WO2018223843A1 - 用于dna编辑的系统及其应用 - Google Patents

用于dna编辑的系统及其应用 Download PDF

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WO2018223843A1
WO2018223843A1 PCT/CN2018/088105 CN2018088105W WO2018223843A1 WO 2018223843 A1 WO2018223843 A1 WO 2018223843A1 CN 2018088105 W CN2018088105 W CN 2018088105W WO 2018223843 A1 WO2018223843 A1 WO 2018223843A1
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rna
dna
crrna
seq
gene
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PCT/CN2018/088105
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French (fr)
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张必良
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广州市锐博生物科技有限公司
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Priority to US16/615,828 priority Critical patent/US20210189435A1/en
Priority to JP2019523070A priority patent/JP7010941B2/ja
Priority to AU2018279569A priority patent/AU2018279569B2/en
Priority to EP18814269.9A priority patent/EP3636760A4/en
Publication of WO2018223843A1 publication Critical patent/WO2018223843A1/zh

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Definitions

  • the present invention relates to systems for DNA editing and their applications in the field of biotechnology.
  • the CRISPR–Cas system is an adaptive immune defense mechanism formed by bacteria and archaea during long-term evolution and can be used against invading viruses and foreign DNA.
  • the CRISPR–Cas system can be divided into Class I, Class II and Class III systems. These three types of systems can be further divided into more subclasses based on their genes encoding the Cas protein.
  • Class I and Class III CRISPR–Cas systems require only the involvement of both the crRNA and Cas proteins, while the class II CRISPR–Cas system includes the crRNA, tracrRNA and Cas proteins.
  • the CRISPR/Cas9 system provides immunity by integrating fragments of invading phage and plasmid DNA into CRISPR and using the corresponding CRISPR RNAs (crRNAs) to direct the degradation of homologous sequences.
  • This system works by the fact that crRNA (CRISPR-derived RNA) binds to tracrRNA (trans-activating RNA) by base pairing to form a tracrRNA/crRNA complex, which directs the nuclease Cas9 protein to cleave at the target site paired with the crRNA. Cut double-stranded DNA.
  • Cas9 has two key domains: HNH and RuvC, which cut a single strand in a DNA duplex, respectively.
  • DNA double-strand breaks (DSB), cell-priming repair mechanisms, cells can generate site-directed mutagenesis through inaccurate repair of non-homologous end joining (NHEJ), or can be repaired by homologous recombination (HR) Gene site insertion or gene replacement.
  • Cas9 has successfully performed genomic engineering studies in bacteria, human cells, zebrafish and mice.
  • the present invention first provides a system for DNA editing, the system being a CRISPR-Cas system, including A or B:
  • RNA of the name RNA-2 or a biological material associated with the RNA-2 an RNA of the name RNA-2 or a biological material associated with the RNA-2;
  • the RNA-2 is an RNA of the formula I;
  • Nx is a spacer sequence (Spacer) in the CRISPR/Cas system
  • ncrRNA is any one of the following a1) to a4):
  • the biological material associated with the RNA-2 is any one of the following A1) to A5):
  • A1 a DNA molecule encoding the RNA-2;
  • A2) an expression cassette comprising the DNA molecule of A1);
  • A3 a recombinant vector comprising the DNA molecule of A1), or a recombinant vector comprising the expression cassette of A2);
  • A5 a cell line comprising the DNA molecule of A1), or a cell line comprising the expression cassette of A2);
  • RNA-1 is any one of the following b1) to b4):
  • B2 an RNA obtained by adding one or several nucleotides at the 5' end and/or the 3' end of b1);
  • the biological material associated with the RNA-1 is any one of the following B1) to B5):
  • B2 an expression cassette comprising the DNA molecule of B1);
  • B3 a recombinant vector comprising the DNA molecule of B1), or a recombinant vector comprising the expression cassette of B2);
  • B4 a recombinant microorganism comprising the DNA molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);
  • B5) a cell line comprising 1) the DNA molecule, or a cell line comprising the expression cassette of B2);
  • ncrRNA is complementary to the RNA-1 partial fragment to form a structure including a stem region and a loop region;
  • Nx is an RNA sequence complementary to the fragment of the DNA of interest for editing, meeting the requirements for a spacer sequence in the CRISPR/Cas system.
  • N can be a ribonucleotide or ribonucleotide modification; x is the number of N.
  • x is a non-zero natural number.
  • x may specifically be any of the following e1)-e3):
  • the RNA-2 can form a complex with a partial fragment of the RNA-1 via ncrRNA, and the Cas protein (ie, Cas nuclease) in the CRISPR-Cas system can bind to the complex to effect editing of the DNA.
  • the Cas protein ie, Cas nuclease
  • nucleotides may specifically be from one to ten nucleotides.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 85% or higher, or 90% or higher, or 95% or higher identity to the nucleotide sequence set forth in SEQ ID NO. 1 or SEQ ID NO. 5 of the present invention. Nucleotide sequence. Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
  • the stringent conditions are hybridization in a solution of 2 ⁇ SSC, 0.1% SDS at 68 ° C for 2 times, 5 min each time, and in a solution of 0.5 ⁇ SSC, 0.1% SDS, at 68° C.
  • the membrane was washed twice for 15 minutes each time; or, in a solution of 0.1 ⁇ SSPE (or 0.1 ⁇ SSC) and 0.1% SDS, the membrane was hybridized and washed at 65 °C.
  • the above 85% or more identity may be 85%, 90% or 95% or more identity.
  • SEQ ID NO. 1 or SEQ ID NO. 5 of the present invention can readily mutate SEQ ID NO. 1 or SEQ ID NO. 5 of the present invention using known methods, such as directed evolution and point mutation methods. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of SEQ ID NO. 1 or SEQ ID NO. 5 of the present invention, as long as they have the same function, are derived from The nucleotide sequence of the invention is also equivalent to the sequence of the invention.
  • the cell line does not include propagation material.
  • the RNA may be any one of the following a21) to a23):
  • RNA of 12-18 nt in length A23 (RNA of 12-18 nt in length.
  • RNA may be any one of the following b21) to b24):
  • the ribonucleotide can be A, U, C or G.
  • the ribonucleotide modification may be a substance obtained by modifying A, U, C or G in a ribose, a phosphate backbone and/or a base;
  • the modification is a substance obtained by modifying at least one nucleotide of the RNA in a ribose, a phosphate backbone, and/or a base.
  • the modification may be a 2'-O-methyl modification, a 2'-deoxy modification, a 2'-fluoro modification or a cholesterol modification.
  • the DNA may be any one of the following M1)-M5):
  • RNA of a21 may be the RNA represented by SEQ ID NO. 2 in the sequence listing;
  • the RNA may be the RNA shown in SEQ ID NO. 3 in the Sequence Listing;
  • the RNA may be the RNA shown by SEQ ID NO. 4 in the Sequence Listing.
  • the RNA may be the RNA shown in SEQ ID NO. 6 in the Sequence Listing;
  • the RNA may be the RNA shown in SEQ ID NO. 7 in the Sequence Listing;
  • the RNA may be the RNA shown in SEQ ID NO. 8 in the Sequence Listing.
  • the modification may be a 2'-O-methyl modification, a 2'-deoxy modification, a 2'-fluoro modification or a cholesterol modification.
  • the DNA may be a VEGFA (Vascular endothelial growth factor A) gene, an EMX1 (Empty spiracles homeobox 1) gene, an Oct4 (organic cation/carnitine transporter 4) gene, a beta thalassemia gene, a TP53 (tumor protein p53) gene or a TP53 gene. Promoter.
  • VEGFA Vascular endothelial growth factor A
  • EMX1 Extracellular cation/carnitine transporter 4
  • beta thalassemia gene a TP53 (tumor protein p53) gene or a TP53 gene. Promoter.
  • the RNA-2 may be the RNA shown in any one of SEQ ID NOS. 9-29.
  • the RNA-2 is an RNA represented by at least one of SEQ ID NO. 9-12, SEQ ID NO. 28 and SEQ ID NO.
  • the RNA-2 is an RNA represented by at least one of the sequences of SEQ ID NO. 13-15;
  • the RNA-2 is an RNA represented by at least one of SEQ ID NO. 16-19;
  • the RNA-2 is the RNA set forth in SEQ ID NO. 20-22;
  • the RNA-2 is the RNA set forth in SEQ ID NO. 23 and/or 24;
  • the RNA-2 is an RNA represented by at least one of the sequences of SEQ ID NOS. 25-27.
  • the system can be a CRISPR-Cas system, specifically a Type II CRISPR-Cas system, such as a CRISPR/Cas9 system.
  • the above system may further comprise N3), N3) being a Cas9 nuclease or a biological material associated with a Cas9 nuclease;
  • the biomaterial associated with the Cas9 nuclease is any one of the following C1) to C7):
  • C1 a nucleic acid molecule encoding a Cas9 nuclease
  • C2 an expression cassette comprising the nucleic acid molecule of C1);
  • the Cas9 nuclease may be derived from R1) or R2):
  • the Streptococcus bacterium is specifically Streptococcus pyogenes.
  • the Staphylococcus bacterium may specifically be Staphylococcus aureus.
  • the Cas9 nuclease may specifically be a protein represented by Genbank ID: AQM52323.1.
  • the system may also include other reagents and/or instruments required for DNA editing in addition to crRNA, tracrRNA, and Cas9 nucleases using the CRISPR/Cas9 DNA editing method.
  • RNA-1 When the system contains RNA-2 and RNA-1, the molar ratio of RNA-1 to RNA-2 can be 1: (1-1.5).
  • the molar ratio of Cas9 to tracrRNA to crRNA can be 1: (1-20): (1:20), specifically 1: (1-8): (1: 8), further may be 1: (1-4): (1: 4), and further may be 1: (2-4): (2-4).
  • RNA-2 in the above molar ratio is based on the total concentration of the plurality of RNAs.
  • the system may consist solely of the RNA-2, also from the above N1) and N2), or may be composed of the above N1), N2) and N3).
  • the invention also provides the following O1) or O2):
  • RNA-2 O1 the RNA-2 or a biological material associated with the RNA-2;
  • RNA-1 O2
  • O2 the RNA-1 or a biological material associated with the RNA-1.
  • the invention also provides any of the following applications:
  • RNA-1 as a tracrRNA
  • RNA-1 the use of the RNA-1 or the biological material associated with the RNA-1 in DNA editing
  • RNA-2 or the biological material associated with the RNA-2 in DNA editing
  • the product may be a CRISPR-Cas system, specifically a Type II CRISPR-Cas system, such as a CRISPR/Cas9 system.
  • the invention also provides a method of editing DNA, the method comprising: processing DNA using the system to effect editing of the DNA.
  • the concentration of RNA-2 may be 100-800 nM, specifically 200-400 nM.
  • the concentration of RNA-1 may range from 100 to 800 nM, specifically from 200 to 400 nM.
  • the CRISPR/Cas9 system may also contain a Cas9 nuclease or a biological material associated with a Cas9 nuclease.
  • the concentration of Cas9 nuclease may range from 20 to 400 nM, specifically from 100 to 400 nM, and further may range from 100 to 200 nM.
  • the vector may be used in an amount of from 1 to 200 ⁇ g, further from 20 to 100 ⁇ g, and further preferably from 30 to 50 ⁇ g or from 1 to 5 ⁇ g.
  • the carrier is preferably used in an amount of 3 ⁇ g.
  • the ratio of the cell to the RNA-2 in the system may be 0.5 million to 60,000 cells/100 nM RNA-2, and further may be 0.75. A million to 70,000 cells/100 nM RNA-2, and further may be 1-1.25 million cells/100 nM RNA-2.
  • the cell can be a eukaryotic cell.
  • the cell may further be a mammalian cell, such as a 293T cell, a HeLa cell, a THP1 cell, a HUVEC cell, or a C2C12 mouse myoblast.
  • the invention also provides a method of making the system, the method comprising separately packaging each substance in the system separately.
  • the method of making the system can also include synthesizing each RNA of the system.
  • the synthesis can be synthesized using chemical methods.
  • the DNA may be any one of the following M1)-M5):
  • the editing efficiency is at least 1% or more, such as 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70% , 80%, 85% or 90%.
  • the first position of each sequence is the 5' end of the sequence.
  • the DNA editing can be the cleavage of DNA.
  • the system for DNA editing of the present invention can be used as a CRISPR/Cas9 system editing DNA, wherein the length of the ncrRNA portion of RNA-2 as a crRNA is 14 nt (that is, when ncrRNA2), Cas9 has the highest efficiency, in vitro enzyme.
  • the cut effect was 89.2%, and the intracellular digestion effect was 29.4%.
  • the Cas9 digestion effect was the best, the in vitro digestion effect was 90.7%, and the intracellular digestion effect was 29.8%. .
  • RNA-2 was modified, there was no significant change in the enzymatic cleavage effect of Cas9 in the CRISPR/Cas9 system.
  • RNA-2 and RNA-1 can be used in the CRISPR/Cas9 system component to edit the DNA of interest using the CRISPR/Cas9 system, DNA editing is not affected by the cells and the target DNA, and DNA editing can be realized.
  • Direct editing of DNA in animals when editing DNA using the CRISPR/Cas9 system, it is also possible to improve editing efficiency by using multiple RNA-2s with different sequences, and to improve DNA editing efficiency by stably expressing Cas9 cells.
  • RNA-2 and RNA-1 sequences of the present invention are short and are easily obtained by chemical synthesis, which is advantageous for improving the purity and scale production of RNA, and simplifying the preparation and application of the CRISPR/Cas9 system.
  • chemically synthesized RNA elements do not require plasmid expression, which avoids the problem of plasmid construction (such as inter-plasmid compatibility, plasmid vector carrying capacity, difficulty in constructing multiple plasmid systems, etc.), and is easier to chemically modify, suitable for in vivo application). It is shown that RNA-2 and RNA-1 of the present invention can be utilized as the crRNA and tracrRNA elements in the CRISPR/Cas9 system for editing DNA of interest.
  • Figure 1 shows the in vitro digestion effect of Cas9 under different crRNA.
  • FIG. 1 shows the intracellular digestion effect of Cas9 under different crRNA.
  • Figure 3 shows the in vitro enzymatic cleavage effect of Cas9 under different tracrRNA.
  • Figure 4 shows the intracellular digestion effect of Cas9 under different tracrRNA.
  • Figure 5 shows the effect of modification of crRNA on the in vitro digestion of Cas9.
  • Figure 6 shows the effect of modification of crRNA on the intracellular digestion of Cas9.
  • Figure 7 is a Cas9 nuclease cleavage effect in different cell lines directed against the VEGFA gene.
  • Figure 8 is a Cas9 nuclease cleavage effect at different Oct4-E-T1-crRNA concentrations. Among them, 800, 400 and 200 are the concentrations of Oct4-E-T1-crRNA in nM.
  • Figure 9 shows the in vitro cleavage effect of Cas9 nuclease on the EMX1 gene DNA template under different crRNA.
  • E1R, E7F, Ee3F and E2F represent E1R-crRNA, E7F-crRNA, Ee3F-crRNA and E2F-crRNA, respectively.
  • Figure 10 shows the in vitro cleavage effect of Cas9 nucleases of different crRNAs.
  • Beta-3-T1, Beta-3-T3 and Beta-5-T3 represent Beta-3-T1-crRNA, Beta-3-T3-crRNA and Beta-5-T3-crRNA, respectively.
  • FIG 11 shows the intracellular cleavage effect of the Oct4 gene under different crRNA.
  • E-T1, E-T2 and E-T3 represent Oct4-E-T1-crRNA, Oct4-E-T2-crRNA and Oct4-E-T3-crRNA, respectively.
  • Figure 12 shows the effect of Cas9 nuclease on the intracellular cleavage of the EMX1 gene at different Ee3F-crRNA levels. Among them, 400, 200, 100 and 50 are the amounts of Ee3F-crRNA in ng.
  • Figure 13 shows the intracellular cleavage effect of Cas9 nuclease on genes under different crRNA.
  • Beta-3-T1, Beta-3-T3 and Beta-5-T3 represent Beta-3-T1-crRNA, Beta-3-T3-crRNA and Beta-5-T3-crRNA, respectively.
  • Figure 14 shows the cleavage effect of VEGFA gene in mouse cells (C2C12) and in vivo.
  • Figure 15 shows the intracellular cleavage effect of the promoter and exon regions of the TP53 fragment.
  • Figure 16 shows the in vitro cleavage effect of the promoter and exon regions of the TP53 fragment.
  • Figure 17 is the in vitro cleavage effect of Cas9 nuclease on the Oct4 gene under multiple crRNAs.
  • Mix represents the experimental results of Oct4-E-T1-crRNA, Oct4-E-T2-crRNA and Oct4-E-T3-crRNA, and ET2 represents Oct4-E-T2-crRNA.
  • Figure 18 is the effect of intracellular cleavage of the Oct4 gene by Cas9 nuclease under multiple crRNAs.
  • Mix represents the experimental results of Oct4-E-T1-crRNA, Oct4-E-T2-crRNA and Oct4-E-T3-crRNA, and ET2 represents Oct4-E-T2-crRNA.
  • Figure 19 shows the enzymatic cleavage effects of different Cas9 donors.
  • Figure 20 shows the enzymatic cleavage effects of different Cas9 concentrations.
  • the sequence of the Cas9 nuclease is the sequence shown in Genbank ID: AQM 52323.1 on NCBI. This protein is derived from Streptococcus pyogenes.
  • Cas9-293T cells following embodiment Examples, Cas9-HeLa cells, Cas9-THP-1 cells and Cas9-HUVEC cells using Guangzhou-Jin Biotechnology Ltd. Genome-TALER TM & Genome-CRISPR TM human AAVS1 Safe Harbor
  • the gene knock-in kit (Catalog# SH-AVS-K100) introduces cells derived from the coding gene of Cas9 into 293T cells, HeLa cells, THP-1 cells, and HUVEC cells, respectively.
  • VEGFA-F1 TGCCGCTCACTTTGATGTCT
  • VEGFA-R1 GAGCCTCAGCCCCTCCA
  • VEGFA-F3 ATGGCACATTGTCAGAGGGA
  • VEGFA-R3 GGGAGCAGGAAAGTGAGGTTA
  • VEGFA-outer-F1 (mouse) GGTCAGAGAGAGCGCGCGGG
  • VEGFA-outer-R1 GGGCACGACCGCTTACCTTGG VEGFA-inner-F1 (mouse) GACCAGTCGCTGACGGACAGA VEGFA-inner-R1 (mouse) CGCGGCTCGCGCTCCCTCT Oct4-outer-F1 CTTCTGAGACATGATGCTCTTCCT Oct4-outer-R1 TCATGGGTGAGGGTAGTCTG Oct4-inner-F1 CATCCCTTGGATGTGCCAGT Oct4-inner-R1 TTAGAGGGGAGATGCGGTCA E1-1R/2F-WF/NF TGCGTGTCAAGGAATGGAGAG E1-1R/2F-WR GACAGCCTTTCCAGTGATTCAG E1-1R/2F-NR AGGCTCTAAAGACGGGCTTGAC E1-e3F-NF TCAGGAGGCCCAACCCAGAT E1-e3F-NR ACACGCAGAAGCAGCCTGAC E1-7-F-NF CTGG
  • This example provides that the CRISPR/Cas9 system elements for DNA editing are crRNA and tracrRNA.
  • crRNA is an RNA of formula I;
  • N is any one of a ribonucleotide and a ribonucleotide modification
  • the ribonucleotide is any one of A, U, C and G
  • the ribonucleotide modification is A, U a substance obtained by 2'-O-methyl modification or cholesterol modification by C or G;
  • x is the number of N, x is 20;
  • ncrRNA is any one of ncrRNA1, ncrRNA2, ncrRNA2y, ncrRNA3 and ncrRNA4;
  • ncrRNA1 is the RNA shown in SEQ ID NO. 1 in the Sequence Listing;
  • ncrRNA2 is the RNA shown in SEQ ID NO. 2 in the Sequence Listing;
  • ncrRNA2y is a substance obtained by subjecting the 10th, 11th and 13th nucleotides at the 5' end of ncrRNA2 to 2'-O-methyl modification, respectively;
  • ncrRNA3 is the RNA shown in SEQ ID NO. 3 in the Sequence Listing;
  • ncrRNA4 is the RNA shown in SEQ ID NO. 4 in the Sequence Listing.
  • tracrRNA is tracrRNA1, tracrRNA2, tracrRNA3 and tracrRNA4;
  • tracrRNA1 is the RNA shown in SEQ ID NO. 5 in the Sequence Listing;
  • tracrRNA2 is the RNA shown in SEQ ID NO. 6 in the Sequence Listing;
  • tracrRNA3 is the RNA shown in SEQ ID NO. 7 in the Sequence Listing;
  • tracrRNA4 is the RNA shown in SEQ ID NO. 8 in the Sequence Listing.
  • the first position of each sequence in the sequence listing is the 5' end of the sequence.
  • Phusion enzyme Phusion High-Fidelity DNA Polymerase
  • VEGFA Vascular endothelial growth factor A target gene fragment
  • the primer used in the first PCR amplification was VEGFA-F1/VEGFA-R1
  • the second PCR amplification was performed using the first PCR amplification product as a template, using the primer VEGFA-F3/VEGFA-R3 for amplification, and the second PCR.
  • the amplified product ie, DNA template was 485 bp in length.
  • the Cas9 nuclease, 10 ⁇ Cas9 buffer, crRNA, tracrRNA and H 2 O were mixed, incubated at 37 ° C for 15 min, then the DNA template of step 1 was added, and the mixture was incubated at 37 ° C for 70 min.
  • the reaction system is shown in Table 3.
  • the crRNA was replaced with H 2 O as a negative control (neg.).
  • the crRNA is VEGFA-crRNA1, VEGFA-crRNA2, VEGFA-crRNA3 and VEGFA-crRNA4, and each reaction system is a crRNA, and the structural formula of VEGFA-crRNA1, VEGFA-crRNA2, VEGFA-crRNA3 and VEGFA-crRNA4 is as in the formula 1
  • the Nx portions are identical, and the ncrRNAs are ncrRNA1, ncrRNA2, ncrRNA3 and ncrRNA4 of Example 1, respectively, and the sequence of Nx is: 5'-CCACAGGGAAGCUGGGUGAA-3', which is complementary to a partial sequence of the VEGFA gene; tracrRNA is implemented Example 1 tracrRNA2.
  • the Agilent 2200 Nucleic Acid Automated Electrophoresis System detects DNA fragment distribution and concentration results, and uses the calculation formula to obtain the enzyme digestion efficiency.
  • f(cut) (b+c)/(a+b+c); a is the molar concentration (pmol/l) of the uncut fragment, and b and c are the molar concentrations of the cut fragments (pmol/ l).
  • Figure 1 shows that when the crRNA is 32 nt (VEGFA-crRNA1), 34 nt (VEGFA-crRNA2), 36 nt (VEGFA-crRNA3) and 38 nt (VEGFA-crRNA4), the in vitro digestion system including crRNA, tracrRNA and Cas9 can be 485 bp.
  • the DNA template was cut.
  • the Cas9 digestion effect was optimal (Indel% was 89.2%), that is, when the length of the ncrRNA portion was 14 nt (that is, when ncrRNA2), Cas9 had the highest efficiency.
  • Cas9-293T cells were cultured in an incubator using DMEM medium containing 10% fetal bovine serum at 37 ° C, 5% CO 2 as a culture condition. Among them, Cas9-293T cells stably introduced the coding gene of Cas9 nuclease by gene editing, and expressed Cas9 nuclease (sequence is the sequence shown by Genbank ID: AQM52323.1 on NCBI).
  • Cas9-293T cell cells stably expressing Cas9 nuclease were plated in 24-well plates one day prior to transfection.
  • crRNA is VEGFA-crRNA1, VEGFA-crRNA2, VEGFA-crRNA3 and VEGFA-crRNA4 of step one, one crRNA per reaction system, and tracrRNA is tracrRNA2 of Example 1.
  • the crRNA was replaced with H 2 O as a negative control (neg.).
  • the specific method is as follows:
  • the DMEM medium containing 10% fetal bovine serum was added to 1 ml, and the cells were returned to the incubator for 48 hours to extract the genomic DNA of the cells.
  • DNA was extracted according to the TIANGEN Blood/tissue/cell genomic DNA extraction kit instructions.
  • the VEGFA target gene was amplified using Phusion enzyme.
  • the primer used for the first PCR amplification was VEGFA-F1/VEGFA-R1, and the second PCR amplification was performed using the primer VEGFA-F3/VEGFA-R3 using the first PCR amplification product as a template.
  • buffer2 is T7Endonuclease I (NEB, item number: M0302L):
  • the annealing reaction was carried out on a PCR machine, and the annealing conditions were as follows:
  • the annealing product of step 3 was digested with NEB T7 endonuclease I (T7EI), and after incubation at 37 ° C for 40 min, 1 ⁇ l of proteinase K was added and incubated at 37 ° C for 5 min.
  • T7EI NEB T7 endonuclease I
  • the digested samples were used for detection by the Agilent 2200 Nucleic Acid Automated Electrophoresis System.
  • the results are shown in Figure 2.
  • the results indicate that Cas9 can target 485 bp of target DNA when the length of crRNA is 32 nt (VEGFA-crRNA1), 34 nt (VEGFA-crRNA2), 36 nt (VEGFA-crRNA3) and 38 nt (VEGFA-crRNA4).
  • the fragment is cut open.
  • the Cas9 digestion effect was optimal (Indel% was 29.4%), that is, when the length of the ncrRNA portion was 14 nt (that is, when ncrRNA2), Cas9 had the highest efficiency.
  • Example 3 the effect of different tracrRNA lengths on DNA editing efficiency
  • the crRNA was detected as VEGFA-crRNA5, and the tracrRNA was the tracrRNA1, tracrRNA2, tracrRNA3 and tracrRNA4 of Example 1, respectively, including the enzymatic cleavage efficiency of the in vitro digestion system of crRNA, tracrRNA and Cas9.
  • the reaction system was a tracrRNA and replaced tracrRNA with water as a negative control (neg.).
  • VEGFA-crRNA5 is UGACUGCCGUCUGCACACCC GUUUUAGAGCUAUG (SEQ ID NO. 28).
  • the distribution and concentration results of DNA fragments detected by the Agilent 2200 Nucleic Acid Automated Electrophoresis System indicate that cas9 is tracrRNA when the length is 64 nt (tracrRNA1), 66 nt (tracrRNA2), 68 nt (tracrRNA3) and 70 nt (tracrRNA4).
  • a 485 bp DNA template can be cleaved. From the Indel% results, it was found that when the length of tracrRNA was 66 nt (that is, when tracrRNA2), the Cas9 digestion effect was optimal, and the Indel% was 90.7%.
  • the crRNA is the VEGFA-crRNA5 of step one
  • the tracrRNA is the tracrRNA1, tracrRNA2, tracrRNA3 and tracrRNA4 of Example 1, respectively, the cleavage efficiency of Cas9, a tracrRNA of each reaction system, and The tracrRNA was replaced with water as a negative control (neg.).
  • the distribution and concentration results of the DNA fragments detected by the Agilent 2200 Nucleic Acid Automated Electrophoresis System indicate that cas9 is tracrRNA when the length is 64 nt (tracrRNA1), 66 nt (tracrRNA2), 68 nt (tracrRNA3) and 70 nt (tracrRNA4).
  • a 485 bp target fragment can be cleaved. It can be seen from the Indel% results that when the length of tracrRNA is 66 nt (that is, when tracrRNA2), the Cas9 digestion effect is optimal, and the Indel% is 29.8%.
  • the target gene VEGFA fragment was amplified using the 293T cell genome as a template, and the specific method was the same as in Example 1.
  • the crRNA, the tracrRNA of the trafRNA, the tracrRNA of the trafRNA, the cleavage efficiency of the in vitro digestion system of the crRNA, the tracrRNA and the Cas9, respectively, were detected.
  • VEGFA-crRNA2-1 is a substance obtained by subjecting the 2nd, 30th, 31st, and 33rd positions of the crRNA5 of Example 3 to 2'-O-methyl modification, as follows:
  • m represents 2'-O-methyl modification
  • VEGFA-crRNA2-2 is a substance obtained by modifying cholesterol of crRNA2, as follows:
  • Chol represents cholesterol modification.
  • the VEGFA-crRNA2-1 and VEGFA-crRNA2-2 were respectively detected as the step 1 in the crRNA, and the tracrRNA of the tracrRNA was the cleavage efficiency of the Cas9, and a crRNA in each reaction system. .
  • the crRNA was replaced with H 2 O as a negative control (neg.).
  • Example 5 Different cell lines were detected by enzyme digestion
  • Cas9-293T cells were replaced with Cas9-HeLa cells, Cas9-THP-1 cells and Cas9-HUVEC cells according to the method of step 2 in Example 2. The other steps were unchanged, and the efficiency of Cas9 digestion was determined by different cell lines. Affects each cell of a single reaction system.
  • crRNA VEGFA-crRNA2 2 as an example of embodiment, it is tracrRNA2 tracrRNA Example 1, using H 2 O as a negative control replaced tracrRNA (neg.).
  • Target gene Oct4 (organic cation/carnitine transporter4) gene, EMX1 (Empty spiracles homeobox 1) gene, Beta-3 gene and Beta-5 gene, among which Beta-3 and Beta-5 are beta thalassemia genes, Beta-3
  • the gene contains the ⁇ thalassemia gene mutation site CD17 (HBB: c.52A>T), and the Beta-5 gene contains the ⁇ thalassemia gene mutation site CD17 (HBB: c.52A>T).
  • the crRNA used for the Oct4 gene were Oct4-E-T1-crRNA, Oct4-E-T2-crRNA and Oct4-E-T3-crRNA, and the tracrRNA was the tracrRNA2 of Example 1.
  • the structure of the crRNA is as shown in the formula I of Example 1, and the ncrRNA portions of each crRNA are identical, and are all ncrRNA2 of Example 1, and the Nx portions are different.
  • the sequences of each crRNA are as follows:
  • Oct4-E-T1-crRNA GGUUAUUUCUAGAAGUUAGG GUUUUAGAGCUAUG (SEQ ID NO. 13)
  • Oct4-E-T2-crRNA CUUCUACAGACUAUUCCUUG GUUUUAGAGCUAUG (SEQ ID NO. 14)
  • Oct4-E-T3-crRNA GGAAAGGGGAGAUUGAUAAC GUUUUAGAGCUAUG (SEQ ID NO. 15).
  • the crRNA used for the EMX1 gene was E1R-crRNA, E7F-crRNA, Ee3F-crRNA and E2F-crRNA, and the tracrRNA was the tracrRNA2 of Example 1.
  • the structure of the crRNA is as shown in the formula I of Example 1, and the ncrRNA portions of each crRNA are identical, and are all ncrRNA2 of Example 1, and the Nx portions are different.
  • the sequences of each crRNA are as follows:
  • E1R-crRNA AAGGCCGUGCGGAUCCGCUU GUUUUAGAGCUAUG (SEQ ID NO. 16)
  • E7F-crRNA AGGUCCGACGUGUUGGAGUG GUUUUAGAGCUAUG (SEQ ID NO. 17)
  • Ee3F-crRNA CGAUGUCACCUCCAAUGACU GUUUUAGAGCUAUG (SEQ ID NO. 18)
  • E2F-crRNA UCUCGCCCUCGCAGCUGCUG GUUUUAGAGCUAUG (SEQ ID NO. 19)
  • the crRNA used for the Beta-3 gene was Beta-3-T1-crRNA and Beta-3-T3-crRNA, and the tracrRNA was the tracrRNA2 of Example 1.
  • the structure of the crRNA is as shown in the formula I of Example 1, and the ncrRNA portions of each crRNA are identical, and are all ncrRNA2 of Example 1, and the Nx portions are different.
  • the sequences of each crRNA are as follows:
  • Beta-3-T1-crRNA GUGUGGCAAAGGUGCCCUUG GUUUUAGAGCUAUG (SEQ ID NO. 20)
  • Beta-3-T3-crRNA ACCAAUAGAAACUGGGCAUG GUUUUAGAGCUAUG (SEQ ID NO. 21)
  • the crRNA used for the Beta-5 gene was Beta-5-T3-crRNA, and the tracrRNA was the tracrRNA2 of Example 1.
  • the structure of crRNA is as shown in Formula I of Example 1, and the sequence is as follows:
  • Beta-5-T3-crRNA CGUAAAUACACUUGCAAAGG GUUUUAGAGCUAUG. (SEQ ID NO. 22)
  • the 293T cell genome was used as a template to amplify a fragment of the target gene Oct4 gene, EMX1 gene, Beta-3 gene and Beta-5 gene, that is, a DNA template.
  • the primers used for the first amplification of the Oct4 gene DNA template were Oct4-outer-F1 and Oct4-outer-R1, and the primers used for the second amplification were Oct4-inner-F1 and Oct4-inner-R1.
  • the EMX1 gene DNA templates were EMX1-E2F, EMX1-E1R, EMX1-E7F and EMX1-Ee3F.
  • the primers used in each template were as follows:
  • EMX1-E2F The primers used for the first amplification of the DNA template were E1-1R/2F-WF/NF and E1-1R/2F-WR, and the primers used for the second amplification were E1-1R/2F-WF/NF and E1-1R/2F-NR.
  • EMX1-E1R The primers used for the first amplification of the DNA template were E1-1R/2F-WF/NF and E1-1R/2F-WR, and the primers used for the second amplification were E1-1R/2F-WF/NF and E1-1R/2F-NR.
  • EMX1-E7F The primers used for the first amplification of the DNA template were E1-7-F-WF and E1-7-F-WR, and the primers used for the second amplification were E1-7-F-NF and E1-7-. F-NR.
  • EMX1-Ee3F The primers used for the first amplification of the DNA template were E1-e3F-WF and E1-e3F-WR, and the primers used for the second amplification were E1-e3F-NF and E1-e3F-NR.
  • the primers used for the first amplification of the Beta-3 gene DNA template were Beta-outer-F1 and Beta-outer-R1, and the primers used for the second amplification were Beta3-inner-F1 and Beta3-inner-R1.
  • the primers used for the first amplification of the Beta-5 gene DNA template were Beta-outer-F1 and Beta-outer-R1, and the primers used for the second amplification were Beta5-inner-F1 and Beta5-inner-R1.
  • the DNA template of step 1 was digested with an in vitro digestion system containing crRNA, tracrRNA and Cas9 according to the method of 2-5 in the first step of Example 2, and each reaction system was a crRNA.
  • the crRNA was replaced with H 2 O as a negative control (neg.).
  • the enzymatic cleavage efficiency of Cas9 in vitro at different concentrations was measured.
  • concentration of tracrRNA in each system varied with the concentration of crRNA, and remained in the same reaction system.
  • the concentration of tracrRNA is the same as the concentration of crRNA, and the concentrations of other substances are the same as in Table 3.
  • Enzyme digestion efficiency of EMX1 gene DNA template for detection of Cas9 from different crRNAs (E1R-crRNA, E7F-crRNA, Ee3F-crRNA and E2F-crRNA), EMX1 gene DNA template EMX1-E2F, EMX1-E1R, EMX1-E7F
  • the crRNA corresponding to EMX1-Ee3F are E2F-crRNA, E1R-crRNA, E7F-crRNA and Ee3F-crRNA, respectively, and each system is the same as Table 3.
  • Beta-3 gene DNA template can be cleaved by Cas9 in the presence of Beta-3-T1-crRNA and Beta-3-T3-crRNA
  • Beta-5 gene DNA can be obtained by Cas9 in the presence of Beta-5-T3-crRNA.
  • the template is cut open ( Figure 10).
  • the enzymatic cleavage efficiency of the Cas9 nuclease against the Oct4 gene, the EMX1 gene, the Beta-3 gene and the Beta-5 gene was examined according to the method of the second step of Example 2, and each reaction system was a crRNA.
  • the crRNA was replaced with H 2 O as a negative control (neg.).
  • the crRNA used for the Oct4 gene are Oct4-E-T1-crRNA, Oct4-E-T2-crRNA and Oct4-E-T3-crRNA, and the method is the same as that in the second step of the second embodiment.
  • the primers used for the Oct4 gene are: Oct4-outer-F1 and Oct4-outer-R1 for the first amplification, and Oct4-inner-F1 for the second amplification. And Oct4-inner-R1.
  • the results showed that the Oct4 gene could be cleaved by the Cas9 nuclease in the presence of different crRNAs (Fig. 11).
  • the crRNA used in the EMX1 gene is Ee3F-crRNA, and the amount of Ee3F-crRNA is 50 ng, 100 ng, 200 ng, and 400 ng, respectively.
  • the concentration of tracrRNA in the same reaction system is the same as that of the crRNA.
  • the amount and method of the other substances are the same as in the second step of the second embodiment.
  • the primers used for the EMX1 gene were: E1-e3F-WF and E1-e3F-WR for the first amplification, and E1-e3F-NF for the second amplification.
  • E1-e3F-NR The results showed that the EM9 gene could be cleaved by the Cas9 nuclease at different amounts of crRNA (Fig. 12).
  • the crRNA used in the Beta-3 gene is Beta-3-T1-crRNA and Beta-3-T3-crRNA
  • the crRNA used in the Beta-5 gene is Beta-5-T3-crRNA
  • the method is the same as that in the second step of the second embodiment.
  • the primers used in the Beta-3 gene are: the primers used for the first amplification are Beta-outer-F1 and Beta-outer-R1, and the primers used for the second amplification are Beta3-inner.
  • Beta-5 genes were: Beta-outer-F1 and Beta-outer-R1 for the first amplification and Beta5-inner-F1 for the second amplification. And Beta5-inner-R1.
  • the results showed that the Beta-3 gene can be cleaved by Cas9 nuclease in the presence of Beta-3-T1-crRNA and Beta-3-T3-crRNA, and Beta-5 can be used in the presence of Beta-5-T3-crRNA. Gene incision ( Figure 13).
  • Cas9-293T cells were replaced with C2C12 mouse myoblasts containing Cas9 according to the method of step 2 of Example 2. The other steps were unchanged. The efficiency of Cas9 on the VEGFA gene in C2C12 mouse myoblasts containing Cas9 was detected.
  • the crRNA used was a modified VEGFA-crRNA6, VEGFA-crRNA6-1, and the tracrRNA was tracrRNA2 of Example 1. The crRNA was replaced with H 2 O as a negative control (neg.).
  • VEGFA-crRNA6 AAGAGGAGAGGGGGCCGCAG GUUUUAGAGCUAUG (SEQ ID NO. 29),
  • VEGFA-crRNA6-1 is a substance obtained by 2'-O-methyl modification of the 2nd, 30th, 31st and 33rd positions of VEGFA-crRNA6, as follows:
  • C2C12 mouse myoblast containing Cas9 The preparation method of C2C12 mouse myoblast containing Cas9 is as follows:
  • C57BL/6 mice (Beijing Weitong Lihua Experimental Animal Co., Ltd.) were injected with sodium pentobarbital anesthesia: 30 mg/kg intravenously or intraperitoneally.
  • the biceps femoral genomic DNA was extracted 7 days after transfection using the blood/tissue/cell genome extraction kit.
  • the method is the same as in the second step of the second embodiment.
  • the primers used were VEGFA-outer-F1 (mouse), VEGFA-outer-R1 (mouse), VEGFA-inner-F1 (mouse) and VEGFA-inner-R1 (mouse).
  • Enzyme digestion was carried out using NEB T7 endonuclease I (T7EI) in the same manner as in Example 2, Step 2.
  • the digested samples were used for detection by the Agilent 2200 Nucleic Acid Automated Electrophoresis System.
  • the calculation method is the same as in the second embodiment.
  • the efficiency of TP53 (tumor protein p53) gene and exon was detected.
  • the crRNA used were TP53-P-T1, TP53-P-T2, TP53-E-T1, TP53-E-T2 and TP53-E-T3.
  • Each reaction system is a crRNA, and the tracrRNA used is the tracrRNA2 of Example 1.
  • the crRNA was replaced with H 2 O as a negative control (neg.).
  • the sequence of the crRNA used is as follows:
  • TP53-P-T1 CAAUUCUGCCCUCACAGCUC GUUUUAGAGCUAUG (SEQ ID NO. 23)
  • TP53-P-T2 CCCCAAAAUGUUAGUAUCUA GUUUUAGAGCUAUG (SEQ ID NO. 24)
  • TP53-E-T1 CCCUCCCAUGUGCUCAAGAC GUUUUAGAGCUAUG (SEQ ID NO. 25)
  • TP53-E-T2 UGGGAGCGUGCUUUCCACGA GUUUUAGAGCUAUG (SEQ ID NO. 26)
  • TP53-E-T3 CCAGUCUUGAGCACAUGGGA GUUUUAGAGCUAUG (SEQ ID NO. 27)
  • the 293T cell genome was used as a template to amplify the target gene TP53 fragment, a DNA template.
  • the primers used for the first amplification were TP53-outer-FP and TP53-outer-RP, and the primers used for the second amplification were TP53-inner-FP and TP53-inner-RP.
  • the DNA template of step 1 was digested according to the method of 2-5 in the first step of Example 2 using an in vitro digestion system containing crRNA, tracrRNA and Cas9.
  • the cleavage efficiency of the Cas9 nuclease on the TP53 fragment was examined by the method of the second step of Example 2.
  • the crRNA was replaced with H 2 O as a negative control (neg.).
  • the Cas9 nuclease, 10 ⁇ Cas9 buffer, crRNA, tracrRNA and H 2 O were mixed, incubated at 37 ° C for 15 min, then the DNA template was added, and the mixture was incubated at 37 ° C for 70 min. Replace with h 2 O as crRNA as a negative control (neg.).
  • the DNA template was the Oct4 gene DNA template of Example 6, the crRNA used were Oct4-E-T1-crRNA, Oct4-E-T2-crRNA and Oct4-E-T3-crRNA, and the tracrRNA was the tracrRNA2 of Example 1.
  • the in vitro digestion system of Cas9 is as follows.
  • the system contains three kinds of crRNA, namely Oct4-E-T1-crRNA, Oct4-E-T2-crRNA and Oct4-E-T3-crRNA:
  • the experiment was performed using Oct4-E-T2-crRNA alone as the crRNA, and as a control, the concentration of Oct4-E-T2-crRNA was 400 nM.
  • the Cas9 donor is a 293T cell stably expressing Cas9, namely Cas9-293T, and the specific method is the same as in Example 2.
  • the crRNA and tracRNA used were the crRNA2 of Example 2 and the tracrRNA2 of Example 1, respectively.
  • the Cas9 donor is the Cas9 plasmid (ie PX260).
  • the transfection method is the same as (3) in step 1 of Example 2.
  • the crRNA and tracRNA used were the crRNA2 of Example 2 and the tracrRNA2 of Example 1, respectively.
  • the DNA fragment distribution and concentration results were detected by the Agilent 2200 nucleic acid automated electrophoresis system. From Fig. 19, it was found that the transfected plasmid expressed Cas9 in normal 293T cells, and then transfected with crRNA and tracrRNA. It is also possible to digest the DNA fragment of interest, which is less efficient than Cas9 stable cells (Cas9-293T).
  • the enzymatic cleavage efficiency of the in vitro digestion system including crRNA, tracrRNA and different concentrations of Cas9 was respectively detected according to the method of the first step of Example 2.
  • the concentrations of Cas9 were 20 nM, 100 nM, 200 nM, and 400 nM, respectively, and the other components were the same as in Step 2 of Example 2.
  • the crRNA and tracRNA used were VEGFA-crRNA2 of Example 2 and tracrRNA2 of Example 1, respectively.
  • the distribution and concentration results of the DNA fragments detected by the Agilent 2200 Nucleic Acid Automated Electrophoresis System indicate that the enzyme digestion efficiency is above 50% when the protein concentration is 20-400 nM, and the protein concentration is 100-400 nM.
  • the enzymatic cleavage efficiency is above 80%; when the protein concentration is 100-200 nM, the enzymatic cleavage efficiency is above 85%.

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Abstract

提供了一种用于DNA编辑的系统，其包括crRNA与tracrRNA，crRNA为式Ⅰ所示RNA：Nx-ncrRNA(式Ⅰ)，Nx为间隔序列，ncrRNA为序列表中SEQ ID NO.1-4所示的RNA，tracrRNA为序列表中SEQ ID NO.5-8所示的RNA。

Description

用于DNA编辑的系统及其应用 技术领域
本发明涉及生物技术领域中，用于DNA编辑的系统及其应用。
背景技术
CRISPR–Cas系统是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御机制，可用来对抗入侵的病毒及外源DNA。根据功能元件的不同，CRISPR–Cas系统可以分为Ⅰ类系统、Ⅱ类系统和Ⅲ类系统。这三类系统又可以根据其编码Cas蛋白的基因不同而分为更多的亚类。Ⅰ类系统和Ⅲ类CRISPR–Cas系统只需要crRNA和Cas蛋白两种元件的参与，而Ⅱ类CRISPR–Cas系统包括crRNA、tracrRNA和Cas蛋白三种元件。
CRISPR/Cas9系统通过将入侵噬菌体和质粒DNA的片段整合到CRISPR中，并利用相应的CRISPR RNAs(crRNAs)来指导同源序列的降解，从而提供免疫性。此系统的工作原理是crRNA(CRISPR-derived RNA)通过碱基配对与tracrRNA(trans-activating RNA)结合形成tracrRNA/crRNA复合物，此复合物引导核酸酶Cas9蛋白在与crRNA配对的靶位点剪切双链DNA。
Cas9有两个关键的结构域：HNH和RuvC，它们分别切开DNA双链中的一条单链。产生DNA双链断裂(DSB)，细胞启动修复机制，细胞可通过非同源末端连接(NHEJ)这种不精确的修复方式产生基因定点突变，也可以通过同源重组(HR)方式修复实现精确的基因定点插入或基因替换。Cas9已经在细菌、人类细胞、斑马鱼和小鼠中成功进行基因组工程研究。
发明内容
本发明的目的是提供用于DNA编辑的CRISPR/Cas系统。
本发明首先提供了一种用于DNA编辑的系统，所述系统为CRISPR-Cas系统，包括甲或乙：
甲、下述N1)和N2)：
N1)名称为RNA-2的RNA或与所述RNA-2相关的生物材料；
N2)名称为RNA-1的RNA或与所述RNA-1相关的生物材料；
所述RNA-2为式Ⅰ所示RNA；
Nx-ncrRNA
式Ⅰ；
其中，Nx为CRISPR/Cas系统中的间隔序列(Spacer)；
所述ncrRNA为如下a1)至a4)中的任一种：
a1)序列表中SEQ ID NO.1所示的RNA；
a2)在a1)的5′端和/或3′端添加一个或几个核苷酸得到的RNA；
a3)与a1)或a2)限定的RNA具有85％以上的同一性的RNA；
a4)序列表中SEQ ID NO.1所示的RNA的修饰物；
与所述RNA-2相关的生物材料为下述A1)至A5)中的任一种：
A1)编码所述RNA-2的DNA分子；
A2)含有A1)所述DNA分子的表达盒；
A3)含有A1)所述DNA分子的重组载体、或含有A2)所述表达盒的重组载体；
A4)含有A1)所述DNA分子的重组微生物、或含有A2)所述表达盒的重组微生物、或含有A3)所述重组载体的重组微生物；
A5)含有A1)所述DNA分子的细胞系、或含有A2)所述表达盒的细胞系；
所述RNA-1为如下b1)至b4)中的任一种：
b1)序列表中SEQ ID NO.5所示的RNA；
b2)在b1)的5′端和/或3′端添加一个或几个核苷酸得到的RNA；
b3)与b1)或b2)限定的RNA具有85％以上的同一性的RNA；
b4)序列表中SEQ ID NO.5所示的RNA的修饰物；
与所述RNA-1相关的生物材料为下述B1)至B5)中的任一种：
B1)编码所述RNA-1的DNA分子；
B2)含有B1)所述DNA分子的表达盒；
B3)含有B1)所述DNA分子的重组载体、或含有B2)所述表达盒的重组载体；
B4)含有B1)所述DNA分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述 重组载体的重组微生物；
B5)含有1)所述DNA分子的细胞系、或含有B2)所述表达盒的细胞系；
所述ncrRNA与所述RNA-1部分片段互补形成包括茎区和环区的结构；
乙、所述RNA-2。
所述RNA-2的Nx部分位于5′端，所述RNA-2的ncrRNA部分位于3′端，二部分通过核苷间键连接。Nx为RNA序列，与进行编辑的目的DNA的片段互补，满足为CRISPR/Cas系统中的间隔序列的要求。
N可为核糖核苷酸或核糖核苷酸修饰物；x为N的个数。
其中，x为非零自然数。x具体可为下述e1)-e3)中的任一种：
e1)15～22间的任一整数；
e2)19～21间的任一整数；
e3)20。
所述RNA-2可通过ncrRNA与所述RNA-1的部分片段形成复合物，CRISPR-Cas系统中的Cas蛋白(即Cas核酸酶)可与该复合物结合实现对DNA的编辑。
所述添加一个或几个核苷酸具体可为添加一至十个核苷酸。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的SEQ ID NO.1或SEQ ID NO.5所示的核苷酸序列具有85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
所述严格条件是在2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；或，0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
上述85％以上同一性，可为85％、90％或95％以上的同一性。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的SEQ ID NO.1或SEQ ID NO.5进行突变。那些经过人工修饰的，具有与本发明的SEQ ID NO.1或SEQ ID NO.5的核苷酸序列75％或者更高同一性的核苷酸，只要且具有相同功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
所述细胞系不包括繁殖材料。
上述系统中，a2)所述RNA可为如下a21)至a23)中的任一种：
a21)长度为12-14nt的RNA；
a22)长度为12-16nt的RNA；
a23)长度为12-18nt的RNA。
b2)所述RNA可为如下b21)至b24)中的任一种：
b21)长度为64-66nt的RNA；
b22)长度为64-68nt的RNA；
b23)长度为64-70nt的RNA；
b24)长度为64-86nt的RNA。
所述核糖核苷酸可为A、U、C或G。所述核糖核苷酸修饰物可为对A、U、C或G在核糖、磷酸骨架和/或碱基进行修饰得到的物质；
所述修饰物为对所述RNA中的至少一个核苷酸在核糖、磷酸骨架和/或碱基进行修饰得到的物质。
所述修饰可为2′-O-甲基修饰、2′-脱氧修饰、2′-氟代修饰或胆固醇修饰。
所述DNA可为下述M1)-M5)中的任一种：
M1)真核生物的DNA；
M2)动物的DNA；
M3)哺乳动物的DNA；
M4)人的DNA；
M5)小鼠的DNA。
上述系统中，a21)所述RNA可为序列表中SEQ ID NO.2所示的RNA；
a22)所述RNA可为序列表中SEQ ID NO.3所示的RNA；
a23)所述RNA可为序列表中SEQ ID NO.4所示的RNA。
b21)所述RNA可为序列表中SEQ ID NO.6所示的RNA；
b22)所述RNA可为序列表中SEQ ID NO.7所示的RNA；
b23)所述RNA可为序列表中SEQ ID NO.8所示的RNA。
所述修饰可为2′-O-甲基修饰、2′-脱氧修饰、2′-氟代修饰或胆固醇修饰。
所述DNA可为VEGFA(Vascular endothelial growth factor A)基因、EMX1(Empty spiracles homeobox 1)基因、Oct4(organic cation/carnitine transporter4)基因、β地中海贫血基因、TP53(tumor protein p53)基因或TP53基因的启动子。
上述系统中，所述RNA-2可为SEQ ID NO.9-29中任一序列所示的RNA。
在所述DNA为VEGFA基因时，所述RNA-2为SEQ ID NO.9-12、SEQ ID NO.28和SEQ ID NO.29中至少一条序列所示的RNA；
在所述DNA为Oct4基因时，所述RNA-2为SEQ ID NO.13-15中至少一条序列所示的RNA；
在所述DNA为EMX1基因时，所述RNA-2为SEQ ID NO.16-19中至少一条序列所示的RNA；
在所述DNA为β地中海贫血基因时，所述RNA-2为SEQ ID NO.20-22所示的RNA；
在所述DNA为TP53基因时，所述RNA-2为SEQ ID NO.23和/或24所示的RNA；
在所述DNA为TP53基因的启动子时，所述RNA-2为SEQ ID NO.25-27中至少一条序列所示的RNA。
所述系统可为CRISPR-Cas系统，具体可为Ⅱ类CRISPR-Cas系统，如CRISPR/Cas9系统。
上述系统还可包括N3)，N3)为Cas9核酸酶或与Cas9核酸酶相关的生物材料；
所述与Cas9核酸酶相关的生物材料为下述C1)至C7)中的任一种：
C1)编码Cas9核酸酶的核酸分子；
C2)含有C1)所述核酸分子的表达盒；
C3)含有C1)所述核酸分子的重组载体、或含有C2)所述表达盒的重组载体；
C4)含有C1)所述核酸分子的重组微生物、或含有C2)所述表达盒的重组微生物、或含有C3)所述重组载体的重组微生物；
C5)含有C1)所述核酸分子的细胞系、或含有C2)所述表达盒的细胞系。
上述系统中，所述Cas9核酸酶可来源于R1)或R2)：
R1)细菌；
R2)链球菌属、葡萄球菌属、罗氏菌属、奈瑟菌属、细小棒菌属或乳杆菌属。
所述链球菌属细菌具体可为脓链球菌(Streptococcus pyogenes)。所述葡萄球菌属细菌具体可为金黄色葡萄球菌。所述Cas9核酸酶具体可为Genbank ID：AQM52323.1所示的蛋白质。
所述系统还可包括利用CRISPR/Cas9DNA编辑方法进行DNA编辑中除crRNA、tracrRNA和Cas9核酸酶外所需要的其他试剂和/或仪器。
当所述系统含有RNA-2和RNA-1时，RNA-1和RNA-2的摩尔比可为1:(1-1.5)。
当所述系统含有Cas9和tracrRNA和crRNA时，Cas9和tracrRNA和crRNA的摩尔比可为1：(1-20):(1:20),具体可为1：(1-8):(1:8)，进一步可为1：(1-4):(1:4)，更进一步可为1：(2-4):(2-4)。
当RNA-2为至少两条RNA时，上述摩尔比中的RNA-2以多条RNA的总浓度计。
具体来说，所述系统可仅为所述RNA-2，也由上述N1)和N2)组成，也可由上述N1)、N2)和N3)组成。
本发明还提供了下述O1)或O2)：
O1)所述RNA-2或与所述RNA-2相关的生物材料；
O2)所述RNA-1或与所述RNA-1相关的生物材料。
本发明还提供了下述任一应用：
X1、所述ncrRNA在制备crRNA中的应用；
X2、所述ncrRNA在DNA编辑中的应用；
X3、所述ncrRNA在制备DNA编辑产品中的应用；
X4、所述RNA-1在作为tracrRNA中的应用；
X5、所述RNA-1或与所述RNA-1相关的生物材料在DNA编辑中的应用；
X6、所述RNA-1或与所述RNA-1相关的生物材料在制备DNA编辑产品中的应用；
X7、所述RNA-2在作为crRNA中的应用；
X8、所述RNA-2或与所述RNA-2相关的生物材料在DNA编辑中的应用；
X9、所述RNA-2或与所述RNA-2相关的生物材料在制备DNA编辑产品中的应用；
X10、所述系统在DNA编辑中的应用；
X11、所述系统在干扰体内或体外基因的表达中的应用；
X12、所述系统在建立DNA发生编辑的动物、植物或细胞模型中的应用。
所述产品可为可为CRISPR-Cas系统，具体可为Ⅱ类CRISPR-Cas系统，如CRISPR/Cas9系统。
本发明还提供了编辑DNA的方法，所述方法包括：利用所述系统处理DNA实现所述DNA的编辑。
利用所述系统进行DNA进行编辑的体系中，在所述系统含有RNA-2时，RNA-2的浓度可为100-800nM，具体可为200-400nM。在所述系统含有RNA-1时，RNA-1的浓度可为100-800nM，具体可为200-400nM。所述CRISPR/Cas9系统还可含有Cas9核酸酶或与Cas9核酸酶相关的生物材料。
在所述体系含有Cas9核酸酶时，Cas9核酸酶的浓度可为20-400nM，具体可为100-400nM，进一步可为100-200nM。在所述体系含有含有Cas9核酸酶编码基因的载体时，所述载体的用量可为1-200μg，进一步可为20-100μg，更进一步可为30-50μg或1-5μg。所述载体的用量优选为3μg。
在利用所述CRISPR/Cas9系统对细胞中的基因进行编辑时，所述体系中所述细胞与RNA-2的配比可为0.5万-6万个细胞/100nM RNA-2，进一步可为0.75万-4万个细胞/100nM RNA-2，更进一步可为为1-1.25万个细胞/100nM RNA-2。
所述细胞可为真核细胞。所述细胞进一步可为哺乳动物细胞，如293T细胞、HeLa细胞、THP1细胞、HUVEC细胞或C2C12小鼠成肌细胞。
本发明还提供了所述系统的制备方法，所述方法包括将所述系统中的各物质分别独立包装。
所述系统的制备方法还可包括合成所述系统的各RNA。所述合成可利用化学方法合成。
本发明中，所述DNA均可为下述M1)-M5)中的任一种：
M1)真核生物的DNA；
M2)动物的DNA；
M3)哺乳动物的DNA；
M4)人的DNA；
M5)小鼠的DNA。
所述编辑的效率为至少1％或1％以上，如2％、3％、5％、10％、15％、20％、25％、30％、40％、50％、60％、70％、80％、85％或90％。
本发明中，每条序列的第1位均为该条序列的5′末端。
所述DNA编辑可为对DNA的切割。
实验证明，本发明的用于DNA编辑的系统可作为CRISPR/Cas9系统编辑DNA，其中作为crRNA的RNA-2中ncrRNA部分的长度为14nt时(即为ncrRNA2时)Cas9酶切效率最高，体外酶切效果达89.2％，细胞内酶切效果达29.4％；作为tracrRNA的RNA-1的长度为66nt时Cas9酶切效果为最优，体外酶切效果达90.7％，细胞内酶切效果达29.8％。另外，RNA-2经过修饰后，CRISPR/Cas9系统中的Cas9的酶切效果无明显变化。在RNA-2和RNA-1均可用于CRISPR/Cas9系统元件利用CRISPR/Cas9系统编辑目的DNA时，DNA的编辑不受细胞和目的DNA的影响，均可实现DNA的编辑，利用该系统还可以直接对动物体内DNA进行编辑，在利用该CRISPR/Cas9系统编辑DNA时，还可以通过利用多条序列不同的RNA-2提高编辑效率，利用稳定表达Cas9的细胞可以提高DNA编辑效率。本发明的RNA-2和RNA-1序列短，易于通过化学合成获得，有利于提升RNA的纯度和规模生产，并简化CRISPR/Cas9系统的制备与应用。另外化学合成的RNA元件无需质粒表达，规避了质粒构建的问题(如质粒间相容性、质粒载体承载能力、多个质粒体系构建困难等)，且更易于化学修饰，适宜体内应用)。表明，可以利用本发明的RNA-2和RNA-1作为CRISPR/Cas9系统中的crRNA和tracrRNA元件用于编辑目的DNA。
附图说明
图1为不同crRNA下Cas9的体外酶切效果。
图2为不同crRNA下Cas9的细胞内酶切效果。
图3为不同tracrRNA下Cas9的体外酶切效果。
图4为不同tracrRNA下Cas9的细胞内酶切效果。
图5为crRNA的修饰对Cas9的体外酶切效果的影响。
图6为crRNA的修饰对Cas9的细胞内酶切效果的影响。
图7为针对VEGFA基因的不同细胞系中的Cas9核酸酶切割效果。
图8为不同Oct4-E-T1-crRNA浓度的Cas9核酸酶切割效果。其中，800、400和200均为Oct4-E-T1-crRNA的浓度，单位均为nM。
图9为不同crRNA下Cas9核酸酶对EMX1基因DNA模板的体外切割效果。其中，E1R、E7F、Ee3F和E2F分别表示E1R-crRNA、E7F-crRNA、Ee3F-crRNA和E2F-crRNA。
图10为不同crRNA的Cas9核酸酶体外切割效果。其中，Beta-3-T1、Beta-3-T3和Beta-5-T3分别表示Beta-3-T1-crRNA、Beta-3-T3-crRNA和Beta-5-T3-crRNA。
图11不同crRNA下Oct4基因的细胞内切割效果。其中，E-T1、E-T2和E-T3分别表示Oct4-E-T1-crRNA、Oct4-E-T2-crRNA和Oct4-E-T3-crRNA。
图12为不同Ee3F-crRNA用量下Cas9核酸酶对EMX1基因的细胞内切割效果。其中，400、200、100和50均为Ee3F-crRNA的用量，单位均为ng。
图13为不同crRNA下Cas9核酸酶对基因的细胞内切割效果。其中，Beta-3-T1、Beta-3-T3和Beta-5-T3分别表示Beta-3-T1-crRNA、Beta-3-T3-crRNA和Beta-5-T3-crRNA。
图14为小鼠细胞(C2C12)和小鼠体内(in vivo)VEGFA基因切割效果。
图15为TP53片段的启动子和外显子区域细胞内切割效果。
图16为TP53片段的启动子和外显子区域体外切割效果。
图17为多条crRNA下Cas9核酸酶对Oct4基因的体外切割效果。其中，Mix表示Oct4-E-T1-crRNA、Oct4-E-T2-crRNA和Oct4-E-T3-crRNA共同的实验结果，ET2表示Oct4-E-T2-crRNA。
图18为多条crRNA下Cas9核酸酶对Oct4基因的细胞内切割效果。其中，Mix表示Oct4-E-T1-crRNA、Oct4-E-T2-crRNA和Oct4-E-T3-crRNA共同的实验结果，ET2表示Oct4-E-T2-crRNA。
图19为不同Cas9供体的酶切效果。
图20为不同Cas9浓度的酶切效果。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
表1、试剂来源
Figure PCTCN2018088105-appb-000001
其中Cas9核酸酶的序列为NCBI上Genbank ID：AQM52323.1所示的序列。该蛋白质来源于酿脓链球菌(Streptococcus pyogenes)。
下述实施例中的Cas9-293T细胞、Cas9-HeLa细胞、Cas9-THP-1细胞和Cas9-HUVEC细胞为利用广州易锦生物技术有限公司的Genome-TALER TM& Genome-CRISPR TM人类AAVS1 Safe Harbor基因敲入试剂盒(Catalog# SH-AVS-K100)分别向293T细胞、HeLa细胞、THP-1细胞和HUVEC细胞中导入Cas9的编码基因得到的细胞。
表2、引物序列表
引物 序列(5′-3′)
VEGFA-F1 TGCCGCTCACTTTGATGTCT
VEGFA-R1 GAGCCTCAGCCCCTCCA
VEGFA-F3 ATGGCACATTGTCAGAGGGA
VEGFA-R3 GGGAGCAGGAAAGTGAGGTTA
VEGFA-outer-F1(小鼠) GGTCAGAGAGAGCGCGCGGG
VEGFA-outer-R1(小鼠) GGGCACGACCGCTTACCTTGG
VEGFA-inner-F1(小鼠) GACCAGTCGCGCTGACGGACAGA
VEGFA-inner-R1(小鼠) CGCGGCTCGCGCTCCCTCT
Oct4-outer-F1 CTTCTGAGACATGATGCTCTTCCT
Oct4-outer-R1 TCATGGGTGAGGGTAGTCTG
Oct4-inner-F1 CATCCCTTGGATGTGCCAGT
Oct4-inner-R1 TTAGAGGGGAGATGCGGTCA
E1-1R/2F-WF/NF TGCGTGTCAAGGAATGGAGAG
E1-1R/2F-WR GACAGCCTTTCCAGTGATTCAG
E1-1R/2F-NR AGGCTCTAAAGACGGGCTTGAC
E1-e3F-NF TCAGGAGGCCCAACCCAGAT
E1-e3F-NR ACACGCAGAAGCAGCCTGAC
E1-7-F-NF CTGGGCCTGGTGGGAAATAG
E1-7-F-NR GTAAGGTCCGCCGAAGAAAG
E1-e3F-WF TTTCGGAGCAGTCGAGTG
E1-e3F-WR GTCTGAAGCTGCGACCTTG
E1-7-F-WF CTGGCTTTGCTGCTGTTCCTG
E1-7-F-WR GCAGTTAGAACAGCCCAGCTAGT
Beta-outer-F1 CTGAGACTTCCACACTGATGC
Beta-outer-R1 GCATATTCTGGAGACGCAGGA
Beta3-inner-F1 TGGTGTCTGTTTGAGGTTGCTA
Beta3-inner-R1 CATATTCTGGAGACGCAGGAAGA
Beta5-inner-F1 AAACATCAAGCGTCCCATAGA
Beta5-inner-R1 ACTGTGTTCACTAGCAACCTCA
TP53-outer-FP GGGTGTGATATTACGGAAAGCC
TP53-outer-RP TACACCCAGGTCTCCCAACA
TP53-inner-FP CCAGCTGAGAGCAAACGCAA
TP53-inner-RP AATACACGGAGCCGAGAGCC
实施例1、用于DNA编辑的CRISPR/Cas9系统元件的制备
本实施例提供了用于DNA编辑的CRISPR/Cas9系统元件为crRNA和tracrRNA。
crRNA为式Ⅰ所示RNA；
Nx-ncrRNA
式Ⅰ；
其中，N为核糖核苷酸和核糖核苷酸修饰物中的任一种，核糖核苷酸为A、U、C和G中的任一种，核糖核苷酸修饰物为对A、U、C或G进行2′-O-甲基修饰或胆固醇修饰得到的物质；
x为N的个数，x为20；
ncrRNA为ncrRNA1、ncrRNA2、ncrRNA2y、ncrRNA3和ncrRNA4中的任一种；
ncrRNA1为序列表中SEQ ID NO.1所示的RNA；
ncrRNA2为序列表中SEQ ID NO.2所示的RNA；
ncrRNA2y为分别对ncrRNA2的5′端始的第10、11和13位核苷酸进行2′-O-甲基修饰得到的物质；
ncrRNA3为序列表中SEQ ID NO.3所示的RNA；
ncrRNA4为序列表中SEQ ID NO.4所示的RNA。
tracrRNA为tracrRNA1、tracrRNA2、tracrRNA3和tracrRNA4；
tracrRNA1为序列表中SEQ ID NO.5所示的RNA；
tracrRNA2为序列表中SEQ ID NO.6所示的RNA；
tracrRNA3为序列表中SEQ ID NO.7所示的RNA；
tracrRNA4为序列表中SEQ ID NO.8所示的RNA。
序列表中各序列的第1位均为该条序列的5′末端。
实施例2、不同ncrRNA长度对DNA编辑效率的影响
一、体外酶切检测
1、DNA模板制备
以293T细胞基因组DNA为模板,使用Phusion酶(即Phusion High-Fidelity DNA Polymerase)进行二次PCR扩增得到VEGFA(Vascular endothelial growth factor A)靶基因片段，即DNA模板。第一次PCR扩增所用引物为VEGFA-F1/VEGFA-R1，第二次PCR扩增以第一次PCR扩增产物为模板利用引物VEGFA-F3/VEGFA-R3进行扩增，第二次PCR扩增产物(即DNA模板)长度为485bp。
2、Cas9体外酶切
将Cas9核酸酶，10×Cas9 buffer，crRNA，tracrRNA和H 2O混匀，37℃孵育15min，之后加入步骤1的DNA模板，混匀后37℃孵育70min，反应体系具体如表3所示。以用H 2O替换crRNA作为阴性对照(neg.)。其中，crRNA为VEGFA-crRNA1、VEGFA-crRNA2、VEGFA-crRNA3和VEGFA-crRNA4，每个反应体系一种crRNA，VEGFA-crRNA1、VEGFA-crRNA2、VEGFA-crRNA3和VEGFA-crRNA4的结构式如实施例1式Ⅰ所示，Nx部分相同，ncrRNA分别为实施例1的ncrRNA1、ncrRNA2、ncrRNA3和ncrRNA4，Nx的序列均为：5′-CCACAGGGAAGCUGGGUGAA-3′，该序列与VEGFA基因的部分序列互补；tracrRNA为实施例1的tracrRNA2。
表3、Cas9的体外酶切反应体系
试剂 体积 终浓度
Cas9核酸酶(1.2μM) 2.5μl 100nM
10×Cas9Buffer 3μl 1×
crRNA(4μM) 3μl 400nM
tracrRNA(4μM) 3μl 400nM
DNA模板 2μl(192ng) 20nM
H 2O 16.5μl ——
总计 30μl ——
3、磁珠纯化
1)将步骤2的酶切产物30μl转至EP管中；
2)将两倍体积的磁珠与EP管中的样品涡旋震荡充分混匀，静置5-10min；
3)将EP管放置磁力架上，待磁珠全都吸附到侧壁时，吸走上清；
4)向EP管中加入200μl 80％(体积百分比)的乙醇水溶液，洗脱磁珠；
5)吸走上清液，室温下静置3-5min，待贴壁的磁珠干燥到出现裂痕时，加入20μl水溶解；
6)将加水后的EP管从磁力架拿出，静置3-5min；
7)EP管放回磁力架，上清即回收样品。
4、Agilent2200核酸自动化电泳系统检测
取1-2μl样品和等体积染料上机检测。
5、酶切结果
(1)计算方法
Agilent2200核酸自动化电泳系统检测得到DNA片段分布和浓度结果，利用计算公式得出酶切效率。
计算公式：
Figure PCTCN2018088105-appb-000002
f(cut)＝(b+c)/(a+b+c)；a为未切开的片段的摩尔浓度(pmol/l)，b、c分别为切开的片段的摩尔浓度(pmol/l)。
(2)结果分析
图1表明crRNA长度为32nt(VEGFA-crRNA1)、34nt(VEGFA-crRNA2)、36nt(VEGFA-crRNA3)和38nt(VEGFA-crRNA4)时，包括crRNA、tracrRNA、Cas9的体外酶切体系可将485bp的DNA模板切开。crRNA长度为34nt时，Cas9酶切效果为最优(Indel％为89.2％)，即当ncrRNA部分的长度为14nt时(即为ncrRNA2时)Cas9酶切效率最高。
二、Cas9细胞内酶切检测
1、细胞转染
(1)Cas9-293T细胞以37℃，5％CO 2为培养条件，利用含有10％胎牛血清的DMEM培养基在培养箱内培养。其中，Cas9-293T细胞通过基因编辑，稳定导入了Cas9核酸酶的编码基因，能表达Cas9核酸酶(序 列为NCBI上Genbank ID：AQM52323.1所示的序列)。
(2)转染前一天将稳定表达Cas9核酸酶的Cas9-293T细胞细胞铺板于24孔板中。
(3)调整细胞密度为4×10 4-5×10 4个/mL左右，利用lipo2000(Lipofectamine2000)转染crRNA和tracrRNA。其中crRNA为步骤一的VEGFA-crRNA1、VEGFA-crRNA2、VEGFA-crRNA3和VEGFA-crRNA4，每个反应体系一种crRNA，tracrRNA为实施例1的tracrRNA2。以用H 2O替换crRNA作为阴性对照(neg.)。具体方法如下：
A.取50μl opti-MEM培养基于1.5ml EP管中，加入1μl lipo2000，轻轻混匀，室温静置5min；
B.取50μl opti-MEM培养基于1.5ml EP管中，加入200ng crRNA和300ng tracrRNA，充分混匀；
C.将A和B中得到的溶液混合，室温静置20min；
D.将(1)中24孔培养板中培养基吸出一部分，孔中剩余培养基在300μl左右，将C得到的溶液加入培养孔中混匀；
E.3-6h后向培养孔中补加含有10％胎牛血清的DMEM培养基至1ml，细胞放回培养箱培养48h后提取细胞基因组DNA。
2、细胞基因组DNA提取
(1)按照TIANGEN血液/组织/细胞基因组DNA提取试剂盒说明书提取DNA。
(2)琼脂糖凝胶电泳检测DNA提取效果和测定DNA浓度及纯度。
3、巢式PCR扩增目的DNA片段
(1)巢式PCR扩增
使用Phusion酶扩增VEGFA靶基因。第一次PCR扩增所用引物为VEGFA-F1/VEGFA-R1，第二次PCR扩增以第一次PCR扩增产物为模板利用引物VEGFA-F3/VEGFA-R3进行扩增。
(2)步骤(1)得到的第二次的PCR产物退火，退火产物直接用于下一步酶切。
退火体系如下，其中buffer2为T7Endonuclease I(NEB，货号：M0302L)：
Figure PCTCN2018088105-appb-000003
PCR仪上进行退火反应，退火条件如下：
Figure PCTCN2018088105-appb-000004
4、T7EI酶切效率检测
使用NEB T7核酸内切酶I(T7EI)进行酶切步骤3的退火产物，37℃孵育40min后，加1μl蛋白酶K，37℃孵育5min。
酶切体系(20μl)：
Figure PCTCN2018088105-appb-000005
5、电泳检测
酶切样品用于Agilent2200核酸自动化电泳系统检测。
6、酶切结果
(1)计算方法同步骤一。
(2)分析
结果如图2，结果表明，crRNA长度为32nt(VEGFA-crRNA1)、34nt(VEGFA-crRNA2)、36nt(VEGFA-crRNA3)和38nt(VEGFA-crRNA4)时，Cas9均可将485bp的目的DNA的目的片段切开。crRNA长度为34nt时，Cas9酶切效果为最优(Indel％为29.4％)，即当ncrRNA部分的长度为14nt时(即为ncrRNA2时)Cas9酶切效率最高。
实施例3、不同tracrRNA长度对DNA编辑效率的影响
一、体外酶切检测
1、方法
按照实施例2步骤一的方法分别检测crRNA为VEGFA-crRNA5，tracrRNA分别为实施例1的tracrRNA1、 tracrRNA2、tracrRNA3和tracrRNA4时，包括crRNA、tracrRNA、Cas9的体外酶切体系的酶切效率，每种反应体系一种tracrRNA，并利用水替换tracrRNA作为阴性对照(neg.)。
其中，VEGFA-crRNA5的序列为UGACUGCCGUCUGCACACCC GUUUUAGAGCUAUG(SEQ ID NO.28)。
2、结果
通过Agilent2200核酸自动化电泳系统检测得到的DNA片段分布和浓度结果(图3)，可得出结论：tracrRNA长度为64nt(tracrRNA1)、66nt(tracrRNA2)、68nt(tracrRNA3)和70nt(tracrRNA4)时，Cas9均可将485bp的DNA模板切开。从Indel％结果中可知，tracrRNA长度为66nt时(即为tracrRNA2时)，Cas9酶切效果为最优，Indel％为90.7％。
二、细胞内酶切检测
1、方法
按照实施例2步骤二的方法分别检测crRNA为步骤一的VEGFA-crRNA5，tracrRNA分别为实施例1的tracrRNA1、tracrRNA2、tracrRNA3和tracrRNA4时，Cas9的酶切效率，每种反应体系一种tracrRNA，并利用水替换tracrRNA作为阴性对照(neg.)。
2、结果
通过Agilent2200核酸自动化电泳系统检测得到的DNA片段分布和浓度结果(图4)，可得出结论：tracrRNA长度为64nt(tracrRNA1)、66nt(tracrRNA2)、68nt(tracrRNA3)和70nt(tracrRNA4)时，Cas9均可将485bp的目的片段切开。从Indel％结果中可看出，tracrRNA长度为66nt时(即为tracrRNA2时)，Cas9酶切效果为最优，Indel％为29.8％。
实施例4、不同修饰的酶切检测
一、体外酶切检测
1、方法
以293T细胞基因组为模板,扩增靶基因VEGFA片段，具体方法同实施例一。
按照实施例2步骤一的方法分别检测crRNA分别为VEGFA-crRNA2-1和VEGFA-crRNA2-2，tracrRNA为实施例1的tracrRNA2时，包括crRNA、tracrRNA、Cas9的体外酶切体系的酶切效率，每个反应体系一种crRNA。利用实施例2的VEGFA-crRNA2作为对照，利用H 2O替换crRNA作为阴性对照(neg.)。
其中，VEGFA-crRNA2-1为对实施例3的crRNA5的第2位、第30位、第31位和第33位进行2′-O-甲基修饰得到的物质，具体如下：
UGmACUGCCGUCUGCACACCCGUUUUAGAGCmUmAUmG，m表示2′-O-甲基修饰；
VEGFA-crRNA2-2为对crRNA2进行胆固醇修饰得到的物质，具体如下：
Chol-UGACUGCCGUCUGCACACCCGUUUUAGAGCUAUG，Chol表示胆固醇修饰。
2、结果
Agilent2200核酸自动化电泳系统检测得到的DNA片段分布和浓度结果，从图5可知当对crRNA进行化学修饰(甲基化修饰VEGFA-crRNA2-1,胆固醇修饰VEGFA-crRNA2-2)后，Cas9核酸酶均可将485bp的目的片段切开，与VEGFA-crRNA2相比酶切效率无明显变化。
二、细胞内酶切检测
1、方法
按照实施例2步骤二的方法分别检测crRNA分别为步骤一的VEGFA-crRNA2-1和VEGFA-crRNA2-2，tracrRNA为实施例1的tracrRNA2时，Cas9的酶切效率，每个反应体系一种crRNA。利用实施例2的VEGFA-crRNA2作为对照，利用H 2O替换crRNA作为阴性对照(neg.)。
2、结果
Agilent2200核酸自动化电泳系统检测得到的DNA片段分布和浓度结果，从图6可知当对crRNA进行化学修饰(甲基化修饰VEGFA-crRNA2-1,胆固醇修饰VEGFA-crRNA2-2)后，均可将485bp的DNA模板切开，与VEGFA-crRNA2相比酶切效率无明显变化。
实施例5、不同细胞系酶切检测
1、方法
按照实施例2步骤二的方法，将Cas9-293T细胞分别替换为Cas9-HeLa细胞、Cas9-THP-1细胞和Cas9-HUVEC细胞，其他步骤均不变，检测不同细胞系对Cas9酶切效率的影响，每个反应体系一种细胞。所用crRNA为实施例2的VEGFA-crRNA2，tracrRNA为实施例1的tracrRNA2，利用H 2O替换tracrRNA作为阴性对照(neg.)。
2、结果
通过Agilent2200核酸自动化电泳系统检测得到的DNA片段分布和浓度结果，从图7可知在不同类型的细胞系中，crRNA与tracrRNA转染后Cas9核酸酶均可将目的片段切开，并且各阴性对照的结果也显示阴性对照的酶切效率均为0。
实施例6、不同基因的酶切效率检测
靶基因：Oct4(organic cation/carnitine transporter4)基因、EMX1(Empty spiracles homeobox 1)基因、Beta-3基因和Beta-5基因，其中Beta-3和Beta-5均为β地中海贫血基因，Beta-3基因含有β地中海贫血基因突变位点CD17(HBB:c.52A>T)，Beta-5基因含有β地中海贫血基因突变位点CD17(HBB:c.52A>T)。
Oct4基因所用crRNA为Oct4-E-T1-crRNA、Oct4-E-T2-crRNA和Oct4-E-T3-crRNA，tracrRNA为实施例1的tracrRNA2。crRNA的结构如实施例1式Ⅰ所示，各crRNA的ncrRNA部分相同，均为实施例1的ncrRNA2，Nx部分不同，各crRNA的序列如下：
Oct4-E-T1-crRNA：GGUUAUUUCUAGAAGUUAGG GUUUUAGAGCUAUG(SEQ ID NO.13)
Oct4-E-T2-crRNA：CUUCUACAGACUAUUCCUUG GUUUUAGAGCUAUG(SEQ ID NO.14)
Oct4-E-T3-crRNA：GGAAAGGGGAGAUUGAUAAC GUUUUAGAGCUAUG(SEQ ID NO.15)。
EMX1基因所用crRNA为E1R-crRNA、E7F-crRNA、Ee3F-crRNA和E2F-crRNA，tracrRNA为实施例1的tracrRNA2。crRNA的结构如实施例1式Ⅰ所示，各crRNA的ncrRNA部分相同，均为实施例1的ncrRNA2，Nx部分不同，各crRNA的序列如下：
E1R-crRNA：AAGGCCGUGCGGAUCCGCUU GUUUUAGAGCUAUG(SEQ ID NO.16)
E7F-crRNA：AGGUCCGACGUGUUGGAGUG GUUUUAGAGCUAUG(SEQ ID NO.17)
Ee3F-crRNA：CGAUGUCACCUCCAAUGACU GUUUUAGAGCUAUG(SEQ ID NO.18)
E2F-crRNA：UCUCGCCCUCGCAGCUGCUG GUUUUAGAGCUAUG(SEQ ID NO.19)
Beta-3基因所用crRNA为Beta-3-T1-crRNA和Beta-3-T3-crRNA，tracrRNA为实施例1的tracrRNA2。crRNA的结构如实施例1式Ⅰ所示，各crRNA的ncrRNA部分相同，均为实施例1的ncrRNA2，Nx部分不同，各crRNA的序列如下：
Beta-3-T1-crRNA：GUGUGGCAAAGGUGCCCUUG GUUUUAGAGCUAUG(SEQ ID NO.20)
Beta-3-T3-crRNA：ACCAAUAGAAACUGGGCAUG GUUUUAGAGCUAUG(SEQ ID NO.21)
Beta-5基因所用crRNA为Beta-5-T3-crRNA，tracrRNA为实施例1的tracrRNA2。crRNA的结构如实施例1式Ⅰ所示，序列如下：
Beta-5-T3-crRNA：CGUAAAUACACUUGCAAAGG GUUUUAGAGCUAUG。(SEQ ID NO.22)
一、体外酶切检测
1、DNA模板制备
以293T细胞基因组为模板，扩增靶基因Oct4基因、EMX1基因、Beta-3基因和Beta-5基因的片段，即DNA模板。
Oct4基因DNA模板第一次扩增所用引物为Oct4-outer-F1和Oct4-outer-R1，第二次扩增所用引物为Oct4-inner-F1和Oct4-inner-R1。
EMX1基因DNA模板为EMX1-E2F、EMX1-E1R、EMX1-E7F和EMX1-Ee3F，各模板所用引物如下：
EMX1-E2F：DNA模板第一次扩增所用引物为E1-1R/2F-WF/NF和E1-1R/2F-WR，第二次扩增所用引物为E1-1R/2F-WF/NF和E1-1R/2F-NR。
EMX1-E1R：DNA模板第一次扩增所用引物为E1-1R/2F-WF/NF和E1-1R/2F-WR，第二次扩增所用引物为E1-1R/2F-WF/NF和E1-1R/2F-NR。
EMX1-E7F：DNA模板第一次扩增所用引物为E1-7-F-WF和E1-7-F-WR，第二次扩增所用引物为E1-7-F-NF和E1-7-F-NR。
EMX1-Ee3F:DNA模板第一次扩增所用引物为E1-e3F-WF和E1-e3F-WR，第二次扩增所用引物为E1-e3F-NF和E1-e3F-NR。
Beta-3基因DNA模板第一次扩增所用引物为Beta-outer-F1和Beta-outer-R1，第二次扩增所用引物为Beta3-inner-F1和Beta3-inner-R1。
Beta-5基因DNA模板第一次扩增所用引物为Beta-outer-F1和Beta-outer-R1，第二次扩增所用引物为Beta5-inner-F1和Beta5-inner-R1。
2、Cas9体外酶切
按照实施例2步骤一中2-5的方法，利用含有crRNA、tracrRNA和Cas9的体外酶切体系对步骤1的各DNA模板进行酶切，每个反应体系一种crRNA。利用H 2O替换crRNA作为阴性对照(neg.)。
2.1Oct4基因
检测Oct4-E-T1-crRNA在不同浓度(200nM、400nM和800nM)下的Cas9的体外酶切体系的酶切效率，各体系中tracrRNA的浓度随crRNA浓度的不同而不同，保持同一反应体系中tracrRNA的浓度与crRNA浓度相同，其他物质的浓度均同表3。
结果显示，Oct4-E-T1-crRNA在浓度为200nM、400nM和800nM时Cas9均可将Oct4基因DNA模板切开(图8)。
2.2EMX1基因
检测不同crRNA(E1R-crRNA、E7F-crRNA、Ee3F-crRNA和E2F-crRNA)的Cas9的体外酶切EMX1基因DNA模板的酶切效率，EMX1基因DNA模板EMX1-E2F、EMX1-E1R、EMX1-E7F和EMX1-Ee3F对应的crRNA分别为E2F-crRNA、E1R-crRNA、E7F-crRNA和Ee3F-crRNA，各体系均同表3。
结果显示，不同的crRNA均可将EMX1基因DNA模板切开(图9)。
2.3Beta-3基因和Beta-5基因
检测Beta-3-T1-crRNA和Beta-3-T3-crRNA存在下Cas9的体外酶切Beta-3基因DNA模板的酶切效率，以及Beta-5-T3-crRNA存在下Cas9的体外酶切Beta-5基因DNA模板的酶切效率。
结果显示，Beta-3-T1-crRNA和Beta-3-T3-crRNA存在下Cas9均可将Beta-3基因DNA模板切开，Beta-5-T3-crRNA存在下Cas9可将Beta-5基因DNA模板切开(图10)。
二、细胞内酶切检测
按照实施例2步骤二的方法，检测Cas9核酸酶对Oct4基因、EMX1基因、Beta-3基因和Beta-5基因的酶切效率，每个反应体系一种crRNA。利用H 2O替换crRNA作为阴性对照(neg.)。
Oct4基因所用crRNA为Oct4-E-T1-crRNA、Oct4-E-T2-crRNA和Oct4-E-T3-crRNA，方法均同实施例2步骤二。在巢式PCR扩增目的DNA片段中，Oct4基因所用引物为：第一次扩增所用引物为Oct4-outer-F1和Oct4-outer-R1，第二次扩增所用引物为Oct4-inner-F1和Oct4-inner-R1。结果显示，不同的crRNA存在下Cas9核酸酶均可将Oct4基因切开(图11)。
EMX1基因所用crRNA为Ee3F-crRNA，Ee3F-crRNA的用量分别为50ng、100ng、200ng、400ng，保持同一反应体系中tracrRNA的浓度与crRNA浓度相同，其余物质用量及方法均同实施例2步骤二。在巢式PCR扩增目的DNA片段中，EMX1基因所用引物为：第一次扩增所用引物为E1-e3F-WF和E1-e3F-WR，第二次扩增所用引物为E1-e3F-NF和E1-e3F-NR。结果显示，不同crRNA的用量下Cas9核酸酶均可将EMX1基因切开(图12)。
Beta-3基因所用crRNA为Beta-3-T1-crRNA和Beta-3-T3-crRNA，Beta-5基因所用crRNA为Beta-5-T3-crRNA，方法均同实施例2步骤二。在巢式PCR扩增目的DNA片段中，Beta-3基因所用引物为：第一次扩增所用引物为Beta-outer-F1和Beta-outer-R1，第二次扩增所用引物为Beta3-inner-F1和Beta3-inner-R1；Beta-5基因所用引物为：第一次扩增所用引物为Beta-outer-F1和Beta-outer-R1，第二次扩增所用引物为Beta5-inner-F1和Beta5-inner-R1。结果显示，Beta-3-T1-crRNA和Beta-3-T3-crRNA存在下Cas9核酸酶均可将Beta-3基因切开，Beta-5-T3-crRNA存在下Cas9核酸酶可将Beta-5基因切开(图13)。
实施例7、小鼠基因组VEGFA基因酶切检测
一、细胞内酶切检测
按照实施例2步骤二的方法，将Cas9-293T细胞替换为含有Cas9的C2C12小鼠成肌细胞，其他步骤均不变，检测含有Cas9的C2C12小鼠成肌细胞中Cas9对VEGFA基因酶切效率的影响，所用crRNA为经过修饰的的VEGFA-crRNA6，即VEGFA-crRNA6-1，tracrRNA为实施例1的tracrRNA2。利用H 2O替换crRNA作为阴性对照(neg.)。
VEGFA-crRNA6：AAGAGGAGAGGGGGCCGCAG GUUUUAGAGCUAUG(SEQ ID NO.29)，
VEGFA-crRNA6-1为对VEGFA-crRNA6的第2位、第30位、第31位和第33位进行2′-O-甲基修饰得到的物质，具体如下：
AAmGAGGAGAGGGGGCCGCAG GUUUUAGAGCmUmAUmG，m表示2′-O-甲基修饰。
含有Cas9的C2C12小鼠成肌细胞的制备方法如下：
1、Lipo2000转染质粒PX260
(1)取50μl opti-MEM培养基于1.5ml EP管中，加入1μl lipo2000，混匀后室温静置5min；
(2)取50μl opti-MEM培养基于1.5ml EP管中，加入1μg PX260质粒，混匀；
(3)混合(1)和(2)中得到的溶液，室温静置20min；
(4)将培养C2C12小鼠成肌细胞的24孔培养板中培养基吸出一部分，孔中剩余培养基在300-500μl 左右，将(3)的样品加入培养孔中，3-6h后向培养孔中补加含有10％胎牛血清的DMEM培养基至1ml，细胞放回培养箱培养。
2、质粒转染24h后Lipo2000转染crRNA和tracrRNA，转染方法同实施例2步骤一中1的(3)。
二、C57bl/6小鼠体内(in vivo)酶切检测
1、C57bl/6小鼠体内转染：
(1)C57BL/6小鼠(北京维通利华实验动物有限公司)注射戊巴比妥钠麻醉：30mg/kg静脉或腹腔注射。
(2)制剂混合
将5μg Cas9质粒(即PX260)、4μM crRNA(20μl)和4μM tracrRNA(20μl)溶解在200μl的生理盐水中，震荡混匀。加入6μl转染试剂(即TurboFect in vivo Transfection Reagent)后充分混合。将上述混合液在室温放置15-20分钟，作为转染制剂注射肌肉。所用crRNA为经过修饰的的VEGFA-crRNA6，即VEGFA-crRNA6-1，tracrRNA为实施例1的tracrRNA2。
2.2小鼠肌肉注射
向小鼠股二头肌注射步骤2.1的转染制剂200μl。
2.3小鼠肌肉组织基因组DNA提取
转染后7天用血液/组织/细胞基因组提取试剂盒提取股二头肌基因组DNA。
2.4巢式PCR扩增目的DNA片段
方法同实施例2步骤二中3。所用引物为VEGFA-outer-F1(小鼠)、VEGFA-outer-R1(小鼠)、VEGFA-inner-F1(小鼠)和VEGFA-inner-R1(小鼠)。
2.5T7EI酶切效率检测
使用NEB T7核酸内切酶I(T7EI)进行酶切，具体方法同实施例2步骤二中4。
2.6电泳检测
酶切样品用于Agilent2200核酸自动化电泳系统检测。
3、结果分析
计算方法同实施例2。
通过Agilent2200核酸自动化电泳系统检测得到的DNA片段分布和浓度结果，可知在小鼠中，crRNA、tracrRNA、和Cas9质粒导入小鼠细胞或小鼠体内，均可将526bp目的DNA目的片段切开为2条DNA链(图14)。
实施例8、启动子、外显子酶切检测
检测TP53(tumor protein p53)基因和外显子的酶切效率，所用crRNA分别为TP53-P-T1、TP53-P-T2、TP53-E-T1、TP53-E-T2和TP53-E-T3，每个反应体系一种crRNA，所用tracrRNA为实施例1的tracrRNA2。利用H 2O替换crRNA作为阴性对照(neg.)。所用crRNA的序列如下：
TP53-P-T1：CAAUUCUGCCCUCACAGCUC GUUUUAGAGCUAUG(SEQ ID NO.23)
TP53-P-T2：CCCCAAAAUGUUAGUAUCUA GUUUUAGAGCUAUG(SEQ ID NO.24)
TP53-E-T1：CCCUCCCAUGUGCUCAAGAC GUUUUAGAGCUAUG(SEQ ID NO.25)
TP53-E-T2：UGGGAGCGUGCUUUCCACGA GUUUUAGAGCUAUG(SEQ ID NO.26)
TP53-E-T3：CCAGUCUUGAGCACAUGGGA GUUUUAGAGCUAUG(SEQ ID NO.27)
(P表示启动子，E表示外显子)
一、体外酶切检测
1、DNA模板制备
以293T细胞基因组为模板，扩增靶基因TP53片段，即DNA模板。
第一次扩增所用引物为TP53-outer-FP和TP53-outer-RP，第二次扩增所用引物为TP53-inner-FP和TP53-inner-RP。
2、Cas9体外酶切
按照实施例2步骤一中2-5的方法，利用含有crRNA、tracrRNA和Cas9的体外酶切体系对步骤1的DNA模板进行酶切。
二、细胞内酶切检测
按照实施例2步骤二的方法，检测Cas9核酸酶对TP53片段的酶切效率。利用H 2O替换crRNA作为阴性对照(neg.)。
通过Agilent2200核酸自动化电泳系统检测得到的DNA片段分布和浓度结果，从图15-图16可知，crRNA、tracrRNA、Cas9核酸酶系统可将TP53片段的启动子和外显子区域切开。
实施例9、多条crRNA酶切检测
一、体外酶切检测
1、Cas9体外酶切
将Cas9核酸酶，10×Cas9buffer，crRNA，tracrRNA和H 2O混匀，37℃孵育15min，之后加入DNA模板，混匀后37℃孵育70min。以用H 2O替换为crRNA作为阴性对照(neg.)。DNA模板为实施例6的Oct4基因DNA模板，所用crRNA为Oct4-E-T1-crRNA、Oct4-E-T2-crRNA和Oct4-E-T3-crRNA，tracrRNA为实施例1的tracrRNA2。Cas9体外酶切体系如下，该体系中含有三种crRNA，即含有Oct4-E-T1-crRNA、Oct4-E-T2-crRNA和Oct4-E-T3-crRNA：
Figure PCTCN2018088105-appb-000006
利用Oct4-E-T2-crRNA单独作为crRNA进行实验，作为对照，Oct4-E-T2-crRNA的浓度为400nM。
2、磁珠纯化
同实施例2步骤一中3。
3、Agilent2200核酸自动化电泳系统检测
同实施例2步骤一中4。
二、细胞内酶切检测
按照实施例2步骤二的方法，利用Oct4-E-T1-crRNA、Oct4-E-T2-crRNA和Oct4-E-T3-crRNA以及实施例1的tracrRNA2一起进行转染，crRNA6的用量均为66.7ng，tracrRNA2的用量为300ng。利用Oct4-E-T2-crRNA单独作为crRNA进行实验，作为对照，Oct4-E-T2-crRNA的用量为200ng。
通过Agilent2200核酸自动化电泳系统检测得到的DNA片段分布和浓度结果，从图17-18可知在crRNA用量一致的情况下，针对Oct 4基因的多条crRNA的酶切效率高于单条crRNA，体外和细胞分别提升了1倍和2倍以上。
实施例10、不同的Cas9供体的细胞酶切检测
一、稳定表达Cas9的细胞
Cas9供体为稳定表达Cas9的293T细胞，即Cas9-293T，具体方法同实施例2中一。所用crRNA和tracRNA分别为实施例2的crRNA2和实施例1的tracrRNA2。
2、Cas9质粒
Cas9供体为Cas9质粒(即PX260)。
2.1Lipo2000转染质粒PX260
(1)取50μl opti-MEM培养基于1.5ml EP管中，加入1μl lipo2000，混匀后室温静置5min；
(2)取50μl opti-MEM培养基于1.5ml EP管中，加入1μg PX260质粒，混匀；
(3)混合(1)和(2)中得到的溶液，室温静置20min；
(4)将培养293T细胞的24孔培养板中培养基吸出一部分，孔中剩余培养基在300-500μl左右，将(3)的样品加入培养孔中，3-6h后向培养孔中补加含有10％胎牛血清的DMEM培养基至1ml，细胞放回培养箱培养。
2.2质粒转染24h后Lipo2000转染crRNA和tracrRNA，转染方法同实施例2步骤一中1的(3)。所用crRNA和tracRNA分别为实施例2的crRNA2和实施例1的tracrRNA2。
按照实施例步骤二中2-6的方法，通过Agilent2200核酸自动化电泳系统检测得到的DNA片段分布和浓度结果，从图19可知正常的293T细胞中转染质粒表达Cas9，再转染crRNA和tracrRNA后同样可以酶切目的DNA片段，效率低于Cas9稳转细胞(Cas9-293T)。
实施例11、不同的Cas9浓度的体外酶切检测
1、方法
按照实施例2步骤一的方法分别检测包括crRNA、tracrRNA、不同浓度的Cas9的体外酶切体系的酶切效率。Cas9的浓度分别为20nM、100nM、200nM、400nM，其他组分含量同实施例2步骤一。
所用crRNA和tracRNA分别为实施例2的VEGFA-crRNA2和实施例1的tracrRNA2。
2、结果
通过Agilent2200核酸自动化电泳系统检测得到的DNA片段分布和浓度结果(图20)，可得出结论：蛋白浓度为20-400nM时，酶切效率均在50％以上；蛋白浓度为100-400nM时，酶切效率均在80％以上；蛋白浓度为100-200nM时，酶切效率在85％以上。

Claims (10)

  1. 用于DNA编辑的系统，包括甲或乙：
    甲、下述N1)和N2)：
    N1)名称为RNA-2的RNA或与所述RNA-2相关的生物材料；
    N2)名称为RNA-1的RNA或与所述RNA-1相关的生物材料；
    所述RNA-2为式Ⅰ所示RNA；
    Nx-ncrRNA
    式Ⅰ；
    其中，Nx为CRISPR/Cas系统中的间隔序列；
    所述ncrRNA为如下a1)至a4)中的任一种：
    a1)序列表中SEQ ID NO.1所示的RNA；
    a2)在a1)的5′端和/或3′端添加一个或几个核苷酸得到的RNA；
    a3)与a1)或a2)限定的RNA具有85％以上的同一性的RNA；
    a4)序列表中SEQ ID NO.1所示的RNA的修饰物；
    与所述RNA-2相关的生物材料为下述A1)至A5)中的任一种：
    A1)编码所述RNA-2的DNA分子；
    A2)含有A1)所述DNA分子的表达盒；
    A3)含有A1)所述DNA分子的重组载体、或含有A2)所述表达盒的重组载体；
    A4)含有A1)所述DNA分子的重组微生物、或含有A2)所述表达盒的重组微生物、或含有A3)所述重组载体的重组微生物；
    A5)含有A1)所述DNA分子的细胞系、或含有A2)所述表达盒的细胞系；
    所述RNA-1为如下b1)至b4)中的任一种：
    b1)序列表中SEQ ID NO.5所示的RNA；
    b2)在b1)的5′端和/或3′端添加一个或几个核苷酸得到的RNA；
    b3)与b1)或b2)限定的RNA具有85％以上的同一性的RNA；
    b4)序列表中SEQ ID NO.5所示的RNA的修饰物；
    与所述RNA-1相关的生物材料为下述B1)至B5)中的任一种：
    B1)编码所述RNA-1的DNA分子；
    B2)含有B1)所述DNA分子的表达盒；
    B3)含有B1)所述DNA分子的重组载体、或含有B2)所述表达盒的重组载体；
    B4)含有B1)所述DNA分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
    B5)含有1)所述DNA分子的细胞系、或含有B2)所述表达盒的细胞系；
    所述ncrRNA与所述RNA-1部分片段互补；
    乙、所述RNA-2。
  2. 根据权利要求1所述的系统，其特征在于：a2)所述RNA为如下a21)至a23)中的任一种：
    a21)长度为12-14nt的RNA；
    a22)长度为12-16nt的RNA；
    a23)长度为12-18nt的RNA；
    和/或，b2)所述RNA为如下b21)至b24)中的任一种：
    b21)长度为64-66nt的RNA；
    b22)长度为64-68nt的RNA；
    b23)长度为64-70nt的RNA；
    b24)长度为64-86nt的RNA；
    和/或，所述核糖核苷酸修饰物为对核糖核苷酸在核糖、磷酸骨架和/或碱基进行修饰得到的物质；
    所述修饰物为对所述RNA中的至少一个核苷酸在核糖、磷酸骨架和/或碱基进行修饰得到的物质；
    和/或，
    所述DNA为下述M1)-M5)中的任一种：
    M1)真核生物的DNA；
    M2)动物的DNA；
    M3)哺乳动物的DNA；
    M4)人的DNA；
    M5)小鼠的DNA。
  3. 根据权利要求1或2所述的系统，其特征在于：
    a21)所述RNA为序列表中SEQ ID NO.2所示的RNA；
    a22)所述RNA为序列表中SEQ ID NO.3所示的RNA；
    a23)所述RNA为序列表中SEQ ID NO.4所示的RNA；
    和/或，b21)所述RNA为序列表中SEQ ID NO.6所示的RNA；
    b22)所述RNA为序列表中SEQ ID NO.7所示的RNA；
    b23)所述RNA为序列表中SEQ ID NO.8所示的RNA；
    和/或，
    所述修饰为2′-O-甲基修饰、2′-脱氧修饰、2′-氟代修饰或胆固醇修饰；
    和/或，
    所述DNA为VEGFA基因、EMX1基因、Oct4基因、Beta-3基因、Beta5基因、TP53基因或TP53基因的启动子。
  4. 根据权利要求1-3所述的系统，其特征在于：
    所述DNA为VEGFA基因，所述RNA-2为SEQ ID NO.9-12、SEQ ID NO.28和SEQ ID NO.29中至少一条序列所示的RNA；
    所述DNA为Oct4基因，所述RNA-2为SEQ ID NO.13-15中至少一条序列所示的RNA；
    所述DNA为EMX1基因，所述RNA-2为SEQ ID NO.16-19中至少一条序列所示的RNA；
    所述DNA为β地中海贫血基因，所述RNA-2为SEQ ID NO.20-22所示的RNA；
    所述DNA为TP53基因，所述RNA-2为SEQ ID NO.23和/或24所示的RNA；
    所述DNA为TP53基因的启动子，所述RNA-2为SEQ ID NO.25-27中至少一条序列所示的RNA。
  5. 根据权利要求1-4所述的系统，其特征在于：所述系统还包括Cas9核酸酶或与Cas9核酸酶相关的生物材料；
    所述与Cas9核酸酶相关的生物材料为下述C1)至C7)中的任一种：
    C1)编码Cas9核酸酶的核酸分子；
    C2)含有C1)所述核酸分子的表达盒；
    C3)含有C1)所述核酸分子的重组载体、或含有C2)所述表达盒的重组载体；
    C4)含有C1)所述核酸分子的重组微生物、或含有C2)所述表达盒的重组微生物、或含有C3)所述重组载体的重组微生物；
    C5)含有C1)所述核酸分子的细胞系、或含有C2)所述表达盒的细胞系。
  6. 根据权利要求5所述的系统，其特征在于：所述Cas9核酸酶来源于R1)或R2)：
    R1)细菌；
    R2)链球菌属、葡萄球菌属、罗氏菌属、奈瑟菌属、细小棒菌属或乳杆菌属。
  7. 下述O1)或O2)：
    O1)权利要求1-4中任一所述RNA-2或与所述RNA-2相关的生物材料；
    O2)权利要求1-3中任一所述RNA-1或与所述RNA-1相关的生物材料。
  8. 下述任一应用：
    X1、权利要求1-4中任一所述ncrRNA在制备crRNA中的应用；
    X2、权利要求1-4中任一所述ncrRNA在DNA编辑中的应用；
    X3、权利要求1-4中任一所述ncrRNA在制备DNA编辑产品中的应用；
    X4、权利要求1-3中任一所述RNA-1在作为tracrRNA中的应用；
    X5、权利要求1-3中任一所述RNA-1或与所述RNA-1相关的生物材料在DNA编辑中的应用；
    X6、权利要求1-3中任一所述RNA-1或与所述RNA-1相关的生物材料在制备DNA编辑产品中的应用；
    X7、权利要求1-4中任一所述RNA-2在作为crRNA中的应用；
    X8、权利要求1-4中任一所述RNA-2或与所述RNA-2相关的生物材料在DNA编辑中的应用；
    X9、权利要求1-4中任一所述RNA-2或与所述RNA-2相关的生物材料在制备DNA编辑产品中的应用；
    X10、权利要求1-6中任一所述系统在DNA编辑中的应用；
    X11、权利要求1-6中任一所述系统在干扰体内或体外基因的表达中的应用；
    X12、权利要求1-6中任一所述系统在建立DNA发生编辑的动物、植物或细胞模型中的应用。
  9. 编辑DNA的方法，包括：利用权利要求1-6中任一所述系统处理DNA实现所述DNA的编辑。
  10. 权利要求1-6中任一所述系统的制备方法，包括将所述系统中的各物质分别独立包装。
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