multiplier tubes (PMTs) placed after wavelength filters (ET520/60M-2P for green, ET620/60M-2p for red, Chroma).
  • Experiments were performed at 32 °C in a solution containing (in mM): 127 NaCI, 2.5 KCI, 25 NaHC03, 1 .25 NaH2P04, 4 CaCI2, 25 glucose, 0.001 tetrodotoxin (Tocris) and 4 MNI-caged L-glutamate bubbled with 95% 02 and 5% C02. Secondary branches of apical dendrites of layer 2/3 pyramidal neurons at 15- 17 days in vitro were examined. Images were analyzed with MATLAB and ImageJ.
  • SLENDR single-cell labeling of endogenous proteins by CRISPRCas9-mediated homology-directed repair
  • sgRNAs Specific single-guide RNAs targeting the vicinity of the stop and start codon of CaMKIIa and ⁇ , respectively, were designed. Also corresponding single-stranded oligodeoxynucleotides (ssODNs) (-200 bases) were designed to integrate the HA tag sequence into the genome just upstream of the stop codon of CaMKIIa and downstream of the start codon of ⁇ ( Figures 1A and 1 C).
  • the target sequences were selected throughout this study so that the CRISPR-Cas9 cleavage sites were located either in the non-coding region upstream of the start codon or within 10 bp from the stop codon. Thus, although if the CRISPR-Cas9-mediated cleavage sites were located upstream of the start codon, the HA integrated sites were located just downstream of the start codon.
  • the target sequences were also selected and ssODNs were designed so that Cas9 could not recognize the loci after HDR was completed.
  • pyogenes Cas9 or SpCas9 and sgRNA expressing vectors and ssODNs were introduced together with hyperactive piggyBac transposase and piggyBac transposon vectors expressing monomeric EGFP (mEGFP) as a marker of transfection (Chen, 2012, J Neurosci Methods 207 172-80; Loulier, 2014, Neuron 81 505-20; Yusa, 201 1 , Proc Natl Acad Sci U S A 108 1531 -6) to neuro-progenitor cells using IUE to target pyramidal neurons in the cerebral cortex.
  • the transposon system induces genomic integration of transgenes, preventing the dilution of mEGFP during cell divisions. Following IUE at embryonic day 12 (E12), immunostaining of brain slices was performed at postnatal days 14-48 (P14-48) using anti-HA antibody together with anti-NeuN antibody to label neurons.
  • HA signals were observed in a sparse subset of neurons, suggesting that HDR was successfully induced in these cells.
  • Immunofluorescence signal was localized mostly in cytosol and excluded from the nucleus, consistent with previously reported distribution of CaMKII (Lee, 2009, Nature 458 299-304).
  • Most of HA positive neurons were found in layer 2/3 ( Figures 1 B, 1 D and 1 E).
  • Layer 2/3 comprises the external granular/pyramidal layers, containing small to medium size pyramidal neurons. As can be seen in Fig. 1 , layer 2/3 can be identified with DAPI staining by labeling the nucleus in these cells.
  • mEGFP/NeuN CaMKIIa, 48.2 ⁇ 6.7 %; CaMKII , 40.0 ⁇ 2.9 %).
  • HA/mEGFP CaMKIIa, 7.5 ⁇ 1.2 %; CaMKII , 4.5 ⁇ 0.8 %), providing a few percent of overall knock-in efficiency (HA/NeuN: CaMKIIa, 3.4 ⁇ 0.2 %; CaMKII , 1 .8 ⁇ 0.5 %) (Table 3).
  • HA/NeuN CaMKIIa, 3.4 ⁇ 0.2 %; CaMKII , 1 .8 ⁇ 0.5 %)
  • a smaller population of HA positive neurons was also found in layer 4-6 (HA/mEGFP: CaMKIIa, 1 .8 ⁇ 0.1 %; CaMKIIp, 0.2 ⁇ 0.1 %) ( Figures 1 B, 1 D and 1 E).
  • SLENDR is Scalable to Various Endogenous Proteins
  • SLENDR was performed to fuse the HA tag to either the N- or C- terminus of a variety of proteins including nuclear, cytoskeletal, vesicular, cytosolic and membrane proteins ( Figures 2A-2R and 8-11 and Table 3).
  • MeCP2 a chromatin-associated protein that regulates gene transcription
  • MeCP2 was selected for the following reasons.
  • the endogenous subcellular localization can be imaged by traditional immunostaining approaches using a specific, characterized antibody.
  • MeCP2 is known to accumulate in heterochromatin in the nucleus, allowing for the contrast needed for staining of the endogenous protein in tissue. Therefore, double- immunostaining for the SLENDR inserted HA tag and endogenous MeCP2 enabled further validation of the SLENDR approach.
  • MeCP2-HA and HA-MeCP2 were distributed exclusively in the nucleus at P7 ( Figures 2A, 2B, 10B and 10C).
  • MeCP2-HA was accumulated in the heterochromatin regions (labeled with DAPI) ( Figure 2B) and well co-localized with endogenous MeCP2 ( Figure 10A), confirming the specificity of SLENDR. Also potential effects of SLENDR in transfected cells that did not undergo HDR were evaluated.
  • SLENDR was applied to insert the HA tag to endogenous ⁇ -Actin, a major cytoskeletal protein in dendritic spines.
  • ⁇ -Actin a major cytoskeletal protein in dendritic spines.
  • immunostaining using the anti-HA antibody showed that ⁇ - ⁇ -Actin was highly accumulated in dendritic spines in layer 2/3 pyramidal neurons and microfilaments in astrocytes in the cortex at P28 (Yuste, 2004, Nat Rev Neurosci 5 24-34) ( Figures 2C and 2D).
  • DCX Doublecortin
  • HA-DCX was detected in the cortical migrating neurons and accumulated in the growth cone as early as 60 h after IUE at E12 ( Figures 2E and 2F), suggesting that HDR occurred rapidly, possibly within one or two days after IUE.
  • HA-DCX was detected at both E14 and E18 ( Figures 2F and 1 1 B), suggesting SLENDR is suitable for studying protein localization in the embryonic brain.
  • Rab11 a a small GTPase involved in the endosomal recycling of proteins, was localized to numerous, small dispersed vesicles throughout the soma and dendrites in cortical neurons and astrocytes at P23, consistent with the pattern expected from the localization of recycling endosomes (Hutagalung, 201 1 , Physiol Rev 91 1 19-49) ( Figures 2G, 2H and 11 C).
  • CaV1 .2 the a1 C subunit of the L-type voltage-gated calcium channel, was distributed in clusters on cell bodies and proximal dendrites in layer 2/3 pyramidal neurons at P16 ( Figures 2I and 2J) (Hell, 1993, J Cell Biol 123 949-62).
  • Immunofluorescence signal of CaV1 .2-HA was also detected in the nucleus. This signal may represent the C-terminus of CaV1.2-HA, which functions as a calcium channel associated transcription regulator (Gomez-Ospina, 2006, Cell 127 591 -606).
  • FMRP a polyribosome-associated RNA-binding protein that regulates translation of a large number of mRNAs (Contractor, 2015, Neuron 87 699-715), was found in puncta, which likely reflect FMRP-associated mRNA granules, in the soma as well as dendrites in pyramidal neurons at P7 and P28 ( Figures 2M, 2N and 1 1 D).
  • Arc an immediate early gene product involved in synaptic plasticity, was localized both in the nucleus and cytoplasm at P29 ( Figures 20 and 2P) (Korb, 2013, Nat Neurosci 16 874-83; Shepherd, 2011 , Nat Neurosci 14 279-84).
  • PKC protein kinase C
  • HA-positive cells have never been observed when incorrect ssODNs-sgRNA pairs were used ( Figures 8 and 10).
  • precise genome editing was confirmed by PCR amplification of the targeted loci followed by DNA sequencing of the PCR products ( Figure 9).
  • all HA-positive cells in the same region showed similar HA-staining pattern.
  • Immunoelectron microscopy allows nanoscale visualization of endogenous proteins with defined ultrastructures in cells.
  • the lack of reliable antibodies compatible with electron microscopy imaging limits its application to a variety of proteins.
  • SLENDR could be applied to ultrastructural imaging of endogenous proteins using immunoelectron microscopy.
  • cortical tissue in which endogenous CaMKIIp is fused with HA using SLENDR were used ( Figures 1 C and 1 D).
  • Pre-embedding staining technique was applied: the tissue was incubated with HA-antibody and secondary antibody conjugated with gold, followed by silver enhancement and tissue embedding.
  • serial thin- section (50 nm) of the tissue were prepared using the automatic tape-collecting ultramicrotome (ATUMtome).
  • ATUMtome permits rapid and automated cutting and collection of serial thin sections onto a continuous reel of tape (Kasthuri, 2015, Cell 162 648-61 ; Schalek, et al. 201 1 Microscopy and Microanalysis 17 966-7).
  • a number of serial sections was imaged on the tape by scanning electron microscopy and reconstructed three-dimensional images of SLENDR generated knock-in cells ( Figures 3A-3D). It was found that CaMKIIp was localized near the postsynaptic density (PSD) (mode -80 nm) in dendritic spines (Figure 3F).
  • PSD postsynaptic density
  • Figure 3F dendritic spines
  • SLENDR is Scalable to Various Cell Types in Various Brain Regions
  • SLENDR allows labeling of two different species of proteins with different tags in single cells, providing a valuable tool for co-localization assays of a pair of endogenous proteins.
  • SLENDR constructs were simultaneously introduced to insert the HA tag to ⁇ -Actin and CRISPR constructs to induce NHEJ-mediated gene knockout of MeCP2 in progenitors of hippocampal neurons at E13 ( Figure 5C) ((Incontro, 2014, Neuron 83 1051 -7; Straub, 2014, PLoS One 9 ⁇ 105584; Swiech, 2015, Nat Biotechnol 33 102-6).
  • Visualization of HA-tagged ⁇ -Actin by SLENDR enables visualization of dendritic spine morphology, providing a useful tool to study effects of MeCP2 gene deletion on dendritic spines in single cells.
  • SLENDR can be used to insert a long sequence encoding a fluorescent protein into genes of interest.
  • mEGFP to the sequence just upstream of the stop codon of CaMKIIa or downstream of the start codon of ⁇ in the genome ( Figures 6A and 6C).
  • the SLENDR constructs together with the transposon vectors expressing DsRed2 were introduced to progenitors of layer 2/3 neurons through IUE at E12.
  • SLENDR has been developed, which allows in vivo genome editing in the mammalian brain for single-cell labeling of endogenous proteins. It was demonstrated that SLENDR is a simple and efficient technique to rapidly determine the subcellular localization of endogenous proteins with the resolution of micro- to nanometers in brain tissue. Importantly, the technique is generalizable to a broad spectrum of proteins and various cell types in widespread brain regions. SLENDR also can be used for multiplex labeling of different proteins or for mosaic analysis by combining labeling with single-cell knockout. Furthermore, SLENDR is capable of inserting a long sequence such as that encoding mEGFP in vivo and thus enables live imaging of endogenous proteins during biological processes in the brain.
  • HDR-mediated genome editing has been a challenge in the brain due to the lack of HDR activity in postmitotic neurons and the inefficient delivery of HDR machinery into the brain. This has limited its application in the field of neuroscience (Heidenreich, 2016, Nat Rev Neurosci 17 36-44). These problems were circumvented by targeting mitotic progenitors in the embryonic brain using IUE. In the experiments provided herein, the knock-in efficiency for the HA tag insertion was sufficient to image the subcellular localization of a protein of interest in single cells in brain tissue. It has also been shown that SLENDR can be used to knock-in a long mEGFP sequence, albeit at lower efficiency. To extend the SLENDR technique to broader applications for multiplexed labeling and fluorescent protein fusion, HDR efficiency may be increased.
  • RNPs pre-assembled Cas9 protein-guide RNA ribonucleoprotein complexes
  • SLENDR time window for SLENDR
  • RNPs were recently reported to provide rapid action of the nuclease with high efficiency and low off-target effects for genome editing
  • genetic or pharmacological inhibition of the NHEJ pathway may increase the HDR efficiency (Chu, 2015, Nat Biotechnol 33 543-8; Maruyama, 2015, Nat Biotechnol 33 538-42), although potential side effects must be addressed.
  • sgRNAs were designed by unbiased genome-wide analysis to minimize the potential off-target cleavages by Cas9 (Ran, 2013, Nat Protoc 8 2281-2308). Therefore, an online tool was used (the CRISPR design tool, http://crispr.mit.edu/).
  • CRISPR design tool http://crispr.mit.edu/.
  • control experiments were performed by using correct or incorrect sgRNAs to detect genomic insertion or the expression of the HA tag.
  • distinct sgRNAs targeting the identical gene were used and similar HA staining patterns were observed.
  • the localization of HA staining was consistent with that previously reported based on immunohistochemical, biochemical or electron microscopic studies.
  • SLENDR One of the merits of SLENDR is that high-quality antibodies can be used for the detection of tags.
  • SLENDR can be applied to image various tagged proteins without extensive optimization.
  • SLENDR allows protein labeling in a sparse subset of cells in the tissue, the specificity of immunostaining can be easily validated by examining surrounding negative control cells in the same specimen.
  • mice e.g. knockout mice
  • SLENDR Single epitope tag by SLENDR is sufficient to detect relatively low abundant proteins such as CaV1 .2 and Arc ( Figures 2J and 2P).
  • the sensitivity could be further increased by inserting multiple copies of epitopes or more antigenic probes such as Spaghetti- monster (Viswanathan, 2015, Nat Methods 12 568-76).
  • SLENDR will provide a generalizable and reliable platform for exploring localization of uncharacterized endogenous proteins using light and electron microscopy.
  • the target when the target is a secreted or a type I membrane protein with a signal sequence, one may select a target sequence for CRISPR-Cas9 mediated cleavage to minimize the possibility of deletion or mislocalization of the gene products.
  • a target sequence for CRISPR-Cas9 mediated cleavage to minimize the possibility of deletion or mislocalization of the gene products.
  • Immunodetection of a fused epitope tag may be difficult for some proteins, because the accessibility of antibodies may be sterically limited. For example, it was failed to detect the HA signal with immunostaining in tissue targeting to the C-terminus of PSD-95 with HA by SLENDR, whereas PCR detected the HA knock-in allele at the DNA level (data not shown). This is likely due to high protein density of PSD, which is known to often prevent antibodies to access to targeted proteins in the structure (Fukaya, 2000, J Comp Neurol 426 572-86). This limitation of immunostaining can be overcome by fusing a fluorescent protein tag with SLENDR and directly observing the fluorescence (Fortin, 2014, J Neurosci 34 16698-712). These limitations would not detract the impact of SLENDR, as the technique should be applicable to most proteins with little optimization as shown in this study.
  • SLENDR should allow large-scale, potentially genome-wide, determination of precise subcellular localization of endogenous proteins in various cell types and ages, providing a new level of understanding of protein and cellular function in the brain.
  • the numbers of cells analyzed are indicated in parentheses.
  • the numbers of cells analyzed are indicated in parentheses.
  • the present invention refers to the following nucleotide and amino acid sequences.
  • SEQ ID NO: 34 ssODN sequence (5'-3', upper case: HA tag sequence), CaMKIIa (N-terminus)
  • SEQ ID NO: 35 ssODN sequence (5'-3', upper case: HA tag sequence), CaMKIIp (N-terminus)
  • gg g g SEQ ID NO: 63 PCR primer sequence (5'-3'), 14-3-3s-F1 caggcggaagtcccggattgag
  • SEQ ID NO: 64 PCR primer sequence (5'-3'), 14-3-3s-R1 acaccagatcctcccgatcatc
  • SEQ ID NO: 65 PCR primer sequence (5'-3'), Arc-F1 cactcgctaagctcctccg
  • SEQ ID NO: 66 PCR primer sequence (5'-3'), Arc-R1 gatcacattgggtttggcgg
  • SEQ ID NO: 67 PCR primer sequence (5'-3'), PKCa-F1 gaggcaagaggtggttggg
  • SEQ ID NO: 68 PCR primer sequence (5'-3'), PKCa-R1 agatgaagtcggtgcagtgg
  • SEQ ID NO: 72 tracrRNA sequence TAGCAAGTTAAAATAAGGCTAGTCCGTTTTT

Abstract

La présente invention concerne un procédé d'édition de génome in vivo, comprenant : (a) l'administration d'une machinerie d'édition de génome (par exemple, des vecteurs de plasmide codant pour une nucléase Cas9 et un ARN de guidage unique; ainsi qu'un polynucléotide de matrice donneur), à des cellules progénitrices mitotiques d'un embryon non humain par électroporation in utero; et (b) la modification d'au moins une séquence cible (c'est-à-dire, une séquence d'ADN dans le génome des cellules progénitrices) au moyen d'une réparation dirigée par homologie (HDR). La machinerie d'édition de génome peut comprendre en outre un témoin de transfection, comprenant un transposon, et un polynucléotide codant pour une transposase. Un autre aspect de l'invention concerne un embryon non humain ou un animal non humain comportant une séquence cible modifiée dans son génome, ledit embryon non humain ou animal non humain étant produit par le procédé d'édition de génome in vivo de l'invention. Un autre aspect du procédé d'édition de génome in vivo de l'invention concerne une cellule modifiée obtenue à partir dudit embryon non humain ou dudit animal non humain.
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