WO2016082135A1 - 一种利用定点切割系统对猪h11位点定点插入的方法 - Google Patents
一种利用定点切割系统对猪h11位点定点插入的方法 Download PDFInfo
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Definitions
- the invention belongs to the technical field of genetic engineering, and specifically relates to a method for point-specific insertion of pig H11 sites by means of a point-directed cutting system.
- H11 locus a good gene insertion site on mouse chromosome 11, named hipp11 locus, or H11 locus for short.
- the H11 locus is located in the gap between Eif4enif1 and Drg1 genes, adjacent to exon 19 of Eif4enif1 gene and exon 9 of Drg1 gene, and the size is about 5kb. Since the H11 site is located between two genes, it is safer, has no gene silencing effect, and has a broad-spectrum cell expression activity. Experiments confirmed that the growth and development of mice modified by Hipp11 site-specific gene and wild-type mice are no different.
- the Ros26 locus is similar at present, but this locus is a gene, and its promoter is systemic and broad-spectrum expression, which is difficult to achieve tissue-specific expression. However, there is no similar difficulty at the H11 locus, because it is located between the two There are no promoters between genes, so you can select the promoters required for the experiment to complete the specific expression of the target gene in time and space, and better achieve the task goal. If a safe and effective genetic modification site such as hipp11 is located in the genome of pigs, it will be helpful to stabilize the technical system of transgenic pig breeding.
- Sequence-specific nucleases are mainly formed by linking a DNA recognition domain with an endonuclease domain that can cut DNA non-specifically.
- the main principle is to first recognize and bind to the DNA to be modified by the DNA recognition domain To On the fragment, the DNA is then cleaved by the non-specific endonuclease domain connected to the DNA, causing double-strand break (DSB) of DNA.
- DSB double-strand break
- Zinc Finger Nuclease is the precise gene modification technology described in the previous paragraph. It consists of a specific DNA recognition domain and a specific DNA recognition domain. Non-specific endonuclease composition. In the ZFN recognition domain, a zinc finger structure can specifically recognize multiple (usually 3) consecutive bases, and multiple zinc finger structures can recognize a series of bases.
- the amino acid sequence of the zinc finger recognition domain is the key point, especially how to design how to connect multiple lysine 2-histidine 2 (Cys2-His2) zinc finger proteins in series, and how to change The 16 amino acid residues of the alpha helix determine the specific triplet bases recognized by each zinc finger protein.
- ZFN technology in gene targeted modification makes it widely used in genetic modification at the individual and cellular levels.
- people have achieved targeted gene modification at the cellular level by using ZFN technology.
- Sangamo first realized ZFN-mediated gene targeting in human cultured cell lines in 2005.
- the same ZFN was used to achieve gene-directed insertion through homologous recombination genes.
- people have used ZFN to achieve gene targeted mutations in human iPS and ES cells, respectively.
- transcription activator-like effector nucleases have more advantages. It is another new type of genome that can efficiently modify the genome after zinc finger nuclease technology. technology. There is a protein in the transcription factor activation effector family (TALEs) that can recognize and bind DNA. The specific binding of TALE to DNA sequence is mainly mediated by 34 constant amino acid sequences in the TAL structure. Connect TALEs with the cleavage domain of FokI endonuclease to form TALEN, which can modify the double strands of genomic DNA at specific sites.
- TALEs transcription factor activation effector family
- the repeat domain (Repeat Domain) is responsible for identifying specific DNA sequences.
- Each repeat sequence is basically the same, except for two variable amino acids, namely Repeat-Variable Diresidues (RVD).
- RVD Repeat-Variable Diresidues
- the mechanism of TALE's recognition of DNA is that the RVD on a repetitive sequence can recognize a nucleotide on the DNA target, and then fusion To FokI endonuclease, combined into TALEN.
- TALEN is a heterodimeric molecule (two units of TALEDNA binding domain fused to one unit of catalytic domain), which can cut two closely spaced sequences, thereby enhancing specificity.
- the advantages of the enzyme such as high efficiency, low toxicity, short preparation period, and low cost, are becoming more and more obvious.
- CRISPR CRISPR-associated
- Cas9 CRISPR-associated (Cas) is an adaptive immune defense mechanism of bacteria and archaea.
- CRISPR/Cas9 uses a small RNA to recognize and cut DNA to degrade foreign nucleic acid molecules.
- Cong et al. and Mali et al. also proved that the Cas9 system can effectively target restriction digestion in 293T, K562, iPS and other cells.
- the efficiency of non-homologous recombination (NHEJ) and homologous recombination (HR) is 3-25%. In between, the effect is equivalent to TALEN digestion. They also proved that multiple targets can be targeted at the same time.
- the efficiency of traditional targeting technology is very low, which mainly depends on the random exchange of homologous recombination inside the cell, and the efficiency is very low.
- the above-mentioned targeted cutting technology it will provide a good support for animal and plant gene function research and breeding.
- An object of the present invention is to provide a method for fixed-point insertion of pig H11 site by means of a fixed-point cutting system, so as to solve the defects of current technology such as random insertion, complicated steps, and high price.
- the method provided by the present invention includes the following steps: 1) Determine the target sequence targeted by the targeted cutting system in the porcine target genome sequence; 2) Design and construct the corresponding cutting system targeting according to the target site Sequence; 3) Construction of targeting vector; 4) Transfection of cells, PCR amplification and identification of insertion results.
- the targeted cutting system described in step 1) is TALEN targeted cutting system or CRISPR/Cas targeted cutting system.
- nucleotide cleavage enzyme used in the CRISPR/Cas targeted cleavage system is csa9 or cas9n.
- the target sequence targeted by the targeted cleavage system described in step 1) is the target sequence targeted by the TALEN targeted cleavage system, the CRISPR/Cas9 targeted cleavage system, or the CRISPR/Cas9n targeted cleavage system.
- the target sequence is the target sequence targeted by the TALEN targeted cleavage system, the CRISPR/Cas9 targeted cleavage system, or the CRISPR/Cas9n targeted cleavage system.
- step 1) is specifically as shown in 1), 2) or 3):
- the TALEN targeted cutting system targets a pair of sites, whose nucleotide sequence is as shown in the sequence table To Sequence 1 and Sequence 4, Sequence 2 and Sequence 4 in the Sequence Listing, Sequence 3 and Sequence 4 in the Sequence Listing, Sequence 1 and Sequence 5 in the Sequence Listing, Sequence 2 and Sequence 5 in the Sequence Listing or in the Sequence Listing Shown in sequence 3 and sequence 5;
- the target sequence targeted by the CRISPR/Cas9 targeted cutting system is shown in sequence 6 or sequence 7 in the sequence table.
- the CRISPR/Cas9n targeted cutting system targets a pair of sites, the nucleotide sequences of which are shown in sequence 8 and sequence 9 in the sequence table.
- the targeting sequence described in step 2) above is the polypeptide sequence of TALEN targeted cleavage system, the nucleotide sequence of CRISPR/Cas9 targeted cleavage system, or a pair of nucleotide sequences of CRISPR/Cas9n targeted cleavage system .
- polypeptide sequence of the TALEN targeted cleavage system includes polypeptide A and polypeptide B, and the specific sequence is as shown in 1), 2), 3), 4), 5) or 6):
- sequence of polypeptide A is specifically shown in sequence 10 in the sequence listing, and the sequence of polypeptide B is specifically shown in sequence 13 in the sequence listing;
- sequence of polypeptide A is specifically shown in sequence 11 in the sequence listing, and the sequence of polypeptide B is specifically shown in sequence 13 in the sequence listing;
- sequence of polypeptide A is specifically shown in sequence 12 in the sequence listing, and the sequence of polypeptide B is specifically shown in sequence 13 in the sequence listing;
- sequence of polypeptide A is specifically shown in sequence 10 in the sequence listing, and the sequence of polypeptide B is specifically shown in sequence 14 in the sequence listing;
- sequence of polypeptide A is specifically shown in sequence 11 in the sequence listing, and the sequence of polypeptide B is specifically shown in sequence 14 in the sequence listing;
- sequence of polypeptide A is specifically shown in sequence 12 in the sequence listing, and the sequence of polypeptide B is specifically shown in sequence 14 in the sequence listing.
- nucleotide sequence of the sgRNA of the CRISPR/Cas9 targeted cleavage system described in step 2) above includes the nucleoside identifying the specific DNA sequence fragment on the chromosome and the backbone RNA fragment, and identifying the specific DNA sequence fragment on the chromosome
- the acid sequence is as follows 1) or 2):
- nucleotide sequence of 1) has undergone one or several base substitutions and/or deletions and/or additions and has a nucleotide sequence that has the same function as the nucleotide sequence of 1).
- the nucleotide sequence of the sgRNA of the CRISPR/Cas9n targeted cutting system described in step 2) consists of sgRNA-L and sgRNA-R.
- the sequences of sgRNA-L and sgRNA-R respectively include identifying specific DNA on the chromosome Sequence fragments and backbone RNA fragments;
- nucleotide sequence of sgRNA-L that recognizes the specific DNA sequence fragment on the chromosome is as follows 1) or 2):
- nucleotide sequence of 1) has undergone one or several base substitutions and/or deletions and/or additions and has a nucleotide sequence that has the same function as the nucleotide sequence of 1);
- nucleotide sequence of sgRNA-R that recognizes the specific DNA sequence fragment on the chromosome is as follows 3) or 4):
- nucleotide sequence of 3 undergoes one or several base substitutions and/or deletions and/or additions and has a nucleotide sequence that has the same function as the nucleotide sequence of 3).
- DNA sequence encoding the polypeptide sequence of the TALEN targeted cleavage system described in step 2) includes DNA molecule A and DNA molecule B.
- the specific sequence is as follows 1), 2), 3), 4), 5) or 6) :
- DNA molecule encoding the nucleotide sequence of the sgRNA of the CRISPR/Cas9 targeted cleavage system described in step 2) is the molecule encoding the sequence 15 or the molecule encoding the sequence 16, and its specific nucleotides The sequence is shown in 1) or 2):
- the DNA molecule encoding the sgRNA of the CRISPR/Cas9n targeted cleavage system described in step 2) is composed of a DNA molecule encoding the sgRNA-L and a DNA molecule encoding the sgRNA-L sgRNA-R is composed of DNA molecule B;
- nucleotide sequence of DNA molecule A is shown in sequence 26 in the sequence listing
- nucleotide sequence of DNA molecule B is shown in sequence 27 in the sequence listing.
- the construction of the targeting vector described in step 3) includes the construction of a targeting vector for site-directed cutting and a targeting vector to be inserted into a gene.
- the steps of constructing a targeting vector for the gene to be inserted into the targeted cutting system are as follows: 1) Design the 5'end homology arm and 3'end homology arm of the knocked-out gene and the corresponding universal primers; 2) Combine the above homology The arm, the universal primer, the marker gene and/or the gene to be inserted are introduced into the vector to obtain the targeting vector.
- the 5'end homology arm and 3'end homology arm described in step 1) of constructing the targeting vector of the gene to be inserted wherein the nucleotide sequence of the 5'end homology arm is as shown in the sequence list 28, the nucleotide sequence of the corresponding universal primer is shown in sequence 29 in the sequence list; the nucleotide sequence of the 3'end homology arm is shown in sequence 30 in the sequence list, and the corresponding universal primer The nucleotide sequence of is shown in sequence 31 in the sequence listing.
- the sequence of the targeting vector constructed for the targeted cutting system to insert the gene includes the above To 5'end homology arm sequence, 5'end homology arm universal primer sequence, gene sequence to be inserted, 3'end homology arm universal primer sequence, 3'end homology arm sequence.
- nucleotide sequence of the targeting vector constructed for the site-directed cutting system to insert the gene is shown in sequence 32 in the sequence table.
- nucleotide sequences of the PCR amplification primers used in the insertion result of PCR amplification identification in step 4) are shown in sequence 33, sequence 34, sequence 35, sequence 36, sequence 37, and sequence 38 in the sequence listing.
- Another object of the present invention is to also provide the application of the above method in the targeted modification of pig H11 gene.
- Another object of the present invention is to provide the application of the above method in constructing a pig H11 gene mutation library.
- the invention provides a method for point insertion of pig H11 site by means of a point cutting system, which realizes simple, fast and efficient gene point insertion.
- the present invention relies on the cutting system to design a targeting vector for pig H11 site, which can accurately introduce foreign genes into pig H11 site, so as to solve the problem of low efficiency of traditional targeting technology, inconvenient design of PCR detection primers, and high detection difficulty. Problems and high efficiency.
- the universal detection primer designed for this site greatly reduces the difficulty of screening and detection.
- the targeting vector is transfected into cells, and positive clones are screened through a medium containing a drug corresponding to the gene being screened.
- the resulting positive clones are highly enriched, the cell screening method is simple, and does not require a lot of manpower and material resources.
- the earth facilitates the subsequent cryopreservation and identification of cells, greatly reduces the cost of gene targeting, and at the same time enables the stable expression of foreign genes in H11, which builds a stable platform for transgenes.
- FIG. 1 is a schematic diagram of the structure of the targeting carrier of the present invention.
- Figure 2 shows the results of DNA PCR amplification and identification of recombinant cells constructed by TALEN targeted cutting system
- Figure 3 is a diagram showing the results of DNA PCR amplification and identification of recombinant cells constructed by the CRISPR/cas9n targeted cutting system
- Figure 4 is a diagram showing the results of sequencing, detecting and analyzing the DNA digestion vector of recombinant cells constructed by the CRISPR/cas9 targeted cutting system;
- Figure 5 is a diagram showing the results of PCR amplification and identification of cells constructed by using the CRISPR/cas9 targeted cutting system to insert the green fluorescent protein into the pig H11 site;
- Fig. 6A and Fig. 6B are fluorescence excitation images of positive clones;
- Fig. 6A is a microscopic view of cells under visible light To Observation diagram,
- Figure 6B is a microscopic observation diagram of cells under ultraviolet light.
- a method for site-directed insertion of pig H11 sites by means of a site-directed cutting system first constructs TALEN targeted cutting system, CRISPR/Cas targeted cutting system, and CRISPR/cas9n targeted cutting system.
- the three cutting systems constructed in the present invention can all efficiently recognize the H11 site of pigs, and use corresponding nucleases to cut the sequence genes of the pig H11 site.
- a targeting vector was designed for the pig H11 site.
- the targeting vector is a homology arm with the knocked-out gene attached to both ends and corresponding universal primers, and the gene to be inserted is introduced into pLHG-4 Obtained in.
- Recombinant cells can be obtained by transfecting the above-mentioned targeting vector into cells.
- the targeting vector obtained by the above method contains universal primers, which greatly reduces the difficulty and workload of screening and detection.
- the two homology arms of the design do not have a promoter that initiates the expression of positive screening genes, and there are negative screening genes on the outside of the homology arms.
- the above-mentioned targeting vector transfects cells, and the positive clones are screened through the medium containing the drug corresponding to the gene being screened, and the positive clones obtained are highly enriched, the cell screening method is simple, does not require a lot of manpower and material resources, and greatly facilitates the subsequent cell freezing. Storage and identification greatly reduces the cost of gene targeting, and at the same time allows foreign genes to be stably expressed at the H11 locus, which builds a stable platform for transgenes.
- Example 1 Construction of three kinds of pig H11 site-directed cutting systems
- the present invention firstly based on the gene sequence of pig H11 locus as follows:
- the TALEN system uses the endonuclease activity of FokI to interrupt the target gene, because FokI needs to form a dimer to be active. In actual operation, it is necessary to select two adjacent places in the target gene (14-18 bases apart) The target sequence (generally more than a dozen bases) was constructed separately for TAL recognition module.
- L1 5'-TTCTTATGTTCCTGGAAG-3'T vector: L15, the structure of the vector is: cmv-sp6-NLS-TAL-T-IRES-puro-pA, which was purchased from Shanghai Stansai Biotechnology Co., Ltd.;
- L2 5'-TCTTATGTTCCTGGAAGT-3'T vector: L15, the structure of the vector is: cmv-sp6-NLS-TAL-T-IRES-puro-pA, which was purchased from Shanghai Stansai Biotechnology Co., Ltd.;
- L3 5'-CTTATGTTCCTGGAAGTT-3'T vector: L15, the structure of the vector is: cmv-sp6-NLS-TAL-T-IRES-puro-pA, which was purchased from Shanghai Stansai Biotechnology Co., Ltd.;
- R1 3’-GTAGCCTATAAAACCCAG-5’A vector: R10, the structure of the vector is cmv-sp6-NLS-TAL-A-pA, the vector was purchased from Shanghai Stansai Biotechnology Co., Ltd.;
- R2 3’-AGCCTATAAAACCCAGAG-5’C vector: R12, the structure of the vector is cmv-sp6-NLS-TAL-C-pA, which was purchased from Shanghai Stansai Biotechnology Co., Ltd.;
- R1 5’-GTAGCCTATAAAACCCAG-3’A vector: R10
- R2 5’-AGCCTATAAAACCCAGAG-3’C vector: R12
- step 2 1) Take 10 ⁇ L of the final ligation product of step 4 and add to it and mix well.
- sgRNA target site position 1 (named H11-sg1): 5'-GTTCCTGGAAGTTTAGATCAGGG-3', the nucleotide sequence identifying the target in the corresponding sgRNA sequence is shown in sequence 15 in the sequence table; encoding the above sequence The DNA sequence of is shown in sequence 24 in the sequence listing.
- sgRNA target site position 2 (named H11-sg2): 5'-AGATCAGGGTGGGCAGCTCTGGG-3', the nucleotide sequence identifying the target site in the corresponding sgRNA sequence is shown in sequence 16 in the sequence table; encoding the above sequence The DNA sequence is shown in sequence 25 in the sequence listing.
- Oligonucleotide dimers are inserted into the vector separately
- reaction is carried out in the following reaction system:
- step (3) Take 5 ⁇ L of the final product of step (3) (carrier Cas9/gRNA-H11-sg1, Cas9/gRNA-H11-sg2) and add it to 50 ⁇ L of DH5a competent cells that have just been thawed, flick and mix, and then ice bath for 30 minutes , Heat shock at 42°C for 90 seconds, let it stand on ice for 2 minutes, and apply directly to an ampicillin resistant plate.
- the sequencing primer is: 5'-TGAGCGTCGATTTTTGTGATGCTCGTCAG-3' to obtain the sequencing results of Cas9/gRNA-H11-sg1 and Cas9/gRNA-H11-sg2.
- the above sequencing results are shown in sequence 39 and sequence 40 in the sequence table. This result shows that the DNA sequence encoding sgRNA (ie the sequence of target site 1 and target site 2) can be smoothly inserted into the Cas9/gRNA vector backbone through the above operation.
- the H11 locus of the mouse find the Eif4 and Drg genes of the pig (the locus of the mouse is located in the middle of the two genes), and call the middle region in the NCBI to find the H11 locus of the pig.
- PAM sequence PAM sequence is NGG
- Select the sgRNA target for gene knockout as follows: 5'-TACTGAAATGTGACCTACTTTCTTATGTTCCTGGAAGTTTAGATCAGGGTGGGCAGCTCTGGG-3',
- SgRNA target designed for gene knockout sgRNA-L target site position 1 (named H11-sgL2): 5'-AGATCAGGGTGGGCAGCTCTGGG-3', the corresponding nucleoside in the sgRNA-L sequence that recognizes the target
- the acid sequence is shown in sequence 17 in the sequence listing; the DNA sequence encoding the above sequence is shown in sequence 26 in the sequence listing.
- sgRNA-R target site position 2 (named H11-sgR1): 5'-TTCCAGGAACATAAGAAAGTAGG-3', the nucleotide sequence identifying the target site in the corresponding sgRNA sequence is shown in sequence 18 in the sequence table; encoding the above
- the DNA sequence of the sequence is shown in sequence 27 in the sequence listing.
- the two target sequences are arranged in a "head-to-head” arrangement, and the two target sequences are 4bp apart, that is, there is To 4bp interval.
- H11-sgL2-F 5’-CACCGAGATCAGGGTGGGCAGCTCT-3’
- H11-sgL2-F and H11-sgL2-R are annealed to obtain a double-stranded DNA fragment H11-sgL2 with sticky ends.
- the pX335 addgene, Plasmid 42335) vector (its nucleotide sequence is shown in sequence 41 in the sequence list) )
- the fragment was recovered by digestion with BbsI, and H11-sgL2 was connected to the fragment to obtain pX335-sgRNA-H11-L vector; H11-sgR1-F and H11-sgR1-R were annealed to obtain a double-stranded DNA fragment H11 with sticky ends -gR1, the pX335 vector is digested with Bbs I to recover the fragment, and H11-gR1 is connected to the fragment to obtain the pX335-sgRNA-H11-R vector.
- PEF cells were isolated from aborted pig fetuses (see the literature for isolation methods: Li Hong, Wei Hongjiang, Xu Chengsheng, Wang Xia, Qing Yubo, Zeng Yangzhi; establishment of the fetal fibroblast cell line of the Banna mini-pig inbred line and its biology Scientific characteristics; Journal of Hunan Agricultural University (Natural Science Edition); Volume 36, Issue 6; December 2010; 678-682).
- the recombinant plasmid TALEN-H11-L1 and TALEN-H11-R1, TALEN-H11-L2 and TALEN-H11-R1, TALEN-H11-L3 and TALEN-H11-R1, TALEN-H11 in Example 1 (1) -L1 and TALEN-H11-R2, TALEN-H11-L2 and TALEN-H11-R2, TALEN-H11-L3 and TALEN-H11-R1, TALEN-H11 in Example 1 (1) -L1 and TALEN-H11-R2, TALEN-H11-L2 and TALEN-H11-R2, TALEN-H11-L3 and TALEN-H11-R2, 2.5 ⁇ g each were co-transfected into PEF cells by electrotransformation, and 5 Recombinant cells; 4 ⁇ g each of the recombinant plasmids Cas9/gRNA-H11-sg1 and Cas9/
- transfection using a nuclear transfer instrument (Amaxa, model: AAD-1001S) and a matching mammalian fibroblast transfection kit (Amaxa, product number: VPI-1002).
- Amaxa product number: VPI-1002
- 8 kinds of recombinant cells can be obtained through step 2, among which 5 kinds of recombinant cells can be obtained in the TALEN targeted site-directed cleavage system, 2 kinds of recombinant cells can be obtained in the CRISPR/Cas9 targeted site-directed cleavage system, and CRISPR/Cas9n targeted site-directed cleavage system
- 5 kinds of recombinant cells can be obtained in the TALEN targeted site-directed cleavage system
- 2 kinds of recombinant cells can be obtained in the CRISPR/Cas9 targeted site-directed cleavage system
- CRISPR/Cas9n targeted site-directed cleavage system One type of recombinant cell was obtained, and the above eight types of recombinant cells were cultured at 37°C for 48 hours, and then the cells were collected.
- the specific steps are: first use 0.1% trypsin (Gibco, article number: 610-5300AG) to digest adherent cells, use fetal bovine serum (Gibco, article number: 16000-044) to terminate the digestion, and phosphate buffer solution (Gibco, article number: 10010). -023) Wash the cells twice, and add 200 microliters of cell lysate GA (component of TIANGEN DNA extraction kit DP304). Refer to the kit instructions to extract the genomic DNA of the 8 recombinant cells.
- primer H11-F (5'-GCGAGAATTCTAAACTGGAG-3') and primer H11-R (5'-GATCTGAGGTGACAGTCTCAA-3') to use the 5 types of recombinant cells obtained in the TALEN targeted site-directed cutting system in step 3, respectively DNA was used as a template, PCR amplification was performed, and a 387bp fragment was recovered; using primer H11-F: 5'-GCGAGAATTCTAAACTGGAG-3' and primer H11-R: To 5'-GATCTGAGGTGACAGTCTCAA-3', using the genomic DNA of the two recombinant cells in the CRISPR/Cas9 targeted site-directed cleavage system collected in step 3 as a template for PCR amplification, and recovering a PCR amplification product of about 370 bp; using primers H11-F: 5'-GCGAGAATTCTAAACTGGAG-3'; and primer H11-R: 5'-GATCTGAGGTGACAGTCTCAA
- mutant DNA and wild-type DNA were mixed according to the following system, and subjected to heat denaturation, annealing and renaturation treatments (95°C for 5 min, natural cooling to room temperature).
- Figure 1 in Figure 2 is TALEN-H11-L1 and TALEN-H11-R1
- 2 is TALEN- H11-L2 and TALEN-H11-R1
- 3 are TALEN-H11-L3 and TALEN-H11-R1
- 4 are TALEN-H11-L1 and TALEN-H11-R2
- 5 are TALEN-H11-L2 and TALEN-H11- R2 and 6 are TALEN-H11-L3 and TALEN-H11-R2
- P is transfection-positive Cas9n
- N is blank cells.
- the target will cut out a 160bp+230bp band, and target 2 will cut out 170bp+220bp To Bands, you can see the digested fragments after cutting from the above figure, and the bands 3, 4, 5, and 6 are brighter, and the cutting efficiency is higher than that of groups 1 and 2.
- T7EI restriction diagram 1 is TALEN-H11-L1 and TALEN-H11-R1, 2 is TALEN-H11-L2 and TALEN-H11-R1, 3 is TALEN-H11-L3 and TALEN-H11-R1, 4 is TALEN-H11-L3 and TALEN-H11-R1 -H11-L1 and TALEN-H11-R2, 5 is TALEN-H11-L2 and TALEN-H11-R2, 6 is TALEN-H11-L3 and TALEN-H11-R2, P is transfection positive Cas9n (introduced by another patent ), N is a blank cell.
- the target will cut a 160bp+230bp band, and the target 2 will cut a 170bp+220bp band.
- the pair of gRNA has a certain activity. This pair of specific sgRNAs is very specific when cutting the H11 target site, which can effectively reduce the off-target phenomenon in the CRISPR/Cas9 system, greatly increase the efficiency of site-specific insertion of foreign genes, and thereby reduce the genome caused by non-specific cleavage The impact of mutations at non-target sites.
- the cleavage result identification steps of CRISPR/Cas9 targeted site-directed cleavage system recombinant cells are as follows:
- the PCR amplified product is connected with the PMD-18T vector (Bao Biological, Item No.: D101A) to obtain the ligation product.
- the kit instructions For the specific operation steps, see the kit instructions .
- the ligation product obtained above was transformed into E. coli DH5 ⁇ competent cells, and then spread on LB solid medium plates containing 500 mg/ml ampicillin for cultivation. 40 clones were randomly selected from the two groups and sequenced to calculate the mutation The proportion of clones to the total number of clones, so as to calculate the efficiency of recombinant plasmid Cas9/gRNA-H11-sg1 and Cas9/gRNA-H11-sg2 plasmid.
- the method for site-specific insertion of green fluorescent protein gene into pig H11 site using the CRISPR/Cas9 targeted site-directed cleavage system constructed by the target site 1 described in Example 1 (2) includes the following steps:
- the synthesized fragment is as follows:
- Design 5'homology arm (shown in sequence 28) according to the DNA sequence of pig H11 site, corresponding universal primer (shown in sequence 29), add RFP coding sequence, polyA sequence, and add restriction sites at both ends: AscI (GGCGCGCC), PacI(TTAATTAA), the synthetic fragments are as follows:
- PEF cells were isolated from aborted pig fetuses.
- isolation methods please refer to the literature: Li Hong, Wei Hongjiang, Xu Chengsheng, Wang Xia, Qing Yubo, Zeng Yangzhi; The establishment of the fetal fibroblast cell line of the Banna mini-pig inbred line and its Biological characteristics.
- Recombinant plasmid Cas9/gRNA-H11-g1 and linearized pLHG-H11 each 2.5 ⁇ g were transfected into PEF cells by electrotransformation to obtain recombinant cells.
- the specific steps of transfection are: transfection using a nuclear transfer instrument (Amaxa, model: AAD-1001S) and a matching mammalian fibroblast transfection kit (Amaxa, product number: VPI-1002).
- the obtained recombinant cells were cultured at 30°C for 72 hours, and then the cells were collected. Dilute the cells, spread a certain number of cells in each 10cm petri dish, and change the medium every 2-3 days.
- Figure 2 shows the clones at 6 days of plating.
- the amplified sequence is:
- FIG. 5 P1 represents the fragments amplified by primers H11-L-F1 and H11-L-R1, the size is 1.2kb, and P2 represents H11-L-F2 and H11- L-R2 amplified fragments, P3 represents H11-R-F3 and H11-R-R3 amplified fragments.
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Abstract
一种利用定点切割系统对猪H11位点定点插入的方法，包括以下步骤：1）在猪目标基因组序列中确定靶向切割系统所靶向的靶序列；2）根据靶位点设计、构建相应的切割系统的打靶序列；3）构建打靶载体；4）转染细胞，PCR扩增鉴定定点插入效率。该方法依托猪H11位点定点切割系统，将目的基因定点插入到靶位点，以解决传统打靶技术效率低下，PCR检测引物不便设计，检测难度大的问题。该定点插入方法能让外源基因在H11位点处稳定表达，为转基因猪制作搭建了高效的平台。
Description
本发明属于基因工程技术领域，具体涉及一种借助定点切割系统对猪H11位点定点插入的方法。
已知在生物技术研究中，将目的基因插入到染色体基因组中，可以采用同源重组的办法或使用转座子的办法，但实践表明，同源重组的效率较低，操作麻烦，并且由于目的基因的插入而使原有的基因遭受破坏；使用转座子的方法，也存在插入到染色体的部位是随机的，而且使用的操作转座酶价格昂贵。
因此，由于上述技术使用的局限性，在培育猪的优良品种时，主要采用随机整合的方式使外源基因随机插入猪基因组中，相应的使用该技术得到的重组体使后续的繁育与表型分析非常繁琐。
2010年，斯坦福大学的Simon Hippenmeyer及其研究团队在小鼠的第11号染色体上分离并鉴定出了一个良好的基因插入位点，命名为hipp11位点，简称H11位点。H11位点位于Eif4enif1与Drg1两个基因的间隙，与Eif4enif1基因的19号外显子和Drg1基因的9号外显子相邻，大小约5kb。H11位点由于位于两个基因之间，因此安全性较高，无基因沉默效应，具有广谱的细胞表达活性。实验证实Hipp11位点定点基因修饰的小鼠与野生型小鼠生长发育无区别。目前类似的还有Ros26位点，但是该位点为一个基因，其启动子为全身性广谱表达，难以做到组织特异性表达，然而H11位点则不存在类似的困难，由于其位于两基因之间，并且不存在启动子，所以可以选择实验所需的启动子完成目的基因的时空特异性表达，更好的达到任务目标。如果在猪的基因组中定位hipp11这样安全有效的基因修饰位点，将有利于稳定转基因猪培育的技术体系。
近年来发展的主要方法为依托序列特异的核酸酶进行基因的精确修饰。序列特异的核酸酶主要由一个DNA识别域与一个能非特异性切割DNA的内切酶结构域连接而成。其主要原理为先由DNA识别域识别并结合到需要改造的DNA
片段上，然后由与DNA相连的非特异性内切酶结构域对DNA进行切割，造成DNA的双链断裂(Double-strand break，DSB)，DSB会激活DNA的自我修复而引起基因的突变从而促进该位点的同源重组。
ZFN和TALEN打靶技术是目前研究较为成熟的两种定点突变技术，锌指核酸酶技术(Zinc Finger Nuclease，ZFN)就是前一段所述的基因精确修饰技术，由一个特异性的DNA识别域和一个非特异性核酸内切酶构成。在ZFN识别结构域中，一个锌指结构可以特异识别多个(通常是3个)连续的碱基，多个锌指结构能够识别一连串的碱基。所以，在ZFN的设计过程中，锌指识别结构域的氨基酸序列是重点，特别是设计如何将多个赖氨酸2-组氨酸2(Cys2-His2)锌指蛋白串联，以及如何通过改变α螺旋的16氨基酸残基决定每个锌指蛋白识别的特定三联体碱基。
ZFN技术在基因靶向修饰方面的可行性使得其广泛应用于个体水平和细胞水平的基因修饰。首先人们通过利用ZFN技术实现了细胞水平的基因定向修饰。如Sangamo公司于2005年首次在人类培养细胞系中实现ZFN介导的基因打靶，2007年应用同样的ZFN通过同源重组基因实现了基因定点插入。最近，人们利用ZFN分别在人的iPS和ES细胞中实现了基因的靶向突变。
相比之下，转录激活子样效应因子核酸酶(transcription activator-like effector nucleases，TALEN)有更多优势，它是继锌指核酸酶技术以来的另一种能够对基因组进行高效定点修饰的新技术。转录因子激活效应物家族中有一种蛋白(TALEs)能够识别、结合DNA。TALE与DNA序列特异性结合主要是由TAL结构内34个恒定氨基酸序列介导。将TALEs与FokI核酸内切酶的切割域相连接，形成TALEN，从而可以实现对基因组DNA双链在特定位点进行修饰。
在TALE的中央存在着一个重复区域，这个区域通常是由33-35个氨基酸的数量可变的重复单元构成。重复序列结构域(Repeat Domain)负责识别特异性的DNA序列。每个重复序列基本上都是一样的，除了两个可变的氨基酸，即重复序列可变的双氨基酸残基(Repeat-Variable Diresidues,RVD)。TALE识别DNA的机制在于一个重复序列上的RVD能够识别DNA靶点上的一个核苷酸，再融合
FokI核酸内切酶，组合成TALEN。TALEN是一种异源二聚体分子(两单位的TALEDNA结合结构域融合到一单位的催化性结构域)，能够切割两个相隔较近的序列，从而使得特异性增强。该酶的效率高，毒性小，制备周期短，成本低等优势越来越明显。
(CRISPR)/CRISPR-associated(Cas)是细菌和古细菌一种不断进化适应的免疫防御机制。近些年来，研究者发现CRISPR/Cas9利用一段小RNA来识别并剪切DNA以降解外来核酸分子。Cong等及Mali等还证明Cas9系统能在293T、K562、iPS等多种细胞中，进行有效的靶向酶切，非同源重组(NHEJ)、同源重组(HR)效率在3-25％之间，与TALEN酶切效果相当。他们还证明，多个靶点可以同时进行靶向酶切。
传统的打靶技术效率非常的低，其主要是依赖细胞内部的同源重组随机交换完成的，效率非常低。借助于上述的靶向切割技术，将会为动植物基因功能研究及育种提供一个良好的支持。
发明内容
本发明的一个目的是提供了一种借助定点切割系统对猪H11位点定点插入的方法，以解决目前的技术随机插入、步骤繁琐、价格昂贵等缺陷。
为实现上述目的，本发明所提供的方法包括以下步骤：1)在猪目标基因组序列中确定靶向切割系统所靶向的靶序列；2)根据靶位点设计、构建相应的切割系统的打靶序列；3)构建打靶载体；4)转染细胞，PCR扩增鉴定插入结果。
其中，步骤1)中所述的靶向切割系统是TALEN靶向切割系统或CRISPR/Cas靶向切割系统。
其中，所述的CRISPR/Cas靶向切割系统用的核苷酸切割酶是csa9或cas9n。
其中，步骤1)中所述的靶向切割系统所靶向的靶序列是TALEN靶向切割系统所靶向的靶序列、CRISPR/Cas9靶向切割系统或CRISPR/Cas9n靶向切割系统所靶向的靶序列。
其中，步骤1)中所述的靶序列具体如1)、2)或3)所示：
1)TALEN靶向切割系统所靶向的是一对位点，其核苷酸序列如序列表中的
序列1和序列4、序列表中的序列2和序列4、序列表中的序列3和序列4、序列表中的序列1和序列5、序列表中的序列2和序列5或序列表中的序列3和序列5所示；
2)CRISPR/Cas9靶向切割系统所靶向的靶序列如序列表中的序列6或序列7所示。
3)CRISPR/Cas9n靶向切割系统所靶向的是一对位点，其核苷酸序列如序列表中的序列8和序列9所示。
其中，上述的步骤2)中所述的打靶序列是TALEN靶向切割系统的多肽序列、CRISPR/Cas9靶向切割系统的核苷酸序列或CRISPR/Cas9n靶向切割系统的一对核苷酸序列。
其中，上述的TALEN靶向切割系统的多肽序列包括多肽甲和多肽乙，具体序列如1)、2)、3)、4)、5)或6)所示：
1)多肽甲的序列具体如序列表中的序列10所示，多肽乙的序列具体如序列表中的序列13所示；
2)多肽甲的序列具体如序列表中的序列11所示，多肽乙的序列具体如序列表中的序列13所示；
3)多肽甲的序列具体如序列表中的序列12所示，多肽乙的序列具体如序列表中的序列13所示；
4)多肽甲的序列具体如序列表中的序列10所示，多肽乙的序列具体如序列表中的序列14所示；
5)多肽甲的序列具体如序列表中的序列11所示，多肽乙的序列具体如序列表中的序列14所示；
6)多肽甲的序列具体如序列表中的序列12所示，多肽乙的序列具体如序列表中的序列14所示。
其中，上述的步骤2)中所述的CRISPR/Cas9靶向切割系统的sgRNA的核苷酸序列包括识别染色体上特定的DNA序列片段和骨架RNA片段，识别染色体上特定的DNA序列片段的核苷酸序列如下1)或2)：
1)序列表中的序列15或序列16所示的核苷酸序列；
2)将所述1)的核苷酸序列经过一个或几个碱基的取代和/或缺失和/或添加且具有与1)中的核苷酸序列具有同样功能的核苷酸序列。
其中，步骤2)中所述的CRISPR/Cas9n靶向切割系统的sgRNA的核苷酸序列由sgRNA-L和sgRNA-R组成，sgRNA-L和sgRNA-R其序列分别包括识别染色体上特定的DNA序列片段和骨架RNA片段；
sgRNA-L的识别染色体上特定的DNA序列片段的核苷酸序列如下1)或2)：
1)序列表中的序列17所示的核苷酸序列；
2)将所述1)的核苷酸序列经过一个或几个碱基的取代和/或缺失和/或添加且具有与1)中的核苷酸序列具有同样功能的核苷酸序列；
sgRNA-R的识别染色体上特定的DNA序列片段的核苷酸序列如下3)或4)：
3)序列表中的序列18所示的核苷酸序列；
4)将所述3)的核苷酸序列经过一个或几个碱基的取代和/或缺失和/或添加且具有与3)中的核苷酸序列具有同样功能的核苷酸序列。
其中，编码步骤2)中所述的TALEN靶向切割系统多肽序列的DNA序列，包括DNA分子甲和DNA分子乙，具体序列如下1)、2)、3)、4)、5)或6)：
1)编码序列表中序列10所示多肽的DNA分子甲的具体序列如序列表中的序列19所示，编码序列表中序列13所示多肽的DNA分子乙的具体序列如序列表中的序列22所示；
2)编码序列表中序列11所示多肽的DNA分子甲的具体序列如序列表中的序列20所示，编码序列表中序列13所示多肽的DNA分子乙的具体序列如序列表中的序列22所示；
3)编码序列表中序列12所示多肽的DNA分子甲的具体序列如序列表中的序列21所示，编码序列表中序列13所示多肽的DNA分子乙的具体序列如序列表中的序列22所示；
4)编码序列表中序列10所示多肽的DNA分子甲的具体序列如序列表中的序列19所示，编码序列表中序列14所示多肽的DNA分子乙的具体序列如序列表中的序列23所示；
5)编码序列表中序列11所示多肽的DNA分子甲的具体序列如序列表中的序列20所示，编码序列表中序列14所示多肽的DNA分子乙的具体序列如序列表中的序列23所示；
6)编码序列表中序列12所示多肽的DNA分子甲的具体序列如序列表中的序列21所示，编码序列表中序列14所示多肽的DNA分子乙的具体序列如序列表中的序列23所示。
进一步的，编码步骤2)中所述的CRISPR/Cas9靶向切割系统的sgRNA的核苷酸序列的DNA分子是编码所述序列15的分子或编码所述序列16的分子，其具体核苷酸序列如1)或2)所示：
1)序列表中的序列24所示的核苷酸序列；
2)序列表中的序列25所示的核苷酸序列。
12、根据权利要求9所述的方法，其特征在于，编码步骤2)中所述的CRISPR/Cas9n靶向切割系统的sgRNA的DNA分子由编码所述sgRNA-L的DNA分子甲和编码所述sgRNA-R的DNA分子乙组成；
其中：DNA分子甲的核苷酸序列如序列表中的序列26所示，DNA分子乙的核苷酸序列如序列表中的序列27所示。
其中，步骤3)中所述的构建打靶载体包括构建定点切割的打靶载体和欲插入基因的打靶载体。
其中，针对定点切割系统构建欲插入基因的打靶载体的步骤如下：1)设计被敲除基因的5’端同源臂和3’端同源臂以及相应的通用引物；2)将上述同源臂、通用引物、标记基因和/或欲插入的基因引入到载体中，得到打靶载体。
其中，在构建欲插入基因的打靶载体的步骤1)中所述的5’端同源臂和3’端同源臂，其中5’端同源臂的核苷酸序列如序列表中的序列28所示，其相应的通用引物的核苷酸序列如序列表中的序列29所示；3’端同源臂的核苷酸序列如序列表中的序列30所示，其相应的通用引物的核苷酸序列如序列表中的序列31所示。
其中，针对定点切割系统构建的欲插入基因的打靶载体其序列包括上述的
5’端同源臂序列、5’端同源臂通用引物序列、欲插入的基因序列、3’端同源臂通用引物序列、3’端同源臂序列。
其中，针对定点切割系统构建的欲插入基因的打靶载体其核苷酸序列如序列表中的序列32所示。
其中，步骤4)中PCR扩增鉴定插入结果中所用到的PCR扩增引物的核苷酸序列如序列表中序列33、序列34、序列35、序列36、序列37、序列38所示。
本发明的另一个目的是还提供了上述方法在靶向修饰猪H11基因中的应用。
本发明的再一个目的是提供上述方法在构建猪H11基因突变库中的应用。
本发明提供的一种借助定点切割系统对猪H11位点定点插入的方法，实现了简单、快捷、高效的基因定点插入。本发明依托切割系统对猪H11位点设计的打靶载体，它能够将外源基因精确的引入到猪的H11位点中，以解决传统打靶技术效率低下，PCR检测引物不便设计，检测难度大的问题，且效率高，同时通过针对该位点设计的通用检测引物，使筛选检测难度大大降低。
此外通过实施例可知，所述打靶载体转染细胞,通过含有与正筛选基因相应药物的培养基筛选阳性克隆,得到的阳性克隆富集效率高,细胞筛选方法简单,不需大量人力物力,极大地方便了后续的细胞冻存和鉴定,大大降低了基因打靶的成本，同时能让外源基因稳定在H11表达，为转基因搭建了稳定的平台。
说明书附图
图1为本发明的打靶载体结构示意图；
图2为TALEN靶向切割系统构建的重组细胞的DNA PCR扩增鉴定结果图；
图3为CRISPR/cas9n靶向切割系统构建的重组细胞的DNA PCR扩增鉴定结果图；
图4为测序检测分析CRISPR/cas9靶向切割系统构建的重组细胞的DNA酶切载体结果图；
图5为利用CRISPR/cas9靶向切割系统构建的绿色荧光蛋白定点插入猪H11位点后细胞PCR扩增鉴定结果图；以及
图6A和图6B为阳性克隆的荧光激发图；其中图6A为可见光下的细胞显微观
察图，图6B为紫外光下的细胞显微观察图。
以下实施例用于进一步说明本发明，但不应理解为对本发明的限制。在不背离本发明精神和实质的前提下，对本发明所作的修饰或者替换，均属于本发明的范畴。
如背景技术中所提到的，在培育猪的优良品种时，主要采用随机整合的方式使外源基因随机插入猪基因组中，为后续分析带来麻烦，为了克服上述缺陷，在本发明一种典型的实施方式中，提供了一种借助定点切割系统对猪H11位点定点插入的方法，该方法首先构建了TALEN靶向切割系统、CRISPR/Cas靶向切割系统、CRISPR/cas9n靶向切割系统，本发明构建的三种切割系统均能高效的识别猪的H11位点，并且利用相应的核酸酶对猪H11位点的序列基因进行切割。
然后依托上述靶向切割系统对猪H11位点设计了一种打靶载体，该打靶载体是将两端连有被敲除基因的同源臂以及相应通用引物和将欲插入基因引入到pLHG-4中获得的。将上述打靶载体转染细胞即可得到重组细胞，利用该方法构建定点基因突变库时我们只需要将我们感兴趣的基因插入到同源臂间就可以完成我们欲插入基因的定点插入。
上述方法获得的打靶载体中含有通用引物，大大降低了筛选检测的难度与工作量。并且设计的两同源臂的内部不具有启动正筛选基因表达的启动子,在所述同源臂的外侧还具有负筛选基因。上述打靶载体转染细胞,通过含有与正筛选基因相应药物的培养基筛选阳性克隆,得到的阳性克隆富集效率高,细胞筛选方法简单,不需大量人力物力,极大地方便了后续的细胞冻存和鉴定,大大降低了基因打靶的成本，同时能让外源基因稳定在H11位点表达，为转基因搭建了稳定的平台。
下面将结合具体的实施例来说明本发明的有益效果。
实施例1：三种猪H11位点定点切割系统的构建
一、TALEN靶向定点切割系统的构建
1、目标序列的构建
在基因库中调出猪H11位点的序列。本发明首先根据猪H11位点的基因序列，如下：
5’-TACTGAAATGTGACCTACTTTCTTATGTTCCTGGAAGTTTAGATCAGGGTGGGCAGCTCTGGG-3’
2、TALEN位点设计
目前，TALEN系统利用FokI的内切酶活性打断目标基因，因为FokI需形成2聚体方能发挥活性，在实际操作中需在目标基因中选择两处相邻(间隔14-18碱基)的靶序列(一般十几个碱基)分别进行TAL识别模块构建。
根据该靶点设计TALEN系统切割的位点，示意图见图1，具体序列如下：
L1：5’-TTCTTATGTTCCTGGAAG-3’T载体：L15，该载体的结构为：cmv-sp6-NLS-TAL-T-IRES-puro-pA，该载体购于上海斯丹赛生物技术有限公司；
L2：5’-TCTTATGTTCCTGGAAGT-3’T载体：L15，该载体的结构为：cmv-sp6-NLS-TAL-T-IRES-puro-pA，该载体购于上海斯丹赛生物技术有限公司；
L3：5’-CTTATGTTCCTGGAAGTT-3’T载体：L15，该载体的结构为：cmv-sp6-NLS-TAL-T-IRES-puro-pA，该载体购于上海斯丹赛生物技术有限公司；
R1：3’-GTAGCCTATAAAACCCAG-5’A载体：R10，该载体的结构为：cmv-sp6-NLS-TAL-A-pA，该载体购于上海斯丹赛生物技术有限公司；
R2：3’-AGCCTATAAAACCCAGAG-5’C载体：R12，该载体的结构为：cmv-sp6-NLS-TAL-C-pA，该载体购于上海斯丹赛生物技术有限公司；
3、利用上海斯丹赛生物技术有限公司的FastTALETM TALEN快速构建试剂盒(Cat.No.1802-030)对TALEN进行构建，构建的步骤为：
(一)设计，根据所选择的位点选择合适的模块，设计结果如下：
L1：5’-TTCTTATGTTCCTGGAAG-3’T载体：L15
所选模块：TT1 CT2 TA3 TG4 TT5 CC6 TG7 GA8 AG9
L2：5’-TCTTATGTTCCTGGAAGT-3’T载体：L15
所选模块：TC1 TT2 AT3 GT4 TC5 CT6 GG7 AA8 GT9
L3：5’-CTTATGTTCCTGGAAGTT-3’T载体：L15
所选模块：CT1 TA2 TG3 TT4 CC5 TG6 GA7 AG8 TT9
R1：5’-GTAGCCTATAAAACCCAG-3’A载体：R10
所选模块：GT1 AG2 CC3 TA4 TA5 AA6 AC7 CC8 AG9
R2：5’-AGCCTATAAAACCCAGAG-3’C载体：R12
所选模块：AG1 CC2 TA3 TA4 AA5 AC6 CC7 AG8 AG9
(二)添加模块
按照第1步中选择的模块在200ulPCR管中分别依次加入所需模块(共5管)，模块所对应位置如下：
表1：模块1
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| AA1 | CA1 | AA2 | CA2 | AA3 | CA3 | AA4 | CA4 | AA5 | CA5 | AA6 | CA6 | A |
| AT1 | CT1 | AT2 | CT2 | AT3 | CT3 | AT4 | CT4 | AT5 | CT5 | AT6 | CT6 | B |
| AC1 | CC1 | AC2 | CC2 | AC3 | CC3 | AC4 | CC4 | AC5 | CC5 | AC6 | CC6 | C |
| AG1 | CG1 | AG2 | CG2 | AG3 | CG3 | AG4 | CG4 | AG5 | CG5 | AG6 | CG6 | D |
| TA1 | GA1 | TA2 | GA2 | TA3 | GA3 | TA4 | GA4 | TA5 | GA5 | TA6 | GA6 | E |
| TT1 | GT1 | TT2 | GT2 | TT3 | GT3 | TT4 | GT4 | TT5 | GT5 | TT6 | GT6 | F |
| TC1 | GC1 | TC2 | GC2 | TC3 | GC3 | TC4 | GC4 | TC5 | GC5 | TC6 | GC6 | G |
| TG1 | GG1 | TG2 | GG2 | TG3 | GG3 | TG4 | GG4 | TG5 | GG5 | TG6 | GG6 | H |
表2：模块
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| AA7 | CA7 | AA8 | CA8 | AA9 | CA9 | A1 | T1 | C1 | G1 | A | ||
| AT7 | CT7 | AT8 | CT8 | AT9 | CT9 | A2 | T2 | C2 | G2 | B | ||
| AC7 | CC7 | AC8 | CC8 | AC9 | CC9 | A3 | T3 | C3 | G3 | C | ||
| AG7 | CG7 | AG8 | CG8 | AG9 | CG9 | A4 | T4 | C4 | G4 | D | ||
| TA7 | GA7 | TA8 | GA8 | TA9 | GA9 | A5 | T5 | C5 | G5 | E | ||
| TT7 | GT7 | TT8 | GT8 | TT9 | GT9 | A6 | T6 | C6 | G6 | F | ||
| TC7 | GC7 | TC8 | GC8 | TC9 | GC9 | A7 | T7 | C7 | G7 | G | ||
| TG7 | GG7 | TG8 | GG8 | TG9 | GG9 | H |
(三)加样
按照如下体系分别加入试剂盒中其他溶液，体系如下：
表3：反应体系
| 体系 | |
| 模块 | 1.5μL×9 |
| 溶液1 | 1μL |
| 溶液2 | 1μL |
| 溶液3 | 2μL |
| 载体 | 1.5μL |
| ddH2O | 1μL |
| 总体积 | 20μL |
(四)连接
1)将上述混合液分别置于PCR仪上完成连接，反应程序如下：
2)取出上步反应液，分别加入1μL溶液4，0.5μL溶液5(总体积21.5μL)后，置于37℃孵育60分钟。
(五)转化
1)取出试剂盒中的感受态，冰上放置10min使其融化。
2)取10μL步骤4最后的连接产物加入中，混匀。
3)冰上静置20min。
4)42℃热激60s。
5)冰浴3min。
6)加入500μL SOC，37℃摇床复苏30min。
7)4000rpm，离心5min，将上清倒掉大部分(留约150μL)。
8)将菌体重悬，均匀涂布在kna抗性的LB平板上。
9)37℃培养16h。
(六)挑克隆
在培养好的平板上挑10个克隆，在37℃摇床中培养过夜(超过16h)。利用
引物305(5’-CTCCCCTTCAGCTGGACAC-3’)与306(5’-AGCTGGGCCACGATTGAC-3’)送往公司(北京天一辉远有限公司)测序，选择正确克隆得到TALEN：TALEN-H11-L1、TALEN-H11-L2、TALEN-H11-L3、TALEN-H11-R1与TALEN-H11-R2，提取质粒，完成下一步实验。
二、CRISPR/Cas9靶向定点切割系统的构建
1、在基因库中调出猪的H11位点序列，根据PAM序列(PAM序列为NGG)选择用于基因敲除的sgRNA靶点，如下：5’-TACTGAAATGTGACCTACTTTCTTATGTTCCTGGAAGTTTAGATCAGGGTGGGCAGCTCTGGG-3’，
sgRNA靶位点位置1(命名为H11-sg1):5’-GTTCCTGGAAGTTTAGATCAGGG-3’，相对应的sgRNA序列中识别该靶点的核苷酸序列如序列表中序列15所示；编码该上述序列的DNA序列如序列表中的序列24所示。
sgRNA靶位点位置2(命名为H11-sg2):5’-AGATCAGGGTGGGCAGCTCTGGG-3’，相对应的sgRNA序列中识别该靶点核苷酸序列如序列表中序列16所示；编码该上述序列的DNA序列如序列表中的序列25所示。
2、sgRNA表达质粒构建
使用唯尚力德公司的cas9/gRNA构建试剂盒(Catalog.No.VK001-01)完成构建，构建过程如下：
(1)根据前面所述的两个靶点序列，设计相应的引物序列，由北京天一辉远公司合成，具体序列见表4：
表4 两个sgRNA靶点引物序列
| 核苷酸名称 | 序列(5’-3’) |
| H11-sg1-F | AAACACCGGTTCCTGGAAGTTTAGATCA |
| H11-sg1-R | CTCTAAAACTGATCTAAACTTCCAGGAAC |
| H11-sg2-F | AAACACCGAGATCAGGGTGGGCAGCTCT |
| H11-sg2-R | CTCTAAAACAGAGCTGCCCACCCTGATCT |
(2)寡核苷酸二聚体(oligoduplex)的形成
将合成的oligo分别稀释成10μM，分别按如下比例混合
分别混匀后，按照如下程序处理：95℃3min；将样品管放在95℃水中使上述混合物由95℃到25℃缓慢冷却；再在16℃下处理5min，最终获得寡核苷酸二聚体-1。
分别混匀后，按照如下程序处理：95℃3min；将样品管放在95℃水中使上述混合物由95℃到25℃缓慢冷却；再在16℃下处理5min，最终获得寡核苷酸二聚体-2。
(3)寡核苷酸二聚体分别插入到载体中
在以下反应体系中进行反应：
充分混合后，室温(25℃)静置5min，获得载体Cas9/gRNA-H11-sg1。
充分混合后，室温(25℃)静置5min，获得载体Cas9/gRNA-H11-sg2。
(4)转化
分别取步骤(3)的最终产物(载体Cas9/gRNA-H11-sg1、Cas9/gRNA-H11-sg2)5μL加入到刚解冻的50μL DH5a感受态细胞中，轻弹混匀，冰浴30分钟后，42℃热激90秒，冰上静置2分钟，直接涂于氨苄抗性的平板。
(5)验证
挑5个白色菌落摇菌，提取质粒DNA进行测序。测序引物为：5’-TGAGCGTCGATTTTTGTGATGCTCGTCAG-3’，得到Cas9/gRNA-H11-sg1与Cas9/gRNA-H11-sg2的测序结果，上述测序结果见序列表中的序列39和序列40。该结果表明，通过上述操作能顺利将编码sgRNA的DNA序列(即靶位点1和靶位点2的序列)插入到Cas9/gRNA载体骨架中。
三、CRISPR/Cas9n靶向定点切割系统的构建
1、sgRNA靶点设计
根据小鼠的H11位点，找到猪的Eif4与Drg基因(小鼠的位点位于该两基因中间)，在NCBI中调出中间区域找出猪H11位点，根据PAM序列(PAM序列为NGG)选择用于基因敲除的sgRNA靶点，如下：5’-TACTGAAATGTGACCTACTTTCTTATGTTCCTGGAAGTTTAGATCAGGGTGGGCAGCTCTGGG-3’，
设计用于基因敲除的sgRNA靶点：sgRNA-L靶位点位置1(命名为H11-sgL2):5’-AGATCAGGGTGGGCAGCTCTGGG-3’，相对应的sgRNA-L序列中识别该靶点的核苷酸序列如序列表中的序列17所示；编码该上述序列的DNA序列如序列表中的序列26所示。
sgRNA-R靶位点位置2(命名为H11-sgR1):5’-TTCCAGGAACATAAGAAAGTAGG-3’，相对应的sgRNA序列中识别该靶点核苷酸序列如序列表中序列18所示；编码该上述序列的DNA序列如序列表中的序列27所示。两个靶序列呈“头对头”的排布方式，二者相距4bp，即有
4bp的间隔。
2、sgRNA表达质粒对的构建
首先根据靶序列设计引物序列，后送至北京天一辉远生物科技有限公司合成单链寡核苷酸，具体序列如下：
(1)H11-sgL2:
H11-sgL2-F：5’-CACCGAGATCAGGGTGGGCAGCTCT-3’
H11-sgL2-R:5’-AAACAGAGCTGCCCACCCTGATCTC-3’
(2)H11-sgR1:
H11-sgR1-F:5’-CACCGTTCCAGGAACATAAGAAAGT-3’
H11-sgR1-R:5’-AAACACTTTCTTATGTTCCTGGAAC-3’
其中H11-sgL2-F与H11-sgL2-R退火获得带粘性末端的双链DNA片段H11-sgL2，将pX335(addgene，Plasmid 42335)载体(其核苷酸序列如序列表中的序列41所示)经BbsⅠ酶切回收片段，将H11-sgL2连入该片段中，获得pX335-sgRNA-H11-L载体；H11-sgR1-F与H11-sgR1-R退火获得带粘性末端的双链DNA片段H11-gR1，将pX335载体经Bbs Ⅰ酶切回收片段，将H11-gR1连入该片段中，获得pX335-sgRNA-H11-R载体。两个质粒均送北京天一辉远生物科技有限公司测序验证，测序引物bbsR的序列为：5’-GACTATCATATGCTTACCGT-3’，测序结果分别如序列表中的序列42和序列43所示。该结果表明，通过上述操作能顺利将sgRNA的靶位点1和靶位点2的sgRNA编码序列插入到pX335载体骨架中。
实施例2 三种针对猪H11位点定点切割方法的效率验证
1、分离猪胎儿成纤维细胞。
从流产的猪胎儿中分离得到PEF细胞(分离方法参见文献：李红，魏红江，许成盛，汪霞，卿玉波，曾养志；版纳微型猪近交系胎儿成纤维细胞系的建立及其生物学特征；湖南农业大学学报(自然科学版)；第36卷第6期；2010年12月；678-682)。
2、核转染
将实施例1(一)中的重组质粒TALEN-H11-L1与TALEN-H11-R1，TALEN-H11-L2与TALEN-H11-R1，TALEN-H11-L3与TALEN-H11-R1，TALEN-H11-L1与TALEN-H11-R2，TALEN-H11-L2与TALEN-H11-R2，TALEN-H11-L3与TALEN-H11-R2，各2.5μg通过电转化的方式分别共转染PEF细胞，得到5种重组细胞；将实施例1(二)中取得的重组质粒Cas9/gRNA-H11-sg1与Cas9/gRNA-H11-sg2各4μg通过电转化的方式分别转染PEF细胞，得到2种重组细胞；将实施例1(三)中获得的重组质粒pX335-sgRNA-H11-L与pX335-sgRNA-H11-R各2μg通过电转化的方式共同转染PEF细胞，得到1种重组质粒。转染的具体步骤是：使用核转仪(Amaxa，型号：AAD-1001S)及配套的哺乳动物成纤维细胞转染试剂盒(Amaxa，货号：VPI-1002)进行转染。首先使用0.1％胰蛋白酶(Gibco，货号：610-5300AG)消化贴壁细胞，用胎牛血清(Gibco，货号：16000-044)终止消化，磷酸盐缓冲液(Gibco，货号：10010-023)洗涤细胞两次，添加转染试剂，使用程序T-016转染细胞。
3、DNA的提取
通过步骤2可以获得8种重组细胞，其中在TALEN靶向定点切割系统中可获得5种重组细胞、CRISPR/Cas9靶向定点切割系统中获得2种重组细胞、CRISPR/Cas9n靶向定点切割系统中获得1种重组细胞，将上述8种重组细胞37℃培养48小时，然后收集细胞。具体步骤是：首先使用0.1％胰蛋白酶(Gibco，货号：610-5300AG)消化贴壁细胞，用胎牛血清(Gibco，货号：16000-044)终止消化，磷酸盐缓冲液(Gibco，货号：10010-023)洗涤细胞两次，添加200微升细胞裂解液GA(TIANGEN公司DNA提取试剂盒DP304中的组分)。参考试剂盒说明书步骤分别提取上述8种重组细胞基因组DNA。
4、PCR酶切效率验证
(1)利用引物H11-F(5’-GCGAGAATTCTAAACTGGAG-3’)与引物H11-R(5’-GATCTGAGGTGACAGTCTCAA-3’)分别以步骤3中的TALEN靶向定点切割系统中获得的5种重组细胞DNA为模板，进行PCR扩增，回收387bp片段；利用引物H11-F：5’-GCGAGAATTCTAAACTGGAG-3’与引物H11-R：
5’-GATCTGAGGTGACAGTCTCAA-3’，分别以步骤3中收集的CRISPR/Cas9靶向定点切割系统中的2种重组细胞的基因组DNA作为模板进行PCR扩增，回收约370bp的PCR扩增产物；利用引物H11-F：5’-GCGAGAATTCTAAACTGGAG-3’'；和引物H11-R：5’-GATCTGAGGTGACAGTCTCAA-3’组成引物对，以收集的CRISPR/Cas9n靶向定点切割系统中的重组细胞的基因组DNA作为模板进行PCR扩增，回收387bp片段。
将上述TALEN靶向定点切割系统和CRISPR/Cas9n靶向定点切割系统重组细胞的PCR结果利用唯尚力德公司T7核酸内切酶I(T7endonuclease I,T7E1)(货号：#E001L)进行酶切鉴定。具体步骤为：
(2)突变体DNA与野生型DNA的PCR产物按如下体系进行混合，进行加热变性、退火复性处理(95℃5min，自然冷却至室温)。
表5：PCR扩增反应体系
(3)上述反应体系分别加入0.5ul T7E1酶，37℃反应30min后，跑2％的琼脂糖凝胶电泳检测分析酶切结果，TALEN靶向定点切割系统的重组细胞的酶切结果的电泳图见图2，CRISPR/Cas9n靶向定点切割系统重组细胞的酶切结果的电泳图见图3.其中，图2中图中1为TALEN-H11-L1与TALEN-H11-R1，2为TALEN-H11-L2与TALEN-H11-R1，3为TALEN-H11-L3与TALEN-H11-R1，4为TALEN-H11-L1与TALEN-H11-R2，5为TALEN-H11-L2与TALEN-H11-R2，6为TALEN-H11-L3与TALEN-H11-R2，P为转染阳性Cas9n，N为空白细胞。如果TALEN有效果，靶点将切出160bp+230bp条带，靶点2将切出170bp+220bp
条带，从上图中可以看到切割后的酶切片段，并且3、4、5、6组合条带较亮，切割效率高于1、2组。T7EI酶切图：1为TALEN-H11-L1与TALEN-H11-R1，2为TALEN-H11-L2与TALEN-H11-R1，3为TALEN-H11-L3与TALEN-H11-R1，4为TALEN-H11-L1与TALEN-H11-R2，5为TALEN-H11-L2与TALEN-H11-R2，6为TALEN-H11-L3与TALEN-H11-R2，P为转染阳性Cas9n(另一专利介绍)，N为空白细胞。如果TALEN有效果，靶点将切出160bp+230bp条带，靶点2将切出170bp+220bp条带，从上图中可以看到酶切片段，并且3、4、5、6组合条带较亮，估计效率在2％-3％左右。
从图3的结果来看，如果sgRNA有效果，靶位置1将切出160bp+230bp条带，靶位置2将切出170bp+220bp条带，从图中3可以看到模糊的酶切片段，因此该对gRNA均有一定活性。该对特异性的sgRNA在切割H11靶位点时特异性非常强，能有效减少CRISPR/Cas9系统存在的脱靶现象，能大大增加外源基因定点插入的效率，进而减少非特异性切割所引起的基因组非靶向位点的突变带来的影响。
而CRISPR/Cas9靶向定点切割系统重组细胞的切割结果鉴定步骤如下：将PCR扩增产物并与PMD-18T载体(宝生物，货号：D101A)连接，得到连接产物，具体操作步骤参见试剂盒说明书。
将上述得到的连接产物转化大肠杆菌DH5α感受态细胞，然后涂布于含500mg/ml氨苄青霉素的LB固体培养基平板上进行培养，两组分别随机挑取40个克隆并进行测序，计算突变的克隆占总体克隆数的比例，从而算出重组质粒Cas9/gRNA-H11-sg1与Cas9/gRNA-H11-sg2质粒的效率。
实验结果见图4，结果显示：Cas9/gRNA-H11-sg1的效率为:63％(11个克隆里有7个发生突变)Cas9/gRNA-H11-sg2质粒的效率为58％(40个克隆里有23个发生突变)。上述结果表明：该sgRNA能高效的识别猪H11位点，并借助Cas9酶对该位点进行高效的定点切割。从基因组DNA的H11位点的突变率我们可以看出，针对Cas9/gRNA-H11-sg1，其效率为63％,说明在基因组中100条染色体的H11位点中有63条的H11位点被该sgRNA识别，并切割。同理，
Cas9/gRNA-H11-sg2的效率也非常高。其为后面的针对猪H11位点高效定点整合实验打下了坚实的基础。
实施例3 定点插入绿色荧光蛋白基因的方法
借助实施例1(二)中所述的靶位点1构建的CRISPR/Cas9靶向定点切割系统系统对猪H11位点定点插入绿色荧光蛋白基因的方法，包括如下步骤：
1、打靶载体构建
(1)合成片段
依据猪H11位点的DNA序列设计3’端同源臂(序列30所示)、相应的通用引物(序列31所示)以及在两端分别加酶切位点：MluI(ACGCGT)与FseI(GGCCGGCC)加入，合成片段如下：
5’-ACGCGTttcccgaggctGagttagttgGtccagccagtgattgagttgcgtgcggagggcttcttatcttagTTTTATAGGCTACACTGTTAACACTCAGGCTGTTTTCTACCGTTTAGTCAAAATATAGTCACCTTGCCTGCTTCACCTGTCCATCAGAGAATGGCCTCATTAATTGACTCTCTAGTATGAAGTCAAAGTAGCTTTGGTGGCCCTAAATGGACAAGTATCAAGAGACTGGGTGAATTGAGGAGCTTGAGACTGTCACCTCAGATCGAAAAGACTGAAAAATCACCTCAGATCAAAAAGACTGAAAAATCTTCAGTCTGGAAAGGGGACTCAAAACCATAATTAGAGTATTCTGGTAGAATCCTTTTCTCCACTGTTATTCATACAGTTAAGGTGAATAACTAAAAGTAATTGTGAGCTGAGGAGTAAGATACAACACACAAGGAATCAGTTAACAGAGTCTCGAGTGAAATTATAAATGGAAAGAATTATGACTTGAATCATAACTCTGAGGCCCCATTTTCCCTAACAACTTTTGTCCCAATAAACGTGGGTATTTGTTTGGGAGAAACTATCATATACATGATTACCCAGTAAACAGACTGTTTACTAAGTGGGTTTAATTTTAGAAATTGCGCGCTGCAATCTGGTATTAACCATACAACTACCTACCTATAGGGTCAGCCCAGCCTGAACTATCCCATTGGGGTCTTTATTAAGGCTCAAGAAACGGCCATAGCTTCTTCCTTTAAAATGAGTGTTTATTTCTATGAGCTTTAAAGAAAAAAACAGATAATTTCCCTCAACCTACTGAAGAGGAAGGGATTCAGGAAGAAATAAACACAACAATGCCATTCACTTCAGGCCGGCC-3’
(2)将上一步所得DNA片段用MluI(ACGCGT)与FseI(GGCCGGCC)酶切连入载体pLHG-4(回收9kb左右大小片段，pLHG-4序列见序列表中序列
44所示)中(pLHG-4构建步骤参见李和刚博士学位论文)，得载体命名为
pLHG-H11-AR，序列如下：
5’-CTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG
GCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTACTTAAGGGCGCGCCATGAGATGAACTGCTCTGGGATGCCTAGGTAAATTTCTCTGCATTTCAGTTTCTTTTTAGGAAAGTCAGAACTGTTCCTTGCAAGATGAGTTCTGAGAACAGAATGTGTTGCAGAAAGTACTGGAGTCTTTCTAAAAATTTATCCTATGATATTTCCAAGAGACATGGTCACCCTTAAGCAAAGTTATACAAGTATTCATGGTCAATTAATACCATTTGGGGGGGTGTCTTTTTTCTAGGGCTGCACCCATAGCATAAGGAGGTTCCCAGGAGGTGTGGCCGTCAGCTTATGCCACAACCACAGAAACACCAGATCCAAGCGGCATCTGTGACCTATACCACAGCTCATAGCAACGCCAGATCCTTAGCCCCCTTGATTAAAGCCAGGGATCAAACCTGCCTCCTCAAGGATGCTAGTCAGACTCGTTTACTCTGAGCCACGACAGGAACTCCAAGTAATACCATTTTTAATCTGGAAAAAAATCTAAATATCATTAAATCCAACCTTGTTATTATAAAAGAAGGTACCCCATAGCAAAGGTAGCTAATTCATTCAACTAATGTGCAGCTCATTAAGGGTGGAGCTGGGAAGTGAGATCTCCTACTTAGCGTCACATGCCACCTTGCCTAATAATGATGTATTTGTCTATCAAATGCCTACAAAGACATACAGAGTCTCTCCCTGGACAGTTTTCATTTTATTATGTGATCGTTACTACCCCAAAGATTTCTTTCTTGATTTTATTTTGTCCCTCATATTCTGTCTGTCATCCCTACATTCAGATATCAGAGGTGGGGGTATTGGGGAGGGGGAGATGAGGAGAGGAAAAGGATTGGTTGGTGCATGGCCAGTCAAGTTGAAGATGACTGCAACAATCACGAGAAATCTCTGCAAAACTATAAAAGCTTCCTGGGGTGCCTTCTGAAAAAGTCTGATCCAAGTTGCTTTATTAGGGCCTGGACCATTTCTAGAAGTAGATGAATGCATTCCTTTCATTGGCTAGGAGGTGGGGATGGGGCAGAGAGCATACTTCTGTTTCTGCAGCTGAGACCTGGACATGGTGAACCTGGAGTAGCTACCCATATGGCATGGACAGGTCCAACTGCTGCCCCCTCCTTTGTCCCCCAAGAAGCCAGCAGGGGCAGGATGAAGGCCACCTTGGGGCTGCCCTGAGCCTCCTGCAGTATGCCTGGCAACTACTTTCTTAGCCATCTTTAAGGCCCAATCTTGGGTAAAATACTACTCAACCCATTCTTTAGCCACCTTCTCCAAATGCTTCTAGAAAGCGGCCCCCACAAGTAGGTTCTCTGCAGCAGCACAGTGCAAATGGAGGAACACGACCTCAGTAATTATTTTGTCACTGCAAAGTATCTACAACCTTTGCTATAAAAATTAACACCTTGCTTTCCCTGAAAAATAGCCCAGTCATATCCAGCATTTTCCAGCATCCAGGGCAGAGTGCTTGCTCCTCCCCCAGTCAACAGGACTGTTCATACCGAGGAAATGATTTGAGGGTTCTTTAAGCATTTACGCTGTTAATGCTAAAGCTTTCACGACTTCTACCTGAGGGGGGCTTGAGGGAGGGGGGAGGTTTATGTCCCTGCACCGCCAGGAGCCTGGTCTTTGGTAGGAACGCAGAGGCAGCCGGCGACCTTCCACCCTCAGTGTGTCCTTCCCCAGGAGTTTAGGGAAGTGAATCCCTAGATCCAGCCAACATTTCCACTCCCATTTTCAAGAGATTAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAAGCATCGGCAGGTCAGCAAACCAGCAGTTCTCCATCCTTGGGATCTTAGCAGCCGACGACCTTAATTAAACGCGGTGGCGGCCGCATTACCCTGTTATCCCTAGAATTCGATGCTGAAGTTCCTATAGTTTCTAGAGTATAGGAACTTCGGTCATAACTTCGTATAGCATACATTATACGAAGTTATTCCGGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTGGGGTGGGCGAAGAACTCCAGCATGAGATCCCCGCGCTG
GAGGATCATCCAGCCGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAGAAGGCGGCGGTGGAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGTCATTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATGCTAGCTTATCATCGTGTTTTTCAAAGGAAAACCACGTCCCCGTGGTTCGGGGGGCCTAGACGTTTTTTTAACCTCGACTAAACACATGTAAAGCATGTGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATCCTTCAGCCCCTTGTTGAATACGCTTGAGGAGAGCCATTTGACTCTTTCCACAACTATCCAACTCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGTGGCTTTTGGCCGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTCGCTACAGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCACGACATTCAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGCTCGTCAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAATATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACGTTAGGGGGGGGGGGGGAGAGGGGCGGAATTGGATCCGATATCTTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGGCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAACTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATCTTAAGGATCTGACGGTTCACTAAACCAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGT
ACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATGAACTAATGACCCCGTAATTGAGATCTGAAGTTCCTATAGTTTCTAGAGTATAGGAACTTCGGTCATAACTTCGTATAGCATACATTATACGAAGTTATACGCGTttcccgaggctGagttagttgGtccagccagtgattgagttgcgtgcggagggcttcttatcttagTTTTATAGGCTACACTGTTAACACTCAGGCTGTTTTCTACCGTTTAGTCAAAATATAGTCACCTTGCCTGCTTCACCTGTCCATCAGAGAATGGCCTCATTAATTGACTCTCTAGTATGAAGTCAAAGTAGCTTTGGTGGCCCTAAATGGACAAGTATCAAGAGACTGGGTGAATTGAGGAGCTTGAGACTGTCACCTCAGATCGAAAAGACTGAAAAATCACCTCAGATCAAAAAGACTGAAAAATCTTCAGTCTGGAAAGGGGACTCAAAACCATAATTAGAGTATTCTGGTAGAATCCTTTTCTCCACTGTTATTCATACAGTTAAGGTGAATAACTAAAAGTAATTGTGAGCTGAGGAGTAAGATACAACACACAAGGAATCAGTTAACAGAGTCTCGAGTGAAATTATAAATGGAAAGAATTATGACTTGAATCATAACTCTGAGGCCCCATTTTCCCTAACAACTTTTGTCCCAATAAACGTGGGTATTTGTTTGGGAGAAACTATCATATACATGATTACCCAGTAAACAGACTGTTTACTAAGTGGGTTTAATTTTAGAAATTGCGCGCTGCAATCTGGTATTAACCATACAACTACCTACCTATAGGGTCAGCCCAGCCTGAACTATCCCATTGGGGTCTTTATTAAGGCTCAAGAAACGGCCATAGCTTCTTCCTTTAAAATGAGTGTTTATTTCTATGAGCTTTAAAGAAAAAAACAGATAATTTCCCTCAACCTACTGAAGAGGAAGGGATTCAGGAAGAAATAAACACAACAATGCCATTCACTTCAGGCCGGCCTCTAGAATGCATGTTTAAACAGGCCGCGGGAATTCGATTATCGAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGGTCCTCGCCATGGATCCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAAACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAAAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGATTGGAAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGTAGATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGACGTATCCAGGACTGACGAAGGTTCTCGCACTAAAAGTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAACCGTTGATGGAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGTGCTTCGCGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATTAATAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACCCGTGGAAAACGTGGCCAAGATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAATCGTGTCAGGCGATCTCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCAGATCCTAGAGCTCGCTGATCAGCCTCG
ACTGTGCCTTCTAGTTGCCAGCCATCTATTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAGGGGGGGCCCGGTACCCAATTCGCC-3’
(3)合成片段
依据猪H11位点的DNA序列设计5’同源臂(序列28所示)、相应的通用引物(序列29所示)加入RFP编码序列、polyA序列以及在两端分别加入酶切位点：AscⅠ(GGCGCGCC)、PacⅠ(TTAATTAA)，合成片段如下：
5’-GGCGCGCCCATTGAGCCACGAACAGAACTCCCTCTTACCAACTTATTACTACTAACTTCCCAAGTACTGGCTGCTCAGCTGCTTCCTTGGGCATGGGGGAGGGAGCACTATTTTTTCCTCTCCTGACTTCATCCTCTTCCTTTTAATTTCCATAAGGTTCCCTGTGGCCCTGTGCTTTTTTATTTTGAGGCCTTGCACATCCTTCTGGCCCTGATTGCTTCTCAACTCATCTTGTGCCTGCTGGACTTCCACCGTTGTTTCATGTATCTCGTTAGCTGAGATAGCACTTCCTCCTGCCCTTACCCTTTATCTGGCTCTTAGCTCCTGAAAACTGCATTATTAGCTTCCTCTTTTGCCTCTACTCTTACTCAACCAAAATTGTTTTAAGATCTGTGGATCTAGCTTCTGCTGTGCTATTCTTAGGAACACTTTTATTTCCTCTTAGCTCCATCTCACCAGTTATTGGCTAATGGCTTTGCTTGGTACCTACATCTGTACATTTCTTTCGTACTAGCTTCTAGACTGAAAAAGGACTGTTGGTTCAACATGAAAGGGAAGGAGGTAAAAGAGGACACACAGGAAAGATGGATTGGGATTCAGGTCTCTGCTGTTGTTACTTGAGATTGCTTTCTAGATTCTACTTGTGGAAACAAAAAGCCTTTGCGAGAATTCTAAACTGGAGTATTTCTGTAATTGAGGAGTCTTGCTCAGCAAATCCCACTTAGGGGACTAATGAAGTACCAGGAAGAGACAGACCATGCTCAATCCACAAAGCCAGGTTTTACTGAAATGTGACCTACTTTCTTATGCGATCGCCTgccgaaagagtaatgTtggCCgagataggagaagacGatgatatcacgctacgacggaaacAGTACTATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCCAGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGATGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAAGACCGACATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGCCTAAGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATTAATTAA-3’
(4)AscⅠ(GGCGCGCC)、PacⅠ(TTAATTAA)双酶切载体pLHG-H11-AR(回收8kb大小片段)，与上一步所得DNA片段连接，得终载体pLHG-H11，序
列如序列表中的序列45所示：
2、载体效率验证
(1)分离猪胎儿成纤维细胞。
从流产的猪胎儿中分离得到PEF细胞，具体分离方法参见文献：李红，魏红江，许成盛，汪霞，卿玉波，曾养志；版纳微型猪近交系胎儿成纤维细胞系的建立及其生物学特征。
(2)线性化
利用BclI(NEB,R0160S)对pLHG-H11酶切线性化，利用天根生化科技(北京)有限公司琼脂糖凝胶回收试剂盒(DP209)，回收片段用于下一步实验，具体操作方法参见试剂盒说明书。
(3)核转染
将重组质粒Cas9/gRNA-H11-g1与线性化的pLHG-H11各2.5μg通过电转化的方式分别转染PEF细胞，得到重组细胞。转染的具体步骤是：使用核转仪(Amaxa，型号：AAD-1001S)及配套的哺乳动物成纤维细胞转染试剂盒(Amaxa，货号：VPI-1002)进行转染。首先使用0.1％胰蛋白酶(Gibco，货号：610-5300AG)消化贴壁细胞，用胎牛血清(Gibco，货号：16000-044)终止消化，磷酸盐缓冲液(Gibco，货号：10010-023)洗涤细胞两次，添加转染试剂，使用程序T-016转染细胞。
(4)细胞筛选
电转后，将得到的重组细胞30℃培养72小时，然后收集细胞。对细胞进行稀释，每个10cm培养皿铺一定数量的细胞，每2-3天换一次培养基。图2为铺板6天时的克隆。
铺板约10天后，细胞单克隆开始形成，收集每个单克隆一半的细胞量用于基因组提取，剩下的细胞继续培养。一共收集克隆132个。
5)细胞阳性鉴定
利用下列通用引物进行PCR扩增，扩增序列为：
表6：PCR扩增所使用的引物
请补充做电泳的步骤，电泳结果见图5，图5中P1表示引物H11-L-F1与H11-L-R1扩增出片段，大小为1.2kb，P2表示H11-L-F2与H11-L-R2扩增片段，P3表示H11-R-F3与H11-R-R3扩增片段。
通过经PCR鉴定可得出，132个克隆中得到阳性克隆31个(3对引物均扩出)，所得阳性率为23％，将筛选出的阳性克隆置于紫外光下激发(蓝光)，结果见图6A和图6B，从图6A和6B可见筛选出的阳性克隆均能激发绿色荧光，由此可见该载体能很好的用于H11位点的定点敲入。
Claims (20)
- 一种利用定点切割系统对猪H11位点定点插入的方法，其特征在于，包括以下步骤：1)在猪目标基因组序列中确定靶向切割系统所靶向的靶序列；2)根据靶位点设计、构建相应的切割系统的打靶序列；3)构建打靶载体；4)转染细胞，PCR PCR扩增鉴定定点插入效率。
- 根据权利要求1所述的方法，其特征在于，步骤1)中所述的靶向切割系统是TALEN靶向切割系统或CRISPR/Cas靶向切割系统。
- 根据权利要求2所述的方法，其特征在于，所述的CRISPR/Cas靶向切割系统用的核苷酸切割酶是csa9或cas9n。
- 根据权利要求2所述的方法，其特征在于，步骤1)中所述的靶向切割系统所靶向的靶序列是TALEN靶向切割系统所靶向的靶序列、CRISPR/Cas9靶向切割系统或CRISPR/Cas9n靶向切割系统所靶向的靶序列。
- 根据权利要求4所述的方法，其特征在于，步骤1)中所述的靶序列具体如1)、2)或3)所示：1)TALEN靶向切割系统所靶向的是一对位点，其核苷酸序列如序列表中的序列1和序列4、序列表中的序列2和序列4、序列表中的序列3和序列4、序列表中的序列1和序列5、序列表中的序列2和序列5或序列表中的序列3和序列5所示；2)CRISPR/Cas9靶向切割系统所靶向的靶序列如序列表中的序列6或序列7所示。3)CRISPR/Cas9n靶向切割系统所靶向的是一对位点，其核苷酸序列如序列表中的序列8和序列9所示。
- 根据权利要求1所述的方法，其特征在于，步骤2)中所述的打靶序列是TALEN靶向切割系统的多肽序列、CRISPR/Cas9靶向切割系统的核苷酸序列或CRISPR/Cas9n靶向切割系统的一对核苷酸序列。
- 根据权利要求6所述的方法，其特征在于，步骤2)中所述的TALEN靶向切割系统的多肽序列包括多肽甲和多肽乙，具体序列如1)、2)、3)、4)、5)或6)所示：1)多肽甲的序列具体如序列表中的序列10所示，多肽乙的序列具体如序列表中的序列13所示；2)多肽甲的序列具体如序列表中的序列11所示，多肽乙的序列具体如序列表中的序列13所示；3)多肽甲的序列具体如序列表中的序列12所示，多肽乙的序列具体如序列表中的序列13所示；4)多肽甲的序列具体如序列表中的序列10所示，多肽乙的序列具体如序列表中的序列14所示；5)多肽甲的序列具体如序列表中的序列11所示，多肽乙的序列具体如序列表中的序列14所示；6)多肽甲的序列具体如序列表中的序列12所示，多肽乙的序列具体如序列表中的序列14所示。
- 根据权利要求6所述的方法，其特征在于，步骤2)中所述的CRISPR/Cas9靶向切割系统的sgRNA的核苷酸序列包括识别染色体上特定的DNA序列片段和骨架RNA片段，识别染色体上特定的DNA序列片段的核苷酸序列如下1)或2)：1)序列表中的序列15或序列16所示的核苷酸序列；2)将所述1)的核苷酸序列经过一个或几个碱基的取代和/或缺失和/或添加且具有与1)中的核苷酸序列具有同样功能的核苷酸序列。
- 根据权利要求6所述的方法，其特征在于，步骤2)中所述的CRISPR/Cas9n靶向切割系统的sgRNA的核苷酸序列由sgRNA-L和sgRNA-R组成，sgRNA-L和sgRNA-R其序列分别包括识别染色体上特定的DNA序列片段和骨架RNA片段；sgRNA-L的识别染色体上特定的DNA序列片段的核苷酸序列如下1)或2)：1)序列表中的序列17所示的核苷酸序列；2)将所述1)的核苷酸序列经过一个或几个碱基的取代和/或缺失和/或添加且具有与1)中的核苷酸序列具有同样功能的核苷酸序列；sgRNA-R的识别染色体上特定的DNA序列片段的核苷酸序列如下3)或4)：3)序列表中的序列18所示的核苷酸序列；4)将所述3)的核苷酸序列经过一个或几个碱基的取代和/或缺失和/或添加且具有与3)中的核苷酸序列具有同样功能的核苷酸序列。
- 根据权利要求7所述的方法，其特征在于，编码步骤2)中所述的TALEN靶向切割系统多肽序列的DNA序列，包括DNA分子甲和DNA分子乙，具体序列如下1)、2)、3)、4)、5)或6)：1)编码序列表中序列10所示多肽的DNA分子甲的具体序列如序列表中的序列19所示，编码序列表中序列13所示多肽的DNA分子乙的具体序列如序列表中的序列22所示；2)编码序列表中序列11所示多肽的DNA分子甲的具体序列如序列表中的序列20所示，编码序列表中序列13所示多肽的DNA分子乙的具体序列如序列表中的序列22所示；3)编码序列表中序列12所示多肽的DNA分子甲的具体序列如序列表中的序列21所示，编码序列表中序列13所示多肽的DNA分子乙的具体序列如序列表中的序列22所示；4)编码序列表中序列10所示多肽的DNA分子甲的具体序列如序列表中的序列19所示，编码序列表中序列14所示多肽的DNA分子乙的具体序列如序列表中的序列23所示；5)编码序列表中序列11所示多肽的DNA分子甲的具体序列如序列表中的序列20所示，编码序列表中序列14所示多肽的DNA分子乙的具体序列如序列表中的序列23所示；6)编码序列表中序列12所示多肽的DNA分子甲的具体序列如序列表中的序列21所示，编码序列表中序列14所示多肽的DNA分子乙的具体序列如序列表中的序列23所示。
- 根据权利要求8所述的方法，其特征在于，编码步骤2)中所述的CRISPR/Cas9靶向切割系统的sgRNA的核苷酸序列的DNA分子是编码所述序 列15的分子或编码所述序列16的分子，其具体核苷酸序列如1)或2)所示：1)序列表中的序列24所示的核苷酸序列；2)序列表中的序列25所示的核苷酸序列。
- 根据权利要求9所述的方法，其特征在于，编码步骤2)中所述的CRISPR/Cas9n靶向切割系统的sgRNA的DNA分子由编码所述sgRNA-L的DNA分子甲和编码所述sgRNA-R的DNA分子乙组成；其中：DNA分子甲的核苷酸序列如序列表中的序列26所示，DNA分子乙的核苷酸序列如序列表中的序列27所示。
- 根据权利要求1所述的方法，其特征在于，步骤3)中所述的构建打靶载体包括构建定点切割的打靶载体和欲插入基因的打靶载体。
- 根据权利要求13所述的方法，其特征在于，针对定点切割系统构建欲插入基因的打靶载体的步骤如下：1)设计被敲除基因的5’端同源臂和3’端同源臂以及相应的通用引物；2)将上述同源臂、通用引物、标记基因和/或欲插入的基因引入到载体中，得到在打靶载体。
- 根据权利要求14所述的方法，其特征在于，在构建欲插入基因的打靶载体的步骤1)中所述的5’端同源臂和3’端同源臂，其中5’端同源臂的核苷酸序列如序列表中的序列28所示，其相应的通用引物的核苷酸序列如序列表中的序列29所示；3’端同源臂的核苷酸序列如序列表中的序列30所示，其相应的通用引物的核苷酸序列如序列表中的序列31所示。
- 根据权利要求14所述的方法，其特征在于，针对定点切割系统构建的欲插入基因的打靶载体其序列包括上述的5’端同源臂序列、5’端同源臂通用引物序列、欲插入的基因序列、3’端同源臂通用引物序列、3’端同源臂序列。
- 根据权利要求16所述的方法，其特征在于，针对定点切割系统构建的欲插入基因的打靶载体其核苷酸序列如序列表中的序列32所示。
- 根据权利要求1所述的方法，其特征在于，步骤4)中PCR扩增鉴定插入结果中所用到的PCR扩增引物的核苷酸序列如序列表中序列33、序列34、序列35、序列36、序列37、序列38所示。
- 权利要求1所述的方法在靶向修饰猪H11基因中的应用。
- 权利要求1所述的方法在构建猪H11基因突变库中的应用。
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| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| US12031126B2 (en) | 2020-05-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
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