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WO2019239361A1 - Procédé d'insertion de séquence à l'aide de crispr - Google Patents

Procédé d'insertion de séquence à l'aide de crispr Download PDF

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WO2019239361A1
WO2019239361A1 PCT/IB2019/054934 IB2019054934W WO2019239361A1 WO 2019239361 A1 WO2019239361 A1 WO 2019239361A1 IB 2019054934 W IB2019054934 W IB 2019054934W WO 2019239361 A1 WO2019239361 A1 WO 2019239361A1
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sequence
cells
seq
cas9
dna
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Fanning ZENG
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Novartis AG
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Novartis AG
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/102—Mutagenizing nucleic acids
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present invention generally relates to methods of sequence insertion into a target site within a DNA sequence in a cell of an insertion sequence using the
  • Site-specific genome editing can be achieved by the CRISPR-Cas9 system, which comprises Cas9 nuclease and guide RNA (gRNA).
  • Cas9 creates a double-strand DNA break (DSB) at a target site specified by gRNA sequence.
  • Cells repair DSBs using the non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways.
  • NHEJ non-homologous end joining
  • HDR homology-directed repair
  • the predominant means of repairing DNA DSBs is by NHEJ. It is active in most, if not all cells types, and is not restricted to a certain phase of the cell cycle. The NHEJ system however tends to generate errors with the introduction of variable insertions or deletions (indel) at DSB.
  • the CRISPR-Cas9 system is widely used for gene knock-out, which takes advantage of these indels to create a disruption in the reading frame and thus knock-out gene expression.
  • the constitutive presence of Cas9 and gRNA e.g. through continued expression on vectors
  • Cas9 and gRNA can be used to force errors into the repair process. Even if DNA is correctly repaired by the NHEJ system of the cell, due to the constitutive presence of the Cas9/gRNA, it will be cut again by Cas9, until an error happens resulting in the loss of the gRNA binding site in the targeted site.
  • donor DNA is also required and all three components must be in cells at the same time to ensure a successful genome editing.
  • the donor DNA When the HDR mechanism is utilized to insert a sequence the donor DNA is designed such that it contains an exogenous sequence and two arms homologous to the sequences flanking the DSB. The exogenous sequence can then be integrated into the site of interest via HDR pathway. Both single-stranded DNA (ssDNA) and double- stranded DNA (dsDNA) donors can act as efficient HDR templates.
  • the length of the homology arms may vary depending on the size of DNA fragment that is to be integrated. It may range from around 50 nucleotides (nt) in ssDNA to 500-1500 base pairs (bp) for dsDNA. Synthesis of such donor DNAs can be costly and time consuming.
  • HDR based CRISPR knock-in The efficiency of HDR based CRISPR knock-in is generally very low, often much less than 5%, as the HDR mechanism is intrinsically inefficient. HDR activity is restricted to late S and G2 phases of the cell cycle. Lengthy clonal selection is often required before editing events can be even validated.
  • the NHEJ pathway can also be harnessed for gene knock-in or sequence insertion in a homology-independent manner.
  • dsDNA donor sequences are delivered into cells as mini-circle DNA or as a circular plasmid. These are then integrated into the target site using CRISPR/Cas9 and the NHEJ pathway of the cell.
  • efficiency of these protocols is still unsatisfactory (often around 1-20%) and the methodology has only been shown to work in cell lines (He et al, Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair, Nucleic acid Res. 19: 44(9), May 2016).
  • CRISPR knock-in or sequence insertion method that is efficient and is applicable to use in a variety of cells, including primary cells or non dividing cells.
  • the method is amenable to targeting multiple loci, as well as being affordable and easy to use.
  • the present invention provides a method of sequence insertion into a target site within a DNA sequence in a cell of an insertion sequence, comprising delivery into the cell of a) an endonuclease capable of introducing a double strand break at the target site, b) gRNA and c) donor DNA; wherein the donor DNA comprises the insertion sequence without an additional homology arm or homology arms and the donor DNA is delivered into a cell as linearised dsDNA and the endonuclease is delivered as protein.
  • the cells are selected from the group consisting of cell lines, primary cells or non-dividing cells.
  • the cells are selected from the group consisting of HeLa cells, HCT116 cells, HEK293 cells, SH-SY5Y cells, A549 cells, U20S cells, induced pluripotent stem cells, induced pluripotent stem cell derived iNgn2 neurons, skeletal muscle cells, Schwann cells, tenocytes, hematopoietic stem cells and T-cells.
  • the cells are selected from the group consisting of HeLa cells, HCT116 cells, HEK293 cells, SH-SY5Y cells, A549 cells, U20S cells, induced pluripotent stem cells, induced pluripotent stem cell derived iNgn2 neurons, skeletal muscle cells, Schwann cells and tenocytes.
  • the method of sequence insertion into a target site within a DNA sequence in a cell of an insertion sequence comprises delivery into the cell of
  • the donor DNA comprises the insertion sequence without an additional homology arm or homology arms and the donor DNA is delivered into the cell as linearised dsDNA and the endonuclease is delivered as protein and wherein the cells are selected from the group consisting of induced pluripotent stem cells, primary cells and non-dividing cells.
  • the method does not comprise a method for treatment of the human or animal body by therapy (or surgery). In an embodiment of the method of sequence insertion according to the invention, the method does not comprise a process for modifying the germ line genetic identity of human beings. In an embodiment of the method of sequence insertion according to the invention, the method is performed ex vivo or in vitro.
  • the donor DNA is incorporated into the target site via the non- homologous end joining pathway (NHEJ) pathway.
  • NHEJ non- homologous end joining pathway
  • the cells are primary cells.
  • the primary cells are skeletal muscle cells, Schwann cells, tenocytes, hematopoietic stem cells or T-cells.
  • the primary cells are skeletal muscle cells, Schwann cells, or tenocytes.
  • the cells are induced pluripotent stem cells, iNgn2 neurons, and the primary cells are skeletal muscle cells, Schwann cells, tenocytes, hematopoietic stem cells or T-cells.
  • the cells are induced pluripotent stem cells, iNgn2 neurons, and the primary cells are skeletal muscle cells, Schwann cells, or tenocytes.
  • the cells are skeletal muscle cells, Schwann cells, tenocytes, hematopoietic stem cells or T-cells.
  • the cells are skeletal muscle cells, Schwann cells, or tenocytes. In a specific embodiment the cells are skeletal muscle cells.
  • the donor DNA does not comprise additional nucleotides 5’ or 3’ of the insertion sequence.
  • the linearised dsDNA is blunt ended.
  • the donor DNA is about 3bp to about 1000bp in size, preferably about 9bp to about 700bp in size.
  • the donor DNA is about 3bp to about 120bp in size, preferably about 20bp to about 50bp in size.
  • the efficiency of the sequence insertion is selected from the group consisting of
  • the efficiency of the sequence insertion is at least about 5% in iPS cells or in iNgn2 neurons. In another specific embodiment of the method of sequence insertion, the efficiency of the sequence insertion is up to about 15% in iPS cells or in iNgn2 neurons. In yet another specific embodiment of the method of sequence insertion, the efficiency of the sequence insertion is up to about 12% in iPS cells or up to about 8% in iNgn2 neurons.
  • the efficiency of the sequence insertion is from about 5% to about 15% in iPS cells or in iNgn2 neurons and preferably the donor DNA is about 3 to about 120 bp, more preferably about 20 to about 100 bp in size, particularly preferably about 20 to about 50 bp in size.
  • the efficiency of the sequence insertion is at least about 6% in primary cells, preferably at least about 10% in primary cells.
  • the efficiency of sequence insertion is up to about 70% in primary cells.
  • the primary cells are skeletal muscle cells, Schwann cells or tenocytes.
  • the efficiency of the sequence insertion is between about 6% to about 70% in primary cells and the donor DNA is about 3 to about 120 bp, preferably about 20 to about 100 bp in size, particularly preferably about 20 to about 50 bp in size.
  • the efficiency of the sequence insertion is at least about 6% in Schwann cells. Preferably the efficiency of the sequence insertion is up to about 45% in Schwann cells. In a specific embodiment the efficiency of the sequence insertion is from about 6% to about 12% in Schwann cells and the donor DNA is about 55 to about 100 bp in size. In another specific embodiment the efficiency of the sequence insertion is from about 6% to about 40% in Schwann cells and the donor DNA is preferably about 20 to about 50 bp in size.
  • the efficiency of the sequence insertion is at least about 30% in human muscle skeletal cells. More preferably the efficiency of the sequence insertion is at least about 50% in human muscle skeletal cells. Particularly preferably the efficiency of the sequence insertion is up to about 70% in human muscle skeletal cells. Also preferably the efficiency of the sequence insertion is at least about 30% in human muscle skeletal cells and the donor DNA is about 3 to about 120 bp, preferably about 20 to about 100 bp in size, particularly preferably about 20 to about 50 bp in size. Also yet more preferably the efficiency of the sequence insertion is from about 30% to about 70% in human muscle skeletal cells and the donor DNA is about 20 to about 50 bp in size.
  • the efficiency of the sequence insertion is at least about 20% in tenocytes.
  • the efficiency of the sequence insertion is up to about 30% in tenocytes.
  • the efficiency of the sequence insertion is from about 20% to about 30% in tenocytes and the donor DNA is about 3 to about 120 bp, preferably about 20 to about 100 bp in size, particularly preferably about 20 to about 50 bp in size.
  • the donor DNA is selected from the group consisting of
  • the donor DNA is selected from the group consisting of
  • the donor DNA is a) less than about 120 bp (preferably more than 3 bp) and about 2.5 to about 15 picomolar DNA is delivered or
  • the donor DNA is at least about 3bp in size.
  • the donor DNA is about 3 bp to about 1000 bp in size. More preferably, the donor DNA is about 9 to about 700 bp in size. Yet more preferably, the donor DNA is about 15 to about 380 bp in size.
  • the donor DNA is about 20 to about 120 bp in size, preferably about 20 to about 100 bp in size, particularly preferably about 20 to about 50 bp in size.
  • the donor DNA is less than about 120 bp and at least about 2.5 picomolar DNA is delivered. In another embodiment of the method of sequence insertion according to the invention, the donor DNA is less than about 120 bp and about 2.5 to about 15 picomolar, preferably about 5 to about 10 picomolar, DNA is delivered. In a specific embodiment of the method of sequence insertion according to the invention the donor DNA is less than about 120 bp and about 7.5 picomolar DNA is delivered. In another specific embodiment of the method of sequence insertion according to the invention, the donor DNA is greater than about 500bp, preferably up to 1000bp, and about 0.5 to about 3 picomolar DNA is delivered.
  • the linearised dsDNA is delivered via liposome-mediated transfection or electroporation.
  • the linearised dsDNA is delivered via electroporation.
  • the endonuclease has a NLS sequence and is Cas9 or variants or orthologs thereof which are capable of introducing a double strand break at the target site.
  • the Cas9 is Streptococcus pyogenes wt Cas9.
  • the endonuclease is delivered to the target cell via liposome-mediated transfection or electroporation.
  • the endonuclease is delivered electroporation.
  • the gRNA and endonuclease together are delivered to the target cells as ribonucleoprotein (RNP) particles.
  • RNP ribonucleoprotein
  • the ribonucleoprotein particles are delivered to the target cell via electroporation.
  • the gRNA and endonuclease are delivered together to the target cells as ribonucleoprotein particles and wherein the gRNA and endonuclease are delivered together with the linearised dsDNA by electroporation.
  • the gRNA is delivered as a complex of 2 strands of RNA, one comprising crRNA and the other tracrRNA.
  • the method is used for sequence knock-in. In an alternative aspect of the method of sequence insertion, the method is used for sequence knock-out.
  • the dsDNA donor comprises the sequence of interest and a stop codon from the 5’ to 3’ direction and the sequence of interest and a stop codon from the 3’ to 5’ direction.
  • the insertion sequence is inserted into a target gene and does not introduce frame-shifts of the target gene.
  • the target site in a target gene is towards either the 5’ or 3’ end of the reading frame of the target gene.
  • the target site is in the 5’ untranslated region (5’UTR) of a target gene and the donor DNA comprises an ATG, such that translation of the sequence of interest may begin at this ATG start codon.
  • the PAM is in the 5’UTR and is less than about 50 nt from an ATG codon without an intervening in-frame stop codon.
  • the PAM is in the 5’UTR and is less than about 41 nt from an ATG codon without an intervening in-frame stop codon.
  • the method further comprises the step of generating and then isolating a product from the cells, wherein the product is a nucleic acid, peptide, polypeptide or protein and for nucleic acids comprises the nucleic acid insertion sequence or for peptides, polypeptides or proteins comprises the translated amino acid insertion sequence.
  • the invention in another aspect relates to a method to generate a product with an inserted sequence, comprising the method of sequence insertion according to the invention, wherein the product is a nucleic acid, peptide, polypeptide or protein and for nucleic acids comprises the nucleic acid insertion sequence or for peptides, polypeptides or proteins comprises the translated amino acid insertion sequence.
  • the method further comprises the step of isolating the product with the inserted sequence.
  • the invention provides a product obtainable or obtained from cells which have been modified by the method of sequence insertion according to the invention or generated according to the method to generate a product with an inserted sequence according to the invention, wherein the product is a nucleic acid, peptide, polypeptide or protein and for nucleic acids comprises the nucleic acid insertion sequence or for peptides, polypeptides or proteins comprises the translated amino acid insertion sequence.
  • the invention provides a cell, which has been modified by the method of sequence insertion according to the invention or by the method to generate a product with an inserted sequence according to the invention.
  • the invention provides a cell population obtainable or obtained by the method of sequence insertion according to the invention or by the method to generate a product with an inserted sequence according to the invention.
  • the cells in the cell population are primary cells.
  • the invention provides a product obtainable or obtained by the method according to the invention or a cell population obtainable or obtained by the method according to the invention for use in therapy.
  • the cells in the cell population are primary cells.
  • the invention provides a product obtainable or obtained by the method according to the invention or a cell population obtainable or obtained by the method of sequence insertion according to the invention for use in diagnostics (such as in vivo diagnostics) or surgery.
  • the cells in the cell population are primary cells.
  • the invention provides a composition comprising
  • the invention provides a method of treatment of a disease or disorder, preferably wherein the disease or disorder is associated with aberrant gene expression, using the method of sequence insertion according to the invention or method to generate a product with an inserted sequence according to the invention.
  • the invention provides a method of treatment of a disease or disorder, preferably wherein the disease or disorder is associated with aberrant gene expression, comprising administering to the subject a therapeutically effective amount of the product obtainable from cells which have been modified by the method of sequence insertion according to the invention or cell population obtainable or obtained by the method of sequence insertion according to the invention.
  • the invention provides a diagnostic method using the method of sequence insertion according to the invention. Preferbaly the diagnostic method is performed ex vivo.
  • the invention provides a kit for insertion of insertion sequence into a target site within a DNA sequence in a cell, comprising: (a)
  • gRNA and c) donor DNA capable of introducing a double strand break at the target site, b) gRNA and c) donor DNA, wherein the donor DNA comprises the insertion sequence without an additional homology arm or homology arms and the donor DNA is linearised dsDNA and wherein the gRNA and endonuclease are present together as ribonucleoprotein particles.
  • FIG. 1 V5 tag knock-in into HnRNP in HeLa cells
  • Figure 1a Top (SEQ ID NO: 41): Sequences for HNRNPA2B1 locus with the PAM site (shaded) and Cas9 cleavage site (dotted line).
  • FIG. 1 b Immunostaining of V5 antibody on HeLa cells reveals strong V5 signals exclusively localized in the nucleus, which corresponds well with the expected cellular location of HnRNP A2/B. When compared with DAPI staining, it is calculated that 40 ⁇ 1.5% cells are V5 staining positive.
  • FIG. 1c Cell lysates from wild type (WT) and V5 knock-in (Kl) samples were subjected to western blotting with V5 (left panel) and HnRNP (right panel) antibodies. Two V5 positive bands were visualized in Kl samples, which correspond to the predicted chimeras of the different isoforms.
  • Figure 2 V5 tag knock-in into HnRNP in HCT116 cells
  • FIG. 2a Immunostaining with V5 antibody shows that HCT116 cells display positive signals in nucleus. When compared with DAPI staining, it is calculated that 31.3 ⁇ 3.9% cells are V5 staining positive.
  • Figure 2b Lysates from untransfected or V5-HnRNP knock-in HCT116 cells were subjected to western blotting. A2/B1 isoforms with V5 knock-in can be detected with either V5 (left) or HNRNP antibody (right).
  • Figure 2c Graph showing Sanger sequencing results, which clearly suggests editing events after Cas9 cleavage site.
  • FIG. 3 V5 tag knock-in in b-actin, Vimentin and RLP10a in HeLa cells
  • Figure 3a 3 days after transfection, 45.3 ⁇ 1.5% cells display actin filament structures after staining with V5 antibody.
  • Figure 3b After V5 knock-in in Vimentin, intermediate filament structures were observed in 61.8 ⁇ 1.2% cells with V5 antibody staining.
  • FIG. 3c V5 sequence was knocked into RPL10A, which encodes Ribosomal Protein L10a.
  • Cells were stained with V5 antibody: 5.1 ⁇ 0.5% cells display positive signals in cytosol and nucleoli.
  • FIG. 4 knock-in in other common cell lines (HEK293 cells, SH-SY5Ycells, A549 and U20S cells)
  • FIG. 4a For HNRNPA2B1 in HEK293 cells, V5 signals were detected in 50.7 ⁇ 2.2% cells after CRISPR knock-in.
  • Figure 4c V5 knock-in in Vimentin in A549 cells showing 34.9 ⁇ 1.7% efficiency.
  • Figure 5a shows staining of pluripotency marker Nanog antibody in induced pluripotent stem cells (iPS cells).
  • Figure 5b shows V5 antibody staining showing 10.4 ⁇ 1.8% V5 knock-in efficiency in HnRNP in iPS cells.
  • Figure 5c shows staining of neuronal marker NF200 antibody in iNgn2 cells.
  • Figure 5d shows V5 antibody staining, confirming V5 knock-in in HNRNP in iNgn2 cells. The results showed that 5.9 ⁇ 1.6% iNgn2 cells displayed V5-HNRNP signals.
  • Figure 6 knock-in in rat primary Schwann cells
  • FIG. 6a After V5 knock-in, 36.3 ⁇ 2.5% Schwann cells displayed V5 signals as filament structures (Vimentin).
  • FIG. 6b V5 tag was knocked into Sox10, a nuclear protein, in primary rat Schwann cells. The result suggests that in 9.9 ⁇ 0.2% Schwann cells, Sox10 could be labelled by V5 antibody.
  • Figure 6c Genomic sequence for rat SOX10 (at 3’ terminus of its CDS) is shown on the top, with PAM site shaded and Cas9 cutting site indicated with a dotted line.
  • Figure 6c discloses SEQ ID NOS 43, 42, 44, 19, 45 and 39, respectively, in order of appearance.
  • Figure 7 knock-in in human primary skeletal muscle cells
  • V5 knock-in in Vimentin in primary human skeleton muscle cells V5-Vimentin signals could be observed in 66.3 ⁇ 2.5% cells.
  • Figure 8a Large dsDNA sequences (696 or 697bp) encoding mScarlet were knocked into Vimentin or b-actin ( Figure 8b) in HeLa cells. Live cell images were taken 7 days after transfection.
  • Figure 8c A 376bp dsDNA sequence encoding Y-FAST was knocked into b-actin in HeLa cells. 7 days after transfection, cells were stained with 5mM M2-HBR
  • FIG 9 Insertion of multi-stop codon sequence (MSC) to achieve knockout Figure 9a: Top panel shows two PAM sites in VIM locus, and lower panel shows the sequence for the MSC (SEQ ID NO: 23).
  • FIG. 9b CRISPR knock-out efficiency of Vimentin in HeLa cells was assessed by western blots, with KO efficiency corresponding to the reduction in Vimentin signals. While KO efficiencies were 65-75% with single gRNAs (gRNA 1 or 2), they were improved by addition of MSC. The highest KO efficiency was achieved by using both gRNAs and MSC together.
  • Figure 9c CRISPR knock-out efficiency of Vimentin (top bands) in HeLa cells was assessed by western blots. 1) wild type; 2) gRNA1 ; 3) gRNA2; 4) gRNA1 + gRNA2; 5) 2x gRNAs, plus MSC. b-actin was stained (bottom bands) as the loading control.
  • Figure 10 sequence insertion method to modulate cellular location of target proteins in HeLa cells
  • FIG. 10a V5 labelled HnRNP display normal nucleus location, which in Figure 10b is shown to be disrupted by addition of myristoylation sequence to V5-HnRNP.
  • Figure 11 Establish a novel functional assay in primary skeletal muscle cells
  • HiBiT tag was inserted into the C-terminus of IFIT1 in primary skeletal muscle cells.
  • I FIT 1 expression was induced by incubating the cells with IRNb at various concentrations.
  • the expression level of I FIT 1 was estimated by measuring NanoLuc activity. A clear dose dependent response was observed.
  • Figure 12 knock-in in rat primary tenocytes
  • V5 tag was knocked into Vimentin in primary rat tenocytes. The result suggests that in 23.9 ⁇ 0.9% tenocytes, Vimentin could be labelled by V5 antibody.
  • Figure 13 Establish a novel functional assay in primary tenocytes
  • HiBiT tag was inserted into the N-terminus of C-Jun in primary tenocytes. I FIT 1 expression was suppressed by incubating the cells with 20mM forskolin. The expression level of I FIT 1 was estimated by measuring NanoLuc activity. A clear down-regulation was observed.
  • the singular form“a”,“an” and“the” include plural references unless the context clearly dictates otherwise.
  • the term“a cell” includes a plurality of cells, including mixtures thereof.
  • Non-human animals include vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, cats, horses, cows, chickens, dogs, mice, rats, goats, rabbits, and pigs.
  • the subject is human. Except when noted, the terms "patient” or “subject” are used herein interchangeably.
  • the term“treat”,“treating” or “treatment” of any disease or disorder refers to alleviating or ameliorating the disease or disorder (i.e. , slowing or arresting the development of the disease or at least one of the clinical symptoms thereof); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder, including those which may not be discernible to the patient.
  • the term“prevent”,“preventing” or“prevention” of any disease or disorder refers to the prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder.
  • nucleic acid or“polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly
  • polypeptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified
  • polypeptides derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide or cell naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide or cell partially or completely separated from the coexisting materials of its natural state is“isolated.”
  • CRISPR system refers to a set of molecules comprising an RNA-guided nuclease or other effector molecule and gRNA that together are necessary and sufficient to direct and effect modification of nucleic acid at a target sequence by the RNA-guided nuclease or other effector molecule.
  • a CRISPR system comprises gRNA and a Cas protein, e.g., a Cas9 protein.
  • Cas9 systems Such systems comprising a Cas9 or modified Cas9 molecule are referred to herein as“Cas9 systems” or“CRISPR/Cas9 systems.”
  • RNA refers to a set of nucleic acid molecules that promote the specific directing of a RNA-guided nuclease or other effector molecule (typically in complex with the gRNA molecule) to a target sequence.
  • said directing is accomplished through hybridization of a portion of the gRNA to DNA (e.g., through the gRNA targeting domain), and by binding of a portion of the gRNA molecule to the RNA-guided nuclease or other effector molecule (e.g., through at least the gRNA tracr).
  • targeting domain is the portion of the gRNA molecule that recognizes, e.g., is complementary to, a target sequence, e.g., a target sequence within the nucleic acid of a cell, e.g., within a gene.
  • target sequence refers to a sequence of nucleic acids complimentary, for example fully complementary, to a gRNA targeting domain.
  • the target sequence is disposed on genomic DNA.
  • the target sequence is adjacent to (either on the same strand or on the complementary strand of DNA) a protospacer adjacent motif (PAM) sequence recognized by a protein having nuclease or other effector activity, e.g., a PAM sequence recognized by Cas9.
  • PAM protospacer adjacent motif
  • the term“tracr” as used herein in connection with a gRNA molecule refers to the portion of the gRNA that binds to a nuclease or other effector molecule.
  • Cas9 or“Cas9 molecule” refer to an enzyme from bacterial Type II CRISPR/Cas system responsible for DNA cleavage. Cas9 also includes wild-type protein as well as functional and non-functional mutants thereof. In embodiments, the Cas9 is a Cas9 of S. pyogenes.
  • An“indel,” as the term is used herein, refers to a nucleic acid comprising one or more insertions of nucleotides, one or more deletions of nucleotides, or a combination of insertions and deletions of nucleotides, relative to a reference nucleic acid, that results after being exposed to a composition comprising gRNA, for example a CRISPR system. Indels can be determined by sequencing nucleic acid after being exposed to a
  • composition comprising gRNA, for example, by NGS.
  • an indel is said to be“at or near” a reference site (e.g., a site complementary to a targeting domain of a gRNA molecule) if it comprises at least one insertion or deletion within about 10, 9 , 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide(s) of the reference site, or is overlapping with part or all of said reference site (e.g., comprises at least one insertion or deletion overlapping with, or within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides of a site complementary to the targeting domain of gRNA, e.g., gRNA described herein).
  • The“target site” as used herein is the specific locus in the cellular DNA where it is intended to insert exogenous DNA comprising the sequence of interest.
  • The“donor DNA” as used herein is the exogenous DNA sequence introduced into the cell comprising the sequence of interest.
  • the present invention relates to a method of sequence insertion into a target site within a DNA sequence in a cell of an insertion sequence, which utilizes genome editing in eukaryotic cells using a CRISPR/Cas system, e.g., a CRISPR/Cas9 system, and the NHEJ repair pathway of the cell to insert an exogenous sequence into the target site.
  • a CRISPR/Cas system e.g., a CRISPR/Cas9 system
  • the method comprises delivery into the cell of a) an endonuclease capable of introducing a double strand break at the target site, b) gRNA and c) donor DNA; wherein the donor DNA comprises the insertion sequence without an additional homology arm or homology arms and the donor DNA is delivered into a cell as linearised dsDNA and the endonuclease is delivered as protein.
  • the method may be known as Homology independent gene Tagging (HiTag).
  • The“insertion sequence” as used herein therefore relates to the sequence designed to be incorporated into the target site.
  • the insertion sequence comprises the sequence of interest.
  • The“donor DNA” is the exogenous DNA sequence introduced into the cell comprising the sequence of interest.
  • The“target site” as used herein is the specific locus in the cellular DNA where it is intended to insert exogenous DNA comprising the sequence of interest.
  • the donor DNA is typically designed in such a way as to have sequences flanking the insertion sequence in the donor DNA which are homologous to the sequences at or near the target site.
  • the donor DNA sequence from 5’ to 3’ therefore has a 5’ homology arm followed by the exogenous insertion sequence followed by a 3’ homology arm, wherein the 5’ homology arm is homologous to a sequence upstream of the target site and the 3’ homology arm is homologous to a sequence downstream of the target site.
  • a CRISPR/Cas9 system may be used to create a DSB at a specific target site, and when the DSB is repaired by the HDR pathway, this donor DNA then acts as a template, such that after repair the insertion sequence is incorporated into the target site.
  • the length of the homology arms may vary depending on the size of DNA fragment that is to be integrated. It may range from around 50 nucleotides (nt) in ssDNA to 500-1500 base pairs (bp) for dsDNA.
  • Homology arm or arms in the context of the donor DNA refers to sections of DNA which are homologous to sequences upstream or downstream respectively of the target site.
  • the term“homology” or“homologous” as used herein is defined as the percentage of nucleotide residues in the donor DNA that are identical to the nucleotide residues in the corresponding sequence at, or around, the target site, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleotide sequence homology can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2 or Megalign (DNASTAR) software.
  • homology between the 5' homology arm and the corresponding sequence at, or around, the target site in this context is typically at least about any of 80%, 85%, 90%, 95%, 98%, 99%, or 100%.
  • the homology between the 3' homology arm and the corresponding sequence at, or around, the target site, in this context is also typically at least about any of 80%, 85%, 90%, 95%, 98%, 99%, or 100%.
  • the present invention relates to use of the NHEJ pathway to insert the exogenous sequence in a homology independent manner.
  • the donor DNA is designed such that it does not comprise additional sequences flanking the insertion sequence which are homologous to the upstream and/or downstream sequences of the target site.
  • the donor DNA is designed such that it does not comprise additional nucleotides 5’ or 3’ of the insertion sequence.
  • The“insertion sequence” as used herein therefore relates to the sequence designed to be incorporated into the target site.
  • the insertion sequence comprises the sequence of interest.
  • the insertion sequence comprises nucleotides 5’ and/or 3’ of the sequence of interest.
  • some additional indels may be created when incorporating the insertion sequence, such that the final sequence comprises these indels 5’ and/or 3’ of the insertion sequence.
  • the present invention is not limited only to the exact set of sequences described in this example.
  • a small peptide tag at a target site in the DNA sequence of the cell, e.g. in a specific embodiment a V5 tag, SEQ ID NO: 39.
  • the sequence of interest in this example is therefore this V5 tag.
  • the insertion sequence is then designed to incorporate this sequence of interest (e.g. V5 tag) into the target site.
  • the insertion sequence therefore may be designed such that it has additional nucleotides at the 5’ and/or 3’ end of the sequence of interest (e.g. V5 tag) such that once the insertion sequence is incorporated into the target site, the sequence of interest (e.g.
  • V5 tag can be translated in-frame.
  • sequence of interest e.g. V5 tag
  • at the 5’ end it may have nucleotides coding for an ATG sequence, and at the 3’ end it may have an additional nucleotide, e.g. a thymine, so that the final incorporated sequence is in-frame.
  • additional 5’ and/or 3’ nucleotides are designed to be inserted into the target site, in addition to the sequence of interest.
  • the insertion sequence in this example therefore corresponds to SEQ ID NO: 16.
  • the donor DNA in this example corresponds to SEQ ID NO: 16.
  • the donor DNA is however not designed such that it has additional sequences flanking the sequence of interest or the insertion sequence which are homologous to the sequences upstream and downstream of the target site.
  • the insertion sequence In both these forward and reverse incorporations of the insertion sequence, no indels are present in addition to the insertion sequence i.e. the insertion sequence has been incorporated as designed, but either in the forward or reverse orientation. Yet another category of repair is possible, whereby the insertion sequence has been incorporated, but indel(s) are also present. Thus it is possible that the insertion sequence is incorporated with a small insertion or deletion at the 5’ and/or 3’ end.
  • the efficiency of sequence insertion may be measured in relation to the incorporation of the insertion sequence. In an embodiment it is measured by performing Sanger or NGS sequencing on the edited DNA, to measure the percentage of the various categories of incorporation events. In an embodiment the efficiency of the method of sequence insertion relates to the percentage of functional genome integration of the insertion sequence at the DNA level.
  • the efficiency of sequence insertion is measured at the level of protein expression (of the sequence of interest), by well-known techniques in the art such as immunohistochemistry or western blot.
  • the presence of the sequence of interest is assessed by these methods, in comparison to control, where the method of sequence insertion has not been performed.
  • the efficiency of the method of sequence insertion also depends on the targeting and cutting efficiency of gRNA/Cas9 complex; and the detection of the tagged proteins heavily rely on their endogenous expression level.
  • the insertion sequence is synthesised and prepared by methods well known in the art. (for example Letsinger RL, Ogilvie KK, et al. Synthesis of oligothymidylates via phosphotriester intermediates. J Am Chem Soc. 1969 June; 91(12): 3350-3355, 3360- 3365).
  • Commercial providers of oligonucleotides are widely used which custom make oligonucleotides with the desired sequence, such as IDT and Microsynth.
  • the insertion sequence is prepared as blunt-ended, linearised dsDNA.
  • the insertion sequence may be synthesised as oligonucleotides as forward and reverse strands, which are then dissolved in buffer, such as Duplex buffer (30 mM HEPES, pH 7.5; 100 mM potassium acetate). To anneal the oligonucleotides, they may be mixed at equal volume and incubated at 95°C for 5min, followed by cooling down slowly to RT.
  • the dsDNA is delivered into the cell as linearised DNA, and is not delivered as circular or plasmid DNA to the cell.
  • The“Cas9 molecule” can interact with gRNA (e.g., sequence of a domain of a tracr, also known as tracrRNA or trans activating CRISPR RNA) and, in concert with the gRNA, localize (e.g., target or home) to a site which comprises a target sequence and PAM (protospacer adjacent motif) sequence.
  • gRNA e.g., sequence of a domain of a tracr, also known as tracrRNA or trans activating CRISPR RNA
  • Cas9 molecules of, derived from, or based on the Cas9 proteins of a variety of species can be used in the methods and compositions described herein.
  • Cas9 molecules of, derived from, or based on, e.g., S. pyogenes, S. thermophilus, Staphylococcus aureus and/or Neisseria meningitidis Cas9 molecules can be used in the systems, methods and compositions described herein.
  • Additional Cas9 species include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhiz ' obium sp., Brevibacillus latemsporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lad, Candidatus Puniceispirillum, Clostridiu cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter sliiba
  • Neisseria sp. Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tislrella mobilis, Treponema sp., or Verminephrobacter eiseniae.
  • an active Cas9 molecule to interact with and cleave a target nucleic acid is PAM sequence dependent.
  • a PAM (protospacer adjacent motif) sequence is a sequence in the target nucleic acid. It is typically short, for example 2 to 7 base pairs long.
  • cleavage of the target nucleic acid occurs upstream from the PAM sequence.
  • Active Cas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences).
  • an active Cas9 molecule of S. pyogenes recognizes the sequence motif NGG and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Mali el al, SCIENCE 2013; 339(6121):
  • a Cas9 molecule comprises an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with; differs at no more than 1 %, 2%, 5%, 10%, 15%, 20%, 30%, or 40% of the amino acid residues when compared with; differs by at least 1 , 2, 5, 10 or 20 amino acids but by no more than 100, 80, 70, 60, 50, 40 or 30 amino acids from; or is identical to; S. pyogenes Cas9 (UniProt Q99ZW2).
  • the Cas9 molecule is a S.
  • pyogenes Cas9 variant such as a variant described in Slaymaker et al., Science Express, available online December 1 , 2015 at Science DOI: 10.1126/science. aad5227; Kleinstiver et al., Nature, 529, 2016, pp. 490-495, available online January 6, 2016 at
  • a Cas9 molecule comprises an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with; differs at no more than 1 %, 2%, 5%, 10%, 15%, 20%, 30%, or 40% of the amino acid residues when compared with; differs by at least 1 , 2, 5, 10 or 20 amino acids but by no more than 100, 80, 70, 60, 50, 40 or 30 amino acids from; or is identical to; S. pyogenes Cas9:
  • Lys Ala lie Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220
  • Asp Asp Leu Asp Asn Leu Leu Ala Gin lie Gly Asp Gin Tyr Ala Asp 275 280 285
  • Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala lie Leu Leu Ser Asp 290 295 300
  • Lys Lys Ala lie Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560
  • Lys Glu Asp lie Gin Lys Ala Gin Val Ser Gly Gin Gly Asp Ser Leu 705 710 715 720
  • Asp Asp Ser lie Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860
  • Lys Ala Gly Phe lie Lys Arg Gin Leu Val Glu Thr Arg Gin lie Thr 915 920 925
  • the Cas9 molecule is a S. pyogenes Cas9 variant of SEQ ID NO: 3 that includes one or more mutations to positively charged amino acids (e.g., lysine, arginine or histidine) that introduce an uncharged or nonpolar amino acid, e.g., alanine, at said position.
  • the mutation is to one or more positively charged amino acids in the nt-groove of Cas9.
  • the Cas9 molecule is a S.
  • the Cas9 molecule has a mutation only at position 855 of SEQ ID NO: 3, relative to SEQ ID NO: 3, e.g., to an uncharged amino acid, e.g., alanine.
  • the Cas9 molecule is a S.
  • the Cas9 molecule has a mutation only at position 810, position 1003, and position 1060 of SEQ ID NO: 3, relative to SEQ ID NO: 3, e.g., where each mutation is to an uncharged amino acid, for example, alanine.
  • the Cas9 molecule is a S.
  • the Cas9 molecule has a mutation only at position 848, position 1003, and position 1060 of SEQ ID NO: 3, relative to SEQ ID NO: 3, e.g., where each mutation is to an uncharged amino acid, for example, alanine.
  • the Cas9 molecule is a Cas9 molecule as described in Slaymaker et al., Science Express, available online December 1 , 2015 at Science DOI: 10.1126/science. aad5227.
  • the Cas9 molecule is a S. pyogenes Cas9 variant of SEQ ID NO: 3 that includes one or more mutations.
  • the Cas9 variant comprises a mutation at position 80 of SEQ ID NO: 3, e.g., includes a leucine at position 80 of SEQ ID NO: 3 (i.e., comprises, e.g., consists of, SEQ ID NO: 3 with a C80L mutation).
  • the Cas9 variant comprises a mutation at position 574 of SEQ ID NO: 3, e.g., includes a glutamic acid at position 574 of SEQ ID NO: 3 (i.e., comprises, e.g., consists of, SEQ ID NO: 3 with a C574E mutation).
  • the Cas9 variant comprises a mutation at position 80 and a mutation at position 574 of SEQ ID NO: 3, e.g., includes a leucine at position 80 of SEQ ID NO: 3, and a glutamic acid at position 574 of SEQ ID NO: 3 (i.e., comprises, e.g., consists of, SEQ ID NO: 3 with a C80L mutation and a C574E mutation).
  • it is believed that such mutations improve the solution properties of the Cas9 molecule.
  • the Cas9 molecule is a S. pyogenes Cas9 variant of SEQ ID NO: 3 that includes one or more mutations.
  • the Cas9 variant comprises a mutation at position 147 of SEQ ID NO: 3, e.g., includes a tyrosine at position 147 of SEQ ID NO: 3 (i.e., comprises, e.g., consists of, SEQ ID NO: 3 with a D147Y mutation).
  • the Cas9 variant comprises a mutation at position 411 of SEQ ID NO:
  • the Cas9 variant comprises a mutation at position 147 and a mutation at position 411 of SEQ ID NO: 3, e.g., includes a tyrosine at position 147 of SEQ ID NO: 3, and a threonine at position 411 of SEQ ID NO: 3 (i.e., comprises, e.g., consists of, SEQ ID NO: 3 with a D147Y mutation and a P411T mutation).
  • Wthout being bound by theory, it is believed that such mutations improve the targeting efficiency of the Cas9 molecule, e.g., in yeast.
  • the Cas9 molecule is a S. pyogenes Cas9 variant of SEQ ID NO: 3 that includes one or more mutations.
  • the Cas9 variant comprises a mutation at position 1135 of SEQ ID NO: 3, e.g., includes a glutamic acid at position 1135 of SEQ ID NO: 3 (i.e., comprises, e.g., consists of, SEQ ID NO: 3 with a D1135E mutation). Wthout being bound by theory, it is believed that such mutations improve the selectivity of the Cas9 molecule for the NGG PAM sequence versus the NAG PAM sequence.
  • the Cas9 molecule is a S. pyogenes Cas9 variant of SEQ ID NO: 3 that includes one or more mutations that introduce an uncharged or nonpolar amino acid, e.g., alanine, at certain positions.
  • the Cas9 molecule is a S. pyogenes Cas9 variant of SEQ ID NO: 3 that includes a mutation at position 497, a mutation at position 661 , a mutation at position 695 and/or a mutation at position 926 of SEQ ID NO: 3, for example a mutation to alanine at position 497, position 661 , position 695 and/or position 926 of SEQ ID NO: 3.
  • the Cas9 molecule has a mutation only at position 497, position 661 , position 695, and position 926 of SEQ ID NO: 3, relative to SEQ ID NO: 3, e.g., where each mutation is to an uncharged amino acid, for example, alanine. Without being bound by theory, it is believed that such mutations reduce the cutting by the Cas9 molecule at off-target sites.
  • the Cas9 molecule e.g., a Cas9 of S. pyogenes, may additionally comprise one or more amino acid sequences that confer additional activity.
  • the Cas9 molecule may comprise one or more nuclear localization sequences (NLSs), such as at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs.
  • NLS nuclear localization sequences
  • an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, but other types of NLS are known.
  • Non-limiting examples of NLSs include an NLS sequence comprising or derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 4).
  • Other suitable NLS sequences are known in the art (e.g., Sorokin, Biochemistry
  • the Cas9 molecule may additionally (or
  • a tag e.g., a His tag, e.g., a His(6) tag (SEQ ID NO: 5) or His(8) tag (SEQ ID NO: 6), e.g., at the N terminus or the C terminus.
  • the Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an N-terminal NLS, and a C-terminal NLS (e.g., comprises, from N- to C- terminal NLS-Cas9-NLS), e.g., wherein each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 4).
  • the Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an N-terminal NLS, a C-terminal NLS, and a C-terminal His6 tag (SEQ ID NO: 5) (e.g., comprises, from N- to C-terminal NLS-Cas9-NLS-His tag), e.g., wherein each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 4).
  • SEQ ID NO: 5 e.g., comprises, from N- to C-terminal NLS-Cas9-NLS-His tag
  • each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 4).
  • the Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an N-terminal His tag (e.g., His6 tag (SEQ ID NO: 5), an N-terminal NLS, and a C-terminal NLS (e.g., comprises, from N- to C-terminal His tag-NLS-Cas9-NLS), e.g., wherein each NLS is an SV40 NLS
  • the Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an N-terminal NLS and a C-terminal His tag (e.g., His6 tag (SEQ ID NO: 5) (e.g., comprises from N- to C- terminal His tag-Cas9-NLS), e.g., wherein the NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 4).
  • the Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an N-terminal His tag (e.g., His6 tag (SEQ ID NO: 5) and a C-terminal NLS (e.g., comprises from N- to C- terminal NLS-Cas9-His tag), e.g., wherein the NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 4).
  • NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 4).
  • the Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an N-terminal His tag (e.g., His8 tag (SEQ ID NO: 6), an N-terminal cleavage domain (e.g., a tobacco etch virus (TEV) cleavage domain (e.g., comprises the sequence ENLYFQG (SEQ ID NO: 7), an N-terminal NLS (e.g., an SV40 NLS; SEQ ID NO: 4), and a C-terminal NLS (e.g., an SV40 NLS; SEQ ID NO: 4) (e.g., comprises from N- to C- terminal His tag-TEV-NLS-Cas9-NLS).
  • N-terminal His tag e.g., His8 tag (SEQ ID NO: 6
  • an N-terminal cleavage domain e.g., a tobacco etch virus (TEV) cleavage domain
  • TSV tobacco etch virus
  • the Cas9 has the sequence of SEQ ID NO: 3.
  • the Cas9 has a sequence of a Cas9 variant of SEQ ID NO: 3, e.g., as described herein.
  • the Cas9 molecule comprises a linker between the His tag and another portion of the molecule, e.g., a GGS linker.
  • an enzymatically active Cas9 molecule cleaves both DNA strands at the target site and results in a double stranded break.
  • the ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in a plasmid cleavage assay.
  • synthetic or in vitro- transcribed gRNA molecule is pre-annealed prior to the reaction by heating to 95°C and slowly cooling down to room temperature.
  • Native or restriction digest-linearized plasmid DNA (300 ng ( ⁇ 8 nM)) is incubated for 60 min at 37°C with purified Cas9 protein molecule (50- 500 nM) and gRNA (50-500 nM, 1 : 1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KC1 , 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCI2.
  • Cas9 protein molecule 50- 500 nM
  • gRNA 50-500 nM, 1 : 1
  • Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KC1 , 0.5 mM DTT, 0.1 mM EDTA
  • the reactions are stopped with 5X DNA loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA), resolved by a 0.8 or 1% agarose gel electrophoresis and visualized by ethidium bromide staining.
  • the resulting cleavage products indicate whether the Cas9 molecule cleaves both DNA strands, or only one of the two strands.
  • linear DNA products indicate the cleavage of both DNA strands.
  • Nicked open circular products indicate that only one of the two strands is cleaved.
  • Engineered CRISPR gene editing systems typically involve (1) guide RNA (gRNA) comprising a targeting domain (which is capable of hybridizing to the genomic DNA target sequence), and a sequence which is capable of binding to a Cas, e.g., Cas9 enzyme, and (2) a Cas, e.g., Cas9, protein.
  • gRNA guide RNA
  • the sequence which is capable of binding to a Cas protein may comprise a domain referred to as a tracr domain or tracrRNA.
  • the targeting domain and the sequence which is capable of binding to a Cas, e.g., Cas9 enzyme may be disposed on the same
  • each includes a hybridization domain which allows the molecules to associate, e.g., through hybridization.
  • gRNA molecule formats are known in the art.
  • An exemplary gRNA molecule, e.g., dgRNA molecule, as disclosed herein comprises, e.g., consists of, a first nucleic acid having the sequence:
  • nucleotide sequence 5’nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnGUUUUAGAGCUAUGCUGUUUUG 3’ (SEQ ID NO: 8), where the“n”’s refer to the residues of the targeting domain, e.g., as described herein, and may consist of 15-25 nucleotides, e.g., consists of 20 nucleotides;
  • the second nucleic acid molecule may alternatively consist of a fragment of the sequence above, wherein such fragment is capable of hybridizing to the first nucleic acid.
  • An example of such second nucleic acid molecule is:
  • Another exemplary gRNA molecule e.g., a sgRNA molecule, as disclosed herein comprises, e.g., consists of a first nucleic acid having the sequence:
  • nucleotide sequence e.g., as described herein, and may consist of 15-25 nucleotides, e.g., consist of 20 nucleotides, optionally with 1 , 2, 3, 4, 5,
  • the gRNA comprises a targeting domain which is fully complementarity to 15-25 nucleotides, e.g., 20 nucleotides, of a target gene.
  • the 15-25 nucleotides, e.g., 20 nucleotides, of the target gene are disposed immediately 5’ to a protospacer adjacent motif (PAM) sequence recognized by the RNA-guided nuclease, e.g., Cas protein, of the CRISPR gene editing system (e.g., where the system comprises a S. pyogenes Cas9 protein, the PAM sequence comprises NGG, where N can be any of A, T, G or C).
  • PAM protospacer adjacent motif
  • the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme.
  • the degree of complementarity between a guide sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length
  • target sequences can be chosen by identifying the PAM sequence for a Cas9 molecule (for example, relevant PAM e.g., NGG PAM for S. pyogenes, NNNNGATT (SEQ ID NO: 12), or NNNNGCTT PAM (SEQ ID NO: 13), for N. meningitides, and NNGRRT (SEQ ID NO: 14), or NNGRRV PAM (SEQ ID NO: 15), for S.
  • relevant PAM e.g., NGG PAM for S. pyogenes, NNNNGATT (SEQ ID NO: 12), or NNNNGCTT PAM (SEQ ID NO: 13), for N. meningitides, and NNGRRT (SEQ ID NO: 14), or NNGRRV PAM (SEQ ID NO: 15), for S.
  • targeting domains for use in gRNAs for use with S. pyogenes, N. meningitidis and S. aureus Cas9s are identified using a DNA sequence searching algorithm. 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, and/or 24-mer targeting domains are designed for each Cas9. With respect to S. pyogenes Cas9, preferably, the targeting domain is a 20-mer.
  • gRNA design is carried out using a custom gRNA design software based on the public tool cas-offinder (Bae 2014). This software scores guides after calculating their genome-wide off-target propensity.
  • gRNAs may also be designed using the publically available CRISPOR program15.
  • the gRNA may also comprise, at the 3’ end, additional U or A nucleic acids.
  • the gRNA may comprise an additional 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 U nucleic acids at the 3’ end or an additional 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 A nucleic acids at the 3’ end.
  • the gRNA comprises an additional 4 U nucleic acids at the 3’ end.
  • one or more of the polynucleotides of the dgRNA e.g., the polynucleotide comprising the targeting domain and the polynucleotide comprising the tracr
  • the targeting domain comprises a nucleotide sequence that is complementary, e.g., at least 80, 85, 90, 95, or 99% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid.
  • the targeting domain is 5 to 50, e.g., 10 to 40, e.g., 10 to 30, e.g., 15 to 30, e.g., 15 to 25 nucleotides in length. In an embodiment, the targeting domain is 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length.
  • the strand of the target nucleic acid with which the targeting domain is complementary is referred to herein as the target sequence.
  • the target sequence is disposed on a chromosome, e.g., is a target within a gene.
  • the target sequence is disposed within an exon of a gene.
  • the target sequence is disposed within an intron of a gene.
  • the target sequence comprises, or is proximal (e.g., within 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1000 nucleic acids) to a binding site of a regulatory element, e.g., a promoter or transcription factor binding site, of a gene of interest.
  • the gRNA molecule is provided as RNA and the Cas9 molecule is provided as expressed protein.
  • the gRNA and Cas9 molecule are provided as a ribonucleoprotein complex (RNP).
  • RNP ribonucleoprotein
  • the RNP complex is formed outside the cell, and the gRNA and endonuclease is delivered into the cell as a RNP complex.
  • Ribonucleoprotein (RNP) may be formed by incubating Cas9 and gRNA together at room temperature in buffer, such as Duplex buffer (30 mM HEPES, pH 7.5; 100 mM potassium acetate).
  • 4 pg Cas9 and 2 pg gRNA may be incubated at RT for 10min in Duplex buffer.
  • Delivery may be accomplished by, for example, electroporation or other method that renders the cell membrane permeable to nucleic acid and/or polypeptide molecules, such as liposome-mediated transfection.
  • Electroporation and liposome-mediated transfection are standard techniques well known in the art, see for example Green MR, Sambrook J (2012) Molecular Cloning: A Laboratory Manual (4 th ed) Cold Springs Harbor: Cold Springs Harbor Laboratory Press).
  • Commercially available systems and devices for electroporation are available, such as the Neon Transfection System from Invitrogen/ThermoFischer Scientific.
  • the gRNA, endonuclease and donor DNA are delivered simultaneously into the cell.
  • a transfection mixture is prepared for electroporation comprising RNP (e.g. 4 pg Cas9 and 2 pg gRNA), donor DNA, (e.g. 15 pmol donor DNA), and approximately 400,000 cells.
  • RNP e.g. 4 pg Cas9 and 2 pg gRNA
  • donor DNA e.g. 15 pmol donor DNA
  • approximately 400,000 cells approximately 400,000 cells.
  • the cell lines are selected from the group consisting of HeLa cells, HCT116 cells, HEK293 cells, SH-SY5Y cells, A549 cells and U20S cells.
  • the cells are induced pluripotent stem cells or iNgn2 neurons.
  • the primary cells are skeletal muscle cells, Schwann cells, or tenocytes.
  • the Cas systems e.g., one or more gRNA molecules and one or more Cas molecules (e.g., Cas9 molecules), together with donor DNA and the method of sequence insertion described herein are useful for the treatment of disease in a mammal, e.g., in a human.
  • the terms“treat,”“treated,”“treating,” and“treatment,” include the use of the method of sequence insertion described herein to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder.
  • Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease. Treatment can be measured by the therapeutic measures described herein.
  • the methods of “treatment” of the present invention also include administration of cells altered by the introduction of a Cas system (e.g., one or more gRNA molecules and one or more Cas molecules) and donor DNA into said cells according to the methods described herein to a subject in order to cure, reduce the severity of, or ameliorate one or more symptoms of a disease or condition, in order to prolong the health or survival of a subject beyond that expected in the absence of such treatment.
  • a Cas system e.g., one or more gRNA molecules and one or more Cas molecules
  • donor DNA into said cells according to the methods described herein to a subject in order to cure, reduce the severity of, or ameliorate one or more symptoms of a disease or condition, in order to prolong the health or survival of a subject beyond that
  • the present invention provides a method comprising administering a cell of the invention, e.g., a cell which has been modified by the method of sequence insertion as described herein, to a subject.
  • a cell of the invention e.g., a cell which has been modified by the method of sequence insertion as described herein
  • the cell has been altered by the introduction of the insertion sequence at a target site, such that expression of the functional product of a gene of interest is reduced or eliminated relative to an unmodified cell.
  • the methods described herein can be useful for generating gene- modified cells (such as immune cells), which can be useful for cellular therapeutics.
  • a method of generating a genetically modified animal comprising a donor sequence inserted at a predetermined insertion site on the chromosome of the animal, comprising use of the method of sequence insertion as described herein.
  • compositions and Treatments may comprise the product (nucleic acid, peptide, polypeptide or protein) derived from a cell in which the method of sequence insertion has been performed or cell population in which the method of sequence insertion has been performed and a pharmaceutically or physiologically acceptable carrier or additional therapeutic agent.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins such as glucose, mannose
  • compositions may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.
  • compositions to be administered can be determined by a physician with consideration of individual differences in age, weight, disease state, e.g., tumor size, extent of infection or metastasis, and condition of the patient (subject).
  • compositions may also be administered multiple times at these dosages.
  • the optimal dosage and treatment regime for a particular patient can be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • NLS-SpyCAS9-NLS-His6 was expressed in E. coli strain Rosetta 2 (DE3) in pET28a+, using 2xYT media plus 50 pg/ml kanamycin overnight at 18°C.
  • the Cas9 was then purified using Ni-NTA agarose (Qiagen) capture/elution, followed by size exclusion chromatography on a S200 26/600 Superdex column (GE Lifesciences) using modified buffers from Jinek, M. et al. Science 337, 816-821 (2012). Then Cas9 stocks (5.9 mg/ml) were aliquoted and stored at -80°C. Cas9 nuclease is also commercially available from sources such as IDT. Example A2. dsDNA donors.
  • Oligonucleotides were synthesized by Microsynth with PAGE purification. The forward and reverse strands were dissolved in Duplex buffer to 20 mM each. To anneal the oligos, they were mixed at equal volume and incubated at 95°C for 5min, then were allowed to cool down slowly to RT. The final concentration of dsDNA stock was 10mM.
  • RNA and tracrRNA were dissolved with Duplex buffer (IDT) to 169mM each. They were mixed at the equal volume, and the final RNA concentration adjusted to 2 pg/mI by adding
  • RNP Ribonucleoprotein
  • dsDNA donors were ordered as gBLOCK gene fragments (IDT). 500ng DNA was dissolved in 5 mI buffer R (IDT). The constitution of transfection mixtures are listed in Table 3. 10 pi of the transfection mixtures were used for electroporation.
  • the RNP and dsDNA template were delivered into cells by electroporation with Neon Transfection System (ThermoFisher, MPK5000).
  • the 10 pi kit (MPK1096) was used, and transfection protocols for individual cell type are listed in Table 4. After transfection, cells were cultured in antibiotic free medium for at least 24 hours before replacing with normal growth medium. Table 4
  • a 549, HCT116, HEK293, HeLa, SH-SY5Y and U-2 OS cell lines were purchased from ATCC and cultured according to supplier’s recommendation.
  • the primary human skeletal muscle cells (Lonza, CC-2561) were maintained in Skeletal Muscle Growth Medium (Lonza, CC-3246), supplemented with 20% fetal calf serum (FCS).
  • FCS fetal calf serum
  • Schwann cells culture Primary Schwann cells were isolated as described previously by Kaewkhaw, R., Scutt, A.M. & Haycock, J.W. Nat Protoc 7, 1996-2004 (2012). Briefly, Sciatic nerves were isolated from adult rats and epineurium stripped. The tissues were teased with sharp tweezers, and digested with 0.05% collagenase digestion at 37°C for 2 hours. Homogenous cell suspension was obtained by dissociating the tissue with glass Pasteur pipette and filtered through 40 pm cell strainer. The cells were pelleted down and plated in poly-D-lysine coated flasks. The cells were maintained in a custom made D-valine DMEM medium (based on 10313039, L-Valine free,
  • ThermoFisher supplemented with 10% FCS, B27, 10mM Hepes, 2mM glutamate and 5mM Forskolin.
  • FCS ThermoFisher
  • Human Ngn2 cDNA was synthesized using sequence information from the Ensembl database (Ensembl Gene ID ENSG00000178403) and cloned under the control of TRE tight (Tetracycline Response Element) promoter in a PiggyBac/Tet-ON all-in-one vector18.
  • This vector contains a CAG rtTA16 cassette allowing constitutive expression of Tet-ON system and an Hsv- tkNeo cassette for generation of stable IPS clones.
  • iPS cells After trypsinization into single cells with Tryple express reagent (ThermoFisher) approximately 1 x 10 6 iPS cells were nucleofected by Amaxa nuclefector device using Human Stem Cell Nucleofector Kit 1 (Lonza) and Program B-016 with 4 pg of Ngn2 plasmid and 1 pg of the dual helper plasmid. Subsequently cells were replated on matrigel plates with mTeSR medium containing 10 pM of Rock inhibitor. Antibiotic selection (G418 0.1 mg/ml) was applied 48 hours later. Stable clones appear within 1 week.
  • 1 x 106 of iPS cells were plated on a 6 cm matrigel plate in proliferation medium (DMEM/F12 with Glutamax supplemented with 2% B27 and 1 % N2, 10 ng/ml hEGF, 10 ng/ml hFGF, 1 % Pen/Strep (all from ThermoFisher) containing Rock inhibitor (10pM) for 1d and doxycycline (1 ug/ml) for 3d.
  • DMEM/F12 with Glutamax supplemented with 2% B27 and 1 % N2 10 ng/ml hEGF, 10 ng/ml hFGF, 1 % Pen/Strep (all from ThermoFisher) containing Rock inhibitor (10pM) for 1d and doxycycline (1 ug/ml) for 3d.
  • Example A7 Amplicon sequencing Total genomic DNA was isolated from transfected cells using a commercial DNA extraction kit (Qiagen, DNeasy Blood and Tissue kit). The region of theoretical insertion was amplified by PCR using primers hnRNPA2B1 fw (5’-tcccgtgcggaggtgctcctcgcag) (SEQ ID NO: 37) and hnRNPA2B1 re (5’-agctccgcagcctcgctcacgagg) (SEQ ID NO 38). A 500bp fragment could be obtained after 40 amplification cycles with denaturation 95°C for 30s - annealing 60°C for 30s - extension 68°C for 45s using a proofreading
  • Immunostaining was performed according to standard protocols. Briefly, 72 hours after transfection, cells grown on 96 well plates were fixed and permeabilized for 20 mins at 4°C with Fixation/Permeabilization solution (BD Biosciences). Cells were washed 3 times in PBS, then blocked in PBS containing 5% donkey serum, 1 % bovine serum albumin (BSA) and 10% Perm/Wash buffer (BD Biosciences) at RT for 1 hour. For V5 tag staining, cells were incubated at RT for 4 hours with Alexa647 conjugated anti-V5 antibody, followed by rinsing 3 times in PBS before imaging.
  • Fixation/Permeabilization solution BD Biosciences
  • a palindrome-like HiBiT sequence (Table 1 , SEQ ID NO: 25) was knocked into I FIT 1 in SkMCs, and cells expanded for 10 days in the growth medium. The cells were split into a 384 well plate with 5000 cells per well. After cells attached, lnterferon-b (Merck, IF014) were added to the wells at various concentrations. The expression level of I FIT 1-HiBiT was determined with NanoGlo HiBiT lytic assay kit (Promega, N3040). Briefly, Nano-Glo substrate and LgBiT protein were mixed with the lytic buffer (1 :50 and 1 :100 respectively). The volume of culture media was adjusted to 30 mI each well, then equal volume of reagents was added. After 10 minutes, luminescence signals were measured on EnVision Plate Reader (PerkinElmer).
  • HnRNP Heterogeneous nuclear ribonucleoprotein A2/B1
  • GKPIPNPLLGLDST (SEQ ID NO: 39) into HnRNP, a gRNA was designed that targets the junction of the 5’UTR and the CDS, with the Cas9 cutting site 2bp before ATG (Fig.1a, gRNA1 in Table 2). To ensure that the V5 tag can be translated in frame, an ATG was added to its 5’ end, as well as a thymine (T) to 3’ end (SEQ ID NO: 16, Table 1).
  • Cas9/gRNA1 as a RNP complex and V5 donor (SEQ ID NO: 16) were co delivered into HeLa cells by electroporation. 72 hours after transfection, knock-in efficiency was assessed by co-staining with anti-V5 and DAPI: 40 ⁇ 1.5% of the cells displayed strong V5 signals that exclusively localized in the nucleus (Fig.1 b).
  • HCT116 cell line has been favored for the parallel analysis of Kl efficiency by Next Generation Sequencing (NGS) because of its relatively stable chromosomal composition.
  • NGS Next Generation Sequencing
  • V5 tag (SEQ ID NO: 39) knock-in was performed in HCT116 cells.
  • the gRNA (SEQ ID NO: 27) was designed such that it targeted the junction of the 5’UTR and the CDS, with the Cas9 cutting site 2bp before ATG. To ensure that the V5 tag could be translated in frame, an ATG was added to its 5’ end, as well as a thymine (T) to 3’ end (SEQ ID NO: 16, Table 1).
  • Cas9/gRNA1 as a RNP complex and V5 donor (SEQ ID NO: 16) were co delivered into cells by electroporation. 72 hours after transfection, knock-in efficiency was assessed by co-staining with anti-V5 and DAPI: 31.3 ⁇ 3.9% of the cells displayed strong V5 signals that exclusively localized in the nucleus (Fig.2a)
  • Lysates from untransfected or V5-HnRNP knock-in cells were subjected to western blotting. Similar to the HeLa cells, A2/B1 isoforms were detected with either V5 or HNRNP antibody (Fig.2b).
  • g stands for percentage of functional genome integration, which is 16.5 in this case; C represents the percentage of functional V5 expression at cellular level, which is 30.3. The number matches well with the immunostaining result, which suggested 31.3 ⁇ 3.9% cells express V5-HnRNP.
  • a V5 tag (SEQ ID NO: 39) into b-actin
  • a gRNA (gRNA 2: SEQ ID NO: 28) was designed such that it targeted the junction of the 5’UTR and the CDS, with the Cas9 cutting site 38bp before ATG.
  • an ATG was added to its 5’ end, as well as a thymine (T) to 3’ end (SEQ ID NO: 16, Table 1).
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 pi), 2 pg gRNA2 (SEQ ID NO: 28) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. RNP complex and V5 donor (SEQ ID NO: 16) were co-delivered into cells by
  • V5 tag SEQ ID NO: 39
  • a gRNA SEQ ID NO: 29
  • GT two bases
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 pi), 2 pg gRNA3 (SEQ ID NO: 29) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. Then 400,000 cells (in 7.5 mI buffer R), 15 pmol V5 donor DNA (SEQ ID NO: 17) (1.5 mI) and 6 mI buffer R were added to make up the transfection mixtures (Table 3). 10 mI of the transfection mixtures were used for electroporation.
  • V5 tag SEQ ID NO: 39
  • RPL10A Ribosomal Protein L10a
  • SEQ ID NO: 30 a gRNA was designed to target towards 5’ end of CDS, with the Cas9 cutting site right before ATG. An extra ATG was included to its 5’ end of V5 donor sequence (SEQ ID NO: 18). 72 hours after transfection, knock-in efficiency was assessed by co-staining with anti-V5 and DAPI. 5.1 ⁇ 0.5% cells display positive signals in cytosol and nucleoli.
  • Figure 3 shows that all were successfully labelled with a V5 tag, and all displayed the expected cellular location.
  • Example 4 Applications for the sequence insertion method in other cell lines
  • V5 tag SEQ ID NO: 39
  • the gRNA 1 SEQ ID NO: 27
  • an ATG was added to its 5’ end, as well as a thymine (T) to 3’ end (SEQ ID NO: 16).
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 mI), 2 pg gRNA4 (SEQ ID NO: 30) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. RNP complex and V5 donor (SEQ ID NO: 16) were co-delivered into cells by
  • V5-HnRNP signals were detected in 50.7 ⁇ 2.2% HEK293 cells.
  • V5 tag SEQ ID NO: 39
  • the gRNA 1 SEQ ID NO: 27
  • an ATG was added to its 5’ end, as well as a thymine (T) to 3’ end (SEQ ID NO: 16).
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 mI), 2 pg gRNA1 (SEQ ID NO: 27) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. RNP complex and V5 donor (SEQ ID NO: 16) were co-delivered into cells by
  • V5 tag SEQ ID NO: 39
  • a gRNA SEQ ID NO: 29
  • GT two bases
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 mI), 2 pg gRNA3 (SEQ ID NO: 29) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. Then 400,000 cells (in 7.5 mI buffer R), 15 pmol V5 donor DNA (SEQ ID NO: 17) (1.5 mI) and 6 mI buffer R were added to make up the transfection mixtures (Table 3). 10 mI of the transfection mixtures were used for electroporation.
  • V5 tag SEQ ID NO: 39
  • a gRNA SEQ ID NO: 29
  • GT two bases
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 mI), 2 pg gRNA3 (SEQ ID NO: 29) (1 pi) at RT for 10min in a final volume of 5 mI Duplex buffer. Then 400,000 cells (in 7.5 mI buffer R), 15 pmol V5 donor DNA (SEQ ID NO: 17) (1.5 mI) and 6 mI buffer R were added to make up the transfection mixtures (Table 3). 10 pi of the transfection mixtures were used for electroporation.
  • Example 5 CRISPR knock-in in induced pluripotent stem (iPS) cells and iPS derived iNgn2 neurons
  • CRISPR knock-in efficiency is generally very low in induced pluripotent stem (iPS) cells, as HDR mediated pathway is not efficient in these cells.
  • the sequence insertion method was therefore tested in iPS cells to assess the knock-in efficiency. iPS cells
  • V5 tag SEQ ID NO: 39
  • the gRNA 1 SEQ ID NO: 27
  • an ATG was added to its 5’ end, as well as a thymine (T) to 3’ end (SEQ ID NO: 16).
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 mI), 2 pg gRNA1 (SEQ ID NO: 27) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. RNP complex and V5 donor (SEQ ID NO: 16) were co-delivered into cells by
  • the HDR pathway does not exist in post-mitotic cells. To see whether the sequence insertion method could work in post-mitotic cells, it was tested in iPS derived iNgn2 neurons.
  • a V5 tag SEQ ID NO: 39
  • the gRNA 1 SEQ ID NO: 27
  • the gRNA 1 was designed such that it targeted the junction of the 5’UTR and the CDS, with the Cas9 cutting site 2bp before ATG.
  • an ATG was added to its 5’ end, as well as a thymine (T) to 3’ end (SEQ ID NO: 16).
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 mI), 2 pg gRNA1 (SEQ ID NO: 27) (1 pi) at RT for 10min in a final volume of 5 mI Duplex buffer. RNP complex and V5 donor (SEQ ID NO: 16) were co-delivered into cells by
  • the cultures were treated with antimitotic agent 24 hours before and after.
  • nucleus V5 signal could be observed in 5.9 ⁇ 1.6% of the iNgn2 neurons ( Figure 5d)
  • the neuronal status of iNgn2 cells were confirmed with neurofilament 200 staining ( Figure 5c).
  • Example 6 CRISPR knock-in in rat primary Schwann cells
  • Primary cells are generally not suitable for CRISPR knock-in with the methods known in the art, since they can only be expanded to a limited extent and are not suitable for clonal selection.
  • the sequence insertion method as described herein was tested in primary cells.
  • V5 tag was knocked into Vimentin locus in the primary rat Schwann cells.
  • a gRNA SEQ ID NO: 31 was designed to target towards 5’ end of CDS of rat VIM gene, with the Cas9 cutting site 7bp after ATG.
  • two bases GT
  • Ribonucleoprotein RNP was formed by incubating 4 pg Cas9 (0.68 mI), 2 pg gRNA (SEQ I D NO: 31 ) (1 pi) at RT for 10min in a final volume of 5 mI Duplex buffer.
  • a gRNA (SEQ ID NO: 32) was designed to target towards 3’ end of CDS of rat SOX10 gene, with the Cas9 cutting site right before the stop codon TAG.
  • a palindromic donor DNA containing V5 sequence (SEQ ID NO: 19) was designed. To increase the chance of functional V5 tag integration, a dsDNA donor containing the V5 sequence and a stop codon in both directions was used.
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 mI), 2 pg gRNA6 (SEQ ID NO: 32) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. Then 400,000 cells (in 7.5 mI buffer R), 15 pmol V5 donor DNA (SEQ ID NO: 19) (1.5 mI) and 6 mI buffer R were added to make up the transfection mixtures (Table 3). 10 pi of the transfection mixtures were used for electroporation.
  • Example 7 CRISPR knock-in in human primary cells
  • the sequence insertion method was also tested in human primary cells. Primary human skeletal muscle cells were used as the proof of concept, and V5 tag was knocked into Vimentin.
  • a gRNA (SEQ ID NO: 29) was designed to target towards 5’ end of VIM encoding region, with the Cas9 cutting site 10bp after ATG. To ensure that the V5 tag could be translated in frame, two bases (GT) were added to its 5’ end, as well as a T to 3’ end (SEQ ID NO: 17).
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 mI), 2 pg gRNA3 (SEQ ID NO: 29) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. Then 400,000 cells (in 7.5 mI buffer R), 15 pmol V5 donor DNA (SEQ ID NO: 17) (1.5 mI) and 6 mI buffer R were added to make up the transfection mixtures (Table 3). 10 mI of the transfection mixtures were used for electroporation.
  • gRNA3 (SEQ ID NO: 29) was designed to target towards 5’ end of VIM encoding region, with the Cas9 cutting site 10bp after ATG. To ensure that the mScarlet can be translated in frame, for the donor DNA two bases (GT) were added to the 5’ end of the mScarlet coding sequence, as well as a G to 3’ end (SEQ ID NO: 20).
  • GT two bases
  • Ribonucleoprotein was formed by incubating 3 pg Cas9 (0.51 mI), 1.5 pg gRNA3 (0.75 pi) at RT for 10min in a final volume of 3.75 mI Duplex buffer. Then 300,000 cells (in 5.625 mI buffer R), 500ng donor DNA (5 mI) and 0.625 mI buffer R were added to make up the transfection mixtures (Table 3). 10 mI of the transfection mixtures were used for electroporation.
  • FACS fluorescence-activated cell sorting
  • gRNA2 (SEQ ID NO: 28) was designed to target the 5’UTR of ActB gene, with the Cas9 cutting site 38bp before ATG. To ensure that mScarlet can be translated in frame, an ATG was added to its 5’ end, as well as a guanine (G) to 3’ (SEQ ID NO: 21).
  • Ribonucleoprotein was formed by incubating 3 pg Cas9 (0.51 mI), 1.5 pg gRNA2 (0.75 mI) at RT for 10min in a final volume of 3.75 mI Duplex buffer. Then 300,000 cells (in 5.625 mI buffer R), 500ng donor DNA (5 mI) and 0.625 mI buffer R were added to make up the transfection mixtures (Table 3). 10 pi of the transfection mixtures were used for electroporation.
  • FACS fluorescence-activated cell sorting
  • Y-FAST is half the size of the green fluorescent protein, and only becomes fluorescent once bound to its fluorogenic ligand.
  • a 376bp dsDNA fragment contains Y-FAST sequence was knocked into b-actin in HeLa cells.
  • gRNA2 (SEQ ID NO: 28) was used to target the 5’UTR of ActB gene, with the Cas9 cutting site 38bp before ATG.
  • an ATG was added to its 5’ end, as well as a guanine (G) to 3’ end (SEQ ID NO: 22).
  • Ribonucleoprotein was formed by incubating 3 pg Cas9 (0.51 pi), 1.5 pg gRNA2 (0.75 pi) at RT for 10min in a final volume of 3.75 mI Duplex buffer. Then 300,000 cells (in 5.625 mI buffer R), 500ng donor DNA (5 mI) and 0.625 mI buffer R were added to make up the transfection mixtures (Table 3). 10 mI of the transfection mixtures were used for electroporation.
  • Example 9 Sequence insertion method to improve KO efficiency
  • sequence insertion method was also investigated for its applicability to CRISPR knock-out , which is often comprised by in-frame indels (Bae, S., Kweon, J.,
  • a multi-stop codon sequence (MSC, SEQ ID NO: 23) was inserted into the Cas9 cutting site, to improve KO efficiency.
  • Two gRNAs (SEQ ID NO: 33 and 34) were designed to target VIM gene; SEQ ID NO: 33 targets 41 bp after ATG and SEQ ID NO: 34 targets 256 bp after ATG. The release of the fragment in between would result in a frame shift.
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 mI), 1 pg gRNA7 and 8 each (SEQ ID NO: 33 and 34) (0.5mI) at RT for 10min in a final volume of 5 pi Duplex buffer. Then 400,000 cells (in 7.5 mI buffer R), 15 pmol MSC DNA (SEQ ID NO: 23) (1.5 mI) and 6 mI buffer R were added to make up the transfection mixtures (Table 3). 10 pi of the transfection mixtures were used for electroporation. Indeed, as shown in Figure 9b, while knock-out efficiencies of Vimentin were 65-75% with single gRNAs (gRNA 7 or 8), it increased to 85% with two gRNAs.
  • the efficiency could be improved to 89% when both gRNAs and MSC (SEQ ID NO: 23) are used. In fact, this represents a nearly complete knock-down since the transfection efficiency is around 90% with the recommended protocol (manufacturer data).
  • Example 10 Sequence insertion method to change the cellular location of targeted proteins
  • the cellular location is important for functions of the target protein, and is often determined by the cellular targeting sequences, including signal peptides. Therefore, it is possible to affect protein targeting or trafficking by CRISPR Kl.
  • a myristoylation sequence (GCIKSKRKDNLNDDGVDMKT), (SEQ ID NO: 40) which is a membrane targeting peptide, was added to V5-HnRNP in HeLa cells.
  • the donor DNA thus from the 5’ end had an ATG, a myristoylation sequence, a V5 sequence followed by 1 extra bp at the 3’ end (SEQ ID NO: 24).
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 pi), 2 pg gRNA1 (SEQ ID NO: 27) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. Then 400,000 cells (in 7.5 mI buffer R), 15 pmol donor DNAs (SEQ ID NO: 16 or 24) (1.5 mI) and 6 mI buffer R were added to make up the transfection mixtures (Table 3). 10 mI of the transfection mixtures were used for electroporation.
  • Example 11 Develop a novel luciferase assay in primary SkMC via sequence insertion method
  • Antibody based assays such as ELISA, are generally used to measure protein expression level changes in cells. However, sometimes the relevant antibody can be either too expensive or not available. Given the high efficiency of the sequence insertion method, it was tested whether it was possible to convert an ELISA assay into a luciferase assay. The aim was to attach the HiBiT sequence (an 11 amino acid subunit of Nanoluc luciferase) to protein of interest, whose expression level can be then estimated by measuring NanoLuc activity. As proof of concept, I FIT 1 , an interferon induced protein in SkMC was targeted. The HiBiT tag (SEQ ID NO: 25) was inserted to the C-terminus of I FIT 1 by CRISPR.
  • HiBiT sequence an 11 amino acid subunit of Nanoluc luciferase
  • a gRNA (SEQ ID NO: 35) was designed to target towards the 3’ end of CDS of I FIT 1 gene, with the Cas9 cutting site 2bp before the stop codon TAG.
  • a palindromic-like donor DNA (SEQ ID NO: 25) containing HiBiT sequence was designed. SEQ ID NO: 25 thus will work when inserted in either orientation, as it has (from 5’ end) 2bp + 5aa Nnker+HiBiT sequence + stop codon; stop codon + HiBiT sequence +5aa linker+2bp, so works when inserted in either orientation.
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 pi), 2 pg gRNA9 (SEQ ID NO: 35) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. Then 400,000 cells (in 7.5 mI buffer R), 15 pmol V5 donor DNA (SEQ ID NO: 25) (1.5 mI) and 6 mI buffer R were added to make up the transfection mixtures (Table 3). 10 mI of the transfection mixtures were used for electroporation.
  • Example 12 CRISPR knock-in in rat primary tenocytes
  • the sequence insertion method was also tested in primary rat tenocytes.
  • a V5 tag (SEQ ID NO: 39) into Vimentin in tenocytes
  • a gRNA (SEQ ID NO: 31) was designed to target towards the 5’ end of CDS of rat VIM gene, with the Cas9 cutting site 7bp after ATG.
  • two bases (GT) were added to its 5’ end, as well as a T to 3’ end (SEQ ID NO: 17).
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 mI), 2 pg gRNA3 (SEQ ID NO: 31) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. Then 400,000 cells (in 7.5 mI buffer R), 15 pmol V5 donor DNA (SEQ ID NO: 17) (1.5 mI) and 6 mI buffer R were added to make up the transfection mixtures (Table 3). 10 pi of the transfection mixtures were used for electroporation.
  • Example 13 Develop a novel luciferase assay in primary Schwann cells via sequence insertion method
  • the sequence insertion method was also used in Schwann cells to insert a sequence into c-Jun, a master transcription factor.
  • the HiBiT tag was inserted to N- terminus of C-Jun.
  • a gRNA (SEQ ID NO: 36) was designed to target the 5’UTR of C-Jun gene, with the Cas9 cutting site 20bp before ATG. To ensure that the HiBiT tag can be translated in frame, an ATG was added to its 5’ end, as well as an Adenine (A) to 3’ end (SEQ ID NO: 26).
  • Ribonucleoprotein was formed by incubating 4 pg Cas9 (0.68 pi), 2 pg gRNAIO (SEQ ID NO: 36) (1 mI) at RT for 10min in a final volume of 5 mI Duplex buffer. Then 400,000 cells (in 7.5 mI buffer R), 15 pmol HiBiT donor DNA (SEQ ID NO: 26) (1.5 mI) and 6 mI buffer R were added to make up the transfection mixtures. 10 mI of the transfection mixtures were used for electroporation.

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Abstract

La présente invention concerne de manière générale des procédés d'insertion de séquence dans un site cible à l'intérieur d'une séquence d'ADN dans une cellule d'une séquence d'insertion à l'aide du système CRISPR/Cas et de la voie NHEJ, des produits pouvant être obtenus à partir de ceux-ci, comprenant des populations de cellules et leurs procédés d'utilisation. La présente invention concerne un procédé d'insertion de séquence dans un site cible à l'intérieur d'une séquence d'ADN dans une cellule d'une séquence d'insertion, comprenant les étapes consistant à administrer dans la cellule une endonucléase, de l'ARNg et de l'ADN donneur, l'ADN donneur comprenant la séquence d'insertion sans bras d'homologie supplémentaires et l'ADN donneur étant administré dans la cellule sous forme d'ADNdb linéarisé.
PCT/IB2019/054934 2018-06-14 2019-06-13 Procédé d'insertion de séquence à l'aide de crispr Ceased WO2019239361A1 (fr)

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Publication number Priority date Publication date Assignee Title
US12201699B2 (en) 2014-10-10 2025-01-21 Editas Medicine, Inc. Compositions and methods for promoting homology directed repair
US11866727B2 (en) 2015-11-06 2024-01-09 Crispr Therapeutics Ag Materials and methods for treatment of glycogen storage disease type 1A
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
WO2020003006A3 (fr) * 2018-06-28 2020-03-05 Crispr Therapeutics Ag Compositions et procédés d'édition génomique par insertion de polynucléotides donneurs
US11332760B2 (en) 2018-06-28 2022-05-17 Crispr Therapeutics Ag Compositions and methods for genomic editing by insertion of donor polynucleotides
US11827877B2 (en) 2018-06-28 2023-11-28 Crispr Therapeutics Ag Compositions and methods for genomic editing by insertion of donor polynucleotides
US12214023B2 (en) 2018-10-18 2025-02-04 Intellia Therapeutics, Inc. Compositions and methods for expressing factor IX
WO2020179931A1 (fr) * 2019-03-07 2020-09-10 国立大学法人 東京医科歯科大学 Technique d'édition génique à haut débit
WO2023177182A1 (fr) * 2022-03-16 2023-09-21 가톨릭관동대학교산학협력단 LIGNÉE CELLULAIRE PLURIPOTENTE HUMAINE GÉNÉTIQUEMENT MODIFIÉE POUR CO-EXPRIMER LE GÈNE α-MHC ET UN GÈNE RAPPORTEUR FLUORESCENT

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