WO2017173004A1

WO2017173004A1 - A method for in vivo precise genome editing

Info

Publication number: WO2017173004A1
Application number: PCT/US2017/024869
Authority: WO
Inventors: Takayasu MIKUNI; Jun Nishiyama; Ryohei Yasuda
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-03-30
Filing date: 2017-03-29
Publication date: 2017-10-05
Anticipated expiration: 2018-09-30

Abstract

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.

Description

A method for in vivo precise genome editing

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.

Precise mapping of a large number of proteins with subcellular resolution is essential to understand cellular processes. Thus, it is critical to develop a rapid and scalable method to determine the localization of proteins with high specificity, resolution and contrast. Conventionally, either immunostaining of endogenous proteins or overexpression of proteins fused with epitope tags or fluorescent proteins have been used to determine protein localization. These methods, however, have significant problems: immunostaining often suffers from the lack of specific antibodies against a protein of interest and the cross-reaction of antibodies with nontargeted proteins; overexpression often causes mistargeting of the expressed protein and potential changes in cell function. To address some of these issues, 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). However, in dense tissue, such as mammalian brain, it is difficult to obtain images with high contrast in subcellular processes when all cells are labelled. To overcome these problems, 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). However, none of these techniques provides rapid, scalable and high-throughput readouts for the localization of endogenous proteins. Direct, single-cell manipulation of the genome in vivo to insert a tag sequence to a gene of interest would overcome these limitations, providing rapid, specific and sparse labelling of the gene product. 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

Biotechnol 32 347-55; Wang, 2013, Cell 153 910-8; Yang, 2013, Cell 154 1370-9). 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). Although introducing frame-shift knockout mutations through NHEJ at the single-cell level has been established (Straub, 2014, PLoS One 9 e105584; Swiech, 2015, Nat Biotechnol 33 102-6), targeted insertion of a sequence through HDR has not been possible in the mammalian brain in vivo (Heidenreich, 2016, Nat Rev Neurosci 17 36-44; Piatt, 2014, Cell 159 440-55; Xue, 2014, Nature 514 380-4; Yin, 2014 Nat Biotechnol 32 551 -3). This is due to the lack of homologous recombination activity in post-mitotic cells and the inefficient delivery of HDR machinery to target cells (Chu, 2015, Nat Biotechnol 33 543-8; Cox, 2015, Nat Med 21 121 -31 ; Heidenreich, 2016, Nat Rev Neurosci 17 36-44; Hsu, 2014, Cell 157 1262-78; Maruyama, 2015, Nat Biotechnol 33 538-42; Saleh- Gohari, 2004, Nucleic Acids Res 32 3683-8).

In the prior art CRISPR-Cas9 has been used in combination with in utero electroporation in the brain (Straub, 2014, PLOS one, 9(8), e105584; Shinmyo, 2016, Sci Rep, 6: 2061 1). However, all the studies performed only non-homologous end joining (NHEJ)-mediated gene knockout, and HDR has never been performed in the brain.

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. On the other hand, 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. The prior art has shown that 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 ). However, 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). As a result, methods for stimulating HDR- based repair or alternative strategies for efficient gene insertion are urgently needed (Hsu, 2014, Cell, 157: 1262-1278).

Thus, 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 technical problem is solved by provision of the embodiments as provided herein and as characterized in the claims.

Accordingly, the present invention relates to a method for in vivo genome editing, comprising:

(a) delivering a genome editing machinery to mitotic progenitor cells of a non-human embryo through in utero electroporation; and

(b) modifying at least one target sequence by using homology-directed repair (HDR).

Accordingly, in 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.

In the in vivo genome editing method of the present invention 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. Preferably, double strand breaks are introduced by the site- specific nuclease.

In context of the invention zinc finger nucleases (ZEN) or a transcription activator-like effector nucleases (TALEN) may be used as site-specific DNA nucleases, because these molecules have been previously used for HDR-mediated genome editing in the liver etc. (Li, 2011 , Nature 475: 217-221 ; Bedell, 2012, Nature 491 : 1 14-118; Genovese, 2014, Nature 510: 235-240). However, clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) effector proteins, such as Cas9, provide a much more simple and generalizable genome editing method. Thus, in context of the herein provided in vivo genome editing method of the invention, the site-specific DNA nuclease may be:

(i) a zinc-finger nuclease (ZFN);

(ii) a transcription activator-like effector nuclease (TALEN);

(iii) a Cas9 nuclease; or

(iv) a Cpfl nuclease.

A ZFN comprises a zinc-finger DNA binding domain, which should be designed for each target gene, and a Fokl nuclease. Similarly, 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. For example, herein 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. Herein, 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. Indeed, as 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.

Using 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. Similarly, Using 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 ). Therefore, it is preferred in context of the present invention that 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. For example, herein AsCpfl or LbCpfl may be used. Thus, 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. Herein, 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.

Most preferably, a Cas9 nuclease is used as site-specific DNA nuclease in the in vivo genome editing method of the invention. Thus, one aspect of the present invention relates to the herein provided in vivo genome editing method, wherein the Cas9 nuclease is Streptococcus pyogenes Cas9 (SpCas9) or Staphylococcus aureus Cas9 (SaCas9).

Beside SpCas9 and SaCas9 several other Cas9 orthologs are known, which may be used in context of the present invention. These Cas9 orthologs include those derived from Streptococcus thermophilus, Neisseria meningitides and Francisella novicida. Herein, 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. Furthermore, there are SpCas9 mutants that recognize different PAMs (Kleinstiver, 2015; 523: 481-485), which may also be applied in the herein provided method. However, in context of the present invention it is preferred that 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. Herein, 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).

Because ZFNs and TALENs achieve specific DNA binding via protein domains, individual nucleases have to be synthesized for each target site (Heidenreich, 2016, Nature Reviews Neurosciences, 17: 36-44). By contrast, 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). A simple two-component system (consisting of Cas9 and a fusion of the tracrRNA-crRNA duplex to a "single-guide RNA", "sgRNA") or 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") can be engineered for expression in eukaryotic cells and can achieve DNA cleavage at any genomic locus of interest. 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. Hence, 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.

Thus, in the event the herein provided in vivo genome editing method uses Cas9 as site-specific nuclease, the genome editing machinery may further comprise:

(i) 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; ("dual-guide RNA")

(ii) a polynucleotide encoding the RNA molecules of (i);

(iii) 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 ("single-guide RNA", "sgRNA")

(iv) a polynucleotide encoding the chimeric RNA of (iii).

The term "target sequence specific CRISPR RNA (crRNA)", as used herein, is commonly know in the art and described, e.g. in 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.

As used herein, the term "trans-activating crRNA (tracrRNA)" is commonly known in the art and described, e.g., in Hsu, 2014, Cell 157: 1262-78, Yang, 2014, Nature Protocols, 9:1956-1968 and Heidenreich, 2016, Nature Reviews Neurosciences, 17: 36-44. 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. In particular, 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).

The skilled person readily knows how 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) that target a desired target sequence (e.g. a desired protein encoding gene) can be designed. For example, such 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. For example, such 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. Within the chimeric RNA a segment of the DR and the tracrRNA sequence are complementary and are able to hybridize and to form a hairpin structure. A further method to obtain a single-guide RNA is described in Ran, 2013, Nat Protoc 8 2281 -2308. In particular, single-guide 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/).

In a preferred aspect of the herein provided in vivo genome editing method, 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.

As described above, in one embodiment of the invention, the site-specific nuclease is Cpfl. In this embodiment, the genome editing machinery may further comprise:

(i) at least one guide RNA comprising a target sequence specific crRNA molecule; or

(ii) a polynucleotide encoding the RNA molecules of (i).

The generation of guide RNAs for a Cpfl nuclease is commonly known in the art. For example, such 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 ). It is envisaged that 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. Preferably, 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. In context of the present invention 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. Alternatively, 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.

In the herein provided in vivo editing method 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. Thus, one aspect of the present invention relates to the herein provided in vivo genome editing method, wherein the genome editing machinery comprises two or more different guide RNAs or two or more different polynucleotides encoding different guide RNAs. For example, 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. This enables the generation of models of polygenic diseases such as most of psychiatric disorders including schizophrenia, bipolar disorder, ADHD and autism, as well as many heart diseases and cancers. For example, 2 different guide RNAs (i.e. single- or dual-guide RNAs) or 2 different polynucleotides encoding different guide RNAs (i.e. single- or dual-guide RNAs) may be used in context of the present invention.

In the method of the invention 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. This might increase the time window for HDR, as RNPs were recently reported to provide rapid action of the nuclease with high efficiency and low off-target effects for genome editing (Kim, 2014, Genome research, 24: 1012-1019; Lin, 2014, eLife 3, e04766). Thus, 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. 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). In addition or alternatively, 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). After introducing single- or double-strand breaks, 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. Therefore, 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). In the donor template polynucleotide the regions homologous to the target sequence are preferably localized at the 5' and 3' end of the donor nucleic acid sequence.

Herein the term "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. By using this donor template polynucleotide as a template, the genetic information, including the modification(s), is copied into the target sequence within the genome of the embryo. For example, 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. Also plasmid vectors can be used as donor template polynucleotide, i.e. a (plasmid based) double-stranded DNA may be used as donor template polynucleotide.

Herein, the terms "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. Usually, the regions homologous to the target sequence (homology arms) 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). These programs, in addition to providing a pair-wise sequence alignment, also report the sequence identity level (usually in percent identity) and the probability for the occurrence of the alignment by chance (P-value).

Preferably, 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%. The above defined 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. Thus, 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).

As described above, 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. Accordingly, preferably 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.

In context of the present invention the donor template polynucleotide may be a single-stranded oligodeoxynucleotide (ssODN). The term "oligodeoxynucleotide (ODN)" is commonly known in the art and relates to a nucleic acid polymer made up of a sequence of desoxynucleotide residues. 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. Nonetheless, it will be appreciated that 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. For example, to insert a short sequence like 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. In this regard, 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.

To insert a long sequence such as that encoding a fluorescent protein (about 0.7 kb), 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 (e.g. in a desired protein encoding gene) can easily be designed by using routine techniques, e.g. as described in Ran, 2013, Nat Protoc 8 2281 -2308.

In 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. Recent studies show that NHEJ inhibitor (Scr7; Maruyama, 2015, Nat Biotechnol, 33(5): 538-542; Chu, 2015, Nat Biotechnol, 33(5): 543-548) and HDR enhancer (Rad51 mRNA and RS-1 ; Song, 2016, Nat Commun, 7: 10548, doi: 10.1038/ncomms10548) can increase the HDR efficiency. Therefore, the herein provided in vivo genome editing method may comprise adding a NHEJ inhibitor and/or a HDR enhancer to the progenitor cells.

In order to have a marker of transfection, in the in vivo genome editing method of the invention constructs that induce HDR (e.g. a SpCas9/SaCas9 and sgRNA expressing vector and a donor template polynucleotide) 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)) and 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). In context of the present invention 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). 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. For example, 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). 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. However, by using such a transposon system 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.

In context of the present invention the transposon and the transposase may be encoded by one chimeric or by two separate polynucleotides. Thus, one aspect of the invention relates to the herein provided in vivo genome editing method, wherein the genome editing machinery comprises:

(i) a polynucleotide encoding a transposon, and a polynucleotide encoding a transposase; or

(ii) a chimeric polynucleotide encoding a transposon and a transposase.

The polynucleotides encoding the transposon and/or the transposase may include vector sequence. Thus, the polynucleotides encoding the transposon and/or the transposase may be plasmid vector(s). However, 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. Preferably, 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.). In the herein provided in vivo genome editing method the transposon may comprise an epitope tag and/or a fluorescent protein. For example, fluorescent proteins that can be used include CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein) and many other derivatives. Preferably, the fluorescent protein is mEGFP, tdTomato or DsRed2. For example, mEGNF, DsRed2 or tdTomato may be inserted in the piggybac transposon system. For example, 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 only requirement for the selection of Cas9 or Cpf1 target sites is the presence of a protospacer- adjacent motif (PAM) immediately downstream of the target site. The PAM target sequences of various CRISPR nucleases and their variants (e.g. 5'-NGG for SpCas9, 5'-NNGRRT for SaCas9, 5'-TTN for Cpfl) abundantly exist in the mammalian genome. Therefore, most of the genes can be targeted by using the in vivo genome editing method provided herein. Accordingly, one aspect of the invention relates to the herein provided in vivo genome editing method, wherein the target sequence comprises at least one of the following sequences:

(i) 5'-NGG

(ii) 5'-NNGRRT; or

(iii) 5'-TTN.

Herein 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. In order to minimize the possibility of gene knockdown (e.g. by NHEJ), 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. Accordingly, the protein encoding gene may be modified in the 3'-untranslated region and/or in the 5'- untranslated region of the gene. In addition, the target sequences and donor template polynucleotides (e.g. the ssODNs) may be selected so that the site-specific nuclease (e.g. Cas9) cannot recognize the loci after HDR was completed. For example, the donor temple polynucleotide (e.g. the ssODN) may be chosen so that 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. It is indicated although if the cleavage site of the site-specific nuclease (e.g. of Cas9) is located upstream of the start codon, the modification of the target sequence (e.g. integration of an HA-tag) may be located just downstream (i.e. 0-100bp for a tag insertion; 1-1000bp for a point mutation) of the start codon.

However, in context of the present invention, potential effects of NHEJ may be evaluated for each gene depending on the purpose of experiments. For example, 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. In particular, 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.

Accordingly, in context of the present invention, the genome modification (e.g. insertion of an epitope tag or of a fluorescent protein into an endogenous protein), 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. As shown in the appended Examples (see, e.g. FiglOD and 1 1 A), NHEJ was not observed in most of the neurons. In contrast, for 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.

However, in 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. Thus, 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. For example, 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. Thus, one aspect of the invention relates to the herein provided in vivo method, wherein at least two target sequences are modified. Thus, 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.

Labeling endogenous proteins with fluorescent proteins allows imaging of dynamics in live cells without overexpression artifacts. In addition, introduction of different tags into different genes may be used for co- localization assays. Accordingly, 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:

(i) insertion of an epitope tag and/or a fluorescent protein;

(ii) insertion of an epitope tag or fluorescent protein into one target sequence; and insertion of another epitope tag or fluorescent protein into another target sequence;

(iii) introduction of at least one disease-specific mutation;

(iv) knock-out of a gene; or

(v) a combination of (i) and (iii), or of (i) and (iv).

With the combination of (i) and (iv), above (see item (v), above) interactions between the visualized and deleted proteins and cell autonomous effects of a gene of interest on endogenous proteins may be analyzed, and normal and knockout cells may be compared in the same tissue.

Item (iii) above describes that with the herein provided method at least one disease-specific mutation may be introduced in the target sequence (e.g. in the gene to be targeted). Accordingly, disease model animals (e.g. mice) may be prepared by using the herein provided in vivo genome editing method. For example, mutations that contribute to brain tumors, to the Rett syndrome, to muscular dystrophies, such as Duchenne muscular dystrophy (DMD), to nonsyndromic mental retardation, to nonsyndromic mental retardation, or to Fragile X syndrome may be introduced by using the in vivo genome editing method of the invention. Thus, 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. mouse). For example, 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.

In a preferred aspect, 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). Since most 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.

Another brain tumor model may be produced by the herein provided in vivo genome editing method by mutating the BRAF gene. For example, 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).

Regarding 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%) is the most common mutation associated with Rett syndrome, followed by R270X (9%), R255X (8.7%), T158 M (8.3%) and R306C (6.8%) (Bienvenu, 2006, Nature Reviews Genetics, 7: 415-426). Thus, one or more of these mutations (e.g. the R168X mutation or all 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.

Most of Duchenne muscular dystrophies (DMD) are caused by the lack of the dystrophin protein. However, it is often useful to analyze the mutations that generate a mutated or truncated protein for the functional dissection of a particular protein domain. Thus, a missense human mutation (R54L) which locates at the actin-binding domain in the N-terminus of dystrophin may be useful in context of the invention, since this mutation was shown to cause a DMD phenotype with normally sized but abnormal dystrophin (Blake, 2002, Physiological Reviews, 82: 291-329). Accordingly, 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.

For preparing an animal model for nonsyndromic mental retardation, the SYNGAP1 gene may be mutated. In particular, 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). For preparing an animal model for nonsyndromic mental retardation, the IQSEQ2 gene may be mutated by the herein provided in vivo genome editing method. For example, 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).

Also 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).

As described above, in the herein provided in vivo genome editing method the modification of the at least one target sequence (e.g. of at least one protein encoding gene) 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. 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. In a preferred aspect of the invention, a HA-tag is inserted into the at least one target sequence. In one aspect of the invention, multiple copies of epitope tags (e.g. multiple HA-tags) may be inserted. This has the advantage that an increased sensitivity can be gained.

However, in exceptional cases, immunodetection of a fused epitope tag may be difficult because the accessibility of antibodies may be sterically limited (e.g. due to high density of the protein carrying the epitope tag). This limitation of immunostaining can easily be overcome by fusing a fluorescent protein and directly observing the fluorescence (Fortin, 2014, J Neurosci 34 16698-712). Accordingly, in context of the present invention, if the targeted protein cannot be detected via an epitope tag, said protein should be fused with a fluorescent protein. This limitation does not detract the impact of the herein provided in vivo genome editing method, as the technique should be applicable to most proteins with little optimization as shown in the appended Examples.

As described in item (ii), above, 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. For example, a HA-tag may be introduced into one target gene and a FLAG-tag may be introduced into another target gene.

In addition, by combining items (i) and (iv), above, endogenous subcellular protein localization may be examined in the context of a knockout of a different protein. Such a method advantageously provides functional insight into the interactions between the visualized and deleted protein. For example, in context of the invention, 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. 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).

HDR can occur in mitotic cells. Thus, 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. Thus, one aspect of the present invention relates to the herein provided in vivo genome editing method, wherein 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. In context of the present invention it is preferred that the mitotic progenitor cells are progenitor cells of the developing brain (e.g. progenitor cells of neurons).

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. Therefore, in one aspect of the invention 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. As described above, in context of the present invention, the mitotic progenitor cells may also be progenitor cells of muscle cells. These progenitor cells of muscle cells may be progenitor cells of cardiomyocytes or progenitor cells of skeletal muscle cells. However, it is preferred in context of the present invention that the mitotic progenitor cells are progenitor cells of the developing brain (such as progenitor cells of neurons). In context of the present invention the in utero electroporation may be performed at any development stage of the embryo, provided that mitotic progenitor cells are targeted. The term "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). In one aspect of the in vivo genome editing method of the invention 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. Therefore, preferably in the herein provided in vivo genome editing method in utero electroporation is performed from E10 to E13, more preferably at E12, and cortical progenitors for layer 2/3 neurons are targeted. The "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 context of the present invention 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. For targeting progenitor cells of purkinje cells in the cerebellum in utero electroporation may be performed at E10. Herein "targeting (mitotic) progenitor cells" means that within these cells at least one target sequence is modified by using the in vivo genome editing method of the invention. The appended Examples show that HDR is more efficient when in utero electroporation is performed several days before the final (neurogenic) divisions of targeted cells. Therefore, the in utero electroporation is most preferably performed at E12. In accordance with the invention, the non-human embryo may be the embryo of a mammal, of an avian or of a fish. Preferably, the non-human embryo is a non-human mammalian embryo. In utero electroporation has been successfully performed in the mouse (Tabata, 2001 , Neuroscience, 103:865-72; Borrell, 2005, J Neurosci Methods,143:151 -8; Kitazawa, 2014, J Neurosci, 34:11 15-26; Nishiyama, 2012, Eur J Neurosci, 36:2867-76; Soma, 2009, J Comp Neurol, 513:1 13-28), rat (Chen,2012, J Neurosci Methods, 207:172-80) and ferret (Kawasaki, 2012, Mol Brain, 20:5-24), suggesting that the in vivo genome editing method of the invention can be applied to various mammalian animals. In context of the present invention 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). In a more preferred aspect of the invention the 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.

It is clear for the skilled person that the mother animal of the non-human embryo (i.e. the animal who is carrying the uterus wherein the embryo is developing) 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. Thus, 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. In addition or alternatively, 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. Thus, 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).

By performing the herein provided in vivo genome editing method at least one target sequence in the genome of a non-human embryo is modified. Thus, 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). Thus, 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. For example, 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. In the non-human embryo of the invention not all cells (but only selected progenitor or post-mitotic cells) carry a genome modification.

As described above, 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. Thus, 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). Thus, 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. For example, the non-human animal of the invention may comprise a HA-tag sequence at the N-terminus of CamKlla, CamKI^, MeCP2, β-Adin, Rab11a, 14-3-3ε, FMRP, Arc and/or PKCct and/or at the C-terminus of CamKlla, CamKlfi, MeCP2 and/or Cav1.2. In addition or alternatively, 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. In addition or alternatively, 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.

In accordance with the invention 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). Thus, 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. Thus, 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. Alternatively or in addition thereto, the modified cell of the invention may comprise a HA-tag sequence at the N-terminus of CamKlla, CamKltf, MeCP2, β-Adin, Rab11a, 14-3-3ε, FMRP, Arc and/or PKCct and/or at the C-terminus of CamKlla, CamKlfi, MeCP2 and/or Ca_v1.2. In addition or alternatively, 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. In addition or alternatively, the modified cell may comprise a FLAG-tag sequence at the N-terminus of CamK//jS. One embodiment of the invention relates to a non-human animal (e.g. a mouse) comprising the modified cell of the invention.

All the definitions and preferred embodiments defined above with regard to the in vivo method gene editing method of the invention apply mutatis mutandis also to the non-human embryo of the invention, to the non- human animal of the invention and to the modified cell of the invention.

Since HDR enables versatile modification of the genome such as insertion, deletion or replacement of the sequence, 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. As the appended Examples demonstrate that a long sequence (-0.7 kb) can be inserted in the genome by the inventive method, 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.

Accordingly, 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. For example, 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.

In particular, to identify the expression and subcellular localization of endogenous proteins, 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.

To identify the dynamics of endogenous proteins, live cells or tissue slices or the targeted organ (e.g. a brain) may be directly visualized with light microscopy such as two-photon or confocal microscopy, or by electron microscopy.

For example, 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. Thus, 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. However, 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. In addition, once staining conditions have been optimized for a tag, the method of the invention can be applied to image various tagged proteins without extensive optimization. Furthermore, since 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. These features are particularly advantageous for immunoelectron microscopy imaging, which often requires extensive optimization of staining conditions and good control samples (e.g. knockout mice).

For selecting (i.e. screening) and/ evaluating (i.e. assessing) a pharmaceutical agent for therapeutic and side effects, 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. In this regard, 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). In particular, the in vivo genome editing method of the invention can be easily combined with the abundant existing resources of genetically modified animals. In this regard, by performing the herein provided method in human disease model animals (e.g. mice), one will be able to visualize and investigate changes of the expression and distribution of disease-related proteins and/or molecular markers of diseases. 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. In particular, therefore, 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. Although excitatory/inhibitory imbalance is a well-known factor for pathogenesis of autism, little is known about where, when and how the subcellular abnormality of excitatory/inhibitory synapses occurs in disease states.

Furthermore, with the herein provided in vivo genome editing method the detailed effects of candidate therapeutic drugs on diseases can be evaluated in vivo. 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.

Thus, one aspect of the invention relates to a method for screening and/or evaluating drugs, comprising the steps:

(a) modifying the gene(s) encoding the disease-related protein(s) and/or molecular marker(s) in a non- human animal 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 in vivo genome editing method;

(b) administering a candidate drug to the animal; and

(c) using the epitope tag or fluorescent protein to visualize the disease-related protein(s) and/or molecular marker(s).

In 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). In the above-described method for screening and/or evaluating drugs, the non-human animal is preferably a mouse and more preferably a disease model mouse.

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)

LRRK2 R1441 G transgenic mice (Li, 2009, Nature Neurosci. 12:826-828)

Rett syndrome: MeCP2 knockout/conditional knockout mice (Guy, 2001 , Nature Genet. 27:322;

Guy, 2007, Science 315:1 143-1147) Fragile X syndrome: Fmr1 knockout mice (Bakker, 1994, Cell, 78: 23-33)

Autism: Neuroligin-3 R451 C knockin mice (Tabuchi, 2007, Science, 318: 71 -76)

Shank3 knockout mice (Pega, 2011 , Nature, 472: 437-442; Mei, 2016, Nature, 530:481-484)

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.

As demonstrated by the appended Examples, the inventive method advantageously enables specific single-cell labeling of endogenous proteins by either N- or C-terminal epitope tagging. In addition, 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. Moreover, 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. For example, 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. Accordingly, 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. By using the inventive method 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). However, targeting endogenous protein complexes has been challenging due to the lack of a specific antibody to the antigen. Since 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. For example, 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). Thus, the herein provided in vivo genome editing method can be a valuable tool for developing a specific drug with target-directed drug delivery in vivo.

As described herein, the in vivo genome editing method of the present invention can be used to modify mitotic progenitor cells of a non-human embryo. However, in one embodiment of the present invention, said method is used to modify mitotic progenitor cells of a human embryo. Thus, one embodiment of the present invention relates to a method for in vivo genome editing, comprising:

(a) delivering a genome editing machinery to mitotic progenitor cells of a human embryo through in utero electroporation; and

(b) modifying at least one target sequence by using HDR.

Modifying progenitor cells of a human embryo may be used for curing genetic diseases within this embryo. Accordingly, 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. Mutations that contribute to these diseases that may be cured by using the herein provided in vivo genome editing method are described herein. All definitions, examples and preferred features that are herein described in context with the in vivo genome editing method for modifying mitotic progenitor cells of a non-human embryo apply, mutatis mutandis, for the in vivo genome editing method for modifying mitotic progenitor cells of a human embryo. Encompassed by the present invention are the following items:

1 . A method for in vivo genome editing, comprising:

2. The method of item 1 , wherein 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.

3. The method of item 2, wherein the site-specific DNA nuclease is:

(i) a zinc-finger nuclease (ZFN);

(ii) a transcription activator-like effector nuclease (TALEN);

(iii) a Cas9 nuclease; or

(iv) a Cpfl nuclease.

4. The method of item 3, wherein the Cas9 nuclease is Streptococcus pyogenes Cas9 (SpCas9) or Staphylococcus aureus Cas9 (SaCas9).

5. The method of item 3(iii) or 4, wherein the genome editing machinery further comprises:

(i) 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;

(ii) a polynucleotide encoding the RNA molecules of (i);

(iii) 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

(iv) a polynucleotide encoding the chimeric RNA of (iii).

6. The method of item 3(iv), wherein the genome editing machinery further comprises:

(i) at least one guide RNA comprising a target sequence specific CRISPR RNA (crRNA) molecule; or

(ii) a polynucleotide encoding the RNA molecules of (i). The method of item 5 or 6, wherein the genome editing machinery comprises two or more different guide RNAs or two or more different polynucleotides encoding different guide RNAs. The method of item 1 , 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. The method of any one of items 1-8, 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 method of item 9, wherein the donor template polynucleotide is a single-stranded oligodeoxynucleotide (ssODN). 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 method of any one of items 1 -1 1 , wherein the genome editing machinery comprises:

(ii) a chimeric polynucleotide encoding a transposon and a transposase. The method of item 12, wherein the polynucleotide(s) include(s) a vector sequence. The method of item 12 or 13, wherein the transposon comprises an epitope tag and/or a fluorescent protein. The method of any one of items 1 -14, wherein the target sequence comprises at least one of the following sequences:

(i) 5'-NGG

(ii) 5'-NNGRRT; or

(iii) 5'-TTN. The method of any one of items 1-15, wherein the target sequence is a protein encoding gene. The method of item 16, wherein the protein encoding gene is modified in step (b) in the coding or non-coding region. The method of any one of items 1-17, wherein the modification of the target sequence is substitution, insertion, or deletion of at least one nucleotide of the target sequence. The method of any one of items 1 -18, wherein at least two target sequences are modified. The method of any one of items 1 -19, wherein the modification of the at least one target sequence is any one of the following modifications:

(i) insertion of an epitope tag and/or a fluorescent protein;

(iii) introduction of at least one disease-specific mutation;

(iv) knock-out of a gene; or

(v) a combination of (i) and (iii), or of (i) and (iv). The method of item 20, wherein the epitope tag is a HA-tag and/or the fluorescent protein is monomeric EGFP (mEGFP). The method of any one of items 1-21 , wherein 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 method of item 22, wherein the progenitor cells in the developing brain or the progenitor cells of the developing spinal cord are progenitor cells of neurons. The method of item 22, wherein 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 method of item 22, wherein the progenitor cells of muscle cells are progenitor cells of cardiomyocytes or progenitor cells of skeletal muscle cells. The method of any one of items 1-25, wherein the non-human embryo is at any stage from embryonic day 10 (E10) to embryonic day 18 (E18). The method of any one of items 1-26, wherein the non-human embryo is a non-human mammalian embryo. The method of item 27, wherein the non-human mammalian embryo is 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. 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 non-human animal, which is produced by the method of item 30, wherein the non-human animal carries a HDR-mediated genome modification in a post-mitotic 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. 35. The modified cell of item 34, wherein said cell is a differentiated neuron or a differentiated muscle cell.

36. A non-human animal comprising the modified cell of any one of items 34-36.

37. Use of the non-human embryo of item 32, the non-human animal of item 33 or 36, or the modified cell of item 34 or 35, 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.

38. The use of item 37, wherein the subcellular localization, distribution and/or dynamics of at least one endogenous protein is analyzed by light or electron microscopy.

In the appended Examples a novel technique called SLENDR (single-cell labeling of endogenous proteins by CRISPRCas9-mediated homology-directed repair) has been developed. This technique allows HDR- mediated genome editing in the mammalian brain in vivo. Since HDR is known to predominantly occur in the S and G2 phases of the cell cycle (Chu, 2015, Nat Biotechnol 33 543-8; Heidenreich, 2016, Nat Rev Neurosci 17 36-44; Hsu, 2014, Cell 157 1262-78; Maruyama, 2015, Nat Biotechnol 33 538-42; Saleh- Gohari, 2004, Nucleic Acids Res 32 3683-8), mitotic neuronal progenitors, which presumably have homologous recombination activity, have been targeted. 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. Thus, 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.

The term "electroporation" (also called "electropermeabilization") is commonly known in the art and 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.

The method "in utero electroporation" is commonly known in the art and described, e.g., in Tabata, 2001 , Neuroscience 103 865-72; Saito, 2001 , Dev. Biol. 240 237-46; Nishiyama, 2012, Eur J Neurosci 36 2867- 76. 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.

Then a pregnant animal (e.g. rodent) may be anesthetized. Next, for postoperative pain relief, an analgesic like buprenorphine may be delivered subcutaneously. Then the abdomen may be shaved and sterilized the incision site. To surgically expose the uterus, a vertical incision along the midline in the skin may be made; and, using scissors, it may be cut through the peritoneum. To prevent the embryos from drying out, the opening may be covered with sterile saline soaked gauze. To relax the myometrium, ritodrine hydrochloride may be applied to the exposed uterine horns. Next, 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. Then, 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). For example, for transfecting the developing brain 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. Then, 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. For example, for electroporation of the developing brain, 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. at 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) may be delivered with forceps-shaped electrodes (e.g. at E10-13, CUY650P3; at E13-15, CUY650P5; Nepa Gene) connected to an electroporator (NEPA21 , 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.

Then, the uterine horn may be placed back into the abdominal cavity. Before closing the incision, 2-3 ml of warm saline may be added to the cavity. Then, the peritoneum may be sewed together with absorbable sutures, and the skin may be closed using staples.

In utero electroporation has been shown to be a useful tool for investigating how specific genes contribute to neural development. 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).

The invention relates to an in vivo genome editing method, "in vivo" is Latin for "within the living". Therefore, herein 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 particular, in the in vivo genome editing method of the present invention, 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.

Herein, the term "genome editing" (also called "genome editing with engineered nucleases (GEEN))" 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. Herein, 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.

Herein a "genome editing machinery" comprises all molecules (i.e. polynucleotide(s) and/or protein(s)) that allow genome editing. For example, 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. If the site-specific nuclease is a Cas9 nuclease, the genome editing machinery further comprises a dual-guide RNA (i.e. a crRNA molecule and a tracrRNA molecule), or a single-guide RNA (i.e. a chimeric RNA molecule comprising a crRNA sequence and a tracrRNA sequence), or a polynucleotide (such as a plasmid vector) encoding these RNA molecules. If the site- specific nuclease is a Cpfl nuclease, the genome editing machinery further comprises a crRNA, or a polynucleotide encoding this RNA. In order to induce homology-directed repair (HDR), 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.

Herein, the term "delivering" or grammatical variations thereof mean "providing" or "introducing". In particular, herein "delivering a genome editing machinery to mitotic progenitor cells" means "providing mitotic progenitor cells with a genome editing machinery" . Or, in other words, "delivering a genome editing machinery to mitotic progenitor cells" means "introducing a genome editing machinery into mitotic progenitor cells". More specifically, in context of the present invention, "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.

Herein "homology-directed repair" or "HDR" refers to a mechanism in cells to repair single or double strand DNA lesions by homologous recombination; see, e.g., Cong, 2013, Science 339 819-23; Pardo, 2009, Cellular and Molecular Life Sciences 66 (6): 1039-1056; Bolderson, 2009, Clinical Cancer Research, 15: 6314-6320. 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. Therefore, HDR-based gene editing is difficult in post-mitotic cells such as neurons, cardiomyocytes or skeletal muscle cells. When the homologue DNA piece is absent, another process called non-homologous end joining (NHEJ) can take place instead. The highly error-prone NHEJ pathway induces insertions and deletions (indels) of various lengths that can result in frameshift mutations and, consequently, gene knockout. By contrast, 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) has to contain a region with sequence homology with the region to be repaired.

The term "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.

Herein "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. Herein 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. of the developing brain (such as progenitor cells of neurons), which are capable of cell division; and/or progenitor cells of muscle cells (such as progenitor cells of cardiomyocytes or progenitor cells of skeletal muscle cells), which are capable of cell division.

Herein "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.

The term "progenitor cells" is commonly known in the art and is described, e.g., in Weissman, 2000, Cell 100 157-168. Herein, "progenitor cells" are cells that undergo at least one additional round of division. Accordingly, herein the term "progenitor cells" also includes stem cells. However, herein the term "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. Also included by the term "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.

Herein an "embryo" is a multicellular diploid eukaryote in an early stage of embryogenesis, or development. In general, in organisms that reproduce sexually, 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. In animals the zygote will begin to divide by mitosis to produce a multicellular organism. The result of this process is an embryo. Herein, all stages from the zygote until birth are considered to be embryonic stages. In humans, a pregnancy is generally considered to be in the embryonic stage of development between the fifth and the eleventh weeks after fertilization, and is considered a fetus from the twelfth week on. However, herein the term "embryo" comprises the human embryo as well as the human fetus. In the herein provided in vivo genome editing method in utero 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. Thus, step (b) of the inventive method results in a modified target sequence. Herein, 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. For example, the modification that is introduced into the target sequence may be at least one substitution, insertion, or deletion. Herein 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. Preferably 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.

The term "modified" or "modifying" includes, but is not limited to, one or more nucleotides that are substituted, inserted and deleted within the target sequence.

The term "substitution", as used herein 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). Also encompassed by the term "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. Preferably, the number of replacement nucleotides corresponds to the number of originally present nucleotides that are substituted.

Herein the term "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". When the number of inserted nucleotides is not dividable by three, 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. When the number of inserted nucleotides is dividable by three, the resulting insertion is an "in-frame insertion". In this case, the reading frame remains intact after the insertion and translation will most likely run to completion if the inserted nucleotides do not code for a stop codon. However, because of the inserted nucleotides, the finished protein may contain, depending on the size of the insertion, one or multiple new amino acids that may affect the function of the protein. In a preferred aspect of the invention, term "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. In another preferred aspect of the invention, the term "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.

Herein the term "deletion" refers to the loss of nucleotides or larger parts of genes, such as exons or introns as well as entire genes. As defined with regard to the term "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.

The above defined modifications (i.e. "substitution", "insertion" and "deletion") 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. Examples of 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. Such 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).

The term "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. Most Cas9 nucleases introduce double strand breaks, but some previous studies used mutant Cas9 to introduce multiple single strand breaks to perform HDR-mediated genome editing in vitro, see below. Site-specific cleavage occurs at locations determined by both base-pairing complementarity between the crRNA and the target DNA and a short motif, referred to as the protospacer adjacent motif (PAM), juxtaposed to the complementary region in the target DNA (Jinek, 2012, Science, 337: 816-821 ).

Any Cas9 nuclease known in the art may be employed in accordance with the present invention. The sequences of several known Cas9 nucleases are shown in WO 2014/131833.

Herein the Cas9 nuclease may be a modified Cas9 protein, wherein the nuclease function of the protein is altered into a nicking endonuclease function. In other words, 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. More specifically, aspartate can for example be replaced against alanine at position 10 of the Streptococcus pyogenes Cas9 as shown in Cong, 2013, Science, 339: 819-823. The use of a modified Cas9 protein having nicking endonuclease function provides the advantage that the thus introduced DNA damage in the genome is more likely to be repaired via homologous recombination, instead of by nonhomologous end joining. In accordance with the method of the invention, the transposase and/or the site-specific nuclease (e.g. the Cas9 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. Also the transposon and/or the guide RNA(s) may be introduced in form of a polynucleotide. It will be appreciated that 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). Means and methods to ensure expression of a functional polypeptide or RNA are well known in the art. For example, 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. Preferably, 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. Such 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. 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. 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.

Herein, the term "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. Thus, 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).

In accordance with the present invention 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. In accordance with the present invention, 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. Typically (but not exclusively), in the case of single strand breaks, such breaks are repaired by homologous recombination, while double strand breaks are typically repaired by either non-homologous end joining (NHEJ) or homologous recombination. In context of the present invention, 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. By using such a donor template polynucleotide targeted modification of a genome can be achieved with high specificity. In the herein provided in vivo genome editing method the genome of an embryo is modified. In context of the invention, said embryo (or the animal developed from the embryo or a modified cell obtained from said embryo or from said animal) may be analyzed for successful modification of the target genome. Methods for analyzing for the presence or absence of a modification are well known in the art and 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). Examples for 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. Examples for 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. Examples for 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.

Instead of analyzing the embryo (or the animal developed from the embryo of the modified cell obtained from said embryo or from said animal) for the presence or absence of the desired modification, a successfully modified embryo (or animal developed from the embryo of modified cell obtained from said embryo or from said animal) 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.

Herein, a number of documents including patent applications and scientific publications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. The present invention is further described by reference to the following non-limiting figures and examples.

The Figures show:

Figure 1. In Vivo Single-Cell Labeling of Endogenous Proteins by SLENDR

(A and C) Graphical representation of the mouse genomic loci of CaMKIIa (A) and ΟθΜΚΙΙβ

(C) showing the target sites for Cas9, sgRNA and ssODNs. The sgRNA targeting sequences are underlined. The protospacer-adjacent motif (PAM) sequences are indicated. The stop and start codons of

CaMKIIa (A) and ΟθΜΚΙΙβ (C), respectively, are marked. The Cas9 cleavage sites are indicated by the arrowheads. PCR primer sets (control and recombination) for PCR genotyping

(E and F) are indicated by the arrows.

(B and D) Confocal microscopic images of the cerebral cortex electroporated at E12 (top and middle) and E15 (bottom) showing the DAPI signal and immunoreactivities for mEGFP and the HA tag fused to the C- terminus of endogenous CaMKIIa (B) and N-terminus of endogenous CaMKIIp (D). Middle panels show negative control experiments in which the sgRNA for CaMKIIa was paired with the ssODNs for CaMKIfi (B) and vice versa (D).

(E) The efficiency of SLENDR for CaMKIIa and CaMKIIp (the ratio of the number of HA/mEGFP double- positive neurons to that of mEGFP positive neurons). CaMKIIa, E12 layer

(L) 2/3, n = 545 neurons/3 mice; E12 L4-6, n = 275/3; E15 L2/3, n = 713/3. CaMKIIp, E12 L2/3, n = 716/3;

E12 L4-6, n = 379/3; E15 L2/3, n = 367/3. **p < 0.01 , Dunnett test, in comparison with E12 L2/3.

(F and G) (left) PCR genotyping using genomic DNA extracted from the electroporated brain.

Recombination primer sets (top; F, CaMKIIa-F1 and HA-R1 ; G, HA-F1 and CaMKIIp-R1) and control primer sets (bottom; F, CaMKIIa-F1 and CaMKIIa-R1 ; G, CaMKIIp-F1 and CaMKIIp-R1) were used for

PCR. (right) DNA sequencing analysis of the PCR products for CaMKIIa-HA (F) and HA-CaMKIfi (G). The

HA tag sequence is marked.

Data are represented as mean ± SEM .

Scale bars, 50 μπτι.

See also Figure 12 and Table 5.

Figure 7. Visualization of CaMKIIa and CaMKI^ by SLENDR Using Different sgRNA Targeting Sites, Related to Figure 1

(A, C and E) 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.

(B, D and F) 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 CaMKIIa (D) and the C-termini of CaMKIIa (B) and ΟβΜΚΙΙβ (F).

Scale bars, 50 μιη. Figure 8. Negative Control Experiments Using Incorrect sgRNA-ssODNs Pairs, Related to Figure 2

(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 sgRNA for CaMKIIa (E, G and H) or ΟθΜΚΙΙβ (A-D and F) was paired with the ssODNs for MeCP2

(A), β-Adin (B), DCX (C), Rab11a (D), 14-3-3ε (E), FMRP (F), Arc (G) or PKCa (H), respectively.

Scale bars, 50 μιη.

Figure 9. Verification of In Vivo Genome Editing for Generation of an HA knock-in allele, Related to Figure 2

(A, D, G, J and M) Graphical representation of the mouse genomic loci of β-Actin (A), CaW.2

(D), 14-3-3ε (G), Arc (J) and PKCa (M) without (upper) or with (lower) recombination showing PCR primer sets (control and recombination) used for PCR genotyping.

(B, E, H, K and N) PCR genotyping using the recombination (top) and control (bottom) primer sets.

(C, F, I, L and 0) DNA sequencing analysis of the PCR product using the recombination primer set in B, E,

H, K and N. The HA tag sequence is marked.

Figure 10. Evaluation of Specificity and Effects on the Protein Expression in the Surrounding Cells that Did Not Undergo HDR, Related to Figure 2

(A) Colocalization of MeCP2-HA and endogenous MeCP2. Confocal microscopic images of the cerebral cortex electroporated at E12 showing the immunoreactivities for the HA tag and endogenous MeCP2.

(B) 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. The sgRNA for N-terminus of MeCP2 (upper) or CaMKIfi (lower) was paired with the ssODNs for MeCP2.

(D) Confocal microscopic images showing the DAPI signal and immunoreactivities for mEGFP and endogenous MeCP2 in layer 2/3 in the cortex. The dotted circles show the nuclear region of mEGFP positive neurons. The intensities of the fluorescent signal of endogenous MeCP2 in mEGFP positive (n=103) and negative (n=100) neurons are shown (right, bottom), p = 0.19,

Student's t test.

Scale bars, 5 μιη (A, D); 50 μιη (C). Figure 11. Further Examples of Mapping of the Subcellular Localization of Endogenous Proteins by SLENDR, Related to Figure 2

(A) Confocal microscopic images showing the mEGFP signal and immunoreactivities for endogenous DCX in layer 2/3 in the cortex. mEGFP-positive neurons are indicated as asterisks.

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.

Figure 12. Verification of In Vivo Genome Editing for Generation of a mEGFP knock-in allele, Related to Figure 6

(A and B) Negative control experiments for Figures 6B and 6D. The sgRNA for CaMKIIa was paired with the plasmid-based donor for ΟθΜΚΙΙβ (A) and vice versa (B). Confocal microscopic images of the cerebral cortex showing the fluorescence of DsRed2 and mEGFP.

(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.

(D) PCR genotyping using the primer set in C.

(E) DNA sequencing analysis of the PCR product in D. The 12 bp linker and the mEGFP

sequences are marked.

Scale bars, 50 μm.

The Examples illustrate the invention. Example 1 : Materials and Methods Animals

All experimental procedures were approved by the Max Planck Florida Institute for Neuroscience Institutional Animal Care and Use Committee and were performed in accordance with guidelines from the US National Institutes of Health. Swiss Webster mice were obtained from Charles River. The day on which the vaginal plug was detected was designated as embryonic day 0 (EO). The first 24 h after birth was referred to as postnatal day 0 (PO). Both of male and female pups were used. However, only female mother animals were used for in utero electroporation.

DNA Constructs

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 ΟθΜΚΙΙβ (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. To minimize off-targeting effects, the CRISPR design tool was used (http://crispr.mit.edu/) (Ran, 2013, Nat Protoc 8 2281-308). To generate sgRNA-expressing plasmids, a pair of annealed oligos (-20 bp) 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. Details of the oligonucleotides are described herein below. To fuse mEGFP (monomeric EGFP, A206K) to the C- or N-terminus of CaMKIIa or CaMKII , respectively, plasmid-based donor templates for HDR were prepared. To 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. To target CaMKII , 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. For the piggyBac transposon system, which was used to avoid episomal plasmid loss upon cell division, 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); and 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 /ΙΚΙΙβ 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. For 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. In Utero Electroporation

In utero electroporation was performed as previously described (Borrell, 2005, J Neurosci Methods 143 151-8; Chen, 2012, J Neurosci Methods 207 172-80; Imamura, 2015, Eur J Neurosci 41 147-56; Kitazawa,

2014, J Neurosci 34 11 15-26; Nishiyama, 2012, Eur J Neurosci 36 2867-76; Soma, 2009, J Comp Neurol 513 113-28; Tabata, 2001 , Neuroscience 103 865-72). In brief, mice were deeply anesthetized with 2% isoflurane (Piramal Healthcare). Buprenorphine-SR (0.1 mg/mouse, ZooPharm) was subcutaneously administered for analgesia. To relax the myometrium, ritodorin hydrochloride (0.7-1.4 g/g of body weight; Sigma-Aldrich) was applied to the exposed uterine horns. The final concentration of 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 μς/μΙ, 20 μΜ and 1 μς/μΙ, 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,

2015, Eur J Neurosci 41 147-56; Kitazawa, 2014, J Neurosci 34 11 15-26; Nishiyama, 2012, Eur J Neurosci 36 2867-76; Soma, 2009, J Comp Neurol 513 1 13-28). It should be noted that higher DNA concentration may cause complication in injecting the DNA solution in the ventricle. DNA solution was mixed with Fast Green (0.1 mg/ml, Sigma-Aldrich) and 1-2 μΙ 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. 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). The position and angle of the electrode was set as previously described with some modifications (see Figure 4A) (Borrell, 2005, J Neurosci Methods 143 151-8; Chen, 2012, J Neurosci Methods 207 172- 80; Imamura, 2015, Eur J Neurosci 41 147-56; Kitazawa, 2014, J Neurosci 34 11 15-26; Nishiyama, 2012, Eur J Neurosci 36 2867-76; Soma, 2009, J Comp Neurol 513 1 13-28; Tabata, 2001 , Neuroscience 103 865-72). Table 2: Position and angle of the electrode as well as timing of the in utero electroporation for the transfection of particular progenitor target cells.

Histology

Under deep ketamine-xylazine anesthesia (100 μg of ketamine -10 μg 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 μιη in thickness and 100 μιη in thickness for embryonic brain) were prepared (VT1200, Leica). For immunohistochemistry, sections were permeabilized with 0.3-0.4% Triton X-100 in PBS, blocked with 5% normal goat serum and 2% BSA or 5% normal donkey serum in PBS, and incubated overnight with the following primary antibodies: rabbit anti-HA (1 :1000, Cell Signaling Technology), mouse anti-HA (1 :1000, Covance), rabbit anti-FLAG (1 :1000, Sigma), chicken anti-GFP (1 :1000, Millipore), guinea pig anti-NeuN (1 :1000, Millipore), rabbit anti-GFAP (1 : 1000, DAKO), rabbit anti- MeCP2 (1 :1000, Cell Signaling Technology), rabbit anti-MeCP2 (1 :200, Millipore), goat anti-DCX (1 :200, Santa Cruz) and rabbit anti-calbindin antibodies (1 :1000, Millipore). For the visualization of CaMKIIa-HA, antigen retrieval was performed using pepsin (1 mg/ml in 0.2 N HCI for 1 min, DAKO) (Fukaya and Watanabe, 2000). After 1-3 h incubation with Alexa Fluor-conjugated secondary antibodies (Invitrogen or Jackson Immunoresearch) followed by DAPI staining (0.1 g/ml, Life technologies), the stained slices were imaged using a confocal laser-scanning microscope (LSM780 or LSM880 with Airyscan, Zeiss). 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/). For analyzing mEGFP knock-in cells, coronal vibratome sections were imaged without immunostaining using LSM780. For the spine/dendrite ratio analysis, 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 μιη intervals covering all spines in a dendrite were used to measure the fluorescence intensity of mEGFP-labeled CaMKIIa or CaMKII . To calculate the spine/dendrite ratio of mEGFP-tagged CaMKII intensity, 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.

Preembedding Immunoelectron Microscopy

An 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 μιη 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. Localizing high electron-dense particles larger than 14 nm radius as silver-enhanced gold particles (1.98 μιη3 of a gold-positive dendrite and 2.35 μιη3 of a surrounding control dendrite, respectively) was used for analysis. As negative controls, a dendrite near the HA-positive dendrite was randomly selected. Images were analyzed with ImageJ and Adobe Photoshop CS6 (Adobe Systems) and three-dimensional reconstruction of the spines was performed using Amira software (version 6.0.1 , FEI).

Genomic PCR and DNA Sequencing

To isolate genomic DNA from the lUE-transduced (mEGFP or DsRed2-positive) brain area, DNeasy Blood & Tissue Kit (Qiagen) was used according to the manufacturer's instruction. Using the extracted DNA as a template, 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.

Organotypic Slice Culture Preparation

Organotypic cortical slice cultures were prepared as described previously (Bolz, 1990, Nature 346 359-62; Stoppini, 1991 , J Neurosci Methods 37 173-82; Yamamoto, 1989, Science 245 192-4). In brief, coronal slices of 400 μιη thickness were dissected using a tissue chopper from the electroporated somatosensory cortex at P1-3. The slices were plated on a membrane filter (Millicell-CM PICMORG50, Millipore). These cultures were maintained at 37 °C in an environment of humidified 95% air and 5% C02. The culture medium was exchanged with fresh medium every other day.

Two-Photon Microscope and Glutamate Uncaging

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). In brief, 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). MNI-caged L- glutamate (4-methoxy-7-nitroindolinyl-caged Lglutamate, Tocris) was uncaged with a train of 6 ms laser pulses (3.5-4 mW under the objective, wave length = 720 nm, 30 times at 1 Hz) near a spine of interest. 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.

Statistical Analysis

All statistical values were presented as mean ± SEM. The Student's t test was used when two independent samples were compared. Dunnett test was used for multiple comparisons. Statistical analysis was performed with GraphPad Prism 6. Differences between data sets were judged to be significant at p < 0.05.

Example 2: In Vivo Single-Cell Labeling of Endogenous Proteins by Homology-Directed Repair (HDR)

In order to label a specific protein with a tag in non-dividing brain cells by HDR-mediated gene editing, it is necessary to introduce HDR machinery into progenitor cells before their final cell division. To test whether this strategy, termed SLENDR (single-cell labeling of endogenous proteins by CRISPRCas9-mediated homology-directed repair), can provide the efficiency sufficient for imaging subcellular localization of a protein of interest, it was first aimed to insert the HA tag into endogenous CaMKIIa and CaMKIIp, two major subunits of the dodecameric Ca2+/CaMdependent kinase II (CaMKII) necessary for neuronal plasticity (Kim, 2015, Neuron 87 813-26; Lee, 2009, Nature 458 299-304; Lisman, 2012, Nat Rev Neurosci 13 169-82).

Specific single-guide RNAs (sgRNAs) targeting the vicinity of the stop and start codon of CaMKIIa and ΟθΜΚΙΙβ, 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 ΟθΜΚΙΙβ (Figures 1A and 1 C). To minimize the possibility of gene knockdown in cells where CRISPR-Cas9-mediated DNA double-strand breaks were repaired through the NHEJ pathway, 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. These constructs (S. 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.

In the stained slices, 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. Among neurons in transfected area in layer 2/3, about a half of population was mEGFP positive (mEGFP/NeuN: CaMKIIa, 48.2 ± 6.7 %; CaMKII , 40.0 ± 2.9 %). Among these mEGFP positive cells, a small population of the cells were found to be HA positive (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).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). When IUE was performed at E15, a smaller population of layer 2/3 neurons were labeled with the HA tag (HA/mEGFP: CaMKIIa, 0.7 ± 0.1 %; CaMKII , 0.7 ± 0.4 %, HA/NeuN: CaMKIIa, 0.1 ± 0.0 %; CaMKII , 0.2 ± 0.1 %) (Figures 1 B, 1 D and 1 E). Given that the superficial cortical layers (layer 2/3) are populated with later-born neurons (Chen, 2012, J Neurosci Methods 207 172-80), these findings suggest that SLENDR is more efficient when IUE is performed several days before the final neurogenic divisions of targeted cells.

To test the specificity of the construct, the incorrect ssODNs-sgRNA pairs, like CaMKIIa ssODNs-CaAf ///3 sgRNA or vice versa were used. Under these conditions, no fluorescence signal was detected in brain slices stained with anti-HA antibody (Figures 1 B and 1 D). These results indicate that the genome editing is specific to the sequence of the sgRNA. It was further confirmed that the expected genome editing occurred at the DNA level by performing PCR amplification of the targeted locus. Electrophoretic analysis revealed the presence of recombined PCR product at a size consistent with the recombined allele in brains transfected with the constructs necessary for HDR (Figures 1 F and 1 G). In contrast, neither untransfected control brains nor brains transfected with incorrect ssODNs-sgRNA pairs showed the corresponding PCR amplification (Figures 1 F and 1 G). Furthermore, DNA sequencing of the amplified PCR products indicated that the HA tag sequence was integrated as expected (Figures 1 F and 1 G). In addition, sgRNAs targeting different sequences in the respective genes produced a similar pattern of HA staining (Figure 7), demonstrating flexibility of construct design. Thus, SLENDR enables specific single-cell labeling of endogenous proteins by either N- or C-terminal epitope tagging.

SLENDR is Scalable to Various Endogenous Proteins

To validate the applicability of SLENDR to visualize the subcellular localization of a broad spectrum of endogenous proteins in brain tissue, 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).

First the HA tag was fused to MeCP2, a chromatin-associated protein that regulates gene transcription (Chen, 2001 , Nat Genet 27 327-31). MeCP2 was selected for the following reasons. First, the endogenous subcellular localization can be imaged by traditional immunostaining approaches using a specific, characterized antibody. Second, 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. As expected, it was found that MeCP2-HA and HA-MeCP2 were distributed exclusively in the nucleus at P7 (Figures 2A, 2B, 10B and 10C). In addition, 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. Since many mEGFP-positive cells were expected to undergo the error-prone, NHEJ-mediated repair of Cas9-induced DNA double-strand breaks, the targeting sgRNA were designed to place the cleavage site in the 5'-untranslated region of MeCP2 gene (Figure 10B). This strategy was designed to minimize potential effects on expression of the targeted gene in these cells. Indeed, the intensities of endogenous MeCP2 detected by an antibody against the C- terminus of MeCP2 was similar between mEGFP-positive and negative cells (Figure 10D; p = 0.19, Student's t test). This indicated that the endogenous expression of MeCP2 was not significantly disturbed by the NHEJ-mediated repair.

Next, SLENDR was applied to insert the HA tag to endogenous β-Actin, a major cytoskeletal protein in dendritic spines. As expected, immunostaining using the anti-HA antibody showed that ΗΑ-β-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). In addition, the high contrast images demonstrate that SLENDR enables the precise evaluation of the number and morphology of dendritic spines without the potential morphological phenotypes caused by overexpression of fluorophore tagged-actin or actin binding proteins (Riedl, 2008, Nat Methods 5 605-7).

To evaluate the timescale of HDR after introducing SLENDR constructs.Doublecortin (DCX), a protein that is expressed early in brain development was targeted. DCX is a microtubule-associated protein expressed in postmitotic migrating and differentiating neurons in the developing brain (Gleeson, 1999, Neuron 23 257-71). Intriguingly, 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. In addition, the effect of NHEJ on the expression of DCX was evaluated by immunostaining using an antibody against the C-terminus of the protein. The images showed that 98.7 % of the mEGFP positive neurons exhibited DCX expression (n = 151 cells) (Figure 11 A). Together with the negligible NHEJ effects on MeCP2 expression (Figure 10D), these data suggest that the strategy of targeting the Cas9-mediated cleavage at 5'-untranslated regions minimizes the effect of NHEJ on the expression of the target gene for N-terminal tagging.

SLENDR was further applied to a variety of endogenous proteins with distinct subcellular localizations. 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). 14-3-3ε, a signaling protein involved in neuronal migration and synaptic plasticity, was diffusely distributed in the cytoplasm both at P0 and P28 in neurons and astrocytes (Qiao, 2014, J Neurosci 34 4801 -8; Toyo-oka, 2014, J Neurosci 34 12168-81 ) (Figures 2K and 2L). 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). Finally, it was found that a isoform of protein kinase C (PKC), a member of a family of serine/threonine kinases implicated in a wide range of cellular functions (Steinberg, 2008 Physiol Rev 88 1341-78), was distributed mostly on the plasma membrane of the soma and dendrites at P9 (Figures 2Q and 2R). Interestingly, PKCa was less accumulated on the plasma membrane at P27, suggesting that endogenous PKCa may be more active at the developing stage (Figures 1 1 E-1 1 G). Thus, SLENDR enables to determine the distribution pattern of proteins that has been undefined or controversial by conventional methods.

Notably, HA-positive cells have never been observed when incorrect ssODNs-sgRNA pairs were used (Figures 8 and 10). In addition, precise genome editing was confirmed by PCR amplification of the targeted loci followed by DNA sequencing of the PCR products (Figure 9). Furthermore, for each target protein, all HA-positive cells in the same region showed similar HA-staining pattern. These results collectively demonstrate that SLENDR enables to label endogenous proteins specifically. Taken together, SLENDR is a highly generalizable technique that can be applied to various species of proteins in the brain from embryonic to adult stages for high-quality mapping of subcellular localization.

Nanometer-Resolution Analysis by SLENDR

Immunoelectron microscopy allows nanoscale visualization of endogenous proteins with defined ultrastructures in cells. However, the lack of reliable antibodies compatible with electron microscopy imaging limits its application to a variety of proteins. Thus, it was tested whether SLENDR could be applied to ultrastructural imaging of endogenous proteins using immunoelectron microscopy. To do so, 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. Then 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). Finally, the specificity of the immunogold labeling was examined by comparing the labeling density in HA-positive and surrounding neurons in the same specimen (Figure 3E; HA-positive, 241.3 ± 34.5 particles / μιη³; surrounding control, 2.6 ± 2.6 particles / μιη³; p < 0.001 , Student's t test). These experiments demonstrate that SLENDR is useful for nanometer scale localization of endogenous proteins.

SLENDR is Scalable to Various Cell Types in Various Brain Regions

It was then tested if SLENDR can be applied to different cell types in different brain regions by targeting distinct progenitor cells present at different locations and timings in the developing brain Borrell, 2005, J Neurosci Methods 143 151 -8; Chen, 2012, J Neurosci Methods 207 172-80; Kitazawa, 2014, J Neurosci 34 1115-26; Nishiyama, 2012, Eur J Neurosci 36 2867-76; Soma, 2009, J Comp Neurol 513 113-28). By adjusting the timing and electroporation angle (Figure 4A), see Tables 1 and 2, various HA-tagged endogenous proteins (β-Actin, CaMKIIa, CaMKII and MeCP2) were observed in various cell types in widespread brain regions (Table 4), including CA1 pyramidal neurons and dentate granule cells in the hippocampus (Figures 4B and 4H), spiny stellate cells in the subiculum (Figures 4C and 4H), granule cells in the olfactory bulb (Figure 4D), medium spiny neurons in the striatum (Figure 4E), basolateral amygdala neurons (Figure 4F), and granule and Purkinje cells in the cerebellum (Figures 4G and 4H). HA-CaMKII in parallel fibers originating from cerebellar granule cells was detected along the dendrites of Purkinje cells in the molecular layer (Figure 4G).

As we showed that staining of endogenous ΗΑ-β-Actin enables clear visualization of dendritic spine morphology in neurons (Figure 2D), we imaged ΗΑ-β-Actin in various cell types in the brain to compare the structure of dendritic spines. Morphological diversity of dendritic spines existed among different cell types including dentate granule cells (Figure 4H) and CA1 pyramidal neurons (Figure 4B) in the hippocampus, layer 2/3 pyramidal neurons in the cortex (Figure 2D), spiny stellate cells in the subiculum (Figure 4H) and cerebellar Purkinje cells (Figure 4H). Thus, SLENDR 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. Labeling Multiple Endogenous Proteins by SLENDR

Multiplex labeling of different endogenous proteins with different tags would enable high resolution co- localization assays. Since CRISPR-Cas9 is able to target multiple genes simultaneously (Cong, 2013, Science 339 819-23; Heidenreich, 2016, Nat Rev Neurosci 17 36-44; Swiech, 2015, Nat Biotechnol 33 102-6), the ability of SLENDR to target two different proteins in the same cell was tested (Figure 5A). Simultaneous labeling of CaMKIIa and CalN ΙΚΙΙβ with the HA and FLAG tag, respectively, were performed by co-introducing SLENDR constructs for HA-CaMKIIa and FLAG-CaMKII into the developing cortex at E13 by IUE (Figure 5A). Immunostaining of brain slices at P14 was performed using anti-HA and anti- FLAG antibodies. HA-CaMKIIa and FLAG-CaMKII were detected in a number of layer 2/3 neurons (HA- positive/mEGFP-positive, 4.5 ± 0.4 %; FLAGpositive/mEGFP-positive, 4.9 ± 0.6 %; n = 577 cells), and a significant fraction of neurons exhibited both HA and FLAG signals (HA and FLAG double- positive/mEGFP-positive, 0.7 ± 0.1 %; n = 577 cells) (Figure 5B). This double labeling efficiency was higher than the simple multiplication of each labeling efficiency (4.5% * 4.9% = 0.2%), demonstrating the practicality of the method for co-localization assay. Thus, 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 in Combination with Single-Cell Knockout

The ability to examine endogenous subcellular protein localization in the context of a knockout of a different protein would provide functional insight into interactions between the visualized and deleted protein. In this regard, combining SLENDR with NHEJ-based single-cell knockout would be of particular interest, since it would allow for the study of cell-autonomous gene function and to compare normal and knockout cells in the same tissue (Zong, 2005, Cell 121 479-92). Taking advantage of the multiplexity of CRISPR-Cas9 (Cong, 2013 Science 339 819-23; Heidenreich, 2016 Nat Rev Neurosci 17 36-44; Swiech, 2015 Nat Biotechnol 33 102-6), SLENDR constructs were simultaneously introduced to insert the HA tag to β-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 Θ105584; Swiech, 2015, Nat Biotechnol 33 102-6). Visualization of HA-tagged β-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.

While knockout efficiency of NHEJ-mediated genome editing has been reported to be very high in neurons (-70-100 %) (Heidenreich, 2016, Nat Rev Neurosci 17 36-44; Incontro, 2014, Neuron 83 1051-7; Straub, 2014, PLoS One 9 e105584; Swiech, 2015, Nat Biotechnol 33 102-6), immunohistochemistry with anti- MeCP2 antibody showed that MeCP2 was absent in 48.8 % of mEGFP-positive CA1 pyramidal neurons at P28. This reduced efficiency was probably due to the dilution of the knockout constructs during cell divisions (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). As expected, both MeCP2 negative and positive neurons were observed in ΗΑ-β-Actin positive CA1 neurons in the same slices (Figure 5D). Notably, the knockout efficiency in ΗΑ-β-Actin positive neurons (24.1 %) was substantially lower than that in whole population (48.8%). This is presumably because the knockout constructs are diluted in a population of cells that divides extensively, and this population of cells likely provides more efficient HDR. A loss of function mutation in MeCP2, which leads to Rett syndrome in humans, has been reported to impair maturation of dendritic spines (Chen, 2001 , Nat Genet 27 327-31 ; Swiech, 2015 Nat Biotechnol 33 102-6). Therefore, the density of dendritic spines between MeCP2 negative and positive neurons were compared (Figures 5D and 5E). Spine density in the apical dendrite of CA1 neurons was comparable in MeCP2 positive neurons to that reported in these neurons in a previous electron microscopic study (Harris, 1992, J Neurosci 12 2685-705). Importantly, spine density was significantly lower in MeCP2 negative neurons, consistent with previous studies (Figure 5E; p = 0.046, Student's t test) (Swiech, 2015, Nat Biotechnol 33 102-6).

Although high-quality antibodies against endogenous proteins are required to distinguish knockout and control cells, our strategy combining SLENDR with NHEJ-mediated gene knockout provides a useful means to investigate the cell-autonomous effects of a gene of interest on endogenous proteins.

Live Imaging of Endogenous Proteins by SLENDR

Labeling endogenous proteins with fluorescent proteins would allow to image protein dynamics in live cells without overexpression artifacts, further expanding the applicability of SLENDR. Thus, it was tested if SLENDR can be used to insert a long sequence encoding a fluorescent protein into genes of interest. For this, we targeted mEGFP to the sequence just upstream of the stop codon of CaMKIIa or downstream of the start codon of ΟθΜΚΙΙβ in the genome (Figures 6A and 6C). A plasmid-based template (-2.3-2.5 kb) containing the -0.7 kb mEGFP sequence was used. The SLENDR constructs together with the transposon vectors expressing DsRed2 were introduced to progenitors of layer 2/3 neurons through IUE at E12. At P14-28, it was found that a sparse subset of layer 2/3 neurons exhibited mEGFP fluorescence fused to endogenous CaMKIIa and CaMKII (Figures 6B and 6D). The efficiency of mEGFP knock-in was lower than that of HA knock-in (mEGFP-positive/DsRed2-positive, < 1 %), consistent with previous studies showing that a long sequence insertion through HDR is less efficient (Cox, 2015, Nat Med 21 121-31). The specificity of mEGFP knock-in was confirmed by control experiments using correct and incorrect template- sgRNA pairs and DNA sequencing following PCR amplification of the targeted locus (Figure 12).

To further verify the benefit of imaging endogenous proteins, the distribution pattern of endogenous and overexpressed mEGFP-tagged CaMKII were compared in layer 2/3 cortical neurons at P14. Since CaMKII has an actin-binding domain (Lisman, 2012, Nat Rev Neurosci 13 169-82), overexpressed CaMKII tends to accumulate in spines to a greater extent than overexpressed CaMKIIa. Notably, the degree of enrichment of endogenous CaMKIIa and CaMKII in spines, as measured by the spine/dendrite ratio of the peak intensity, was significantly higher and lower than that of overexpressed CaMKIIa and CaMKII , respectively (Figures 6E and 6F; CaMKIIa, p < 0.001 ; CaMKII , p = 0.004 , Student's t test). This is perhaps because overexpressed subunits tend to form homomeric enzymes, while endogenous CaMKIIa and CaMKII tend to form heteromers (Lisman, 2012, Nat Rev Neurosci 13 169-82). Thus, the localization of endogenous (heteromeric) subunits would be enriched in spines to an extent that is between homomeric CaMKIIa and CaMKII .

Live imaging was next performed to monitor the synapse-specific translocation of CaMKII after the stimulation of a single dendritic spine (Bosch, 2014, Neuron 82 444-59; Lee, 2009, Nature 458 299-304; Lisman, 2012, Nat Rev Neurosci 13 169-82; Nishiyama, 2015, Neuron 87 63-75). Organotypic cortical slice cultures were prepared from mice in which endogenous CaMKIIa or CalN ΙΚΙΙβ was labeled with mEGFP by SLENDR. When a train of two-photon glutamate uncaging pulses (1 Hz, 30 s) was applied at single spines, mEGFP-tagged CaMKIIa and CaMKII were rapidly accumulated in the stimulated spines and remained for more than 30 min. The translocation was highly restricted to the stimulated spine, with no significant increase in fluorescence intensity in the surrounding spines (Figures 6G-6L; CaMKIIa, p < 0.001 ; CaMKli , p < 0.001 ; Dunnett test). These results demonstrate that SLENDR enables monitoring of the dynamics of endogenous proteins in living tissue.

Discussion

In this study 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.

It is known that the efficiency of HDR highly depends on the cell cycle and is limited by the competition with NHEJ (Chu, 2015, Nat Biotechnol 33 543-8; Lin, 2014, Elife 3 e04766; Maruyama, 2015, Nat Biotechnol 33 538-42). Consistent with this, it was found that SLENDR was efficient in a limited time window when neuronal progenitors were still dividing. CRISPR-Cas9-mediated HDR occurred within a few days after IUE (Figure 2F) and the HDR efficiency was significantly reduced when IUE was performed near the stage of the final cell divisions of target cells (Figures 1 B and 1 D). In this regard, the direct delivery of pre-assembled Cas9 protein-guide RNA ribonucleoprotein complexes (RNPs), rather than expressing these components from plasmids, might increase the time window for SLENDR, as RNPs were recently reported to provide rapid action of the nuclease with high efficiency and low off-target effects for genome editing (Kim, 2014, Genome Res 24 1012-9; Lin, 2014, Elife 3 e04766). Alternatively, 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.

We validated the specificity of the SLENDR-mediated sequence insertion in the genome in several ways. First, 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/). Second, control experiments were performed by using correct or incorrect sgRNAs to detect genomic insertion or the expression of the HA tag. Third, distinct sgRNAs targeting the identical gene were used and similar HA staining patterns were observed. Fourth, the localization of HA staining was consistent with that previously reported based on immunohistochemical, biochemical or electron microscopic studies. Fifth, the localization of HA staining was similar from cell to cell. In addition, although off-target cleavage by Cas9 may be induced in the genome, HDR is unlikely to occur at the off-target sites, because homologous recombination is known to be a highly sequence-specific event. The specificity of SLENDR could be further enhanced by using recently reported Cas9 variants with minimum or no off-target effects while retaining comparable on-target cleavage activity (Kleinstiver, 2016, Nature ; Slaymaker, 2016, Science 351 84-8).

One of the merits of SLENDR is that high-quality antibodies can be used for the detection of tags. In addition, once staining conditions have been optimized for a tag, SLENDR can be applied to image various tagged proteins without extensive optimization. Furthermore, since 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. These features are particularly advantageous for immunoelectron microscopy imaging, which often requires extensive optimization of staining conditions and good control

samples (e.g. knockout mice).

It was demonstrated therein that introducing a 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). Overall, because many studies are constrained by the lack of high-quality antibodies against proteins of interest, SLENDR will provide a generalizable and reliable platform for exploring localization of uncharacterized endogenous proteins using light and electron microscopy.

Multiplexing CRISPR-Cas9 mediated processes provided some insight into how HDR and NHEJ occur following double strand breaks. It was found herein that the cells labeled with a tag with SLENDR, or in other words the cells that underwent HDR, showed less NHEJ-mediated knockout. This result suggests that HDR occurs more frequently in a population of highly dividing cells. Consistent with this, the efficiency of double-labeling via multiple HDRs was higher than that obtained by the simple multiplication of each labeling efficiency. The high efficiency of double-labeling by SLENDR opens the possibility of co- localization assay for a pair of endogenous proteins with high resolution and contrast. Given relatively consistent knock-in efficiency (Table 3), the SLENDR-based double labeling technique would be applicable to many pairs of proteins.

SLENDR might lead to unintended sequence changes through insertion/deletion mutations mediated by NHEJ in the transfected cells. Therefore, throughout this study, the effect of on-target NHEJ was minimized by choosing target sequences at 5'-untranslated region or near the stop codon for N- or C- terminal tagging, respectively. As expected from this design, the expression of DCX was detected in 98.7 % of the mEGFP positive neurons following SLENDR for the N-terminal tagging of DCX (Figure 11 A). In addition, the expression level of MeCP2 was not significantly affected for the N-terminal tagging of MeCP2 (Figure 10D). However, potential effects of NHEJ may be evaluated for each gene depending on the purpose of experiments. For example, 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. In particular, one may select target sequences so that the estimated cleavage site (3 bp upstream of the PAM) can be located in the untranslated region.

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.

Since the 5'-NGG protospacer-adjacent motif (PAM) of SpCas9 abundantly exists in the genome and other CRISPR endonucleases with different PAMs are also available to target different sites in the genome (Cong, 2013, Science 339 819-23; Hsu, 2014, Cell 157 1262-78; Zetsche, 2015 Cell 163 759-71), most proteins should be suitable for SLENDR. Furthermore, its throughput and cost-effectiveness are much higher than previous single-cell protein labeling methods (Fortin, 2014, J Neurosci 34 16698-712; Gross, 2013, Neuron 78 971-85). Thus, 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.

Table 3. The Ratio of HA (+) to mEGFP (+) Neurons, HA (+) to NeuN (+) Neurons and mEGFP (+) to NeuN (+) Neurons for Each Target Protein, Related to Figures 1 and 2.

The numbers of cells analyzed are indicated in parentheses.

Table 4. The Ratio of HA(+) to mEGFP(+) Neurons and mEGFP(+) to NeuN(+) Neurons for Each Cell Type, Related to Figure 4.

The numbers of cells analyzed are indicated in parentheses.

Table 5. DNA Constructs Used in This Study, Related to Figures 1 -12. (C), C-terminus; (N), N- terminus; (Ctrl), negative control; (1), 1 pg/μΙ; PB, piggyBac; hyPBase, hyperactive piggyBac transposase; pX330N, pX330 without the FLAG tag sequence fused to SpCas9.

The present invention refers to the following nucleotide and amino acid sequences.

CTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAG

GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCAC

GCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGA

CCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGA

GCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCG

AGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTA

CTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGC

GGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAA

GAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCT

CCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGA

CATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACC

TATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTG

AGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGAC

GGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAG

CCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGA

AGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACA

TCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGC

GGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGC

AGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAA

CCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAG

GTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATG

AAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCG

AGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCA

CAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGA

AGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCG

AGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTA

CCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAG

CGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGA

TTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGT

GGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGAC

CGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAG

AAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGT

GGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAA

AGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGA

TCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGA

ACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAG

AAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACG GACTATGTAAAAGAAGCAAAGCAGTTATTAAAAGTACAAAAAGCATATCATCAACTTGATCAATCATTTATAGACA

CTTATATTGATTTATTGGAAACAAGAAGAACATATTATGAGGGACCAGGTGAAGGTAGCCCATTTGGATGGAAA

GATATTAAAGAATGGTATGAAATGTTAATGGGACATTGTACGTATTTCCCAGAAGAATTACGTAGTGTGAAATAT

GCCTATAATGCTGATTTATATAATGCGCTGAATGATTTGAACAACTTGGTTATTACACGAGATGAGAATGAGAAG

CTAGAGTATTATGAAAAATTCCAAATTATCGAGAATGTCTTTAAACAAAAGAAAAAGCCGACGCTTAAACAAATTG

CGAAGGAAATCTTGGTGAATGAAGAAGACATCAAAGGCTATCGTGTCACAAGTACAGGTAAACCAGAATTTACA

AACTTGAAAGTTTATCACGATATCAAAGATATTACAGCAAGAAAAGAAATTATCGAGAATGCAGAGCTACTCGAT

CAAATAGCTAAAATATTAACTATTTACCAGTCATCAGAAGATATACAAGAAGAATTAACAAACCTAAATTCAGAAT

TGACACAAGAAGAGATTGAACAAATTTCAAACTTGAAAGGTTATACAGGAACTCATAACCTTTCACTAAAGGCAA

TAAATTTAATATTAGACGAATTGTGGCATACGAACGATAATCAAATAGCTATTTTCAATCGTTTGAAACTTGTACC

TAAAAAGGTAGATTTAAGCCAACAAAAAGAAATTCCTACTACTTTAGTTGATGATTTTATACTGTCTCCAGTAGTG

AAACGTTCATTTATACAATCTATTAAAGTTATTAACGCTATTATTAAAAAATACGGTTTGCCAAATGATATTATTAT

TGAACTTGCGAGAGAAAAGAATTCTAAAGATGCACAAAAAATGATTAATGAAATGCAGAAGAGAAATCGTCAAAC

GAATGAACGTATTGAGGAAATTATAAGAACGACAGGTAAAGAAAATGCTAAATATTTAATTGAAAAAATTAAGCT

GCACGATATGCAAGAAGGGAAATGTTTATACTCGTTAGAAGCAATCCCTCTAGAAGATTTACTTAATAATCCATT

CAATTACGAAGTAGACCATATCATTCCACGTTCTGTTTCTTTCGATAACTCTTTCAATAATAAAGTGTTGGTGAAA

CAAGAAGAAAATAGTAAAAAAGGTAATAGAACGCCATTTCAATATTTAAGTTCTTCAGATTCTAAAATAAGTTATG

AGACATTCAAAAAGCATATTTTAAATCTTGCTAAAGGCAAAGGTAGAATCTCTAAGACGAAAAAAGAATATTTGTT

AGAAGAACGAGATATCAATCGCTTCAGTGTCCAAAAAGATTTTATTAACCGTAACTTAGTAGATACACGCTATGC

GACAAGAGGGTTAATGAACTTATTAAGATCTTATTTTAGAGTGAATAACTTAGATGTCAAAGTGAAATCGATTAAT

GGCGGATTCACAAGTTTCTTAAGAAGGAAATGGAAGTTCAAAAAAGAAAGAAATAAGGGCTACAAACACCATGC

TGAAGATGCACTGATTATTGCGAACGCTGATTTTATTTTCAAAGAATGGAAAAAACTAGATAAAGCTAAAAAAGT

GATGGAAAATCAAATGTTTGAAGAAAAGCAAGCTGAAAGTATGCCTGAAATTGAGACTGAGCAAGAGTATAAAG

AAATTTTTATAACGCCTCATCAAATTAAACATATTAAGGATTTTAAAGATTATAAATATTCACATAGAGTTGATAAA

AAGCCGAATAGAGAGTTAATAAATGATACATTATATTCTACGAGAAAAGATGACAAGGGTAATACATTAATCGTT

AATAACTTAAATGGTTTATACGATAAAGATAATGATAAATTGAAAAAATTAATTAATAAATCACCTGAAAAATTATT

GATGTATCATCATGATCCACAAACATATCAAAAATTAAAATTGATCATGGAACAATATGGCGATGAGAAAAATCC

GCTTTATAAATATTATGAAGAAACAGGCAATTACTTAACAAAATATAGTAAAAAAGATAACGGACCAGTCATCAAA

AAAATTAAATATTATGGTAACAAGCTAAATGCGCATTTAGATATTACGGATGATTATCCAAATAGCAGAAATAAAG

TAGTAAAACTTTCATTAAAACCATATCGCTTTGATGTTTATTTAGATAATGGGGTATATAAATTTGTGACAGTTAAA

AATTTAGATGTTATCAAAAAAGAAAACTACTATGAAGTTAATTCAAAGTGTTATGAAGAAGCAAAAAAACTGAAGA

AAATTAGTAATCAAGCAGAATTTATCGCAAGTTTTTACAATAATGACTTGATTAAGATTAACGGAGAATTATATAG

AGTCATAGGTGTAAATAATGATCTACTTAACAGAATTGAAGTAAATATGATAGACATCACATATAGAGAATATTTA

GAGAACATGAATGATAAAAGACCACCTAGAATAATTAAAACAATAGCAAGCAAAACACAATCTATTAAAAAGTATT

CTACAGATATTCTAGGCAATCTTTATGAAGTGAAGAGTAAAAAGCATCCTCAAATCATAAAGAAAGGATGA

AGGAGAAGGAGATCCTGAAGTCTCAGCTGGACAGCCTGCTGGGCCTGTACCACCTGCTGGACTGGTTTGCCGT

GGATGAGTCCAACGAGGTGGACCCCGAGTTCTCTGCCCGGCTGACCGGCATCAAGCTGGAGATGGAGCCTTC

TCTGAGCTTCTACAACAAGGCCAGAAATTATGCCACCAAGAAGCCCTACTCCGTGGAGAAGTTCAAGCTGAACT

TTCAGATGCCTACACTGGCCTCTGGCTGGGACGTGAATAAGGAGAAGAACAATGGCGCCATCCTGTTTGTGAA

GAACGGCCTGTACTATCTGGGCATCATGCCAAAGCAGAAGGGCAGGTATAAGGCCCTGAGCTTCGAGCCCACA

GAGAAAACCAGCGAGGGCTTTGATAAGATGTACTATGACTACTTCCCTGATGCCGCCAAGATGATCCCAAAGTG

CAGCACCCAGCTGAAGGCCGTGACAGCCCACTTTCAGACCCACACAACCCCCATCCTGCTGTCCAACAATTTC

ATCGAGCCTCTGGAGATCACAAAGGAGATCTACGACCTGAACAATCCTGAGAAGGAGCCAAAGAAGTTTCAGA

CAGCCTACGCCAAGAAAACCGGCGACCAGAAGGGCTACAGAGAGGCCCTGTGCAAGTGGATCGACTTCACAA

GGGATTTTCTGTCCAAGTATACCAAGACAACCTCTATCGATCTGTCTAGCCTGCGGCCATCCTCTCAGTATAAG

GACCTGGGCGAGTACTATGCCGAGCTGAATCCCCTGCTGTACCACATCAGCTTCCAGAGAATCGCCGAGAAGG

AGATCATGGATGCCGTGGAGACAGGCAAGCTGTACCTGTTCCAGATCTATAACAAGGACTTTGCCAAGGGCCA

CCACGGCAAGCCTAATCTGCACACACTGTATTGGACCGGCCTGTTTTCTCCAGAGAACCTGGCCAAGACAAGC

ATCAAGCTGAATGGCCAGGCCGAGCTGTTCTACCGCCCTAAGTCCAGGATGAAGAGGATGGCACACCGGCTG

GGAGAGAAGATGCTGAACAAGAAGCTGAAGGATCAGAAAACCCCAATCCCCGACACCCTGTACCAGGAGCTGT

ACGACTATGTGAATCACAGACTGTCCCACGACCTGTCTGATGAGGCCAGGGCCCTGCTGCCCAACGTGATCAC

CAAGGAGGTGTCTCACGAGATCATCAAGGATAGGCGCTTTACCAGCGACAAGTTCTTTTTCCACGTGCCTATCA

CACTGAACTATCAGGCCGCCAATTCCCCATCTAAGTTCAACCAGAGGGTGAATGCCTACCTGAAGGAGCACCC

CGAGACACCTATCATCGGCATCGATCGGGGCGAGAGAAACCTGATCTATATCACAGTGATCGACTCCACCGGC

AAGATCCTGGAGCAGCGGAGCCTGAACACCATCCAGCAGTTTGATTACCAGAAGAAGCTGGACAACAGGGAGA

AGGAGAGGGTGGCAGCAAGGCAGGCCTGGTCTGTGGTGGGCACAATCAAGGATCTGAAGCAGGGCTATCTGA

GCCAGGTCATCCACGAGATCGTGGACCTGATGATCCACTACCAGGCCGTGGTGGTGCTGGAGAACCTGAATTT

CGGCTTTAAGAGCAAGAGGACCGGCATCGCCGAGAAGGCCGTGTACCAGCAGTTCGAGAAGATGCTGATCGA

TAAGCTGAATTGCCTGGTGCTGAAGGACTATCCAGCAGAGAAAGTGGGAGGCGTGCTGAACCCATACCAGCTG

ACAGACCAGTTCACCTCCTTTGCCAAGATGGGCACCCAGTCTGGCTTCCTGTTTTACGTGCCTGCCCCATATAC

ATCTAAGATCGATCCCCTGACCGGCTTCGTGGACCCCTTCGTGTGGAAAACCATCAAGAATCACGAGAGCCGC

AAGCACTTCCTGGAGGGCTTCGACTTTCTGCACTACGACGTGAAAACCGGCGACTTCATCCTGCACTTTAAGAT

GAACAGAAATCTGTCCTTCCAGAGGGGCCTGCCCGGCTTTATGCCTGCATGGGATATCGTGTTCGAGAAGAAC

GAGACACAGTTTGACGCCAAGGGCACCCCTTTCATCGCCGGCAAGAGAATCGTGCCAGTGATCGAGAATCACA

GATTCACCGGCAGATACCGGGACCTGTATCCTGCCAACGAGCTGATCGCCCTGCTGGAGGAGAAGGGCATCG

TGTTCAGGGATGGCTCCAACATCCTGCCAAAGCTGCTGGAGAATGACGATTCTCACGCCATCGACACCATGGT

GGCCCTGATCCGCAGCGTGCTGCAGATGCGGAACTCCAATGCCGCCACAGGCGAGGACTATATCAACAGCCC

CGTGCGCGATCTGAATGGCGTGTGCTTCGACTCCCGGTTTCAGAACCCAGAGTGGCCCATGGACGCCGATGC

CAATGGCGCCTACCACATCGCCCTGAAGGGCCAGCTGCTGCTGAATCACCTGAAGGAGAGCAAGGATCTGAA

GCTGCAGAACGGCATCTCCAATCAGGACTGGCTGGCCTACATCCAGGAGCTGCGCAACAAAAGGCCGGCGGC

CACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCC

SEQ ID NO: 34: ssODN sequence (5'-3', upper case: HA tag sequence), CaMKIIa (N-terminus)

gtggcccctagttctgggggcagcgcttcagcatcccagccctagttcccagcctaaagcctcgcctgcctgcccagtgccaggatgTACCCATACGATGT

TCCAGATTACGCTgctaccatcacctgcacccgattcacagaagagtaccagctctttgaggaactgggaaagtgagtcacacctcagggagtcctag

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

Plasmid-based donor template sequences (5'-3')

SEQ ID NO: 71 : DR sequence

GTTTTAGAGCTA

SEQ ID NO: 72: tracrRNA sequence TAGCAAGTTAAAATAAGGCTAGTCCGTTTTT

SEQ ID NO: 73: T-rich PAM

TTTN

Claims

1 . A method for in vivo genome editing, comprising:

2. The method of claim 1 , wherein 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.

3. The method of claim 2, wherein the site-specific DNA nuclease is:

(i) a zinc-finger nuclease (ZFN);

(ii) a transcription activator-like effector nuclease (TALEN);

(iii) a Cas9 nuclease; or

(iv) a Cpfl nuclease.

4. The method of claim 3(iii), wherein the genome editing machinery further comprises:

(ii) a polynucleotide encoding the RNA molecules of (i);

(iv) a polynucleotide encoding the chimeric RNA of (iii).

5. The method of claim 3(iv), wherein the genome editing machinery further comprises:

(ii) a polynucleotide encoding the RNA molecules of (i).

6. The method of claim 4 or 5, wherein the genome editing machinery comprises two or more different guide RNAs or two or more different polynucleotides encoding different guide RNAs.

7. The method of claim 1 , 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.

8. The method of any one of claims 1-7, 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.

9. The method of claim 8, wherein the donor template polynucleotide is a single-stranded oligodeoxynucleotide (ssODN).

10. The method of any one of claims 1-10, wherein the genome editing machinery comprises:

(ii) a chimeric polynucleotide encoding a transposon and a transposase.

1 1. The method of claim 10, wherein the polynucleotide(s) include(s) a vector sequence.

12. The method of claim 10 or 1 1 , wherein the transposon comprises an epitope tag and/or a fluorescent protein.

13. A non-human embryo, which is produced by the method of any one of claims 1-12, wherein the non-human embryo carries a HDR-mediated genome modification in a progenitor cell or in a postmitotic cell.

14. A non-human animal, which is produced by the method of any one of claims claim 1-12, wherein the non-human animal carries a HDR-mediated genome modification in a post-mitotic cell.

15. A modified post-mitotic cell that has been obtained from the non-human embryo of claim 13 or from the non-human animal of claim 14, wherein the modified post-mitotic cell carries a HDR- mediated modification in its genome.