WO2018129486A2 - Composition et procédés pour une expression améliorée de gène rapporteur knock-in - Google Patents

Composition et procédés pour une expression améliorée de gène rapporteur knock-in Download PDF

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WO2018129486A2
WO2018129486A2 PCT/US2018/012849 US2018012849W WO2018129486A2 WO 2018129486 A2 WO2018129486 A2 WO 2018129486A2 US 2018012849 W US2018012849 W US 2018012849W WO 2018129486 A2 WO2018129486 A2 WO 2018129486A2
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cell
gene
reporter gene
interest
target sequence
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PCT/US2018/012849
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WO2018129486A3 (fr
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Tim D. Ahfeldt
Lee L. Rubin
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President And Fellows Of Harvard College
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • Co-expression of a gene of interest and a reporter gene is of great value for the study of cell differentiation and cellular biology.
  • Techniques for enhanced expression of the reporter gene with a gene of interest are important for biological and biomedical research. For example, detecting expression of tyrosine hydroxylase in pluripotent stem cells by detecting the co-expression of a reporter gene is of great value for investigators studying midbrain neurons, dopaminergic neurons and Parkinson's disease pathology.
  • the present invention relates to compositions and methods useful for making reporter cells (i.e., cells co-expressing a reporter and a genetic locus of interest).
  • the compositions and methods described herein provide a cell with a knock-in reporter (e.g., a fluorescent protein) and a downstream WPRE element, wherein the cell co-expresses a genetic locus of interest and a reporter gene.
  • a knock-in reporter e.g., a fluorescent protein
  • the invention relates to a nucleic acid targeting vector comprising, in the 5' to 3' direction, a 5' homology arm homologous to a first target sequence in a cell, a reporter gene, an expression enhancer comprising a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) operably linked to the reporter gene, and a 3' homology arm homologous to a second target sequence downstream of the first target sequence.
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • the invention relates to a composition
  • a cell e.g., transgenic cell, a cell line
  • a cell having a genome comprising a nucleotide sequence comprising a reporter gene and a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) operably linked to the reporter gene, wherein the reporter gene is co-expressed with a target sequence of interest (e.g., genetic locus of interest, gene of interest).
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • the invention relates to a method of generating a cell that co- expresses a reporter gene with a gene of interest comprising providing a targeting vector; providing a cell in which at least a portion of the gene of interest is located between the first target sequence and the second target sequence; introducing the targeting vector into the cell; and maintaining the cell under conditions appropriate for integration of the reporter gene and WPRE into the genome of the cell such that the reporter gene is co-expressed with the gene of interest, wherein said portion of the gene of interest is cleaved prior to or subsequent to introducing the targeting vector into the cell.
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • upstream of the reporter gene a 5' homology arm homologous to a first target sequence; and incorporating downstream of the WPRE a 3' homology arm homologous to a second target sequence, wherein the second target sequence is located downstream of the first target sequence and wherein the first target sequence and the second target sequence flank at least a portion of the gene of interest.
  • FIG. 1 is a schematic illustrating the creation of targeting vectors and the insertion of a reporter gene and WPRE element.
  • HR120-PA-1 vector is shown at the top.
  • HR120-p2A-TD-TOM multicistronic vector
  • the copGFP-polyA cassette was removed using EcoRI and Nrul.
  • Vector sequence was restored via G-Block cloning which introduced a P2A cassette.
  • the Xhol site and Gibson assembly was used to introduce the TD-tomato followed by the WPRE element.
  • the copGFP-polyA cassette was removed using EcoRI and Nrul.
  • Vector sequence was restored via G-Block cloning and then the Xhol site and Gibson assembly was used to introduce the Clover followed by the WPRE element.
  • Homology arms were added to the multicistronic and fusion vectors via restriction digest and Gibson assembly.
  • the reporter gene and WPRE were inserted into the genomic locus shown (Genomic Locus TH) via CRISPR.
  • a region of ex on 14 TH gene with PAM sequences and suitable for targeting with a targetable nuclease is shown as sequences labeled px330 CRISPR Guide TH II and px330 CRISPR Guide TH I.
  • the light blue areas of the sequences correspond to the guide RNA sequences and the PAM sequence.
  • the asterisk shows the stop codon in the tyrosine hydroxylase exon 14.
  • the resulting added sequences are shown in "Targeted Locus.” Correct insertion direction was confirmed with an 865 nt PCR product for the 3 ' arm of the insert. Following selection of successful clones, the CRE cassette was excised to result in the "targeted Locus post CRE excision.” Correct insertion was confirmed with an 878 nt PCR product for the 3' arm of the insert.
  • FIGS. 2A-2C shows that correctly targeted clones were differentiated using a protocol based on a previously published protocol (FIG. 2A). Reporter expression can be seen as early as day 7. Cells were stained in embryoid bodies (EBs) using tyrosine hydroxylase (TH) antibody. The number of TH positive cells increased over time in the culture, as seen in the comparison between day 7 and day 30 EBs (FIG. 2B). Near perfect overlap between reporter expression and antibody staining was observed. Cells could be dissociated and plated or purified via FACS sorting which led to a strong enrichment with close to 100% of the cells expression the TD-tomato reporter (FIG. 2C).
  • TH tyrosine hydroxylase
  • FIG. 3 is another schematic illustrating the insertion of a reporter gene and WPRE element into the tyrosine hydroxylase genomic locus (Genomic Locus TH).
  • the reporter gene and WPRE element were inserted into the genomic locus shown (Genomic Locus TH) via CRISPR.
  • a region of ex on 14 TH gene with PAM sequences and suitable for targeting with a targetable nuclease (e.g., Cas9) is shown as sequences labeled px330 CRISPR Guide TH II and px330 CRISPR Guide TH I.
  • the light blue areas of the sequences correspond to the guide RNA sequences and the PAM sequence.
  • the asterisk shows the stop codon in the tyrosine hydroxylase exon 14.
  • the resulting added sequences are shown in "Targeted Locus.” Correct insertion direction was confirmed with an 865 nt PCR product for the 3' arm of the insert and a 626 bp PCR product for the 5' arm of the insert. Following selection of successful clones, the CRE cassette was excised to result in the "targeted Locus post CRE excision.” Correct insertion was confirmed with an 878 nt PCR product for the 3' arm of the insert and a 626 nt PCR product for the 5' arm of the insert.
  • FIGS. 4A-4F- FIGS. 4A through 4F are illustrations of CRISPR mediated knock-out mutagenesis to create isogenic PD lines.
  • FIGS. 5A-5G- FIGS. 5A through 5G are illustrations of early onset PD mutations could result in increased rate of cell death in midbrain DANs in basal culture conditions.
  • FIGS 6A-6E- FIGS 6 A through 6E are illustrations showing that global transcriptional analysis identifies overlapping dysregulated genes and pathways between PARKIN-/- and ATP13A2-/- cell lines.
  • FIGS. 7A-7H- FIGS. 7 A through 7H are illustrations showing differential expression analysis showed a strong increase in the number of differentially expressed proteins during the time course of differentiation in the WT versus PARKIN-/- comparison
  • FIGS 8A-8C- FIGS 8 A through 8C are illustrations showing knockout of DJ- 1 leads to the dysregulation of proteins involved in cell cycle as well as proteins involved in the development of Charcot-Marie-Tooth disease.
  • FIGS. 9A-9E- FIGS. 9 A through 9E are illustrations showing generation and characterization of isogenic tyrosine hydroxylase knock-in reporter cell lines carrying three distinct PD mutations.
  • FIGS. 10A-10D- FIGS. 10A through 10D are illustrations showing that targeting vector was designed to retain a largely unaltered endogenous TH gene product using a bicistronic targeting vector containing tdTomato.
  • FIGS. 12A-12E- FIGS. 12A through 12E are illustration showing loss of PARKIN decreases the number of TH-positive neurons.
  • FIGS. 13A-13C- FIGS. 13A through 13C are illustrations showing Global transcriptional analysis identifies overlapping dysregulated genes and pathways between PARKIN-/- and ATP13A2-/- cell lines.
  • FIGS 14A-14C- FIGS. 14A through 14C are illustrations showing quantitative proteomics reveals overlap in dysregulated pathways in isogenic PD lines.
  • FIGS 15A-15C- FIGS 15A through 15C are illustrations showing quantitative proteomics reveals overlap in dysregulated pathways in isogenic PD lines.
  • RNA interference RNA interference
  • compositions and methods disclosed herein generally relate to
  • compositions and methods useful for making and using reporter cells i.e., cells co-expressing a reporter and a genetic locus of interest.
  • such compositions and methods can provide a cell with a knock-in reporter (e.g., a fluorescent protein) and a downstream WPRE element. This combination dramatically and unexpectedly improves the co-expression of the reporter and genetic locus of interest versus standard methods in the art.
  • a knock-in reporter e.g., a fluorescent protein
  • the reporter and WPRE element are be inserted into embryonic stem cells to co-express with tyrosine hydroxylase, a protein indicative of dopaminergic neurons.
  • compositions and methods disclosed herein provide much higher co-expression of the reporter with the genetic locus of interest, dramatically increasing the robustness and sensitivity of identifying cells expressing the genetic locus of interest.
  • compositions disclosed herein relate to a nucleic acid targeting vector having homology arms flanking a reporter gene and an expression enhancer comprising a WPRE operably linked to the reporter gene.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid and polynucleotide are used interchangeably herein and should be understood to include double-stranded
  • a nucleic acid often comprises standard nucleotides typically found in naturally occurring DNA or RNA (which can include modifications such as methylated nucleobases), joined by phosphodiester bonds.
  • a nucleic acid may comprise one or more non-standard nucleotides, which may be naturally occurring or non-naturally occurring (i.e., artificial; not found in nature) in various embodiments and/or may contain a modified sugar or modified backbone linkage.
  • Nucleic acid modifications e.g., base, sugar, and/or backbone modifications
  • non-standard nucleotides or nucleosides, etc. may be incorporated in various embodiments. Such modifications may, for example, increase stability (e.g., by reducing sensitivity to cleavage by nucleases), decrease clearance in vivo, increase cell uptake, or confer other properties that improve the translation, potency, efficacy, specificity, or otherwise render the nucleic acid more suitable for an intended use.
  • nucleic acid modifications are described in, e.g., Deleavey GF, et al., Chemical modification of siRNA. Curr. Protoc. Nucleic Acid Chem. 2009;
  • nucleic acid or nucleic acid region is given in terms of a number of nucleotides (nt) it should be understood that the number refers to the number of nucleotides in a single-stranded nucleic acid or in each strand of a double-stranded nucleic acid unless otherwise indicated.
  • An "oligonucleotide” is a relatively short nucleic acid, typically between about 5 and about 100 nt long.
  • targeting vector refers to a vector comprising a polynucleotide having homology regions (i.e., homology arms) with sequences that are homologous to sequences present in a host cell genetic locus.
  • the homology arms flank a polynucleotide region (e.g., region containing a reporter gene and WPRE) which becomes integrated into a host cell genetic locus.
  • vector refers to a nucleic acid or a virus or portion thereof (e.g., a viral capsid or genome) capable of mediating entry of, e.g., transferring, transporting, etc., a nucleic acid into a cell.
  • nucleic acid to be transferred is generally linked to, e.g., present in, the vector.
  • a nucleic acid vector may include sequences that direct autonomous replication (e.g., an origin of replication).
  • Useful nucleic acid vectors include, for example, naturally occurring or modified viral genomes or portions thereof or nucleic acids (DNA or RNA) that can be packaged into viral capsids, DNA or RNA plasmids, and transposons. Plasmid vectors typically include an origin of replication and may include one or more selectable marker genes. Viruses or portions thereof that can be used to introduce nucleic acid molecules into cells are referred to as viral vectors.
  • Useful viral vectors include adenoviruses, adeno- associated viruses, retroviruses, lentiviruses, vaccinia virus and other poxviruses,
  • herpesviruses e.g., herpes simplex virus
  • a virus having tropism for a particular cell type e.g., neurons or a particular type of neuron
  • expression vectors that may be used in mammalian cells include, e.g., the pcDNA vector series, pSV2 vector series, pCMV vector series, pRSV vector series, pEFl vector series, Gateway® vectors, and PrecisionXTM HR Targeting Vectors, etc.
  • pcDNA vector series e.g., pSV2 vector series, pCMV vector series, pRSV vector series, pEFl vector series, Gateway® vectors, and PrecisionXTM HR Targeting Vectors, etc.
  • expression enhancer is intended to refer to a polynucleotide region or regions that binds proteins (e.g., transcription factors) to enhance (increase) transcription of a gene. Enhancers may be located some distance away from the promoters and transcription start site (TSS) of genes whose transcription they regulate and may be located upstream or downstream of the TSS. In some embodiments, the expression enhancer is located downstream of the TSS.
  • TSS transcription start site
  • Woodchuck Posttranscriptional Regulatory Element is a
  • WPRE has been shown to act on additional posttranscriptional mechanisms to stimulate expression of heterologous cDNAs (Zufferey et al., "Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors,' J. Virol., 73 (1999), pp. 2886-2892, incorporated herein by reference in its entirety).
  • operably linked refers to a nucleic acid regulatory element and a nucleic acid sequence being appropriately positioned relative to each other so as to place expression of the nucleic acid under the influence or control of the regulatory element(s).
  • an expression enhancer and a reporter gene are considered “operably linked” if they are positioned in such a way in a DNA molecule that the expression enhancer region enhances (increases) transcription of the reporter gene under appropriate conditions.
  • “operably linked” refers to the positional relationship between the regulatory element(s) (e.g., WPRE) and the nucleic acid sequence (e.g., reporter gene).
  • a particular expression enhancer does in fact enhance transcription of an operably linked nucleic acid molecule (e.g., reporter gene), may depend on a variety of factors, such as the presence or absence of appropriate factors and/or the presence or absence of inhibitory substances.
  • reporter refers to a molecule that can be used as an indicator of the occurrence or level of a particular biological process, activity, event, or state in a cell or organism. Reporters typically have one or more properties or enzymatic activities that allow them to be readily measured or that allow selection of a cell that expresses the reporter molecule. In general, a cell can be assayed for the presence of a reporter by measuring the reporter itself or an enzymatic activity of the reporter protein.
  • Detectable characteristics or activities that a reporter may have include, e.g., fluorescence, bioluminescence, ability to catalyze a reaction that produces a fluorescent or colored substance in the presence of a suitable substrate, or other readouts based on emission and/or absorption of photons (light).
  • a reporter is a molecule that is not endogenously expressed by a cell or organism in which the reporter is used.
  • reporter gene refers to a nucleic acid that encodes a reporter.
  • the reporter construct may be assembled in or inserted into a vector.
  • the reporter construct or vector may be transferred into one or more cells.
  • the reporter gene may be integrated into the genome. After transfer, cells are assayed for the presence of the reporter by measuring the reporter or the activity (e.g., enzymatic activity) of the reporter.
  • a reporter gene is codon-optimized for expression in mammalian cells. In some embodiments, a reporter gene is codon-optimized for expression in human cells.
  • homologous means two or more nucleic acid sequences that are either identical or similar enough that they are able to hybridize to each other or undergo intermolecular exchange.
  • sequences are homologous if they are either identical or similar enough that they are able to hybridize to each other under physiological conditions present in a cell (e.g., a mammalian cell).
  • a “homology arm” refers to a region of a nucleic acid targeting vector homologous to a genomic region.
  • At least one of homology arms is homologous to a region of a genetic locus (e.g., gene of interest).
  • the homology arms comprise a 5' homology arm homologous to a first target sequence in a cell and a 3' homology arm homologous to a second target sequence downstream of the first target sequence.
  • the 5' and 3' homology arms may be homologous to a contiguous region of the genome of the cell or homologous to discontinuous regions of the genome of the cell. Using homology arms that are homologous to contiguous genomic regions enables knock-in of a reporter gene without removal of endogenous genomic nucleotide sequence.
  • homology arms that are homologous to discontinuous genomic regions may enable both knock-in of the reporter gene and knock-out of an endogenous genomic nucleotide sequence.
  • “Knock-in” is a genetic modification resulting from the addition of the genetic information encoded in a chromosomal locus with further DNA sequence.
  • “Knock-out” is a genetic modification resulting from the disruption or removal of the genetic information encoded in a chromosomal locus.
  • the 5' homology arm is homologous to a target sequence immediately upstream of a stop codon of a genetic locus (e.g., gene of interest) and the 3' homology arm is homologous to a target sequence comprising the stop codon of the genetic locus (e.g., gene of interest), thereby enabling incorporation of the reporter gene and expression enhancer into a chromosome so that the reporter gene is co-expressed with the genetic locus (e.g., gene of interest) without changing the primary structure of a gene product.
  • the homology arms are both homologous to target sequences partially or fully upstream of a stop codon of the genetic locus (e.g., gene of interest). In some instances, insertion of a reporter gene and expression enhancer within the sequence encoding a gene product does not disrupt the function of the gene product.
  • each of the homology arms may comprise about 40 or more nucleotides.
  • each homology arm comprises about 50-1000 nucleotides, about 100- 800 nucleotides, about 200-500 nucleotides, or about 300-400 nucleotides.
  • each homology arm comprises about 350 nucleotides.
  • the homology arms are between about 100 nt - 200 nt, about 200 nt - 300 nt, about 300 nt - 400 nt, about 400 nt - 500 nt, about 500 nt - 750 nt, about 750 nt -1000 nt, about 1 kb - 1.5 kb, or more.
  • the two homology arms may be about the same length (e.g., within about 50 - 100 nt of each other) or may differ in length by more than about 100 nt. Either or both homology arms can independently fall within any of the afore-mentioned ranges.
  • the homology arms need not be perfectly homologous to the genomic DNA.
  • the homologous region(s) of a donor nucleic acid have at least 50% 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more sequence identity to a genomic sequence with which homologous
  • the homology arms are homologous to regions flanking a targeted nuclease cut site.
  • "flanking" indicates that the homology arms are located on either side of the targeted nuclease cut side, the flanking homology arms may be directly on either side of the cut side (contiguous) or one or both of the flanking homology arms may be some distance away from the cut site (non-contiguous).
  • the targeted nucleic acid cut site is located at the junction between contiguous flanking regions homologous to the homology arms.
  • the targeted nuclease cute site is within about 100 nt of the region homologous to the 3 ' end of the 5' homology arm.
  • the targeted nuclease cute site is within about 100 nt of the region homologous to the 5' end of the 3 ' homology arm. In some embodiments, the targeted nuclease cute site is within about 50 nt of the region homologous to the 3 ' end of the 5' homology arm. In some embodiments, the targeted nuclease cute site is within about 50 nt of the region homologous to the 5' end of the 3 ' homology arm. In some embodiments, the targeted nuclease cute site is within about 10 nt of the region homologous to the 3 ' end of the 5' homology arm.
  • the targeted nuclease cute site is within about 10 nt of the region homologous to the 5' end of the 3 ' homology arm. In some embodiments, the targeted nuclease cute site is within about 5 nt of the region homologous to the 3 ' end of the 5' homology arm. In some embodiments, the targeted nuclease cute site is within about 5 nt of the region homologous to the 5' end of the 3 ' homology arm.
  • the targeted nuclease cut site is within about 0-100 nt of the region homologous to the 5' end of the 3 ' homology arm and about 0-100 nt of the region homologous to the 3 ' end of the 5' homology arm.
  • a guide sequence for the targetable nuclease is not homologous to the targeting vector.
  • a guide sequence for the targetable nuclease is not homologous to a genomic sequence comprising the inserted reporter gene and WPRE.
  • the homology arms are homologous to one or more regions of the human tyrosine hydroxylase locus (Gene ID: 7054; NCBI).
  • the 5' homology arm is homologous to the 5' end of exon 14 of the human tyrosine hydroxylase locus and the 3' homology arm is homologous to a region comprising the human tyrosine hydroxylase stop codon with the region homologous to the 3' homology arm.
  • the 5' homology arm is homologous to the 5' end of exon 14 of the human tyrosine hydroxylase locus and the 3' homology arm is homologous to a region comprising the human tyrosine hydroxylase stop codon that is contiguous with the region homologous to the 3' homology arm.
  • the 5' homology arm comprises, consists essentially, or consists of the nucleotide sequence of SEQ ID NO: 1.
  • the 3' homology arm comprises, consists essentially, or consists of the nucleotide sequence of SEQ ID NO: 2.
  • the nucleic acid targeting vector comprises, in the 5' to 3' direction, (i) a 5' homology arm homologous to a first target sequence in a cell, (ii) a reporter gene, (iii) an expression enhancer comprising a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) operably linked to the reporter gene, and (iv) a 3' homology arm homologous to a second target sequence downstream of the first target sequence.
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • the expression enhancer region of the targeting vector may comprise the WPRE element and further transcription enhancer elements (e.g., SV40 enhancer, LTR). Transcription enhancers increase the likelihood of transcription of a particular gene. Any suitable transcription enhancer may be included in the expression enhancer region with the WPRE.
  • the reporter gene encodes a fluorescent protein. Any suitable fluorescent protein may be used. For instance, fluorescent proteins which may be suitable can be found on the world-wide web at
  • the fluorescent protein is a green fluorescent protein, a red fluorescent protein, or an infrared fluorescent protein.
  • fluorescent proteins include, e.g., GFP, EGFP, Sinus, Azurite, EBFP2, BFP, mTurquoise, ECFP, Cerulean, mTFPl, mUkGl, mAGl, AcGFP, mWasabi, EmGFP, YPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mK02, mOrange, mOrange2, LSSmOrange, PSmOrange, and PSmOrange2, mStrawberry, mRuby, mCherry, mRaspberry, tdTomato, mKate, mKate2, mPlum, mNeptune
  • the fluorescent protein is CLOVER or TD-TOMATO.
  • the reporter gene further comprises a stop codon downstream of the sequence encoding a guide protein.
  • the targeting vector has an IRES element or a sequence encoding a self-cleaving peptide located between the 5' homology arm and the reporter gene.
  • the sequence encoding a self-cleaving peptide encodes p2A, t2A, e2A, f2A.
  • the sequence encoding the self-cleaving peptide also encodes for a GSG sequence at the amino terminus to enhance cleavage efficiency.
  • the targeting vector does not have an IRES element or sequence encoding a self-cleaving peptide between the 5' homology arm and the reporter gene.
  • the targeting vector has an insulator sequence located between the WPRE and the 3' homology arm.
  • the length of the insulator sequence is not limited. In some embodiments, the insulator sequence is about 1-10 nt, about 1-50 nt, about 1-100 nt, or about 1-500 nt in length. In some embodiments, the insulator sequence blocks transcription of the 3' homology arm.
  • the targeting vector may have one or more restriction sites. In some embodiments, the insulator sequence has one or more restriction sites.
  • the targeting vector does not comprise a promoter sequence upstream of and/or operably linked to the reporter gene or WPRE element.
  • the targeting vector may include an expression cassette having positive and/or negative selection or screening markers.
  • Positive selection markers are those polynucleotides that encode a product that enables only cells that carry and express the gene to survive and/or grow under certain conditions. For example, cells that express neomycin resistance (Neo R ) gene are resistant to the compound G418, while cells that do not express Neo R are killed by G418.
  • Positive selection markers are not limited and can include hygromycin resistance, ZeocinTM resistance, and/or Puromycin resistance.
  • Negative selection markers are those polynucleotides that encode a produce that enables only cells that carry and express the gene to be killed under certain conditions.
  • thymidine kinase e.g., herpes simplex virus thymidine kinase, HSV-TK
  • HSV-TK herpes simplex virus thymidine kinase
  • Any known negative selection marker is contemplated and is not limited. Screening markers that may be used can be, for example, flourescent proteins or luciferases (e.g., GFP, mRUBY), or beta-galactosidase. Other screening markers may include sequences encoding polypeptides that will be expressed on the cell surface, allowing for identification with specific antibodies or other ligands to that surface expressed polypeptide.
  • the antibodies or ligands in these assays may be tagged in some manner, for example with a fluorophore, to allow rapid cell screening.
  • the expression cassette having positive and/or negative selection or screening markers further comprises LoxP sites flanking the selection and/or screening markers.
  • the invention is directed towards a composition
  • a composition comprising a cell having a genome comprising a nucleotide sequence having a reporter gene and a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) operably linked to the reporter gene, wherein the reporter gene is co-expressed with a target sequence of interest.
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • the cell is a non-naturally occurring transgenic cell.
  • the cell is a mammalian cell, e.g. a human, non-human primate, rodent (e.g., mouse, rat, rabbit, hamster), ungulate (e.g., ovine, bovine, equine, caprine species), canine, or feline cell.
  • the cell is an avian cell (e.g., chicken).
  • the cell is a somatic cell.
  • the cell is a pluripotent stem cell, and induced pluripotent stem cell or a multipotent stem cell.
  • the cell is a germ cell, stem cell, or zygote. In some embodiments the cell is a primary cell. In some embodiments the cell is a diseased cell. In some embodiments the cell is a cancer cell. In some embodiments the cell is a white blood cell or fibroblast. In some embodiments the cell is a cell that has been isolated from an embryo. In some embodiments, the cell is an embryonic stem cell.
  • Cells of the invention include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.
  • the cell is a neural cell (e.g., meninges, astrocyte, motor neuron, a cell of the dorsal root ganglia or anterior horn motor neuron), a neural lineage cell or a neural stem cell.
  • the cell is a pluripotent stem cell or an induced pluripotent stem cell.
  • the cell is a human pluripotent stem cell or a human induced pluripotent stem cell.
  • the cell is in a non-human transgenic animal. In some embodiments, the transgenic animal is a mouse, rat or non-human primate.
  • co-expressed with a target sequence of interest refers to expression of the reporter gene with the target sequence of interest, usually on the same mRNA.
  • the co-expression may result in a fusion protein or a separate reporter protein and target sequence product.
  • the reporter gene may be any reporter gene as described herein. As used herein co-expression is intended to mean that expression of the reporter gene substantially matches the expression of target sequence of interest.
  • the target sequence of interest (e.g., genomic locus of interest) is not limited.
  • the target sequence of interest is a gene of interest.
  • the gene of interest encodes a transcription factor, a transcriptional co-activator or co-repressor, an enzyme, a chaperone, a heat shock factor, a heat shock protein, a receptor, a secreted protein, a transmembrane protein, a histone (e.g., HI, H2A, H2B, H3, H4), a peripheral membrane protein, a soluble protein, a nuclear protein, a mitochondrial protein, a growth factor, a cytokine (e.g., an interleukin, e.g., any of IL-1 - IL-33), an interferon (e.g., alpha, beta, or gamma), a chemokine (e.g., a CXC, CX3C, C (or XC),
  • a chemokine
  • a chemokine may be CCL1 - CCL28, CXCL1 - CXCL17, XCL1 or XCL2, or CXC3L1).
  • the gene of interest encodes a colony-stimulating factor, a hormone (e.g., insulin, thyroid hormone, growth hormone, estrogen, progesterone, testosterone), an extracellular matrix protein (e.g., collagen, fibronectin), a motor protein (e.g., dynein, myosin), cell adhesion molecule, a major or minor histocompatibility (MHC) gene, a transporter, a channel (e.g., an ion channel), an immunoglobulin (Ig) superfamily (IgSF) gene (e.g., a gene encoding an antibody, T cell receptor, B cell receptor), tumor necrosis factor, an F-kappaB protein, an integrin, a cadherin superfamily member (e.g., a cadherin), a
  • Growth factors include, e.g., members of the vascular endothelial growth factor (VEGF, e.g., VEGF- A, VEGF-B, VEGF-C, VEGF-D), epidermal growth factor (EGF), insulin-like growth factor (IGF; IGF-1, IGF-2), fibroblast growth factor (FGF, e.g., FGF1 - FGF22), platelet derived growth factor (PDGF), or nerve growth factor (NGF) families.
  • VEGF vascular endothelial growth factor
  • EGF epidermal growth factor
  • IGF insulin-like growth factor
  • IGF-1 insulin-like growth factor
  • IGF-2 insulin-like growth factor
  • FGF fibroblast growth factor
  • PDGF platelet derived growth factor
  • NGF nerve growth factor
  • a growth factor promotes proliferation and/or differentiation of one or more hematopoietic cell types.
  • a growth factor may be CSF1 (macrophage colony- stimulating factor), CSF2 (granulocyte macrophage colony- stimulating factor, GM-CSF), or CSF3 (granulocyte colony-stimulating factors, G- CSF).
  • the gene of interest encodes erythropoietin (EPO).
  • the gene of interest encodes a neurotrophic factor, i.e., a factor that promotes survival, development and/or function of neural lineage cells (which term as used herein includes neural progenitor cells, neurons, and glial cells, e.g., astrocytes, oligodendrocytes, microglia).
  • the protein is a factor that promotes neurite outgrowth.
  • the protein is ciliary neurotrophic factor (CNTF) or brain- derived neurotrophic factor (BDNF).
  • the gene of interest is a human tyrosine hydroxylase gene.
  • the gene (e.g., human gene) of interest is SLC6A3, AGRP, POMC, HB9, GFAP, SCN10A, SCN9A, or TRPV1.
  • the cell comprises two or more nucleotide sequences each having a reporter gene and a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) operably linked to the reporter gene, wherein the reporter gene of each nucleotide sequence is co-expressed with a different target sequence of interest.
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • the cell comprises 2, 3, 4, 5 or more nucleotide sequences each having a reporter gene and a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) operably linked to the reporter gene, wherein the reporter gene of each nucleotide sequence is co-expressed with a different target sequence of interest.
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • the reporter gene of each nucleotide sequence may express a different reporter.
  • the nucleotide sequence may further comprise expression enhancers, isolator sequences, restriction sites, sequence encoding selection markers, and/or sequence encoding screening markers as described herein.
  • the reporter gene encodes TD-Tomato or Clover.
  • the expression of the reporter gene is under control of the endogenous promoter of the gene of interest.
  • the promoter e.g., human gene promoter, mouse gene promoter
  • the promoter is not limited.
  • the promoter is the human tyrosine hydroxylase gene promoter ⁇ see Kessler, et al., Brain Res Mol Brain Res. 2003 Apr 10; 112(l-2):8-23).
  • the promoter e.g., human gene promoter
  • the promoter is a SLC6A3 gene promoter, an AGRP gene promoter, a POMC gene promoter, an HB9 gene promoter, a GFAP gene promoter, a SCN10A gene promoter, a SCN9A gene promoter, or a TRPVl gene promoter.
  • the location within the genome of the nucleotide sequence comprising the reporter gene and WPRE element are not limited as long as the reporter gene is co-expressed with the target sequence of interest (e.g., gene of interest).
  • the target sequence of interest e.g., gene of interest
  • the target sequence of interest is a region of a gene encoding for a polypeptide.
  • the nucleotide sequence comprising the reporter gene and WPRE element is located upstream of the '5 end of a stop-codon of the target sequence of interest (e.g., gene of interest).
  • the nucleotide sequence comprising the reporter gene and WPRE element is located at the 3' end of an open reading frame of the target sequence of interest (e.g., gene of interest). In some embodiments, the nucleotide sequence comprising the reporter gene and WPRE element is located at the 3' end of an open reading frame of the target sequence of interest (e.g., gene of interest) and at the 5' end of the stop-codon of the target sequence of interest (e.g., gene of interest).
  • the nucleotide sequence comprising the reporter gene and WPRE element is located within or adjacent to a human gene (e.g., tyrosine hydroxylase, SLC6A3, AGRP, POMC, HB9, GFAP, SCN10A, SCN9A, or TRPVl). In some embodiments, the nucleotide sequence comprising the reporter gene and WPRE element is located upstream of the 5' end of the human gene (e.g., tyrosine hydroxylase, SLC6A3, AGRP, POMC, HB9, GFAP, SCN10A, SCN9A, or TRPVl) stop-codon.
  • a human gene e.g., tyrosine hydroxylase, SLC6A3, AGRP, POMC, HB9, GFAP, SCN10A, SCN9A, or TRPVl
  • the nucleotide sequence comprising the reporter gene and WPRE element is located upstream of the 5' end of the stop-codon human of a gene (e.g., tyrosine hydroxylase, SLC6A3, AGRP, POMC, HB9, GFAP, SCN10A, SCN9A, or TRPVl) and downstream of the 3' end of the open-reading frame of the human gene (e.g., tyrosine hydroxylase, SLC6A3, AGRP, POMC, HB9, GFAP, SCN10A, SCN9A, or TRPVl).
  • a gene e.g., tyrosine hydroxylase, SLC6A3, AGRP, POMC, HB9, GFAP, SCN10A, SCN9A, or TRPVl
  • the nucleotide sequence comprising the reporter gene and WPRE element is located in exon 14 of the human gene (e.g., tyrosine hydroxylase, SLC6A3, AGRP, POMC, HB9, GFAP, SCN10A, SCN9A, or TRPVl).
  • the human gene e.g., tyrosine hydroxylase, SLC6A3, AGRP, POMC, HB9, GFAP, SCN10A, SCN9A, or TRPVl.
  • Another embodiment of the invention is directed towards a method of generating a cell that co-expresses a reporter gene with a target sequence of interest (e.g., gene of interest).
  • the method may comprise providing a nucleic acid targeting vector as disclosed herein and providing a cell with target sequences homologous to the homology arms of the targeting vector, introducing the targeting vector into the cell; and maintaining the cell under conditions appropriate for integration of the reporter gene and WPRE into the genome of the cell such that the reporter gene is co-expressed with the target sequence (e.g., gene) of interest.
  • the cell may be non-naturally occurring or naturally occurring.
  • the cell may be any cell type disclosed herein.
  • the cell may be a pluripotent stem cell or an induced pluripotent stem cell.
  • the cell may be a human pluripotent stem cell or a human induced pluripotent stem cell.
  • the cell is an embryonic stem cell (e.g., human embryonic stem cell).
  • sequences of the 3' homology arm and 5' homology arm are not limited.
  • the homology arms may be any homology arm described herein.
  • At least one of the homology arms is homologous to a region of a genetic locus of interest (e.g., gene of interest).
  • the genetic locus of interest e.g., gene of interest
  • the genetic locus of interest is not limited.
  • the genetic locus of interest (e.g., gene of interest) may be any gene disclosed herein.
  • both homology arms are homologous to regions of a genetic locus of interest (e.g., gene of interest).
  • the 5' homology arm is homologous to a region of a genetic locus of interest (e.g., gene of interest).
  • the 5' homology arm is homologous to a region of genetic locus of interest (e.g., gene of interest) upstream of, proximate to, or adjacent to a stop codon. In some embodiments, a portion of the 3' homology arm is homologous to the stop codon or a portion of the stop codon. In some embodiments, the 5' homology arm is homologous to a region of a genetic locus of interest (e.g., gene of interest) adjacent to and upstream of the stop codon and the 3' homology arm is homologous to a region of the genetic locus of interest (e.g., gene of interest) contiguous with the region homologous to the 5' homology arm and including the stop codon.
  • a region of genetic locus of interest e.g., gene of interest
  • the step of introducing the target vector into the cell is not limited and may be performed by any method known in the art. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, the electroporation is via NucleofectorTM Technology (Lonza Group, Basel, Switzerland).
  • the step of maintaining the cell under conditions appropriate for integration of the reporter gene and WPRE into the genome of the cell is not limited and may be performed by any method known in the art.
  • the conditions comprise providing the cell with a targetable nuclease to generate a DNA break at a target site and incorporating the reporter gene and WPRE into the genome of the cell by homology directed repair (HDR).
  • HDR homology directed repair
  • Targetable nucleases e.g., site specific nucleases
  • DNA breaks e.g., double-stranded DNA breaks
  • HR homologous recombination
  • HDR homology-directed repair
  • Modifications that can be generated using targetable nucleases include insertions, deletions, or substitutions of one or more nucleotides, or introducing an exogenous DNA segment such as an expression cassette (a nucleic acid comprising a sequence to be expressed and appropriate expression control elements, such as a promoter, to cause the sequence to be expressed in a cell) or tag at a selected location in the genome.
  • an expression cassette a nucleic acid comprising a sequence to be expressed and appropriate expression control elements, such as a promoter, to cause the sequence to be expressed in a cell
  • tag at a selected location in the genome.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • RGNs RNA-guided nucleases
  • Cas proteins of the CRISPR/Cas Type II system and engineered meganucleases.
  • ZFNs and TALENs comprise the nuclease domain of the restriction enzyme Fokl (or an engineered variant thereof) fused to a site-specific DNA binding domain (DBD) that is appropriately designed to target the protein to a selected DNA sequence.
  • DBD site-specific DNA binding domain
  • the DNA binding domain comprises a zinc finger DBD.
  • the site-specific DBD is designed based on the DNA recognition code employed by transcription activator- like effectors (TALEs), a family of site-specific DNA binding proteins found in plant-pathogenic bacteria such as Xanthomonas species.
  • TALEs transcription activator- like effectors
  • the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Type II system is a bacterial adaptive immune system that has been modified for use as an RNA-guided endonuclease technology for genome engineering.
  • the bacterial system comprises two endogenous bacterial RNAs called crRNA and tracrRNA and a CRISPR-associated (Cas) nuclease, e.g., Cas9.
  • the tracrRNA has partial complementarity to the crRNA and forms a complex with it.
  • the Cas protein is guided to the target sequence by the crRNA/tracrRNA complex, which forms a RNA/DNA hybrid between the crRNA sequence and the
  • the crRNA and tracrRNA components are often combined into a single chimeric guide RNA (sgRNA or gRNA) in which the targeting specificity of the crRNA and the properties of the tracrRNA are combined into a single transcript that localizes the Cas protein to the target sequence so that the Cas protein can cleave the DNA.
  • the sgRNA often comprises an approximately 20 nucleotide guide sequence complementary to the desired target sequence followed by about 80 nt of hybrid crRNA/tracrRNA.
  • the guide RNA need not be perfectly complementary to the target sequence. For example, in some embodiments it may have one or two mismatches.
  • one or more guide sequences is a naturally occurring RNA sequence, a modified RNA sequence (e.g., a RNA sequence comprising one or more modified bases), a synthetic RNA sequence, or a combination thereof.
  • a "modified RNA” is an RNA comprising one or more modifications (e.g., RNA comprising one or more non-standard and/or non-naturally occurring bases and/or modifications to the backbone, internucleoside linkage(s) and/or sugar). Methods of modifying bases of RNA are well known in the art.
  • modified bases include those contained in the nucleosides 5 -m ethyl cyti dine (5mC), pseudouridine ( ⁇ ), 5- methyluridine, 2'0-methyluridine, 2-thiouridine, N-6 methyladenosine, hypoxanthine, dihydrouridine (D), inosine (I), and 7- methylguanosine (m7G).
  • 5mC nucleosides 5 -m ethyl cyti dine
  • pseudouridine ⁇
  • 5- methyluridine 2-thiouridine
  • N-6 methyladenosine 5- methyluridine
  • 2-thiouridine 2-thiouridine
  • N-6 methyladenosine hypoxanthine
  • dihydrouridine D
  • inosine I
  • 7- methylguanosine m7G
  • an RNA comprises one or more modifications selected from: phosphorothioate, 2'-OMe, 2'-F, 2' -constrained e
  • MS phosphorothioate
  • MSP 2'-OMe 3-thioPACE
  • a modification may stabilize the RNA and/or increase its binding affinity to a complementary sequence.
  • the one or more guide sequences comprise at least one locked nucleic acid (LNA) unit, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA units, such as from about 3-7 or 4-8 LNA units, or 3, 4, 5, 6 or 7 LNA units.
  • LNA locked nucleic acid
  • all the nucleotides of the one or more guide sequences are LNA.
  • the one or more guide sequences may comprise both beta-D-oxy-LNA, and one or more of the following LNA units: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in either the beta-D or alpha-L configurations or combinations thereof.
  • all LNA cytosine units are 5'methyl-cytosine.
  • the one or more guide sequences is a morpholino.
  • Morpholinos are typically synthetic molecules, of about 25 bases in length and bind to complementary sequences of RNA by standard nucleic acid base-pairing. Morpholinos have standard nucleic acid bases, but those bases are bound to morpholine rings instead of deoxyribose rings and are linked through phosphorodiamidate groups instead of phosphates.
  • a guide sequence can vary in length from about 8 base pairs (bp) to about 200 bp.
  • each of one or more guide sequences can be about 9 to about 190 bp; about 10 to about 150 bp; about 15 to about 120 bp; about 20 to about 100 bp; about 30 to about 90 bp; about 40 to about 80 bp; about 50 to about 70 bp in length.
  • each genomic sequence e.g., target sequence of interest, gene of interest
  • the portion of each genomic sequence to which the guide sequence is complementary or homologous to can be about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38 39, 40, 41, 42, 43, 44, 45, 46 47, 48, 49, 50, 51, 52, 53,54, 55, 56,57, 58, 59 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 81, 82, 83, 84, 85, 86, 87 88, 89, 90, 81, 92, 93, 94, 95, 96, 97, 98, or 100 nu
  • each guide sequence can be at least about 70%, 75%, 80%, 85%, 90%, 95%, 100%), etc. identical, complementary or similar to the portion of each genomic sequence.
  • each guide sequence is completely or partially identical, complementary or similar to each genomic sequence.
  • each guide sequence can differ from perfect complementarity or homology to the portion of the genomic sequence by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. nucleotides.
  • one or more guide sequences are perfectly complementary or homologous (100%)) across at least about 10 to about 25 (e.g., about 20) nucleotides of the genomic sequence.
  • the genomic target sequence (e.g., genomic locus of interest, gene of interest, target sequence of interest) should also be immediately followed by a Protospacer Adjacent Motif (PAM) sequence.
  • PAM Protospacer Adjacent Motif
  • the PAM sequence is present in the DNA target sequence but not in an guide sequence.
  • the Cas protein will be directed to any DNA sequence with the correct target sequence followed by the PAM sequence.
  • the PAM sequence varies depending on the species of bacteria from which the Cas protein was derived.
  • the targetable nuclease comprises a Cas9 protein.
  • Cas9 from Streptococcus pyogenes may be used.
  • the PAM sequences for these Cas9 proteins are NGG, NNNNGATT, NNAGAA, NAAAAC, respectively.
  • a number of engineered variants of the site-specific nucleases have been developed and may be used in certain embodiments.
  • engineered variants of Cas9 and Fokl are known in the art.
  • a biologically active fragment or variant can be used.
  • Other variations include the use of hybrid targetable nucleases.
  • CRISPR RNA-guided Fokl nucleases the Fokl nuclease domain is fused to the amino-terminal end of a catalytically inactive Cas9 protein (dCas9) protein.
  • RFNs act as dimers and utilize two guide RNAs (Tsai, QS, et al., Nat Biotechnol . 2014; 32(6): 569- 576).
  • Site-specific nucleases that produce a single-stranded DNA break are also of use for genome editing.
  • Such nucleases can be generated by introducing a mutation (e.g., an alanine substitution) at key catalytic residues in one of the two nuclease domains of a targetable nuclease that comprises two nuclease domains (such as ZFNs, TALENs, and Cas proteins).
  • a mutation e.g., an alanine substitution
  • Examples of such mutations include D10A, N863 A, and H840A in SpCas9 or at homologous positions in other Cas9 proteins.
  • a nick can stimulate HDR at low efficiency in some cell types.
  • nickases targeted to a pair of sequences that are near each other and on opposite strands can create a single-stranded break on each strand (" double nicking" ), effectively generating a DSB, which can be repaired by HDR using a donor DNA template (Ran, F. A. et al. Cell 154, 1380-1389 (2013).
  • donor nucleic acid refers to an exogenous nucleic acid segment that, when provided to a cell, e.g., along with a targetable nuclease, can be used as a template for DNA repair by homologous recombination and thereby cause site-specific genome modification (sometimes termed " genome editing” ).
  • the modifications can include insertions, deletions, or substitutions of one or more nucleotides, or introducing an exogenous DNA segment such as an expression cassette or tag at a selected location in the genome.
  • a donor nucleic acid typically comprises sequences that have homology to the region of the genome at which the genomic modification is to be made.
  • the donor may contain one or more single base changes, insertions, deletions, or other alterations with respect to the genomic sequence, so long as it has sufficient homology to allow for homology-directed repair.
  • the donor nucleic acid is the nucleic acid sequence comprising the reporter gene and WPRE flanked by the homology arms.
  • the homology arms are homologous to genomic sequences flanking a location in genomic DNA at which the insertion is to be made (e.g., DNA break).
  • DNA break e.g., DNA break
  • the homology begins no more than lOObp away from the break, e.g., between 1 and lOObp away, e.g., 1 - 50 bp away, e.g., 1-15 bp away, from the break.
  • Donor nucleic acid can be provided, for example, in the form of DNA plasmids, PCR products, or chemically synthesized oligonucleotides, and may be double- stranded or single-stranded in various embodiments.
  • the size of the donor nucleic can vary from as small as about 40 base pairs (bp) to about 10 kilobases (kb), or more. In some embodiments the donor nucleic is between about 1 kb and about 5 kb long.
  • RNAs e.g., TALENs, or ZFNs
  • a targeting vector e.g., comprising homology arms
  • a targetable nuclease may be targeted to a unique site in the genome of a mammalian cell by appropriate design of the nuclease or guide RNA.
  • a nuclease or guide RNA may be introduced into cells by introducing a nucleic acid that encodes it into the cell. Standard methods such as plasmid DNA transfection, viral vector delivery, transfection with synthetic mRNA (e.g., capped, polyadenylated mRNA), or microinjection can be used. If DNA encoding the nuclease or guide RNA is introduced, the coding sequences should be operably linked to appropriate regulatory elements for expression, such as a promoter and termination signal. In some embodiments a sequence encoding a guide RNA is operably linked to an RNA polymerase III promoter such as U6 or tRNA promoter.
  • RNA polymerase III promoter such as U6 or tRNA promoter.
  • one or more guide RNAs and Cas protein coding sequences are transcribed from the same nucleic acid (e.g., plasmid).
  • multiple guide RNAs are transcribed from the same plasmid or from different plasmids or are otherwise introduced into the cell.
  • the multiple guide RNAs may direct Cas9 to different target sequences in the genome, allowing for multiplexed genome editing.
  • a nuclease protein e.g., Cas9
  • a nuclease protein may be introduced into cells, e.g., using protein transduction.
  • Nuclease proteins, guide RNAs, or both may be introduced using microinjection.
  • Methods of using targetable nucleases, e.g., to perform genome editing, are described in numerous publications, such as Methods in Enzymolog , Doudna JA, Sontheimer EJ. (eds), The use of CRISPR/Cas9, ZFNs, and TALENs in generating site-specific genome alterations. Methods Enzymol. 2014, Vol. 546 (Elsevier); Carroll, D., Genome Editing with Targetable Nucleases, Annu. Rev. Biochem. 2014. 83 :409- 39, and references in either of these. See also U.S. Pat. Pub. Nos. 20140068797,
  • clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acid guide sequences (gRNAs) are present in the cell and the gRNAs direct Cas protein to create a double stranded break in a region between the regions homologous to the 5' homology arm and the 3' homology arm.
  • the reporter gene and WPRE are then integrated into the genome of the cell by homology directed repair.
  • the gRNA sequences do not hybridize with the targeting vector or the genome after integration of the nucleic acid comprising the reporter gene and WPRE.
  • the target polynucleotide sequence is cleaved such that a double-strand break results. In some embodiments, more than one target polynucleotide sequence is cleaved such that a double-strand break results.
  • the method comprises selecting cells with homologous recombination events over non-homologous recombination events via an enrichment step.
  • the enrichment step is not limited. At least two enrichment methods have been developed: the positive-negative selection (PNS) method and the "promoterless" selection method.
  • PNS the first method
  • the second method is a positive selection in genetic terms: it selects for recombination at the correct (homologous) locus by relying on the use of a positively selectable gene whose expression is made conditional on recombination at the homologous target site. See, e.g., Mortensen R., Curr Protoc Mol Biol.
  • zinc finger DNA-binding domains with alterations in at least one zinc coordinating residue such as CCHC zinc fingers. See, e.g., PCT/US2007/025455 (WO/2008/076290). Each of these references is incorporated by reference in its entirety.
  • a cell e.g., a human embryonic stem cell, a human induced pluripotent stem cell
  • Cas9 and a guide RNA homologous to a target sequence of a genomic region encoding a protein of interest under conditions such that Cas9 cleaves the genomic region.
  • a targeting vector as disclosed herein is introduced to the cell, wherein the targeting vector comprises a reporter gene, a WPRE element, a 5' homology arm and a 3' homology arm and wherein one of the homology arms is homologous to a region on one side of the cleavage site of the Cas9 and the other homology arm is homologous to a region on the other side of the cleavage site of the Cas9.
  • the reporter gene and WPRE are integrated into the genome of the cell by homologous recombination upstream of the stop codon of the nucleotide sequence encoding the protein of interest. Correct orientation of the inserted reporter gene and WPRE are confirmed by checking the length of a PCR product from PCR with primers to a region of the inserted sequence and a genomic region.
  • Another embodiment of the invention is directed towards a method of making a targeting vector for integrating a reporter gene and a WPRE in a cell wherein the reporter gene is co-expressed with the target sequence of interest (e.g., gene of interest) comprising: providing a vector comprising, in the 5' to 3' direction, a reporter gene and an expression enhancer comprising a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), wherein the expression enhancer and the reporter gene are operably linked;
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • incorporating upstream of the reporter gene a 5' homology arm homologous to a first target sequence; and incorporating downstream of the WPRE a 3' homology arm homologous to a second target sequence; wherein the second target sequence is located downstream of the first target sequence and wherein the first target sequence and the second target sequence flank at least a portion of the target sequence of interest (e.g., gene of interest).
  • the target sequence of interest e.g., gene of interest
  • the method further comprises making a vector comprising, in the 5' to 3' direction, a reporter gene and an expression enhancer comprising a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), wherein the expression enhancer and the reporter gene are operably linked.
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • a nucleotide sequence coding for a self-cleaving peptide as described herein or an IPER element may also be incorporated upstream of the reporter gene.
  • the vector may include an origin of replication and may include one or more selectable marker genes.
  • the vector may be any appropriate vector as described herein.
  • the vector may be created by any technique known in the art and is not limited.
  • the method comprises providing a vector comprising, in the 5' to 3' direction, a reporter gene and an expression enhancer comprising a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), wherein the expression enhancer and the reporter gene are operably linked.
  • the vector is any suitable vector described herein.
  • the vector may include a nucleotide sequence coding for a self-cleaving peptide as described herein or an IPER element upstream of the reporter gene.
  • the vector may also include an origin of replication and may include one or more selectable marker genes.
  • the vector includes a cassette encoding a reporter gene and/or a selectable marker.
  • the step of incorporating the homology arms into the vector are by any suitable method known in the art and not limited.
  • G-Block Gibson assembly is utilized to add one or both of the homology arms.
  • G-block Gibson assembly can be performed via the method described in Gibson, et al. (2009) "Enzymatic assembly of DNA molecules up to several hundred kilobases," Nature Methods, 6(5):343-345.
  • the vector is digested with a restriction enzyme at a desired location.
  • a double stranded nucleotide sequence comprising the homology arm and about 18-40 bp ends having the same sequence as a cut ends of the vector is provided and both the vector and double stranded nucleotide sequence are subject to 5' exonuclease digestion.
  • the resulting single stranded ends of the homology arm vector are annealed and DNA polymerase is utilized to fill in any missing sequence.
  • Ligase then covalently joins the DNA of adjacent segments, removing any nicks in the DNA.
  • the nucleotide sequences present at either side of the vector restriction site for making the overlapping sequence on the homology arm are shown in Table 1 :
  • both double stranded nucleotide sequence comprising the 5' homology arm and the double stranded nucleotide sequence comprising the 3' homology arm are incorporated by Gibson assembly.
  • the vector is digested with a first restriction enzyme and the double stranded nucleotide sequence comprising the 5' homology arm is incorporated. Then the vector is digested with a second restriction enzyme and the double stranded nucleotide sequence comprising the 3' homology arm is incorporated. In other embodiments, the vector is digested with a first restriction enzyme and the double stranded nucleotide sequence comprising the 3' homology arm is incorporated.
  • the vector is digested with a second restriction enzyme and the double stranded nucleotide sequence comprising the 5' homology arm is incorporated.
  • the first and second restriction enzymes are selected from Nhel, BamHI, and EcoRI but the restriction enzyme is not limited.
  • flanking at least a portion of a gene of interest is intended to mean that at least a portion of the 5' homology arm is homologous to a portion of the gene of interest.
  • the 5' homology arm is homologous to a portion of the gene of interest that is upstream of the stop codon.
  • the 5' homology arm is homologous to a region of the gene of interest comprising a 3' end of the last exon of the gene of interest and not comprising a stop codon.
  • the 5' homology arm is homologous to a region of the gene of interest immediately upstream of a stop codon and the 3' homology arm is homologous to a region comprising the stop codon.
  • the homology arms have sequences to enable insertion of the reporter gene and expression enhancer immediately after the final exon of the gene of interest and prior to the stop codon to enable co-expression of the entire gene of interest followed by expression of the reporter gene.
  • any one or more nucleic acids, polypeptides, cells, species or types of organism, disorders, subjects, or combinations thereof, can be excluded.
  • a composition of matter e.g., a nucleic acid, polypeptide, cell, or non-human transgenic animal
  • methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
  • the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum.
  • Numerical values include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by "about” or “approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by "about” or “approximately”, the invention includes an embodiment in which the value is prefaced by "about” or “approximately”.
  • the final vectors were derived through modifications of the commercially available HR120-PA-1 vector (www.systembio.com/genome-engineering-precisionx-HR- vectors/gene-tagging).
  • HR120-PA-1 vector www.systembio.com/genome-engineering-precisionx-HR- vectors/gene-tagging.
  • the copGFP-polyA cassette was removed using EcoRI and Nrul.
  • Vector sequence was restored via G-Block cloning which introduced a P2A cassette.
  • the following GBlock sequences were used:
  • the Xhol site was cleaved via restriction digest and Gibson assembly was used to introduce the fluorophore CDS followed by the WPRE element.
  • the TD-tomato CDS followed by the WPRE element was amplified using pFUGW-TD-Tomato as a template.
  • the PCR product was inserted via Gibson cloning.
  • the P2A-TD-Tomato-WPRE cassette can be excised through EcoRI restriction digest.
  • Gibson assembly was used to introduce the fluorescent protein CDS followed by the WPRE element.
  • the Clover CDS followed by the WPRE element was amplified using pFUGW-Clover as a template.
  • the PCR product was inserted via Gibson assembly.
  • homology arms for any given gene can be added.
  • the vector was cut with the restriction endonuclease Nhel.
  • a homology arm can be created either by PCR or DNA synthesis utilizing 18-40 bp long overlap sequences between the vector and insert. A template for the DNA generation is given below.
  • Vector and homology arm are enzymatically assembled using Gibson reaction.
  • Bamhl restriction endonuclease linearize the vector to add a 3 ' homology arm.
  • EcoRI for the 5' arm and Bamhl for the 3' arm were used.
  • the 5' homology arm ends before the stop codon of the gene of interest to allow for a fusion protein or multicistronic expression.
  • the necessary overhangs are shown in detail in Table 2.
  • Further CRISPRs can be designed with overlap in both the 5' and 3' arms to avoid cleavage of the homology construct during the targeting.
  • Geltrex coated 96 well dishes were prepared by coating with geltrex coating solution: ⁇ of geltrex/Matrigel in about 10 ml DMEM. 50 ⁇ (1/3) of picked colonies were transferred into pre-labeled PCR tubes for gDNA extraction.
  • PCR cells direct lysis mix was prepared (add 50 ⁇ Proteinase K to 1 ml of Viagen cell lysis reagent). 100 ⁇ of lysis reagent was added to each well containing 50 ⁇ picked colony. Plate sealed with sticky lid and placed on a rocking plate in PCR machine at 55°C for 6h followed by incubation at 85°C for 45 min and then incubation at 4°C. Lysates were stored in a refrigerator.
  • Phusion Hifi mastermix Polymerase was used for PCR Amplification: [0125] Primers designed to amplify region of interest (size 50-250 bp). gDNA concentration/quality determined through a test PCR using 1-5 ⁇ of lysate in a 15 ⁇ PCR reaction. For gDNA primers, NEB TM calculator for Phusion Hifi was used. The same amount of gDNA for all PCRs was used and a touchdown PCR was always performed.
  • PCR products were analyzed for the appearance of one single band and sequencing was performed using forward PCR primers.
  • Isolated clones had a small cytoplasm to nucleus ratio and stained positive for the pluripotency markers OCT4 and TRA- 160 (Figure 9E).
  • the targeting vector was designed to retain a largely unaltered endogenous TH gene product using a bicistronic targeting vector containing tdTomato (Shaner et al., 2004) ( Figure 4C and Figure 10A-D).
  • Our differentiation scheme is based on a modified version of the dual SMAD inhibition protocol followed by patterning by modulating sonic hedgehog and WNT signaling (Figure 5D) (Kriks et al., 2011; Valente et al., 2004).
  • PD is characterized by the disproportionate death of midbrain DANs.
  • TFFtdTomato positive DANs showed significantly higher ROS accumulation than their TFFtdTomato negative counterparts.
  • the increase was significant in all PD lines, but the PARKIN-/- line showed the strongest increase, consistent with the observed cell death phenotype.
  • many different disease mechanisms have been proposed to play a role in the development of PD.
  • KEGG pathways with direct relevance to DANs that were significantly dysregulated in the PARKIN- /- line. These pathways included hsa05032, 'Morphine addiction', hsa04726, 'Serotonergic synapse', hsa04727, 'GABAergic synapse', hsa05030, 'Cocaine addiction', and hsa04080, 'Neuroactive ligand-receptor interaction'.
  • the midbrain contains several DAN populations and selective cell death in the PARKIN-/- line could change its composition relative to that of the other lines.
  • ventral tegmental area (VTA) DANs are known to play a primary role in the reward system and addiction.
  • VTA ventral tegmental area
  • DJ-1 is a multifunctional protein. The role it plays in the development of PD is presently unclear.
  • MCM minichromosome maintenance complexes
  • DJ-1 may act as a cysteine protease, and a valine-lysine-valine-alanine (VKVA) recognition sequence has been identified in target proteins (Mitsugi et al., 2013).
  • PARKIN is broadly expressed throughout the body, including heart, testis, liver and kidney, as well as brain (Kuhn et al., 2004). However, PD patients in general and those carrying loss of PARKIN mutations specifically exhibit the dysfunction and death of midbrain DANs.
  • Several recent publications have shown that PARKIN ubiquitinates many proteins (Bingol et al., 2014; Ordureau et al., 2015; Rose et al., 2016; Sarraf et al., 2013). In our isogenic system, we have been able to study the effect of PARKIN loss on cellular proteomes at three developmental time points.
  • G protein-coupled receptor 50 G protein-coupled receptor 50 (GPR50) was significantly enriched at all three-time points suggesting that it is the cell- type specific environment that is critical in disease progression. Broad dysregulation of protein abundances increased from pluripotent cells to NPCs, but was strongest in the DANs, illustrating the importance of studying loss of PARKIN in the most relevant cell type.
  • Oxidative Stress is a shared phenotype in all EO-PD DANs
  • ECM interactions have been implicated in Alzheimers Disease and PD, and multiple strategies exist to intervene pharmaceutically with ECM interactions, or their metabolizing enzymes (Berezin et al., 2014). However, little is known about the specific dysregulated genes found in our study and their implications in disease. It will be important to analyze if the same genes are found to be dysregulated in PD patients and to understand the role these genes play in the development of PD phenotypes.
  • PD disease pathways are often explained in a network context (Trinh and Farrer, 2013; Verstraeten et al., 2015) but few studies have attempted to investigate, at the molecular level, how mutations in such diverse proteins can all lead to PD.
  • Several studies, including our own, have used transcriptomics data to identify and understand PD relevant genes and pathways.
  • protein stability and degradation are independent of transcriptional activity and strongly contribute to the regulation of protein levels. These processes are widely implicated in neurodegenerative diseases including PD (Caudle et al., 2010; Tai and Schuman, 2008).
  • Dysregulation of a PD protein that in turn, leads to the dysregulation of another PD relevant gene can be seen as a common pathway.
  • SNCA a-synuclein
  • MAT tau protein
  • SNCA was the first specific genetic aberration to have been linked to the development of PD (Polymeropoulos et al., 1997), and accumulation of SNCA aggregates and the formation of Lewy bodies are hallmarks of PD.
  • Several SNCA mutations in PD patients have been investigated, and a gene dosage effect exists.
  • DJ-1 Loss of DJ-1 leads to dysregulation of distinct pathways involving the cell cycle and the neuropathology of Charcot-Marie-Tooth disease
  • NEFL is a major component of neurofilaments and, together with NEFM and heavy neurofilament (NEFH) subunits, form the major intermediate filament in neurons. Mutations in this locus lead to disruption of axonal neurofilament translocation, which affects the transport of mitochondria in axons (Brownlees et al., 2002). Mutant forms of HSP27 induce CMT through deficient retrograde axonal transport of mitochondria (Kalmar et al., 2017). Defects in mitochondrial transport have been suggested to play a role in the pathogenesis of PD, but this has not been conclusively demonstrated. Here we suggest a connection between loss of function mutations in DJ-1 and genes that are known to cause CMT.
  • CMT is also genetically heterogenous, and, recently, a different CMT mutation in the LRSAMl gene was linked to the development of PD in three patients (Aerts et al., 2016).
  • DJ-1 is a multi-functional protein and DJ-1 protease activity has been studied using recombinant DJ-1 and a peptide library.
  • VKVA valine-lysine-valine-alanine
  • McAlister G.C., Huttlin, EX., Haas, W., Ting, L., Jedrychowski, M.P., Rogers, J.C., Kuhn, K., Pike, I, Grothe, R.A., Blethrow, J.D., et al. (2012).
  • Bioconductor package for differential expression analysis of digital gene expression data Bioinformatics 26, 139-140.
  • Movement disorders official journal of the Movement Disorder Society 23, 1850-1859.
  • Parkinson's disease loss of neurons from the ventral tegmental area contralateral to therapeutic surgical lesions.

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Abstract

L'invention concerne des procédés, des vecteurs, des cellules transgéniques et des compositions pour exprimer un marqueur avec un gène d'intérêt. En particulier, l'invention concerne des procédés, des vecteurs, des cellules transgéniques et des compositions pour une expression élevée d'une protéine fluorescente telle que TDTomato avec un gène d'intérêt tel que la tyrosine hydroxylase afin d'évaluer l'expression du gène d'intérêt in vivo et in vitro.
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