WO2017024047A1 - Compositions et procédés d'augmentation des taux de recombinaison induits par la nucléase dans les cellules - Google Patents

Compositions et procédés d'augmentation des taux de recombinaison induits par la nucléase dans les cellules Download PDF

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WO2017024047A1
WO2017024047A1 PCT/US2016/045378 US2016045378W WO2017024047A1 WO 2017024047 A1 WO2017024047 A1 WO 2017024047A1 US 2016045378 W US2016045378 W US 2016045378W WO 2017024047 A1 WO2017024047 A1 WO 2017024047A1
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cell
cas9
chimeric protein
dna
rna
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PCT/US2016/045378
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Lior IZHAR
David Baram
Noam DIAMANT
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Emendobio Inc.
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • Targeted genome modification is a powerful tool that can be used to reverse the effect of pathogenic genetic variations and therefore has the potential to provide new therapies for human genetic diseases.
  • Current genome engineering tools most recently, the RNA-guided DNA endonuclease Cas9, produce sequence-specific DNA breaks in a genome.
  • the modification of the genomic sequence occurs at the next step and is the product of the activity of one of two cellular DNA repair mechanisms, triggered in response to the newly formed DNA break.
  • These mechanisms include: (1) non-homologous end- joining (NHEJ) in which the two ends of the break are ligated together in a fast but also inaccurate manner (i.e.
  • HDR homology-directed repair
  • the present disclosure provides compositions and methods for increasing the efficiency and reducing the off-target effect of Cas9 mediated genome editing.
  • the method comprises the introduction of a chimeric protein, which contains Cas9 fused to a cell-cycle dependent degron and a guide RNA that is complementary to a specific site in the DNA of a genome of a cell.
  • the addition of the cell-cycle dependent degron element to Cas9 results in regulated degradation of the chimeric protein at a specific phase of the cell-cycle.
  • Cas9 mediated genome editing is a two-step process.
  • the first step involves generation of a DNA strand break at a specific genomic locus.
  • the second step involves repair of the DNA break by cellular DNA repair mechanisms which may result in alteration of the original DNA sequence.
  • DNA breaks are repaired by two main cellular repair pathways: (a) non homologous end-joining (NHEJ) , which is error-prone and occurs predominantly during the Gl phase of the cell cycle, and (b) homology directed repair (HDR) which is an accurate repair process and depends on the presence of a homologous DNA donor.
  • NHEJ non homologous end-joining
  • HDR homology directed repair
  • Homologous recombination enables the insertion of DNA from an external source to a specific location in a genome in an accurate manner. Homologous recombination is most predominant during the S, G2 and M phases of the cell-cycle.
  • Cas9 mediated DNA strand breaks that are formed during Gl phase will be repaired by the error-prone pathway of NHEJ.
  • Cas9 mediated DNA breaks that are formed during S, G2 and M phases of the cell-cycle are repaired predominantly by HDR.
  • HDR is the only repair pathway that allows introduction of DNA from an external source to a genome in an accurate manner. Therefore focusing the activity of Cas9 to the S, G2 and M phases of the cell-cycle using the Cas9-degron chimera improves the efficiency of specific genome editing applications. Furthermore, limiting the expression of Cas9 to specific phases of the cell-cycle reduces the extent of time in which Cas9 is active. This in turn reduces the chances for collateral damage in the form of DNA breaks that are generated in low frequency at genomic locations that are not the intended target, a phenomena known as off targeting.
  • the present invention provides a chimeric protein comprising (a) an RNA-guided DNA nuclease and (b) at least one cell-cycle dependent degron.
  • the present invention provides a chimeric protein comprising (a) a Cas9 nuclease and (b) at least one cell-cycle dependent degron.
  • Figure 1 Schematic representation of the DNA construct use for generation of a Cas9-degron chimera with the degron fused to the C-terminal (A) or the N-terminal (B) .
  • FIG. 2 Schematic representation of the PCDNA.D53-hCas9-GMNN vector cloning procedure.
  • Figure 3 Introduction of CRISPR components and GFP correction DNA donor to 293T-IGFP cells results in positive GFP signal.
  • 293T- iGFP cells were transfected with: (A) a mix of the gRNA expression vector pGFP-GUIDl, GFP correction single stranded DNA donor and vector expressing Cas9. (B) Similar to (A) but without the DNA donor. (C) Similar to to (A) but without the Cas9 expression vector. (D) Similar to (A) but without the gRNA expression vector. GFP fluorescence intensity was measured 48hr post transfection using a flow cytometer.
  • FIG. 4 Fusion of GMNN degron to Cas9 result in an increase in the frequency of HDR related gene editing events.
  • 293T-iGFP cells were transfected with a mix of the gRNA expression vector pGFP- GUID1, the DNA donor DD-GFP-C-88ss and either the Cas9 expression vector PCDNA.
  • D53-CAS9 left or the Cas9-GMNN expression vector PCDNA.
  • D53-hCAS9-GMNN (right).
  • HDR mediated repair efficiency of the inactive GFP gene was determined by measuring fluorescence intensity using a flow cytometer 24 hr post transfection. The results are expressed as fold effect and were obtained by normalizing to the GFP positive fraction of cells transfected with the Cas9 only (pCDNA3.1 HA-Cas9) vector.
  • FIG. 5 An increase in frequency of HDR related gene editing events , can be achieved by fusion of GMNN degron to either side of Cas9 and it requires a functional degron elements.
  • 293T-iGFP cells were transfected with a mix of the gRNA expression vector pGFP-GUIDl, the DNA donor DD-GFP-C-88ss and either the Cas9 expression vector pCDNA3.1 HA-Cas9 (left), an N' terminal Gmnn- Cas9 fusion expression vector pCDNA3.1-GMN1-I10-Cas9-HA (mid left), C' terminal Cas9-G N fusion expression vector pCDNA3.1 HA- Cas9-GMNNl-110 (mid right) or its GMN degron mutated version pCDNA3.1 HA-Cas9-GMNN_null (right).
  • HDR mediated repair efficiency of the inactive GFP gene was determined by measuring fluorescence intensity using a flow cytometer 24 hr post transfection. The results are expressed as fold effect and were obtained by normalizing to the GFP positive fraction of cells transfected with the Cas9 only (pCDNA3.1 HA-Cas9) vector.
  • FIG. 6 An increase in frequency of HDR related gene editing events can be achieved by fusing Cas9 to cell-cycle degrons from other proteins.
  • 293T-iGFP cells were transfected with a mix of the gRNA expression vector pGFP-GUIDl, the DNA donor DD-GFP-C- 88ss and either the Cas9 expression vector pCDNA3.1 HA-Cas9 (left) , the C terminal Cas9-Securin degron expression vector pCDNA3.1-HA-Cas9-C'-Securin(l-79) (mid left), or C terminal Cas9-CDC20 degron expression vector pCDNA3.1-HA-Cas9-C ' -CDC20 ( 1- 113) (right) .
  • HDR mediated repair efficiency of the inactive GFP gene was determined by measuring fluorescence intensity using a flow cytometer 24 hr post transfection. The results are expressed as fold effect and were obtained by normalizing to the GFP positive fraction of cells transfected with the Cas9 only (pCDNA3.1 HA- Cas9) vector.
  • Figure 7 An increase in frequency of HDR related gene editing events is achieved by fusion of GMNN degron elements of different sizes to Cas9.
  • 293T-iGFP cells were transfected with a mix of the gRNA expression vector pGFP-GUIDl, the DNA donor DD-GFP-C-88ss and either the Cas9-inactive degron fusion expression vector pCDNA3.1 HA-Cas9-GMNN_null, an expression vector for Cas9 fused to a compact version of the GMNN degron that carries the intact KEN and DEAD Box elements, an expression vector for Cas9 fused to the regular 1-110 aa long GMN degron and an expression vector for Cas9 fused to a longer piece of GMNN N-terminal end (1-131) that includes an extra KEN box sequence.
  • HDR mediated repair efficiency of the inactive GFP gene was determined by measuring fluorescence intensity using a flow cytometer 72 hr post transfection. The results are expressed as fold effect and were obtained by normalizing to the GFP positive fraction of cells transfected with the Cas9 null-Gmnn Degron vector (pCDNA3.1 HA-Cas9-null GMN degron) .
  • a fusion protein comprising a RNA-guided DNA nuclease portion and a cell-cycle dependent degron portion.
  • a fusion protein which comprises any RNA-guided DNA nuclease e.g. Cas9, Cpfl, etc.
  • the present invention provides a chimeric protein comprising (a) a Cas9 nuclease and (b) at least one cell-cycle dependent degron.
  • the chimeric protein has a restricted expression profile to the S, G2 and M phases of the cell cycle.
  • the at least one cell-cycle dependent degron is fused to the N-terminal of the Cas9 nuclease.
  • the at least one cell-cycle dependent degron is fused to the C-terminal of the Cas9 nuclease.
  • the at least one cell-cycle dependent degron is fused to both termini of the Cas9 nuclease.
  • the chimeric protein comprises multiple copies of the cell-cycle dependent degron.
  • the chimeric protein comprises more than one type of cell-cycle dependent degron.
  • the at least one cell-cycle dependent degron contains a D-box. In some embodiments, wherein the at least one cell-cycle dependent degron is of a protein selected from the group consisting of Cyclin A, Cyclin B, Hsll, Cdc6, Finl, p21 and Geminin. In some embodiments, wherein the at least one cell-cycle dependent degron contains a KEN box.
  • the at least one cell-cycle dependent degron is of a protein selected from the group consisting of Cdc20, Sgol, Nek2 and B99.
  • amino acid sequence of the at least one cell-cycle dependent degron is set forth in SEQ ID NO: 11.
  • the Cas9 nuclease is selected from the group consisting of Streptococcus pyogenes Cas9, Streptococcus thermophilus Cas9, Streptococcus pasteurianus Cas9,
  • Staphylococcus aureus Cas9 Neisseria cinerea Cas9, Campylobacterlari Cas9, Corynejacteriufli diphtheria Cas9 and Pavibaculum lavamentivorans Cas9.
  • the Cas9 nuclease is derived from Streptococcus pyogenes.
  • the Cas9 nuclease is derived from Streptococcus thermophilus.
  • a chimeric protein whose amino acid sequence is set forth as SEQ ID NO: 12. In some embodiments, a chimeric protein whose amino acid sequence is set forth as SEQ ID NO: 18.
  • the present invention also provides a polynucleotide comprising a sequence which encodes the chimeric protein of any one of the embodiments described herein.
  • the present invention further provides a method for increasing the rate of insertion of donor DNA to the genome in a cell, the method comprising delivering to the cell: a) the chimeric protein of any one of the embodiments described herein, or a polynucleotide encoding the chimeric protein; and
  • a guide-RNA that is complementary to a target DNA sequence, or a polynucleotide encoding the guide-RNA; wherein the rate of insertion is increased compared to the rate of insertion under the same conditions in a cell expressing the Cas9 nuclease not fused to the cell-cycle dependent degron.
  • the rate of insertion can be compared to the rate of insertion in a cell expressing the Cas9 nuclease not fused to a functional cell- cycle dependent degron, or to the rate of insertion in a cell expressing the native Cas9.
  • the rate of DNA insertion is increased by at least 10%, more preferably at least 50%, more preferably by 100%.
  • off-target excision is reduced by at least 10%, more preferably at least 50%, more preferably by 100% compared to the native Cas9.
  • the off-target excision can be compared to the rate of insertion in a cell expressing the Cas9 nuclease not fused to a functional cell-cycle dependent degron; in particular the Cas9 fused to a non-functional cell-cycle dependent degron.
  • the target DNA sequence is genomic DNA.
  • the target DNA sequence is mitochondrial DNA
  • any of the methods described herein may further include delivering to the cell a donor DNA in addition to the (a) chimeric protein of any one of the embodiments described herein and (b) guide-RNA. Delivery of the donor DNA from an external source, or exogenous donor DNA, may be accomplished by any known method to one of ordinary skill in the art.
  • the present invention also provides a method of genome editing comprising delivering to a cell: a) the chimeric protein of any one of the embodiments described herein, or a polynucleotide encoding the chimeric protein of any one of the embodiments described herein; and
  • RNA so as to induce the genome editing.
  • the genome editing results in an at least 10%, more preferably at least 50%, more preferably at least 100% increase in the rate of DNA insertion compared to native Cas9.
  • the rate of insertion can be compared to the rate of insertion in a cell expressing the Cas9 nuclease not fused to a functional cell-cycle dependent degron, or to the rate of insertion in a cell expressing the native Cas9.
  • the genome editing results in an at least 10%, more preferably at least 50%, more preferably at least 100% reduction of off-target excision compared to native Cas9.
  • the off-target excision can be compared to the rate of excision in a cell expressing the Cas9 nuclease not fused to a functional cell-cycle dependent degron; in particular the Cas9 fused to a non-functional cell-cycle dependent degron.
  • the genome editing inserts DNA from an external source.
  • the present invention also provides a host cell having a DNA sequence edited by the method of any one of the embodiments described herein.
  • the host cell is a mammalian cell.
  • the present invention also provides, a method for reversing the pathogenic effect of disease-causing genetic variations in a cell by the method of any one of the embodiments described herein.
  • the at least one cell-cycle dependent degron is of a Securin protein.
  • amino acid sequence of the at least one cell-cycle dependent degron encodes at least the sequence set forth in SEQ ID NO: 11 of the Geminin protein.
  • the Cas9 nuclease is derived from any available natural source.
  • a chimeric protein whose amino acid sequence is set forth as SEQ ID NO: 19.
  • a chimeric protein whose amino acid sequence is encoded by SEQ ID NO: 22.
  • a chimeric protein whose amino acid sequence is encoded by SEQ ID NO: 23.
  • the host cell is a plant cell.
  • the rate of insertion is increased compared to the rate of insertion in a cell expressing the Cas9 nuclease not fused to a functional cell-cycle dependent degron, or a cell expressing the native Cas9.
  • the present invention provides a chimeric protein comprising: a) an RNA-guided DNA nuclease; and
  • the at least one cell-cycle dependent degron is fused to the N-terminal of the RNA-guided DNA nuclease, or wherein the at least one cell-cycle dependent degron is fused to both termini of the RNA-guided DNA nuclease.
  • the chimeric protein comprises multiple copies of the cell-cycle dependent degron, and/or wherein the chimeric protein comprises more than one type of cell-cycle dependent degron.
  • the at least one cell-cycle dependent degron contains a D-box, preferably wherein the at least one cell- cycle dependent degron is of a protein selected from the group consisting of Securin, Cyclin A, Cyclin B, Hsll, Cdc6, Finl and p21, and/or wherein the at least one cell-cycle dependent degron contains a KEN box, preferably wherein the at least one cell-cycle dependent degron is of a protein selected from the group consisting of Securin, Cdc20, Sgol, Nek2 and B99.
  • RNA-guided DNA nuclease is Cpfl nuclease.
  • the present invention also provides a polynucleotide comprising a sequence which encodes the chimeric protein of any one of the embodiments described herein.
  • the present invention further provides a method for increasing the rate of insertion of donor DNA to the genome in a cell, the method comprising delivering to the cell: a) the chimeric protein of any one of the embodiments described herein, or a polynucleotide encoding the chimeric protein; and
  • a guide-RNA that is complementary to a target DNA sequence, or a polynucleotide encoding the guide-RNA; wherein the rate of insertion is increased compared to the rate of insertion under the same conditions in a cell expressing the RNA-guided DNA nuclease not fused to the cell-cycle dependent degron, or compared to a cell expressing the native RNA-guided DNA nuclease, preferably wherein off-target excision is reduced by at least 10%, more preferably at least 50%, more preferably by 100% compared to a cell expressing the native RNA-guided DNA nuclease, or a cell expressing the RNA-guided DNA nuclease not fused to a functional cell-cycle dependent degron.
  • the present invention further provides a method of genome editing comprising delivering to a cell: a) the chimeric protein of any one of the embodiments described herein, or a polynucleotide encoding the chimeric protein of any one of the embodiments described herein; and b) a guide-RNA, or a nucleic acid sequence encoding the guide- RNA; so as to induce the genome editing, preferably wherein the genome editing results in an at least 10%, more preferably at least 50%, more preferably at least 100% reduction of off-target excision compared to the RNA-guided DNA nuclease not fused to the cell- cycle dependent degron, or compared to a cell expressing the native RNA-guided DNA nuclease.
  • the present invention provides a host cell having a DNA sequence edited by the method of the embodiments described herein, preferably wherein the host cell is a plant cell.
  • the present invention also provides a method for reversing the pathogenic effect of disease-causing genetic variations in a cell by the method of any one of the embodiments described herein.
  • the present invention also provides a use of a chimeric protein of any one of the embodiments described herein for use in the manufacture of a medicament to treat a subject with a genetic disorder.
  • Genetic disorders include any disease whose pathology is caused by a genetic component. For example, many forms of cancer are mediated by mutations in oncogenes or tumor suppressor genes and are thus considered one type of genetic disorder.
  • genetic disorders include, but are not limited to, Achondroplasia, Alpha-1 Antitrypsin Deficiency, Antiphospholipid Syndrome, Autism, Autosomal Dominant Polycystic Kidney Disease, Breast cancer, Charcot-Marie-Tooth, Colon cancer, Cri du chat, Crohn's Disease, Cystic fibrosis, Dercum Disease, Down Syndrome, Duane Syndrome, Duchenne Muscular Dystrophy, Factor V Leiden Thrombophilia, Familial Hypercholesterolemia, Familial
  • Mediterranean Fever Fragile X Syndrome, Gaucher Disease, Hemochromatosis, Hemophilia, Holoprosencephaly, Huntington's disease, Klinefelter syndrome, Marfan syndrome, Myotonic Dystrophy, Neurofibromatosis, Noonan Syndrome, Osteogenesis Imperfecta, Parkinson's disease, Phenylketonuria, Tru Anomaly, Porphyria, Progeria, Prostate Cancer Retinitis Pigmentosa, Severe Combined Immunodeficiency (SCID) , Sickle cell disease, Skin Cancer, Spinal Muscular Atrophy, Tay-Sachs, Thalassemia Trimethylaminuria, Turner Syndrome, Velocardiofacial Syndrome, AGR Syndrome and Wilson Disease.
  • SCID Severe Combined Immunodeficiency
  • the cell-cycle dependent degron may be derived from the Geminin protein. Fusion of a Geminin derived degron to a protein results in degradation of the chimeric protein at the late M to early Gl phase of the cell- cycle .
  • any protein or protein domain which has a cell-cycle dependent degradation property may be fused to Cas9 to achieve cell-cycle dependent degradation of the Cas9- Degron chimeric protein ( Figures 1A and IB) .
  • Many proteins which have cell-cycle dependent degradation and comprise a degron are known in the art.
  • the degron is fused to the N-terminal of Cas9.
  • the degron is fused to the C-terminal of Cas9.
  • the degron comprises at least one D-box.
  • the degron comprises at least one KEN box. In some embodiments the degron comprises at least one D-box and at least one KEN box.
  • Cas9 is derived from Streptococcus pyogenes or Streptococcus thermophilus .
  • nuclease is derived from any available natural source.
  • the use of the Cas9-degron for genome editing results in an at least 10% increase in the rate of DNA insertion .
  • the use of the Cas9-degron for genome editing results in an at least 50% increase in the rate of DNA insertion.
  • the use of the Cas9-degron for genome editing results in an at least 100% increase in the rate of DNA insertion.
  • the use of the Cas9-degron for genome editing results in an at least 400% increase in the rate of DNA insertion.
  • the use of the Cas9-degron for genome editing results in an at least 500% increase in the rate of DNA insertion.
  • the use of the Cas9-degron for genome editing results in 10-500%, preferably 50-500%, 100-500%, 200- 500%, 300-500%, or 400-500% increase in the rate of DNA insertion .
  • the use of the Cas9-degron for genome editing results in an at least 10% reduction of off-target excision of a genome. In some embodiments the use of the Cas9-degron for genome editing results in an at least 50% reduction of off-target excision of a genome. In some embodiments the use of the Cas9-degron for genome editing results in an at least 100% reduction of off-target excision of a genome.
  • the use of the Cas9-degron for genome editing results in an at least 500%, at least 1,000%, at least 2,000%, at least 5,000%, at least 10,000% reduction of off- target excision of a genome.
  • the use of the Cas9-degron for genome editing results in an at least 1,000-fold, at least 10,000- fold, at least 100,000-fold, or at least one million-fold reduction of off-target excision of a genome.
  • Embodiments describing the rate of DNA insertion and off-target effects mediated by the chimeric protein of the subject invention can be measured relative the rate of DNA insertion and off-target effects mediated by a RNA-guided DNA nuclease not fused to a functional cell-cycle dependent degron, in a particular a RNA-guided DNA nuclease fused to a non-functional cell-cycle dependent degron, or a native RNA-guided DNA nuclease.
  • the Cas9-Geminin chimera has the amino acid sequence of SEQ ID NO: 18.
  • the Cas9-Geminin chimera further comprises a tag.
  • the tag is an HA tag the Cas9-Geminin chimera has the amino acid sequence of SEQ ID NO: 12.
  • a method for increasing gene disruption, e.g. deletions and/or additions, of an exogenous nuclease in a cell by restricting the activity of an RNA-guided nuclease by generating a nuclease-degron fusion protein that has a cell-cycle dependent degradation profile wherein the fusion protein is substantially degraded at the Gl phase and is expressed at the S-G2-M phases.
  • the method comprises introducing one or more nuclease-degron fusion protein (and/or mRNAs or expression constructs that express the nuclease (s) and one or more single guide RNA if needed) along with one or more DNA donor molecules into a host cell.
  • a chimeric protein of the subject invention i.e. an RNA-guided DNA nuclease fused to a cell-cycle dependent degron, and its guide-RNA may be directly introduced into a host cell.
  • an RNA guided nuclease and its guide RNA may be encoded on a vector capable of expressing the RNA guided nuclease and its guide RNA in a host cell.
  • Various vector systems and functional promoters operable in host cells are known in the art.
  • the chimeric protein of the subject invention and its guide-RNA may be encoded on the same or separate vectors.
  • the chimeric protein of the subject invention and its guide-RNA, or a vector encoding them may be introduced to a host cell at the same time or at different times relative to each other.
  • a donor molecule may also be introduced at the same time or at a different time from the chimeric protein of the subject invention and its guide- RNA.
  • Introduction of a protein or nucleic acid into a cell can be accomplished by a variety of methods including, but not limited to, transformation and transfection techniques, viral transduction, electroporation, chemically-induced uptake of exogenous nucleic acids, hydrodynamic delivery, lipofection, sonoporation and other methods known to a person of ordinary skill in the field of molecular biology. Any of such method may be used to introduce the chimeric protein of the subject invention, its guide-RNA or a donor molecule. Different methods may be used for each of the chimeric protein of the subject invention, its guide- RNA and a donor molecule.
  • Suitable cells include but not limited to eukaryotic and prokaryotic cells and/or cell lines.
  • Non-limiting examples of such cells or cell lines generated from such cells include COS, CHO (e.g., CH0--S, CH0-K1, CHO-DG44, CHO-DUXBl 1, CHO-DUKX, CHO 1SV) , VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2/0-Agl4, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T) , and perC6 cells, any plant cell (differentiated or undifferentiated) as well as insect cells such as Spodopterafugiperda (Sf) , or fungal cells such as Saccharomyces, Pichia and Schizosaccharomyces .
  • COS COS
  • CHO e.g., CH0--S,
  • the cell line is a CHO-K1, MDCK or HEK293 cell line.
  • primary cells may be isolated and used ex vivo for reintroduction into the subject to be treated following treatment with the RNA-guided DNA nuclease system (e.g. CRISPR/Cas) .
  • Suitable primary cells include peripheral blood mononuclear cells (PBMC) , and other blood cell subsets such as, but not limited to, CD4+ T cells or CD8+ T cells.
  • Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+) , neuronal stem cells and mesenchymal stem cells.
  • Eukaryotic cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells (e.g., T-cells) .
  • Plant cells include, but are not limited to, cells of monocotyledonous (monocots) or dicotyledonous (dicots) plants.
  • monocots include cereal plants such as maize, rice, barley, oats, wheat, sorghum, rye, sugarcane, pineapple, onion, banana, and coconut.
  • dicots include tobacco, tomato, sunflower, cotton, sugarbeet, potato, lettuce, melon, soybean, canola (rapeseed) , and alfalfa.
  • Plant cells may be from any part of the plant and/or from any stage of plant development.
  • the host cells are an established cell line while in other aspects, the host cell is a primary cell isolated from a mammal. In some aspects, the cell is a plant cell where the cell can be from a germplasm or a differentiated cell.
  • nucleic acids encoding RNA-guided DNA nucleases e.g., Cas9 or Cpfl, and/or donors are administered for in vivo or ex vivo gene therapy uses.
  • Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
  • cells are isolated from the subject organism, transfected with an RNA-guided DNA nuclease or a nucleic acid (gene or cDNA) encoding an RNA-guided DNA nuclease, and re- infused back into the subject organism (e.g., patient).
  • RNA-guided DNA nuclease or a nucleic acid (gene or cDNA) encoding an RNA-guided DNA nuclease
  • Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients) .
  • Transgenic animals can include those developed for disease models, as well as animals with desirable traits.
  • Embryos may be treated using the methods and compositions of the invention to develop transgenic animals.
  • suitable embryos may include embryos from small mammals (e.g., rodents, rabbits, etc.), companion animals, livestock, and primates.
  • rodents may include mice, rats, hamsters, gerbils, and guinea pigs.
  • companion animals may include cats, dogs, rabbits, hedgehogs, and ferrets.
  • Non-limiting examples of livestock may include horses, goats, sheep, swine, llamas, alpacas, and cattle.
  • Non-limiting examples of primates may include capuchin monkeys, chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys, squirrel monkeys, and vervet monkeys.
  • suitable embryos may include embryos from fish, reptiles, amphibians, or birds.
  • suitable embryos may be insect embryos, for instance, a Drosophila embryo or a mosquito embryo.
  • Transgenic organisms contemplated by the methods and compositions of this invention also include transgenic plants and seeds.
  • suitable transgenes for introduction include exogenous nucleic acid sequence that may comprise a sequence encoding one or more functional polypeptides (e.g., a cDNA) , with or without one or more promoters and/or may produce one or more RNA sequences (e.g., via one or more shRNA expression cassettes), which impart desirable traits to the organism.
  • Such traits in plants include, but are not limited to, herbicide resistance or tolerance; insect resistance or tolerance; disease resistance or tolerance (viral, bacterial, fungal, nematode) ; stress tolerance and/or resistance, as exemplified by resistance or tolerance to drought, heat, chilling, freezing, excessive moisture, salt stress; oxidative stress; increased yields; food content and makeup; physical appearance; male sterility; drydown; standability; prolificacy; starch quantity and quality; oil quantity and quality; protein quality and quantity; amino acid composition; and the like.
  • exogenous nucleic acid sequence comprises a sequence encoding a herbicide resistance protein (e.g., the AAD
  • CRISPR regularly interspaced short palindromic repeat
  • CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, which target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA) .
  • crRNA CRISPR RNA
  • processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA) , endogenous ribonuclease 3 (rnc) and a Cas9 protein.
  • tracrRNA trans-encoded small RNA
  • rnc endogenous ribonuclease 3
  • Cas9 protein serves as a guide for ribonuclease 3-aided processing of pre-crRNA.
  • Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer.
  • the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5' exonucleolytically .
  • DNA-binding and cleavage typically requires protein and both RNAs .
  • single guide RNAs sgRNA, or simply "gNRA”
  • Cas9 recognizes a short motif known as the PAM or protospacer adjacent motif in order to distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (Jinek et al .
  • Cas9 orthologs have been described in various species, including, but not limited to, Streptococcus pyogenes (Taxonomy ID: 1314, refseq ID: WP_010922251.1 , Streptococcus thermophilus (Taxonomy ID: 1308, refseq ID: WP_011681470.1, Streptococcus pasteurianus (Taxonomy ID: 197614, refseq ID: WP_003065552.1 ) , Staphylococcus aureus (Taxonomy ID: 1280, refseq ID: CCK74173.1), Neisseria cinerea (Taxonomy ID: 483, WP_003676410.1 ), Campylobacter lari (Taxonomy ID: 201, refseq ID:WP_039627869.1) , Corynebacterium diph
  • any CRISPR RNA-guided DNA nuclease e.g., Cas9, Cpfl, etc. can be utilized as the nuclease portion of the fusion protein of the present invention.
  • CRISPR RNA-guided DNA nucleases are the PAM recognition sequence.
  • S. pyogenes Cas9 typically recognizes 5'-NGG-3'
  • Cpfl nuclease of F. novicida recognizes 5'-TTN-3' (Zetsche et al., 2015) or 5'-YTN-3' (Fonfara et al., 2016).
  • a thorough description of the PAM-mediated Cas binding can be found in Sternberg et al., 2014.
  • the choice of which Cas9 or Cpfl ortholog is used in the chimeric protein of the subject invention may depend at least partly on the PAM sequence the nuclease binds and its distance from the desired target site.
  • RNA-guided DNA nucleases have been engineered to recognize PAMs other than their native, canonical PAM (Kleinstiver et al., 2015). For instance, although F. novicida Cas9 naturally recognizes the canonical PAM sequence 5'-NGG-3', it can be modified to recognize the PAM 5'-YG-3', thus creating additional Cas9 targets (Hirano et al. 2016). Engineered RNA- guided DNA nucleases which recognize alternate PAM sequences may be used as the nuclease portion of the chimeric protein of the subject invention. Each of the references above are incorporated by reference into this application.
  • Cas9 protein may be a "functional derivative” of a naturally occurring Cas9 protein.
  • a “functional derivative” of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide.
  • “Functional derivatives” include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide.
  • a biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments.
  • the term “derivative” encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof.
  • Suitable derivatives of a Cas9 polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas9 protein or a fragment thereof.
  • Cas9 protein which includes Cas9 protein or a fragment thereof, as well as derivatives of Cas9 protein or a fragment thereof, may be obtainable from a cell or synthesized chemically or by a combination of these two procedures.
  • the cell may be a cell that naturally produces Cas9 protein, or a cell that naturally produces Cas9 protein and is genetically engineered to produce the endogenous Cas9 protein at a higher expression level or to produce a Cas9 protein from an exogenously introduced nucleic acid, which nucleic acid encodes a Cas9 that is same or different from the endogenous Cas9.
  • the cell does not naturally produce Cas9 protein and is genetically engineered to produce a Cas9 protein.
  • the above description applies to any CRIPSR RNA- guided DNA nuclease e.g., Cpfl.
  • DNA breaks refer to both single strand breaks (SSB) and double strand breaks (DSB) .
  • SSB are breaks that occur in one of the DNA strands of the double helix.
  • DSB are breaks in which both DNA strands of the double helix are severed.
  • Donor DNA refers to a DNA sequence that has homologous overlaps to a DNA region containing a DNA break. Typically, an overlap between 30-3000 base pairs is used. In some embodiments, use of donor DNA allows for cells to utilize HDR instead of NHEJ. Thus, "Donor DNA” or “DNA donor” typically has homology to a target sequence in order to be used as a repair template during HDR.
  • insertion of an exogenous donor DNA may be used to accomplish correction of a mutant gene or for increased expression of a wild-type gene.
  • the donor sequence is typically not identical to the genomic sequence where it is placed.
  • a donor sequence can contain a non- homologous sequence flanked by two regions of homology to allow for efficient HDR at the location of interest.
  • donor sequences can comprise a vector molecule containing sequences that are not homologous to the region of interest in cellular chromatin.
  • a donor molecule can contain several, discontinuous regions of homology to cellular chromatin.
  • sequences not normally present in a region of interest can be present in a donor nucleic acid molecule and flanked by regions of homology to the sequence in the region of interest.
  • an endogenous template which is naturally present in the cell, may be used as the template for HDR.
  • a sister chromatid may be used as a template for recombination after a break is created by the nuclease.
  • a related element upstream or downstream of the break may also be used as template.
  • a ⁇ -globin sequence can be repaired by using a nearby ⁇ -globin sequence as a template.
  • a donor sequence may be an oligonucleotide and be used for gene correction or targeted alteration of an endogenous sequence.
  • the oligonucleotide may be introduced to the cell on a vector, may be electroporated into the cell, or may be introduced via other methods known in the art.
  • the oligonucleotide can be used to 'correct' a mutated sequence in an endogenous gene (e.g., the sickle mutation in beta globin) , or may be used to insert sequences with a desired purpose into an endogenous locus.
  • the donor polynucleotide can be DNA or RNA, single-stranded and/or doublestranded and can be introduced into a cell in linear or circular form. See, e.g., U.S. Patent Publication Nos. 20100047805; 20110281361; and 20110207221. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3' terminus of a linear molecule and/or selfcomplementary oligonucleotides are ligated to one or both ends.
  • Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group (s) and the use of modified intemucleotide linkages such as, for example, phosphorothioates , phosphoramidates, and 0- methyl ribose or deoxyribose residues.
  • the donor may be chemically modified, including but not limited to containing locked-nucleic acids (LNA) or peptide-nucleic acids (PNA) .
  • LNA locked-nucleic acids
  • PNA peptide-nucleic acids
  • An aspect of the invention described herein is a method for increasing HDR following nuclease-mediated cleavage in a cell.
  • the methods of the invention encompass increasing homology- directed recombination in any of its forms or mechanisms.
  • the terms "integrated,” “inserted,” and “copied” are used interchangeably in this application to refer to such recombination.
  • a "Degron” refers to a specific sequence of amino acids in a protein that directs the starting place of degradation.
  • a degron sequence can occur at either the N or C- terminal region, and these are called N-degrons or C-degrons, respectively.
  • a degron may be any amino acid sequence, or combination of sequences, that confers degradation to a protein that it is fused to.
  • a "cell-cycle dependent degron” refers to a particular degron, which leads to degradation of a protein to which it is fused at specific phases of the cell-cycle.
  • the ordered progression through the cell-cycle depends on regulating the abundance of several proteins through ubiquitin-mediated proteolysis .
  • Degradation is precisely timed and specific.
  • One example of such degradation system is the anaphase promoting complex (APC) , a ubiquitin protein ligase.
  • APC is activated both during mitosis and late in mitosis/Gl, by the WD repeat proteins Cdc20 and Cdhl, respectively. These activators target distinct sets of substrates.
  • APC/C- coactivator complexes recognize most of their substrates through recognition of two short motifs: the D (destruction) box degron (RxxLxx (1/V) xN) (D box) and the KEN box degron (a three amino acid motif of K-E-N) .
  • Cdc20-APC requires a D box domain, whereas Cdhl-APC relies on the KEN box degron.
  • Examples of naturally occurring proteins that comprise a D box degron are: cell-cycle dependent cyclins (CDKs) such as Cyclin A and Cyclin B and other cell-cycle regulated proteins such as: Hsll, Cdc6, Finl, p21 and Geminin .
  • proteins that comprise a KEN box degron are: Cdc20, Sgol, Nek2 and B99.
  • Other proteins which contain either a D- box or a KEN box are well-known in the art (Glotzer et al . , 1991, Vietnameser and Kirschner, 2000).
  • any amino acid motif which occurs in a protein and is recognizable by a cell-cycle regulated degradation machinery will constitute a cell-cycle dependent degron.
  • a degron of a protein of any organism may be fused to a RNA-guided DNA nuclease in any cell type to confer degradation to the nuclease
  • the use of a degron of a protein endogenous to a host cell, or a degron of a protein of the most closely related species to the host cell is also contemplated.
  • progression through the cell cycle in plants is regulated by CDKs (De Veylder et al., 2003; Inze, 2005) .
  • a cell-cycle specific degron of a protein endogenous to plants e.g. a plant CDK
  • Degrons are transposable to other proteins.
  • the introduction of a cell-cycle dependent degron to a protein confers a cell-cycle dependent degradation profile to the chimeric protein.
  • the degron may be added to the target protein on its C-terminal or N- terminal or on both termini.
  • the degron may be inserted as a single copy or as multiple copies. Potentially a chimeric protein may comprise more than one type of degron.
  • Target DNA sequence refers to a genomic or mitochondrial DNA site where Cas9 binds.
  • single guide RNAs can be engineered to bind to a target of choice in a genome by commonly known methods known in the art for creating specific RNA sequences. These single guide RNAs are designed to guide the Cas9 to any chosen target site.
  • gRNA binding domain refers to a domain on gRNA configured to target Cas9 to a specific DNA sequence where it unwinds and cleaves the double stranded DNA.
  • “Targeted insertion” as used herein refers to the result of a successful homologous recombination event wherein a desired portion of a donor DNA was inserted or copied into a desired position in the genome of a cell.
  • the use of the nuclease-degron of the present invention for genome editing results in an increase of the rate of targeted insertions. This increase can be calculated by quantifying the percentage of cells in a cell population where a targeted insertion event has occurred as a result of nuclease mediated genome editing.
  • Various assays have been described that enable the determination of targeted insertion rates using the genome editing systems described herein. These assays and other assays that are known in the art may be used to quantify the increase in HDR rate and the corresponding targeted insertion rate as mediated by the nuclease-degron of the present invention.
  • the increase in the rate of targeted DNA "insertion" or "integration" by a fusion protein of the subject invention comprising an RNA-guided DNA nuclease and a cell-cycle dependent degron is defined with relation to a control nuclease without a functional degron under otherwise same conditions.
  • the control RNA-guided DNA nuclease used for relative comparison within the same system may be either the native RNA-guided DNA nuclease alone, i.e. lacking any cell-cycle dependent degron, or the RNA- guided DNA-nuclease fused to a non-functional form of the cell- cycle dependent degron being tested.
  • the increase in the rate of targeted DNA insertion may be represented in any number of ways known to those of skill in the art including, but not limited to, fold-increase or percent increase relative to the controls described above.
  • a RNA-guided nuclease fused to a cell-cycle dependent degron is expected to have a higher rate of insertion via HDR in a given population of cells compared to the same RNA-guided nuclease not fused to the cell-cycle dependent degron in the same population of cells over the same period of time.
  • a population of cells carrying a RNA-guided nuclease fused to a cell-cycle dependent degron is expected to have more targeted insertion events occur relative to the same population of cells carrying the same RNA-guided nuclease not fused to the cell-cycle dependent degron over the same period of time.
  • the chimeric protein of the subject invention increases the efficiency of targeted insertion via HDR relative to an RNA-guided nuclease alone.
  • off-target excision of the genome refers to the percentage of cells in a cell population where the DNA of a cell was excised by a nuclease at an undesired locus during genome editing.
  • the detection and quantification of off-target insertion events can be done by known methods.
  • off-target effect refers to the level of off-target excision, or cleavage, of the genome as detected by suitable assays.
  • suitable assays include genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq) (Tsai et al .
  • a chimeric protein of the present invention i.e., an RNA-guided DNA nuclease fused to a cell-cycle dependent degron
  • a chimeric protein of the present invention may drastically decrease the level of off-target effects relative to the RNA-guided DNA nuclease not tagged to the cell-cycle dependent degron, or tagged to a non-functional form of the cell-cycle dependent degron, as detected by any one of the above-identified assays or others known to a person having ordinary skill in the art.
  • off- target effects may be reduced by ten-fold, one hundred-fold, one thousand-fold, one million fold, or to zero or nearly undetectable levels .
  • Cleavage refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage .
  • sequence in the context of nucleotides refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded.
  • donor sequence refers to a nucleotide sequence that is inserted or copied into a genome.
  • a donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value there between or there above), between about 100 and 1,000 nucleotides in length (or any integer there between) , between about 200 and 500 nucleotides in length.
  • a “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist .
  • a “fusion” molecule is a molecule in which two or more subunit molecules are linked, e.g., but not limited to, covalently.
  • the subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules. Examples of the first type of fusion molecule include, but are not limited to, fusion proteins, also known as chimeric proteins.
  • Fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, wherein the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein.
  • Trans-splicing, polypeptide cleavage and polypeptide ligation can also be involved in expression of a protein in a cell.
  • the disclosed compositions and methods can be used for any application in which it is desired to increase nuclease-mediated genomic modification in any cell type, including clinical applications nuclease-based therapies feasible in a clinical setting as well as agricultural i.e. plant applications.
  • the methods and compositions described herein can be used to generate model organisms and cell lines, including the generation of stable knock-out cells in any given organism.
  • the methods and compositions of the invention can also be used in the production of transgenic organisms.
  • Transgenic animals can include those developed for disease models, as well as animals with desirable traits.
  • Example - A Cas9-cell cycle degron fusion protein system for enhanced CRISPR-Cas directed recombination at a desired location in the genome of human cells .
  • An assay system for determining the recombination rates of donor DNA to a desired location in the genome was generated. The system comprises :
  • piGFP lenti-viral expression vector harbors a GFP gene which comprises two stop codon mutations at locations 96-99 and 102-105 of the GFP coding sequence (SEQ ID NO: 02) . These stop codons lead to premature termination during translation and result in a truncated GFP protein comprising the first 31 amino acids of the GFP protein. This fragment alone lacks any fluorescence property detectable by standard detection methods (e.g light microscopy, flow- cytometry or microplate reading) .
  • the vector also includes a Puromycin resistance gene that enables viability-based selection towards cells in which the vector has been integrated into the genome.
  • the pHAGE-iGFP vector contains the genetic elements required for packaging, transduction, and stable integration of the viral expression construct into genomic DNA as well as Puromycin resistance gene .
  • pLeti6V5-iGFPl is a lenti-based mammalian expression vector with a Blasticidin resistance gene. Like piGFP, it also carries the inactive GFP gene. 3. piGFP or pLeti6V5-iGFPl were transduced into human embryonic kidney 293T cells to produce a stably expressing cell-line as follows:
  • piGFP/pLeti6V5-iGFPl were delivered into 293T producer cells by simultaneous transfection with a mix of three (3) packaging plasmids (pPACKHlT , System Biosciences), using the transfection reagent Lipofectamine®3000 and following the protocol provided by the manufacturer.
  • the resulting pseudo viral particles were collected after 48hr and used to infect a fresh batch of human embryonic kidney 293T cells by applying 50 ⁇ 1 of viral particles containing medium directly on the cells.
  • the medium was replaced with Puromycin/Blaticidin containing medium at a final concentration of ⁇ / ⁇ .
  • Puromycin/Blasticidin resistant cells were collected four (4) days later. The collected cells harbored the inactive GFP construct in their genome and are referred to in this application as 293T-iGFP.
  • D53-CAS9 (SEQ ID NO: 03) is a Cas9 expression vector. It is prepared by insertion of a DNA fragment containing the humanized S. pyogenes CAS9 fused to a nuclear localization signal (hCas9)
  • the pcDNATM-DEST53 was linearized by PCR using the primers P03 (SEQ ID NO: 08) and P04 (SEQ ID NO: 09).
  • the hCAS9 PCR fragment was then cloned into the linearized pcDNATM-DEST53 with the In-Fuision® Cloning kit (Clontech) according to the manufacturer's instructions.
  • hCas9 was inserted between positions 2660 and 3378 of pcDNATM-DEST53 and the cloning resulted in the deletion of 718bp of the original vector.
  • the resulting PCDNA D53-hCAS9 (SEQ ID NO: 03) vector was then used to construct the expression vector PCDNA.
  • D53-hCAS9-GMNN-cHA as described below.
  • D53-hCAS9-GMN expression vector (SEQ ID NO: 10) was prepared by gateway cloning of the N-terminal fragment (amino acids 1-110) of the human Geminin protein (SEQ ID NO: 11) at the C-terminal of S. pyogenes hCas9 (SEQ ID NO: 04) on PCDNA.
  • D53-hCAS9 resulting in a CAS9-Gmnn fusion protein of 1514 amino acids (SEQ ID NO:12).
  • the construction of this vector was done in two stages. First, an Entry vector carrying the N-terminal fragment of GMNN with HA tag fused to its C-terminal end was made. This was done by a gateway BP cloning reaction in which a chemically synthesized DNA fragment containing the GMNN-HA fragment and attachment sites
  • GMNN-HA was transferred from the pENTR-GMNN-HA Vector into PCDNA.
  • D53-hCAS9-GWA using a LR reaction, giving rise to PCDNA.
  • D53-hCAS9-GMNN as a final product .
  • pGFP-GUIDl is an expression vector carrying a U6 promoter followed by DNA coding for a guide RNA that targets the inactive GFP gene at a location between the two premature stop codons and a trans-activating CRISPR RNA scaffold (SEQ ID NO: 15) .
  • DD-GFP-C-88ss (SEQ ID NO: 16) is a synthetic single stranded DNA of 88 nucleotides with partial homology to the inactive GFP site on piGFP. DD-GFP-ss also contains the correct GFP sequence segment that is missing from the inactive GFP. It was designed to be used as donor DNA in a genome editing reaction to restore the iGFP gene to its original GFP form.
  • DD-GFP-C-80ss (SEQ ID NO: 17) is similar to DD-GFP-C-88ss but contains only 80 nucleotides pCDNA3.1-GMN1-I10-Cas9-HA is a mammalian expression vector. It expresses a Cas9 protein with GMNN degron (amino acids 1- 110) fused to its N-terminal end (SEQ ID NO: 19) . This fusion protein, which also contain HA tag at its C terminal end, was constructed by cloning of a synthetic DNA fragment coding for the Cas9-GMNN (1-110) degron fragment into the mammalian expression vector pCDNA3.1+/C-HA between BamHI and Xhol. 0.
  • pCDNA3.1 HA-Cas9-O4NNl-110 is similar to pCDNA3.1-
  • G Nl-110-Cas9-HA expresses a Cas9 protein with GMNN degron (amino acids 1-110) fused to its C-terminal end (SEQ ID NO: 20).
  • This fusion protein which also contains HA tag at its N-terminal end, was constructed by cloning of the a synthetic DNA fragment coding for the GMNN ( 1-110 ) -Cas9 degron fragment into the mammalian expression vector pCDNA3.1+/N-HA between BamHI and Xhol .
  • pCDNA3.1 HA-Cas9-(3iNN_null is similar to pCDNA3.1 HA-
  • Cas9-GMNNl-110 but expresses a mutated version of the GMNN degron (SEQ ID NO: 21).
  • the mutations introduced to the GMNN ORF resulted in substitutions to alanine and disruption of the KEN and D-box elements along the GMNN degron (KEN ⁇ i3- 'i5)AAA, KE (91-93)AAA, R23A, L26A) .
  • pCDNA3.1-HA-Cas9-C -Securin (1-79) is similar to pCDNA3.1 HA-Cas9-GMNNl-110 but instead of a Cas9-GMNN degron fusion, it expresses a Cas9 fused to a fragment of the Securin protein (NP_001269311.1) (SEQ ID NO: 22).
  • the fused Securin fragment contains the first 79 amino acids and includes a KEN box at position 9-11 and a D-Box at position 61-64 of the original Securin protein seguence.
  • pCDNA3.1-HA-Cas9-C'-CDC20 (1-113) is similar to pCDNA3.1
  • HA-Cas9-GMNNl-110 but instead of Cas9-GMNN degron fusion, it expresses a Cas9 fused to a fragment of the CDC20 protein (NP_001246.2) (SEQ ID NO: 23).
  • the fused CDC20 fragment contains the first 113 amino acids and includes a KEN box at position 97-99. 14.
  • pCDNA3.1 HA-Cas9 is similar to pCDNA3.1-GMN1-I10-Cas9- HA but instead of Cas9-degron fusion protein, it expresses Cas9 alone (SEQ ID NO: 24).
  • pCDNA3.1 HA-Cas9-GMNN compact (1-124; ⁇ 34-80) is similar to pCDNA3.1 HA-Cas9-GMNNl-110 but expresses an edited version of the GMNN degron (SEQ ID NO: 25) .
  • This version carries the first 124 amino acids of the GMNN protein with a deletion of 46 amino acids between position 34 and 80.
  • the sequence contains all of the intact KEN and D-box elements along the GMNN degron.
  • pCDNA3.1 HA-Cas9-GM N long (1-130) is similar to pCDNA3.1 HA-Cas9-GMNNl-110 but carries an extra 21 amino acids of the GMNN sequence containing an extra KEN box element (SEQ ID NO: 26) .
  • An expression vector carrying one of the relevant expression cassettes (i.e. cas9 with or without degron fusion, SEQ ID Nos: 3 , 10, or 19-24), were introduced into 293T-iGFP cells along with the donor DNA oligo DD-GFP-C and the guide RNA expression vector pGFP-GUIDl.
  • Transfected cells were harvested from individual wells of 6 wells plate at 24, 48 or 72hr post transfection. Cell suspensions of each sample were then transferred to a FACS compatible tube for measurement of GFP florescent intensity. Flow cytometry was performed on a BD-LSRII (Becton Dickinson) and analysis was done using FlowJo FACS analysis software.
  • Jinek et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, 2012, Science, Vol. 337, 816-821.
  • the KEN box an APC recognition signal distinct from the D box targeted by Cdhl, 2000, Genes Dev., 14 (6) : 655-665.
  • GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases, 2015, Nat. Biotechnol., Vol. 33, 187-197.

Abstract

La présente invention concerne une composition et un procédé permettant d'augmenter le taux d'insertion d'ADN donneur dans le génome par recombinaison à l'aide du système Cas9. Plus spécifiquement, le procédé utilise une chimère Cas9-degron, qui a un profil d'expression restreint aux phases S, G2 et M du cycle cellulaire, limitant ainsi l'activité du système à ces étapes du cycle cellulaire. La limitation de l'activité de Cas9 aux phases S, G2 et M entraîne une augmentation significative des taux de réparation par recombinaison homologue des cassures de l'ADN induites par Cas9 dans une séquence génomique, ce qui résulte en une plus grande efficacité d'édition du génome.
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