WO2018081978A1 - 提高基因编辑效率的方法和系统 - Google Patents

提高基因编辑效率的方法和系统 Download PDF

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WO2018081978A1
WO2018081978A1 PCT/CN2016/104450 CN2016104450W WO2018081978A1 WO 2018081978 A1 WO2018081978 A1 WO 2018081978A1 CN 2016104450 W CN2016104450 W CN 2016104450W WO 2018081978 A1 WO2018081978 A1 WO 2018081978A1
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protein
pest
sequence
cas9
gene editing
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French (fr)
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王健
胡莹莹
罗舟
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Priority to CN201680088506.XA priority patent/CN109689693B/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention is in the field of biotechnology, and in particular, the present invention relates to methods and systems for improving the efficiency of gene editing.
  • the CRISPR/Cas9 or CRISPR-Cas9 system is a tool for gene editing that has emerged in recent years.
  • the system consists of an sgRNA capable of specifically recognizing a DNA sequence and a Cas9 nuclease capable of cleaving a DNA sequence. Modifications such as site-directed mutagenesis, fragment deletion or inversion can be achieved by Cas9 cleavage of specific sequences on the genome. Site insertion or substitution, including precise genetic modification mediated by homologous recombination, can also be achieved under conditions of addition of exogenous donor DNA.
  • engineered Cas9 can also regulate RNA, transcription levels, and epigenetics.
  • fusion protein having the structure of Formula Ia or Ib:
  • E is an endonuclease protein element
  • P is a PEST protein element
  • the endonuclease protein element is selected from the group consisting of Cas9 protein (including SpCas9, SaCas9, NmCas9, St1Cas9) and variants thereof (eg, VQR, EQR, VRER, etc. of SpCas9) , Cpf1 protein (including AsCpf1, FnCpf1, LbCpf1), C2c2 protein, Argonaute protein family; TALE protein, zinc finger protein, dCas9 and other artificial endonuclease fusion with FokI.
  • Cas9 protein including SpCas9, SaCas9, NmCas9, St1Cas9
  • variants thereof eg, VQR, EQR, VRER, etc. of SpCas9
  • Cpf1 protein including AsCpf1, FnCpf1, LbCpf1
  • C2c2 protein Argonaute protein family
  • TALE protein zinc finger protein
  • the endonuclease protein element is a Cas9 protein.
  • amino acid sequence of the SpCas9 protein is set forth in SEQ ID NO.
  • the PEST protein element is selected from the group consisting of:
  • PEST sequence of ODC1 protein PEST sequence of GCN4 protein, PEST sequence of CLN2/CLN3 protein, PEST sequence of NIMA protein, PEST sequence of Cactus protein, PEST sequence of HDC protein, PEST sequence of CPEB protein, PEST sequence of NPDC1 protein, The PEST sequence of the FOS protein, the PEST sequence of the NFKBIA protein, and the like.
  • amino acid sequence of the PEST protein element is set forth in SEQ ID NO.
  • amino acid sequence of the fusion protein is selected from the group consisting of:
  • (C) a derivative polypeptide which is formed by substitution, deletion or addition of the amino acid sequence of SEQ ID NO. 3 by 1-10 amino acid residues, and which retains the gene editing activity.
  • the peptide bond or peptide linker in the fusion protein is 0-30 amino acids in length.
  • the fusion protein further comprises an optional nuclear localization signal element (NLS); preferably the nuclear localization signal element is added to the N-terminus or C-terminus of the fusion protein, or simultaneously At the N and C ends.
  • NLS nuclear localization signal element
  • an isolated polynucleotide encoding the fusion protein of the first aspect of the invention.
  • the polynucleotide comprises a codon optimized sequence suitable for expression by a prokaryotic or eukaryotic cell.
  • the polynucleotide is DNA or RNA.
  • the polynucleotide is an mRNA.
  • a vector comprising the polynucleotide of the second aspect of the invention is provided.
  • the vector comprises a plasmid, a viral vector or the like.
  • the viral vector comprises: a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, a retroviral vector, a yellow fever virus vector, and a herpesvirus vector.
  • the vector comprises a prokaryotic and eukaryotic expression vector.
  • a genetically engineered cell comprising the vector of the third aspect of the invention, or the polynucleotide of the second aspect of the invention, or the invention A fusion protein as described on the one hand.
  • the genetically engineered cells include prokaryotic cells and eukaryotic cells.
  • the genetically engineered cell is an animal cell, a plant cell, or a microbial cell.
  • the cell is a germ cell or a fertilized egg.
  • a gene editing system comprising the fusion protein of the first aspect of the invention, or the polynucleotide of the second aspect of the invention, or the invention Three aspects of the carrier.
  • the gene editing system is selected from the group consisting of: CRISPR/Cas gene editing system, CRISPR/Cpf1 gene editing system, CRISPR/C2c2 gene editing system, Argonaute/gDNA gene editing system, zinc finger nuclease gene Editing system (ZFNs), and transcriptional activator nuclease gene Editing system (TALENs).
  • an endonuclease-mediated gene editing method comprising the steps of:
  • the target gene is edited using the fusion protein of the first aspect of the invention.
  • a seventh aspect of the invention a method for increasing the efficiency of gene editing of an endonuclease-mediated gene editing system, the method comprising the steps of:
  • a fusion protein that expresses a PEST protein and an endonuclease in a target cell.
  • the coding sequence of the PEST protein is fused to the 5' or 3' end of the endonuclease gene.
  • the method comprises the steps of:
  • the fusion protein of the first aspect of the invention is expressed in a target cell.
  • Figure 1 shows the manner in which an endonuclease protein element is fused to a PEST protein element.
  • the PEST element can be located at the C-terminus or N-terminus of the endonuclease element, wherein the short line represents a peptide bond or a peptide linker between the two, and is 0-30 amino acid residues.
  • Figure 2a and Figure 2b show the amino acid sequence (SEQ NO: 1) and the coding polynucleotide sequence (SEQ ID NO. 4), respectively, of the exemplary SpCas9 protein.
  • Figure 3 shows the amino acid sequence (SEQ ID NO. 2) and the coding polynucleotide sequence (SEQ ID NO. 5) of the PEST domain of the exemplary mouse ODC1 protein.
  • Figure 4a and Figure 4b respectively show an example of the amino acid sequence (SEQ ID NO. 3) and the coding polynucleotide sequence (SEQ ID NO. 6) of the fusion protein obtained by fusing the PEST domain of the mouse ODC1 protein to the C-terminus of the SpCas9 protein. .
  • Figure 5 shows an exemplary Cas9-PEST plasmid map.
  • a complete plasmid map of the PEST coding sequence was added between the Cas9 coding sequence and the nuclear localization signal (NLS) sequence.
  • the box marks the location of the PEST code sequence.
  • Figure 6 shows the structure of the zebrafish gata1a gene and the CRISPR/Cas9 target site information.
  • the gene has 6 exons (boxed, the solid part represents the coding region and the hollow portion represents the non-coding region), and the CRISPR/Cas9 target site selected in the embodiment of the present invention is located on the 5th exon. (The sequence is gray in gray), and the SpeI cleavage site can be used to detect the efficiency of the target site.
  • Figure 7 shows the results of electrophoresis detection of a sgRNA template for preparation of a gata1a target site by PCR, the template size being 120 bp.
  • Figure 8 shows that Cas9-PEST enhances the site-directed mutagenesis efficiency of the zebrafish gata1a gene target site.
  • the known target sites on the zebrafish gata1a gene were selected, the same doses of Cas9 and Cas9-PEST were injected, and the efficiency of site-directed mutagenesis was examined using restriction endonuclease (the uppermost band was a mutated band, indicated by a black triangle).
  • the ratio of the brightness of the strip to the total brightness of the three strips is the mutation efficiency.
  • ImageJ software can To quantify the brightness of the strips, the average efficiencies of the two samples were calculated to be 22.5% and 35.2%, respectively (the control group was uninjected), indicating that PEST helps to improve the efficiency of Cas9.
  • Figure 9 shows the structure of the zebrafish mstnb gene and the CRISPR/Cas9 target site information.
  • the gene has three exons (boxed, the solid portion represents the coding region and the hollow portion represents the non-coding region), and the CRISPR/Cas9 target site selected in the embodiment of the present invention is located on the first exon. (The sequence is gray in gray), and the BslI restriction site can be used to detect the efficiency of the target site.
  • Figure 10 shows that Cas9-PEST can increase the site-directed mutagenesis efficiency of the zebrafish mstnb gene target site from 13.4% to 56.1%.
  • the present inventors have found through extensive and intensive research that the fusion protein formed by the fusion of the PEST short peptide and the Cas9 protein can significantly improve the efficiency of gene editing, and on the basis of this, the present invention has been completed.
  • the problem to be solved by the present invention is to improve the editing efficiency of the CRISPR system in the early stage of fertilized egg splitting, thereby increasing the editing efficiency in adult animals.
  • the method By increasing the rate of translation of Cas9 mRNA, the method enables Cas9 protein to function early in the early development of fertilized eggs and to more efficiently edit targeted genes.
  • the rate of translation of the Cas9 mRNA into a protein can be effectively improved, thereby improving the efficiency of gene editing.
  • the PEST sequence in the ODC1 protein is selectively fused to Cas9, and other sequences or domains which can effectively improve the translation efficiency of Cas9 mRNA, such as PEST sequences of other proteins, may be enhanced in chickens after fusion with Cas9. /Cas9-mediated gene editing efficiency.
  • Cas9 used as an example of the present invention is SpCas9 optimized by zebrafish codon.
  • the invention may also be used to engineer any endonuclease for gene editing, such as SaCas9, NmCas9, St1Cas9, Cpf1, C2c2, Argonaute, TALEN, ZFN, and the like.
  • the PEST protein (PEST short peptide) is a special type of signal peptide rich in proline (P), glutamic acid (E), serine (S) and threonine (T), which can shorten the half-life of the protein.
  • PEST short peptides domains
  • experiments have shown that they are related to the rapid degradation of proteins, and the pathway of degradation may be Pass the proteasome or calpain.
  • the sequence, length, position and secondary structure of the PEST short peptide vary in different proteins, and some proteins contain multiple PEST domains.
  • the PEST protein element is selected from the group consisting of:
  • PEST sequence of ODC1 protein PEST sequence of GCN4 protein, PEST sequence of CLN2/CLN3 protein, PEST sequence of NIMA protein, PEST sequence of Cactus protein, PEST sequence of HDC protein, PEST sequence of CPEB protein, PEST sequence of NPDC1 protein, The PEST sequence of the FOS protein, the PEST sequence of the NFKBIA protein, etc. (for details, see Rogers et al. 1986, Rechsteiner and Rogers 1996, Fleming and Wang 2000, Reverte et al. 2001, Spencer et al. 2004).
  • amino acid sequence of the PEST protein is set forth in SEQ ID NO. 2; the nucleotide sequence encoding the cDNA (from cDNA of mouse Odc1) is set forth in SEQ ID NO.
  • the endonuclease protein element described in the present invention refers to an endonuclease which can be used for gene editing, a variant thereof, and an active fragment thereof.
  • Typical endonucleases include: Cas9 proteins (including SpCas9, SaCas9, NmCas9, St1Cas9) and variants thereof (such as variants of VQR, EQR, VRER, etc. of SpCas9) (Cong et al. 2013, Hou et al. 2013, Mali et al. 2013, Kleinstiver et al. 2015, Ran et al. 2015), Cpf1 proteins (including AsCpf1, FnCpf1, LbCpf1) (Zetsche et al. 2015, Kim et al. 2016, Kleinstiver et al. 2016), C2c2 Protein (Abudayyeh et al.
  • Cas9 proteins including SpCas9, SaCas9, NmCas9, St1Cas9
  • variants thereof such as variants of VQR, EQR, VRER, etc. of SpCas9
  • Cpf1 proteins including AsC
  • Argonaute protein family (Gao et al. 2016); TALE protein, zinc finger protein, dCas9 and other artificial endonucleases fused with FokI (Kim et al. 1996, Bibikova et al. 2002, Miller et al. 2011, Tsai et al. 2014).
  • the CRISPR/Cas system is an acquired immune system that is currently found in most bacteria and all archaea to eliminate foreign plastids or phage, and to leave foreign gene fragments in their genomes as "memory.”
  • the full name is the clustered regular intertemporal short palindromic repeats/CRISPR-associated proteins.
  • CRISPR/Cas systems Three different types have been discovered, present in approximately 40% and 90% of sequenced bacteria and archaea.
  • the composition of the second type is relatively simple.
  • the Cas9 protein and the guide RNA (gRNA) are the core components. Due to its DNA interference (DNAi) characteristics, it is currently actively used in genetic engineering as a gene editing tool.
  • the mechanism of non-homologous end joining (NHEJ) is used in the same manner as zinc finger nuclease (ZFN) and transcription-activator nuclease (TALEN) to generate double-strand breaks of DNA in the genome for editing.
  • Type II CRISPR/Cas has been applied to the genetic editing of mammalian cells and zebrafish through genetic engineering. Its simple design and easy handling are the biggest advantages. The future will be applicable to a variety of different model organisms.
  • CRISPR CRISPR
  • SRSR Short Regularly Spaced Repeats
  • CRISPR/Cas technology refers to the principle gene of CRISPR/Cas system. Transformed gene editing technology.
  • the core of CRISPR/Cas is the Cas9 protein and the guide RNA (gRNA).
  • the core technology for gene editing in different species using the CRISPR/Cas system is to heterologously express Cas9 protein with DNA cleavage activity in this species, and the second step is to obtain gRNA and target homologous sequences. Cas9 is directed to the target for DNA cleavage.
  • the specific method of operation is well known to those skilled in the art.
  • the Cas9 protein derived from Streptococcus pyogenes is a multidomain versatile Cas protein having a RuvC-like nuclease-like domain at its N-terminus and a HNH nuclease domain in its middle.
  • the Cas9 protein binds to gRNA to cleave DNA at a specific site.
  • the CRISPR/Cas system recognition sequence derived from Streptococcus pyogenes is 23 bp and can target 20 bp.
  • the last 3 NGG sequences of the recognition site are called PAM ( Protospacer adjacent motif) sequences, which are important for DNA cleavage.
  • the CRISPR/Cas system of most eukaryotic organisms (including silkworm, Arabidopsis, yeast, nematode, etc.) is originally derived from Streptococcus pyogenes, and the Cas9 protein is mostly humanized or codon optimized by other species.
  • the Cas9 provided by the present invention is derived from Streptococcus pyogenes.
  • the amino acid sequence of the Cas9 protein is as shown in SEQ ID NO.
  • the invention provides a fusion protein of PEST protein and Cas9 protein and a coding sequence thereof (including DNA, mRNA).
  • the fusion protein of the invention may optionally contain a linker peptide.
  • the size and complexity of the linker peptide may affect the activity of the protein.
  • the linker peptide should be of sufficient length and flexibility to ensure that the two proteins attached have sufficient freedom in space to perform their function. At the same time, the effect of the formation of an alpha helix or a beta sheet in the ligation peptide on the stability of the fusion protein is avoided.
  • the length of the linker peptide is generally from 0 to 30 amino acids, preferably from 1 to 5 amino acids.
  • the term also includes derivatives of the fusion proteins of the invention, which refers to polypeptides of the fusion protein of the invention that have been added or replaced with 1-3 amino acids, have 1-2 amino acid deletions, and still have gene editing activity. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • recombinant peptides can be used to obtain the relevant peptide sequences in large quantities. This is usually carried out by cloning into a vector, transferring it to a cell, and then separating the related peptide (fusion protein) from the proliferated host cell by a conventional method.
  • the fusion protein has the amino acid sequence shown in SEQ ID NO. 3, and the coding polynucleotide sequence is shown in SEQ ID NO.
  • the polynucleotide of the present invention is a DNA or RNA sequence encoding the fusion protein of the present invention, which can be artificially synthesized in its entire sequence.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the RNA form includes mRNA.
  • the present invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby.
  • the term "primer” refers to a generic term for an oligonucleotide that is paired with a template and which can be used to synthesize a DNA strand complementary to a template under the action of a DNA polymerase.
  • the primer may be native RNA, DNA, or any form of natural nucleotide.
  • the primer may even be a non-natural nucleotide such as LNA or ZNA.
  • the primer is “substantially” (or “substantially") with a chain on the template A special sequence is complementary.
  • the primer must be sufficiently complementary to a strand on the template to initiate extension, but the sequence of the primer need not be fully complementary to the sequence of the template.
  • a sequence that is not complementary to the template is added to the 5' end of the primer complementary to the template at the 3' end, and such primer is still substantially complementary to the template.
  • the non-fully complementary primers can also form a primer-template complex with the template for amplification.
  • the full-length nucleotide sequence of the fusion protein or element thereof of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed according to published nucleotide sequences, particularly open reading frame sequences, and used as commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art.
  • the template is amplified to obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • a method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.
  • the amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if a signal peptide DNA is expressed as a precursor and is involved in the secretion of a polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; if the ribosome binding site is placed at a position that enables translation, then it is operably linked to the coding sequence.
  • operably linked means adjacent, and for secretory leader sequences means adjacent in the reading frame.
  • vector includes plasmids, cosmids, expression vectors, cloning vectors, and the like.
  • various carriers known in the art such as commercially available carriers can be used.
  • a commercially available vector is selected, and then the nucleotide sequence encoding the novel immunotoxin of the present invention is operably linked to an expression control sequence to form a protein expression vector.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered using the vector or fusion protein coding sequences of the invention, and methods of producing the proteins of the invention by recombinant techniques.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences of the proteins of the invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance Sex as well as green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance Sex as well as green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, NS0, COS7, or 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the protein in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ions Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the gene encoding the fusion protein of the present invention can be transcribed in vitro to obtain the corresponding mRNA, and then the mRNA is injected into the target cell to generate the fusion protein of the present invention, and the target gene is edited under the cooperation of gRNA.
  • the present invention fuses the DNA sequence encoding the PEST short peptide to the 3' end of the Cas9 coding sequence by a seamless cloning method, and then in vitro transcribes Cas9-PEST mRNA for fertilized egg injection.
  • the specific plan is as follows:
  • the pGH-T7-Cas9 vector (this vector reference (Liu et al. 2014), where Cas9 is a zebrafish codon-optimized Cas9 coding sequence) was doubled with two restriction enzymes, SphI and XbaI.
  • the Cas9 C-terminal coding region and nuclear localization signal (NLS) were excised, and the vector backbone was recovered after electrophoresis.
  • Cloning Kit connects the three fragments A, B and C to the vector backbone. Due to the overlapping sequence between the fragments, the three fragments are connected to the vector backbone in the order of B ⁇ A ⁇ C, ie the PEST coding sequence is successfully inserted. Cas9-PEST plasmid was obtained between Cas9 and NLS (Fig. 5).
  • plasmid was verified by sequencing, linearization was performed using XbaI.
  • the linearized product was recovered as a template and subjected to in vitro transcription using T7 RNA polymerase to obtain Cas9-PEST mRNA.
  • the mRNA and sgRNA can be mixed at a final concentration of 200-300 ng/ ⁇ L and 40-60 ng/ ⁇ L, respectively, and the zebrafish fertilized eggs are injected, and each fertilized egg is injected 2nL.
  • the present invention provides a CRISPR/Cas9 system capable of significantly improving the efficiency of gene editing.
  • the present invention is less expensive and more flexible.
  • the present invention can be applied to other endonucleases, and is not limited by synthetic proteins.
  • the CRISPR/Cas9 target site in the fifth exon of the zebrafish gata1a gene (Fig. 6) was used as an example to compare the gene editing efficiency of Cas9 mRNA and Cas9-PEST mRNA in zebrafish embryos. Proceed as follows:
  • sgRNA template for in vitro transcription was first PCR amplified using the pMD19-gRNA scaffold plasmid (Chang et al. 2013) as a template.
  • PCR forward primer T7-gata1aE5-sfd 5'-taatacgactcactataGTAGTGTTGTAGTACTAGTGgttttagagctagaaatagc-3' (where the lowercase part is the T7 promoter sequence and scaffold, fixed; the uppercase part is the target site sequence); the reverse primer tracr rev: 5 '-aaaaaaaagcaccgactcggtgccac-3'.
  • PCR was carried out using 2 ⁇ TaqPlatium Mix (TIANGEN), and after electrophoresis was detected as a single band (Fig. 7), the PCR product was purified using an ultrathin DNA product purification kit (TIANGEN) to obtain a template for in vitro transcription of sgRNA. .
  • TIANGEN 2 ⁇ TaqPlatium Mix
  • TIANGEN ultrathin DNA product purification kit
  • the sgRNA was then transcribed in vitro using the T7 RNA polymerase system (Takara), and the reaction system was generally 20 ⁇ L. After the transcription was completed, 1 ⁇ L of DNase I (Takara) was added to remove the template.
  • the sgRNA was purified by ethanol precipitation: the reaction system was diluted to 200 ⁇ L with RNase-free H 2 O, and then 550 ⁇ L of absolute ethanol and 20 ⁇ L of 3 M sodium acetate were added, mixed uniformly, and left on ice for 10 minutes. Subsequently, the mixture was centrifuged at 13,000 g for 15 minutes in a centrifuge at 4 ° C, and the supernatant was carefully discarded. Then, 500 ⁇ L of RNase-free 70% ethanol was added, and the mixture was centrifuged at 13,000 g for 5 minutes at 4 ° C, and the supernatant was discarded again. After drying at room temperature, 30 ⁇ L of RNase-free H 2 O was added to dissolve to obtain a sgRNA stock solution. The Nanodrop was stored at -20 ° C after concentration and diluted at the time of injection.
  • Two kinds of mRNA were purified by lithium chloride precipitation: 30 ⁇ L of lithium chloride and 30 ⁇ L of RNase-free H 2 O were added to a 20 ⁇ L reaction system, mixed uniformly, and left at -20 ° C for 2 hours or more. Subsequently, the mixture was centrifuged at 13,000 g for 15 minutes in a centrifuge at 4 ° C, and the supernatant was carefully discarded. Then, 500 ⁇ L of RNase-free 70% ethanol was added, and the mixture was centrifuged at 13,000 g for 5 minutes at 4 ° C, and the supernatant was discarded again.
  • Microinjection 5 ⁇ L of each of the two injections, a final concentration of Cas9 mRNA (or Cas9-PEST mRNA) of 200 ng/ ⁇ L, a final concentration of sgRNA of 50 ng/ ⁇ L, and about 5% by volume of phenol red in the injection.
  • the solution serves as an indicator.
  • the zebrafish fertilized eggs were divided into three groups. The first group was injected with Cas9 mRNA+sgRNA, the second group was injected with Cas9-PEST mRNA+sgRNA, and the third group was not injected as a control. Make sure that the injection period is a single-cell period, and the injection volume is 2nL per egg.
  • PCR was performed using 2 ⁇ HotstartTaq PCR StarMix (GenStar), and after electrophoresis as a single band, SpeI (NEB) was used (restriction site contained in the target site and unique on the PCR product)
  • the PCR product was digested, and the digested product was detected by electrophoresis. Due to the site-directed mutagenesis of Cas9 at the target site, the SpeI cleavage site may be disrupted. Therefore, the PCR product of the experimental group cannot be completely cleaved by SpeI.
  • the uncut ratio can represent the efficiency of site-directed mutagenesis, ie the gene editing of Cas9. Efficiency, while the PCR product of the control group was completely cut.
  • the site-directed mutagenesis efficiency of injection of 400pg Cas9 mRNA + 100pg sgRNA was 22.5%, while the injection of the same dose of Cas9-PEST mRNA + sgRNA increased the site-directed mutagenesis efficiency to 35.2% (Fig. 8), and the gene editing efficiency increased by about 56. %.
  • the CRISPR/Cas9 target site in the first exon of the zebrafish mstnb gene was used as an example to compare the gene editing efficiency of Cas9 mRNA and Cas9-PEST mRNA in zebrafish embryos.
  • the procedure is similar to that of Embodiment 1.
  • the site-directed mutagenesis efficiency of injection of 400 pg Cas9 mRNA + 100 pg sgRNA was calculated to be 13.4%, and the injection of the same dose of Cas9-PEST mRNA + sgRNA increased the site-directed mutagenesis efficiency to 56.1% (Fig. 10), compared with the control group. Increased by about 4 times.
  • NPDC-1 a novel regulator of neuronal proliferation, is degraded by the ubiquitin/proteasome system through a PEST degradation motif. J Biol Chem 279(35):37069- 37078.
  • Cpf1 is a single RNA-guided Endonuclease of a class 2 CRISPR-Cas system. Cell 163(3):759-771.

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Abstract

本发明提供了一种提高基因编辑效率的方法和系统，具体地，本发明公开了将PEST短肽和Cas9蛋白融合后形成的融合蛋白能够显著提高基因编辑的效率。

Description

提高基因编辑效率的方法和系统 技术领域
本发明属于生物技术领域，具体地说，本发明涉及提高基因编辑效率的方法和系统。
背景技术
CRISPR/Cas9或CRISPR-Cas9系统是近年来兴起的用于基因编辑的工具。该系统由能够特异性识别DNA序列的sgRNA和能够切割DNA序列的Cas9核酸酶组成。通过Cas9对基因组上特定序列的切割，可以实现定点突变、片段删除或倒位等修饰。在加入外源供体DNA的条件下，还能够实现定点插入或替换，包括同源重组介导的精确的遗传修饰。除了对基因组进行编辑，工程化的Cas9还可以对RNA、转录水平、以及表观遗传进行调控。尽管CRISPR/Cas9技术具有简便、快速、特异等优点，但在细胞、动物、植物的应用中，其效率尚不能充分满足基因编辑，尤其是定点插入或替换的需求。提高这一系统的效率是基因编辑技术领域关注的重点。
因此，本领域技术人员致力于开发提高基因编辑效率的方法及其应用。
发明内容
本发明的目的在于提供一种提高基因编辑效率的方法和系统。
在本发明的第一方面，提供了一种融合蛋白，所述的融合蛋白具有式Ia或Ib所述结构：
E-P  (Ia)
P-E  (Ib)
其中，
E为核酸内切酶蛋白元件；
P为PEST蛋白元件；
“-”表示连接上述各元件的肽键或肽接头。
在另一优选例中，所述的核酸内切酶蛋白元件选自下组：Cas9蛋白(包括SpCas9、SaCas9、NmCas9、St1Cas9)及其变体(如SpCas9的VQR、EQR、VRER等变体)、Cpf1蛋白(包括AsCpf1、FnCpf1、LbCpf1)、C2c2蛋白、Argonaute蛋白家族；TALE蛋白、锌指蛋白、dCas9等与FokI融合构成的人工核酸内切酶。
在另一优选例中，所述核酸内切酶蛋白元件为Cas9蛋白。
在另一优选例中，所述SpCas9蛋白的氨基酸序列如SEQ ID NO.1所示。
在另一优选例中，所述PEST蛋白元件选自下组：
ODC1蛋白的PEST序列、GCN4蛋白的PEST序列、CLN2/CLN3蛋白的PEST序列、NIMA蛋白的PEST序列、Cactus蛋白的PEST序列、HDC蛋白的PEST序列、CPEB蛋白的PEST序列、NPDC1蛋白的PEST序列、FOS蛋白的PEST序列、NFKBIA蛋白的PEST序列等。
在另一优选例中，所述PEST蛋白元件的氨基酸序列如SEQ ID NO.2所示。
在另一优选例中，所述融合蛋白的氨基酸序列选自下组：
(A)具有SEQ ID NO.2所示氨基酸序列的多肽；
(B)具有与SEQ ID NO.2所示氨基酸序列≥80％同源性(优选地，≥90％的同源性；等优选地≥95％的同源性；最优选地，≥97％的同源性，如98％以上，99％以上)的多肽，且所述多肽具有基因编辑活性；
(C)将SEQ ID NO.3所示氨基酸序列经过1-10个氨基酸残基的取代、缺失或添加而形成的，且保留基因编辑活性的衍生多肽。
在另一优选例中，所述融合蛋白(E-P or P-E)中的肽键或肽接头的长度为0-30个氨基酸。
在另一优选例中，所述融合蛋白还包括任选地核定位信号元件(Nuclear location signal,NLS)；优选地所述核定位信号元件添加在融合蛋白的N端或者C端，或者同时添加在N端和C端。
在本发明的第二方面，提供了一种分离的多核苷酸，所述的多核苷酸编码本发明第一方面所述的融合蛋白。
在另一优选例中，所述多核苷酸包括适于原核或真核细胞表达的密码子优化序列。
在另一优选例中，所述多核苷酸为DNA或RNA。
在另一优选例中，所述多核苷酸为mRNA。
在本发明的第三方面，提供了一种载体，所述载体含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述的载体包括质粒、病毒载体等。
在另一优选例中，所述的病毒载体包括：慢病毒载体、腺病毒载体、腺相关病毒载体、逆转录病毒载体、黄热病毒载体和疱疹病毒载体等。
在另一优选例中，所述的载体包括原核和真核表达载体。
在本发明的第四方面提供了一种基因工程细胞，所述基因工程细胞含有本发明第三方面所述的载体，或含有本发明第二方面所述的多核苷酸，或含有本发明第一方面所述的融合蛋白。
在另一优选例中，所述基因工程细胞包括原核细胞和真核细胞。
在另一优选例中，所述基因工程细胞为动物细胞、植物细胞、或微生物细胞。
在另一优选例中，所述细胞为生殖细胞或受精卵。
在本发明的第五方面，提供了一种基因编辑系统，所述基因编辑系统包括本发明第一方面所述的融合蛋白、或本发明第二方面所述的多核苷酸、或本发明第三方面的载体。
在另一优选例中，所述基因编辑系统选自下组：CRISPR/Cas基因编辑系统、CRISPR/Cpf1基因编辑系统、CRISPR/C2c2基因编辑系统、Argonaute/gDNA基因编辑系统、锌指核酸酶基因编辑系统(ZFNs)、和类转录激活因子核酸酶基因 编辑系统(TALENs)。
本发明的第六方面，提供了一种核酸内切酶介导的基因编辑方法，所述方法包括步骤：
使用本发明第一方面所述的融合蛋白对靶基因进行编辑。
在本发明的第七方面，提供了一种提高核酸内切酶介导的基因编辑系统的基因编辑效率的方法，所述方法包括步骤：
在靶细胞中，表达PEST蛋白和核酸内切酶的融合蛋白。
在另一优选例中，所述方法中将所述PEST蛋白的编码序列融合至所述核酸内切酶基因的5’或3’端。
在另一优选例中，所述方法包括步骤：
在靶细胞中，表达本发明第一方面所述的融合蛋白。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了核酸内切酶蛋白元件与PEST蛋白元件融合的方式。PEST元件可以位于核酸内切酶元件的C端或者N端，其中的短线代表两者之间的肽键或肽接头，为0-30个氨基酸残基。
图2a和图2b分别显示了示范SpCas9蛋白的氨基酸序列(SEQ NO:1)和编码多核苷酸序列(SEQ ID NO.4)。
图3显示了示范小鼠ODC1蛋白的PEST结构域的氨基酸序列(SEQ ID NO.2)和编码多核苷酸序列(SEQ ID NO.5)。
图4a和图4b分别显示了一例将小鼠ODC1蛋白的PEST结构域融合至SpCas9蛋白C端所得融合蛋白的氨基酸序列(SEQ ID NO.3)和编码多核苷酸序列(SEQ ID NO.6)。
图5显示了示范Cas9-PEST质粒图谱。在原有的pGH-T7-Cas9载体上，Cas9编码序列与核定位信号(NLS)序列之间加入PEST编码序列的完整的质粒图谱。方框标记了PEST编码序列的位置。
图6显示了斑马鱼gata1a基因的结构和CRISPR/Cas9靶位点信息。该基因具有6个外显子(方框所示，实心部分代表编码区，空心部分代表非编码区)，本发明实施例中所选择的CRISPR/Cas9靶位点位于第5个外显子上(背景为灰色的序列)，可用SpeI酶切位点来检测该靶位点的效率。
图7显示了通过PCR制备gata1a靶位点的sgRNA模板的电泳检测结果，模板大小为120bp。
图8显示了Cas9-PEST提高了斑马鱼gata1a基因靶位点的定点突变效率。选择斑马鱼gata1a基因上的已知靶位点，注射相同剂量的Cas9和Cas9-PEST，使用限制性内切酶法检测定点突变效率(最上面的条带为突变的条带，黑色三角指示。该条带的亮度占三条带总亮度的比值即为突变效率。ImageJ软件可以 量化条带的亮度)，计算两组各6个样品的平均效率分别为22.5％和35.2％(对照组为未注射的样品)，表明PEST有助于提高Cas9的工作效率。
图9显示了斑马鱼mstnb基因的结构和CRISPR/Cas9靶位点信息。该基因具有3个外显子(方框所示，实心部分代表编码区，空心部分代表非编码区)，本发明实施例中所选择的CRISPR/Cas9靶位点位于第1个外显子上(背景为灰色的序列)，可用BslI酶切位点来检测该靶位点的效率。
图10显示了Cas9-PEST可将斑马鱼mstnb基因靶位点的定点突变效率从13.4％提高至56.1％。
具体实施方式
本发明人通过广泛而深入的研究后发现，将PEST短肽和Cas9蛋白融合后形成的融合蛋白能够显著提高基因编辑效率，在此基础上，完成了本发明。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
本发明要解决的问题是提高CRISPR系统在受精卵分裂早期的编辑效率，从而增加在动物成体的编辑效率。本方法通过提高Cas9mRNA翻译的速率，促使Cas9蛋白能够在受精卵发育早期中尽早地发挥功能，且更有效地编辑靶向基因。
通过在Cas9编码区域的3’端融合小鼠ODC1蛋白中PEST序列的编码序列，可有效提高Cas9mRNA翻译成为蛋白的速率，从而提高基因编辑的效率。
本发明中选择将ODC1蛋白中的PEST序列与Cas9进行融合，其他可以有效提高Cas9mRNA翻译效率的序列或者结构域，比如其他蛋白的PEST序列，与Cas9进行融合后，都有可能提高在动物中CRISPR/Cas9介导的基因编辑效率。
另外需指出，作为本发明的一个例子使用的Cas9是经过斑马鱼密码子优化的SpCas9。本发明还可能用于改造SaCas9、NmCas9、St1Cas9、Cpf1、C2c2、Argonaute、TALEN、ZFN等任何用于基因编辑的内切酶。
PEST蛋白
PEST蛋白(PEST短肽)是一类特殊的信号肽，富含脯氨酸(P)、谷氨酸(E)、丝氨酸(S)和苏氨酸(T)，可使蛋白的半衰期缩短。
已知自然界中的多种蛋白如ODC1、HDC、NPDC、CPEB、NFKBIA等含有PEST短肽(结构域)，实验表明它们与蛋白的快速降解有关，降解的途径可能是通 过蛋白酶体或者钙蛋白酶。PEST短肽的序列、长度、位置和二级结构在不同蛋白中都有所变化，有些蛋白含有多个PEST结构域。
在本发明的一个优选地实施方式中，所述PEST蛋白元件选自下组：
ODC1蛋白的PEST序列、GCN4蛋白的PEST序列、CLN2/CLN3蛋白的PEST序列、NIMA蛋白的PEST序列、Cactus蛋白的PEST序列、HDC蛋白的PEST序列、CPEB蛋白的PEST序列、NPDC1蛋白的PEST序列、FOS蛋白的PEST序列、NFKBIA蛋白的PEST序列等(具体可参考文献：Rogers et al.1986,Rechsteiner and Rogers 1996,Fleming and Wang 2000,Reverte et al.2001,Spencer et al.2004)
在本发明的一个优选的实施方式中，所述PEST蛋白的氨基酸序列如SEQ ID NO.2所示；其编码核苷酸序列(来自小鼠Odc1的cDNA)如SEQ ID NO.5所示。
核酸内切酶蛋白元件
本发明中所述的核酸内切酶蛋白元件是指可以用于基因编辑的核酸内切酶、其变体、及其活性片段。
典型的核酸内切酶包括：Cas9蛋白(包括SpCas9、SaCas9、NmCas9、St1Cas9)及其变体(如SpCas9的VQR、EQR、VRER等变体)(Cong et al.2013,Hou et al.2013,Mali et al.2013,Kleinstiver et al.2015,Ran et al.2015)、Cpf1蛋白(包括AsCpf1、FnCpf1、LbCpf1)(Zetsche et al.2015,Kim et al.2016,Kleinstiver et al.2016)、C2c2蛋白(Abudayyeh et al.2016)、Argonaute蛋白家族(Gao et al.2016)；TALE蛋白、锌指蛋白、dCas9等与FokI融合构成的人工核酸内切酶(Kim et al.1996,Bibikova et al.2002,Miller et al.2011,Tsai et al.2014)。
CRISPR/Cas系统
CRISPR/Cas系统为目前发现存在于大多数细菌与所有的古菌中的一种后天免疫系统，以消灭外来的质体或者噬菌体，并在自身基因组中留下外来基因片段作为“记忆”。全名为常间回文重复序列丛集/常间回文重复序列丛集关联蛋白系统(clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins)。
目前已发现三种不同类型的CRISPR/Cas系统，存在于大约40％和90％已测序的细菌和古菌中。其中第二型的组成较为简单，以Cas9蛋白以及向导RNA(gRNA)为核心的组成，由于其对DNA干扰(DNAi)的特性，目前被积极地应用于遗传工程中，作为基因体剪辑工具，与锌指核酸酶(ZFN)及类转录活化因子核酸酶(TALEN)同样利用非同源性末端接合(NHEJ)的机制，于基因体中产生去氧核糖核酸的双股断裂以利剪辑。二型CRISPR/Cas并经由遗传工程的改造应用于哺乳类细胞及斑马鱼的基因体剪辑。其设计简单以及操作容易的特性为最大的优点。未来将可应用在各种不同的模式生物当中。
称为CRISPR的基因组重复丛集，即原核生物拟核DNA链中的丛生重复序列，在1987关于E.coli的一份研究报告中被首次描述。2000年，相似的重复序列在其它真细菌和古细菌中被发现并被命名为短间隔重复序列(Short Regularly Spaced Repeats，SRSR)。2002年SRSR被重命名为CRISPR。其中一部分基因编码 的蛋白为核酸酶和解旋酶。这些关联蛋白(CAS,CRISPR-associated proteins)与CRISPR组成了CRISPR/Cas系统。
CRISPR/Cas技术
本发明所称的“CRISPR/Cas技术”、“CRISPR/Cas基因编辑”、“CRISPR/Cas基因编辑技术”、“CRISPR/Cas基因编辑方法”均指利用CRISPR/Cas系统的原理对目的基因进行改造的基因编辑技术。
Cas9蛋白及其变体
CRISPR/Cas的核心就是Cas9蛋白以及向导RNA(gRNA)。在不同物种中能够利用CRISPR/Cas系统进行基因编辑的核心技术，首要一步即在该物种中异源表达有DNA剪切酶活性的Cas9蛋白，第二步则是获得gRNA和靶点同源序列将Cas9引导至靶点进行DNA剪切。其中第二步中，具体的操作方法是本领域技术人员所熟知的。
来源于Streptococcus pyogenes的Cas9蛋白是一种多结构域多功能的Cas蛋白，其N端具有类RuvC核酸酶的结构域，其中部具有HNH核酸酶结构域。Cas9蛋白与gRNA结合能够实现在特异位点处切割DNA，来源于Streptococcus pyogenes的CRISPR/Cas系统识别序列为23bp,并能靶向20bp,其识别位点最末3位NGG序列被称作PAM(protospacer adjacent motif)序列，其对于DNA切割非常重要。目前大多数真核生物(包括家蚕、拟南芥、酵母、线虫等)的CRISPR/Cas系统最初均源自Streptococcus pyogenes，Cas9蛋白则多是经过人源化改造或其它物种的密码子优化。
较佳地，本发明所提供的Cas9来源于化脓链球菌(Streptococcus pyogenes)。在本发明的一个优选地实施方式中，所述Cas9蛋白的氨基酸序列如SEQ ID NO.1所示；
其编码核酸序列(经过斑马鱼密码子优化)如SEQ ID NO.4所示。
融合蛋白
本发明提供了PEST蛋白和Cas9蛋白的融合蛋白及其编码序列(包括DNA、mRNA)。
本发明的融合蛋白，可任选地含有连接肽。连接肽大小和复杂性可能会影响蛋白的活性。通常，连接肽应当具有足够的长度和柔韧性，以保证连接的两个蛋白在空间上有足够的自由度以发挥其功能。同时避免连接肽中形成α螺旋或β折叠等对融合蛋白的稳定性的影响。连接肽的长度一般为0-30个氨基酸，较佳地1-5个氨基酸。
应理解，所述术语还包括本发明融合蛋白的衍生物，指本发明融合蛋白在经过1-3个氨基酸添加或替换、1-2个氨基酸缺失并仍具有基因编辑活性的多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
一旦鉴定获得了相关的肽序列，就可以用重组法来大批量地获得相关肽序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到相关肽(融合蛋白)。
此外，还可用化学方法直接合成相关肽序列。
在本发明的一个优选地实施方式中，所述融合蛋白的氨基酸序列如SEQ ID NO.3所示，其编码多核苷酸序列如SEQ ID NO.6所示。
多核苷酸
本发明的多核苷酸，是编码本发明融合蛋白的DNA或RNA序列，可以全序列人工合成。
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。RNA形式包括mRNA。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的蛋白质片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码多肽的功能。
如本文所用，术语“引物”指的是在与模板配对，在DNA聚合酶的作用下能以其为起点进行合成与模板互补的DNA链的寡聚核苷酸的总称。引物可以是天然的RNA、DNA，也可以是任何形式的天然核苷酸。引物甚至可以是非天然的核苷酸如LNA或ZNA等。引物“大致上”(或“基本上”)与模板上一条链上的 一个特殊的序列互补。引物必须与模板上的一条链充分互补才能开始延伸，但引物的序列不必与模板的序列完全互补。比如，在一个3'端与模板互补的引物的5'端加上一段与模板不互补的序列，这样的引物仍大致上与模板互补。只要有足够长的引物能与模板充分的结合，非完全互补的引物也可以与模板形成引物-模板复合物，从而进行扩增。
本发明融合蛋白或其元件的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据已公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的基因。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
如本文所用，“可操作地连于”指这样一种状况，即线性DNA序列的某些部分能够影响同一线性DNA序列其他部分的活性。例如，如果信号肽DNA作为前体表达并参与多肽的分泌，那么信号肽(分泌前导序列)DNA就是可操作地连于多肽DNA；如果启动子控制序列地转录，那么它就是可操作地连于编码序列；如果核糖体结合位点被置于能使其翻译的位置时，那么它是可操作地连于编码序列。一般“可操作地连于”意味着相邻近，而对于分泌前导序列则意味着在阅读框中相邻。
如本文所用，术语“载体”包括质粒、粘粒、表达载体、克隆载体等。
在本发明中，可选用本领域已知的各种载体如市售的载体。比如，选用市售的载体，然后将编码本发明新免疫毒素的核苷酸序列可操作地连于表达调控序列，形成蛋白表达载体。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或融合蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述蛋白质的方法。
本领域的技术人员熟知的方法能用于构建含本发明蛋白的编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗 性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞(基因工程细胞)可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属的细菌细胞；真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CHO、NS0、COS7、或293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的蛋白质可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
在基因编辑过程中，可以对编码本发明融合蛋白的基因进行体外转录，获得对应的mRNA，然后将mRNA注射入靶细胞中翻译产生本发明的融合蛋白，在gRNA配合下对靶基因进行编辑。
基因编辑方法
本发明通过无缝克隆的方法将编码PEST短肽的DNA序列融合至Cas9编码序列的3’端，然后体外转录得到Cas9-PEST mRNA用于受精卵注射。具体的方案如下：
1.将pGH-T7-Cas9载体(该载体参考文献(Liu et al.2014)，此处Cas9为经过斑马鱼密码子优化的Cas9编码序列)用SphI和XbaI两种限制性内切酶进行双酶切，切除Cas9C端编码区及核定位信号(NLS)，电泳后回收载体骨架。
2.从小鼠cDNA文库中扩增ODC1蛋白所含的PEST编码序列，在扩增的上、下游引物外侧分别加入与Cas9、NLS重叠的约20bp序列，扩增的产物命名为片段A。
3.从原载体上分别扩增Cas9C端编码区及NLS，扩增时也需要在引物外侧加上与载体骨架或PEST编码序列重叠的约20bp序列，扩增的产物命名为片段B和片段C。
4.使用诺维赞公司的无缝克隆试剂盒(ClonExpressMultiS One Step  Cloning Kit)将A、B、C三个片段与载体骨架连接，由于片段间的重叠序列的设置，三个片段会按照B→A→C的顺序连入载体骨架，即成功将PEST编码序列插入Cas9和NLS之间(图5)，获得Cas9-PEST质粒。
5.质粒经测序验证后，使用XbaI进行线性化。回收线性化产物作为模板，利用T7RNA聚合酶进行体外转录，获得Cas9-PEST mRNA。可将此mRNA与sgRNA按照终浓度分别为200～300ng/μL和40～60ng/μL混合，注射斑马鱼受精卵，每枚受精卵注射2nL。
本发明的主要优点在于：
(1)本发明提供了能够显著提高基因编辑效率的CRISPR/Cas9系统。
(2)与使用Cas9蛋白相比，本发明成本更低，灵活性更高。
(3)本发明可以应用于其他的内切酶，不受合成蛋白的限制。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1
以斑马鱼gata1a基因第5个外显子中的CRISPR/Cas9靶位点(图6)为例，比较Cas9mRNA和Cas9-PEST mRNA在斑马鱼胚胎中的基因编辑效率。步骤如下：
1.制备sgRNA:首先以pMD19-gRNA scaffold质粒(Chang et al.2013)为模板，PCR扩增用于体外转录的sgRNA模板。PCR的正向引物T7-gata1aE5-sfd：5’-taatacgactcactataGTAGTGTTGTAGTACTAGTGgttttagagctagaaatagc-3’(其中小写部分为T7启动子序列和scaffold，固定不变；大写部分为靶位点序列)；反向引物tracr rev：5’-aaaaaaagcaccgactcggtgccac-3’。使用2×TaqPlatium Mix(TIANGEN公司)进行PCR，电泳检测为单一条带后(图7)，使用超薄DNA产物纯化试剂盒(TIANGEN公司)对PCR产物进行纯化，得到用于体外转录sgRNA的模板。该模板的全序列为：
Figure PCTCN2016104450-appb-000001
然后使用T7RNA聚合酶体系(Takara公司)体外转录sgRNA，反应体系一般为20μL。转录完成后加入1μL DNase I(Takara公司)去除模板。
最后通过乙醇沉淀法纯化sgRNA：用RNase-free H2O将反应体系稀释至200μL，再加入550μL无水乙醇和20μL 3M醋酸钠，混合均匀后于冰上放置10分钟。接着在4℃离心机以13000g的转速离心15分钟，小心弃去上清后，加入500μL RNase-free的70％乙醇，以4℃，13000g离心5分钟，再次弃去 上清。室温晾干后，加入30μL RNase-free H2O溶解，得到sgRNA储液。Nanodrop测定浓度后于-20℃保存，注射时进行稀释。
2.制备Cas9mRNA和Cas9-PEST mRNA：将改造前的pGH-T7-Cas9质粒和改造的Cas9-PEST质粒都用XbaI(NEB公司)进行线性化，电泳检测后使用普通DNA产物纯化试剂盒(TIANGEN公司)回收线性化产物。以各1μg线性化产物为模板，用T7mMESSAGE mMACHINE试剂盒(Ambion公司)体外转录两种mRNA，反应体系一般为20μL。转录完成后加入1μL TURBO DNase(Ambion公司)去除模板。
通过氯化锂沉淀法纯化两种mRNA：向20μL反应体系中加入30μL氯化锂和30μL RNase-free H2O，混合均匀后于-20℃放置2小时以上。接着在4℃离心机以13000g的转速离心15分钟，小心弃去上清后，加入500μL RNase-free的70％乙醇，以4℃，13000g离心5分钟，再次弃去上清。室温晾干后，加入30μL RNase-free H2O溶解，得到Cas9mRNA储液和Cas9-PEST mRNA储液。Nanodrop测定浓度后于-20℃保存，注射时进行稀释。
3.显微注射：配置两种注射液各5μL，Cas9mRNA(或Cas9-PEST mRNA)的终浓度为200ng/μL，sgRNA的终浓度为50ng/μL，注射液中加入约5％体积的酚红溶液作为指示剂。将斑马鱼的受精卵分为三组，第一组注射Cas9mRNA+sgRNA，第二组注射Cas9-PEST mRNA+sgRNA，第三组不注射作为对照。确保注射的时期都是单细胞期，注射量都是每枚卵2nL。
4.检测基因编辑效率：待受精卵发育至24小时，从两个实验组中各取6×3枚，对照组中取2×3枚提取基因组。用gata1a基因靶位点的上、下游引物，通过PCR扩增基因组上覆盖靶位点的序列。引物序列信息见表2。
表2实施例中所用基因靶位点的检测引物
Figure PCTCN2016104450-appb-000002
使用2×HotstartTaq PCR StarMix(GenStar公司)进行PCR，电泳检测为单一条带后，用SpeI(NEB公司)(靶位点上含有的限制性内切酶位点且在PCR产物上是唯一的)对PCR产物进行酶切，再通过电泳检测酶切产物。由于Cas9在靶位点的切割引发定点突变，可能破坏SpeI酶切位点，因此实验组的PCR产物不能完全被SpeI切开，未酶切的比例可代表定点突变的效率，即Cas9的基因编辑效率，而对照组的PCR产物则能完全被切开。
通过计算，注射400pg Cas9mRNA+100pg sgRNA引起的定点突变效率为22.5％，而注射相同剂量的Cas9-PEST mRNA+sgRNA可将定点突变效率提升至35.2％(图8)，基因编辑效率提高了约56％。
实施例2
以斑马鱼mstnb基因第1个外显子中的CRISPR/Cas9靶位点(图9)为例，比较Cas9mRNA和Cas9-PEST mRNA在斑马鱼胚胎中的基因编辑效率。步骤与实施例1相似。经计算，注射400pg Cas9mRNA+100pg sgRNA引起的定点突变效率为13.4％，而注射相同剂量的Cas9-PEST mRNA+sgRNA可将定点突变效率提升至56.1％(图10)，与对照组相比突变效率提高了约4倍。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
参考文献
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Claims (10)

  1. 一种融合蛋白，其特征在于，所述的融合蛋白具有式Ia或Ib所述结构：
    E-P     (Ia)
    P-E     (Ib)
    其中，
    E为核酸内切酶蛋白元件；
    P为PEST蛋白元件；
    “-”表示连接上述各元件的肽键或肽接头。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述的核酸内切酶蛋白元件选自下组：Cas9蛋白(包括SpCas9、SaCas9、NmCas9、St1Cas9)及其变体(如SpCas9的VQR、EQR、VRER等变体)、Cpf1蛋白(包括AsCpf1、FnCpf1、LbCpf1)、C2c2蛋白、Argonaute蛋白家族；TALE蛋白、锌指蛋白、dCas9等与FokI融合构成的人工核酸内切酶。
  3. 如权利要求1所述的融合蛋白，其特征在于，所述PEST蛋白元件选自下组：
    ODC1蛋白的PEST序列、GCN4蛋白的PEST序列、CLN2/CLN3蛋白的PEST序列、NIMA蛋白的PEST序列、Cactus蛋白的PEST序列、HDC蛋白的PEST序列、CPEB蛋白的PEST序列、NPDC1蛋白的PEST序列、FOS蛋白的PEST序列、NFKBIA蛋白的PEST序列等。
  4. 一种分离的多核苷酸，所述的多核苷酸编码权利要求1所述的融合蛋白。
  5. 一种载体，所述载体含有权利要求4所述的多核苷酸。
  6. 一种基因工程细胞，其特征在于，所述基因工程细胞含有权利要求5所述的载体，或含有权利要求4所述的多核苷酸，或含有权利要求1所述的融合蛋白。
  7. 如权利要求6所述的基因工程细胞，其特征在于，所述基因工程细胞为动物细胞、植物细胞、或微生物细胞。
  8. 一种基因编辑系统，其特征在于，所述基因编辑系统包括权利要求1所述的融合蛋白、或权利要求4所述的多核苷酸、或权利要求5的载体。
  9. 一种核酸内切酶介导的基因编辑方法，其特征在于，所述方法包括步骤：
    使用权利要求1所述的融合蛋白对靶基因进行编辑。
  10. 一种提高核酸内切酶介导的基因编辑系统的基因编辑效率的方法，其特征在于，所述方法包括步骤：
    在靶细胞中，表达PEST蛋白和核酸内切酶的融合蛋白。
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