A METHOD OF SITE-DIRECTED INSERTION TO H11 LOCUS IN PIGS BY USING SITE-DIRECTED CUTTING SYSTEM

Abstract

The present invention provides a method of site-directed insertion to H11 locus in pigs by using site-directed cutting system, includes the following steps: 1) identify the targeted sequence targeted by the targeted cutting system in the targeted genome sequence of pigs; 2) design and construct the targeting sequence of the corresponding cutting system according to the targeted site; 3) construction of targeting vector; 4) transfect cells, identify the efficiency of fixed-point insertion by PCR amplification. The invention is dependent on the site-directed cutting system of H11 locus in pigs, to insert the target gene into the target site, in order to solve the problems such as low efficiency of traditional shooting technique, inconvenience design of PCR detection primer, harder to detect. The invention provides a method of site-directed insertion which can stably express the foreign gene at the H11 locus, to build an efficient platform for the production of transgenic pigs.

Claims

1. A method of site-directed insertion to H11 locus in pigs by using site-directed cutting system, which is characterized in that said method includes the following steps: 1) identify the targeted sequence targeted by the targeted cutting system in the targeted genome sequence of pigs; 2) design and construct the targeting sequence of the corresponding cutting system according to the targeted site; 3) construction of targeting vector; 4) transfect cells, identify the efficiency of site-directed insertion by PCR amplification.

2. The method according to claim 1, which is characterized in that, said targeted cutting system in step 1 is a TALEN targeted cutting system or CRISPR/Cas targeted cutting system.

3. The method according to claim 2, which is characterized in that, said nucleotide cleaving enzyme using in CRISPR/Cas target cutting system is csa9 or cas9n.

4. The method according to claim 2, which is characterized in that, said targeted sequence targeted by the targeted cutting system in step 1 is the targeted sequence targeted by the TALEN targeted cutting system, CRISPR/Cas9 targeted cutting system or targeted sequence targeted by CRISPR/Cas9n targeted cutting system.

5. The method according to claim 4, which is characterized in that, said targeted sequences in step 1 are shown in 1), 2) or 3): 1) the targeted sequences targeted by the TALEN targeted cutting system are a pair of sites, having nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:4, SEQ ID NO:2 and SEQ ID NO:4, SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:1 and SEQ ID NO:5, SEQ ID NO:2 and SEQ ID NO:5, or SEQ ID NO:3 and SEQ ID NO:5; 2) the targeted sequences targeted by CRISPR/Cas9 targeted cutting system are shown in SEQ ID NO:6 or SEQ ID NO:7; 3) the targeted sequences targeted by CRISPR/Cas9n targeted cutting system is a pair of sites, having nucleotide sequences shown in SEQ ID NO:8 and SEQ ID NO:9.

6. The method according to claim 1, which is characterized in that, said targeted sequences in step 2 are polypeptide sequences of a TALEN targeted cutting system, nucleotide sequences of CRISPR/Cas9 targeted cutting system or a pair of nucleotide sequences of CRISPR/Cas9n targeted cutting system.

7. The method according to claim 6, which is characterized in that, said polypeptide sequences of the TALEN targeted cutting system include polypeptide A and polypeptide B, the specific sequences are shown in 1), 2), 3), 4), 5) or 6): 1) the specific sequences of the polypeptide A are shown in SEQ ID NO:10, specific sequences of the polypeptide B are shown in SEQ ID NO:13; 2) the specific sequences of the polypeptide A are shown in SEQ ID NO:11, specific sequences of the polypeptide B are shown in SEQ ID NO:13; 3) the specific sequences of the polypeptide A are shown in SEQ ID NO:12, specific sequences of the polypeptide B are shown in SEQ ID NO:13; 4) the specific sequences of the polypeptide A are shown in SEQ ID NO:10, specific sequences of the polypeptide B are shown in SEQ ID NO:14; 5) the specific sequences of the polypeptide A are shown in SEQ ID NO:11, specific sequences of the polypeptide B are shown in SEQ ID NO:14; 6) the specific sequences of the polypeptide A are shown in SEQ ID NO:12, specific sequences of the polypeptide B are shown in SEQ ID NO:14.

8. The method according to claim 6, which is characterized in that, said sgRNA nucleotide sequences of CRISPR/Cas9n targeted cutting system in step 2) include identification of specific DNA sequence segments and skeletal RNA fragments on a chromosome, the nucleotide sequences which identify the specific DNA sequence segments are shown in 1) or 2): 1) the nucleotide sequences are shown in SEQ ID NO:15 or SEQ ID NO:16; 2) the nucleotide sequences of the 1) are replaced by one or a few bases and/or deleted and/or added and have the same function as the nucleotide sequences in the 1).

9. The method according to claim 6, which is characterized in that, said sgRNA nucleotide sequences of CRISPR/Cas9n targeted cutting system in step 2) compose of sgRNA-L and sgRNA-R, the sequences of sgRNA-L and sgRNA-R respectively including identification of specific DNA sequence segments and skeletal RNA fragments on a chromosome; the nucleotide sequences of sgRNA-L which identify the specific DNA sequence segments on a chromosome are shown in 1) or 2): 1) the nucleotide sequences are shown in SEQ ID NO:17; 2) the nucleotide sequences of the 1) are replaced by one or a few bases and/or deleted and/or added and have the same function as the nucleotide sequences in the 1); the nucleotide sequences of sgRNA-R which identify the specific DNA sequence segments on a chromosome are shown in 3) or 4): 3) the nucleotide sequences are shown in SEQ ID NO:18; 4) the nucleotide sequences of the 3) are replaced by one or a few bases and/or deleted and/or added and have the same function as the nucleotide sequences in the 3).

10. The method according to claim 7, which is characterized in that, the DNA sequences encoding said polypeptide sequences of the TALEN targeted cutting system in step 2) include DNA molecular A and DNA molecular B, the specific sequences are shown in 1), 2), 3), 4), 5) or 6): 1) the specific sequences of DNA molecular A which encode the polypeptide shown in SEQ ID NO:10 are shown in SEQ ID NO:19, and the specific sequences of DNA molecular B which encode the polypeptide shown in SEQ ID NO:13 are shown in SEQ ID NO:22; 2) the specific sequences of DNA molecular A which encode the polypeptide shown in SEQ ID NO:11 are shown in SEQ ID NO:20, and the specific sequences of DNA molecular B which encode the polypeptide shown in SEQ ID NO:13 are shown in SEQ ID NO:22; 3) the specific sequences of DNA molecular A which encode the polypeptide shown in SEQ ID NO:12 are shown in SEQ ID NO:21, and the specific sequences of DNA molecular B which encode the polypeptide shown in SEQ ID NO:13 are shown in SEQ ID NO:22; 4) the specific sequences of DNA molecular A which encode the polypeptide shown in SEQ ID NO:10 are shown in SEQ ID NO:19, and the specific sequences of DNA molecular B which encode the polypeptide shown in SEQ ID NO:14 are shown in SEQ ID NO:23; 5) the specific sequences of DNA molecular A which encode the polypeptide shown in SEQ ID NO:11 are shown in SEQ ID NO:20, and the specific sequences of DNA molecular B which encode the polypeptide shown in SEQ ID NO:14 are shown in SEQ ID NO:23; 6) the specific sequences of DNA molecular A which encode the polypeptide shown in SEQ ID NO:12 are shown in SEQ ID NO:21, and the specific sequences of DNA molecular B which encode the polypeptide shown in SEQ ID NO:14 are shown in SEQ ID NO:23.

11. The method according to claim 8, which is characterized in that, the DNA molecules encoding said sgRNA nucleotide sequences of CRISPR/Cas9n targeted cutting system in step 2) are the DNA molecules encoding said SEQ ID NO:15 or the DNA molecules encoding said SEQ ID NO:16, the nucleotide sequences of which are show in 1) or 2): 1) the nucleotide sequences are shown in SEQ ID NO:24; 2) the nucleotide sequences are shown in SEQ ID NO:25.

12. The method according to claim 9, which is characterized in that, the DNA molecules encoding said sgRNA of CRISPR/Cas9n targeted cutting system in step 2) compose of the DNA molecules A encoding said sgRNA-L and the DNA molecules B encoding said sgRNA-R; wherein the nucleotide sequences of DNA molecules A are shown in SEQ ID NO:26, and the nucleotide sequences of DNA molecules B are shown in SEQ ID NO:27.

13. The method according to claim 1, which is characterized in that, said construction of targeting vector in step 3) include the construction of targeting vector with site-specific cleavage and the targeting vector to insert the gene.

14. The method according to claim 13, which is characterized in that, the steps of construction of targeting vector to insert the gene aimed at site-specific cleavage system are as follows: 1) design of the 5 terminal homology arm and 3 terminal homology arm with their gene knocked out and the corresponding universal primers; 2) obtain the targeting vector by leading said homology arms, universal primers, marker gene and/or genes to be inserted into the carrier.

15. The method according to claim 14, which is characterized in that, said 5 terminal homology arm and 3 terminal homology arm in the step 1) on construction of targeting vector to insert the gene, wherein the nucleotide sequences of the 5 terminal homology arm are shown in SEQ ID NO:28, and the nucleotide sequences of corresponding universal primers are shown in SEQ ID NO:29; the nucleotide sequences of the 3 terminal homology arm are shown in SEQ ID NO:30, and the nucleotide sequences of corresponding universal primers are shown in SEQ ID NO:31.

16. The method according to claim 14, which is characterized in that, the sequences of targeting vector to insert the gene constructed for site-specific cleavage system include above mentioned the sequences of 5 terminal homology, the universal primers sequences of 5 terminal homology, the gene sequences to be inserted, the universal primers sequences of 3 terminal homology, the sequences of 3 terminal homology.

17. The method according to claim 16, which is characterized in that, the nucleotide sequences of targeting vector to insert the gene constructed for site-specific cleavage system are shown in SEQ ID NO:32.

18. The method according to claim 1, which is characterized in that, the nucleotide sequences of PCR amplified primers used in PCR amplification to identify insertion results in step 4) are shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38.

19. The application of the method of claim 1 in targeted modification of porcine H11 gene.

20. The application of the method of claim 1 in the construction of porcine H11 gene mutation library.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1 is the structure schematic of targeting vector of the present invention;

[0062] FIG. 2 are the identification results of PCR amplification of recombinant cell DNA constructed by TALEN targeted cutting system;

[0063] FIG. 3 are the identification results of PCR amplification of recombinant cell DNA constructed by CRISPR/cas9n targeted cutting system;

[0064] FIG. 4 are the results of sequencing detection and analysis of the DNA enzyme cutting vector of the recombinant cells constructed by CRISPR/cas9n targeted cutting system;

[0065] FIG. 5 are the identification results of PCR amplification of the cells obtained by site-directed insertion to porcine H11 site of the green fluorescent protein constructed by CRISPR/cas9n targeted cutting system; and

[0066] FIG. 6A and FIG. 6B are fluorescence excitation of positive clones; wherein FIG. 6A shows microscopic observation results of cells under visible light, FIG. 6B shows microscopic observation results of cells under UV light.

DETAILED DESCRIPTION

[0067] The following examples are used to further illustrate the invention, but should not be construed as a limitation to the present invention. Under the precondition of without departing from the spirit and the essence of the invention, the modification or the replacement of the invention belongs to the category of the invention.

[0068] As mentioned in the background, in the cultivation of improved varieties of pigs, the foreign genes are randomly inserted into the genome of pigs, with trouble for the following analysis, in order to overcome the defects, in a typical embodiment of the invention, a method of site-directed insertion to H11 locus in pigs by using site-directed cutting system is provided, the method firstly constructs a TALEN targeted cutting system, a CRISPR/Cas targeted cutting system and a CRISPR/cas9n targeted cutting system, the three kinds of cutting system constructed by the invention can effectively identify the porcine H11 site, and use the corresponding nuclease to cut the sequence gene of the porcine H11 site.

[0069] Then a targeting vector is designed to the porcine H11 site using the said targeted cutting system, the said targeting vector is obtained by introduce the homologous arms connected with knockout gene on the two terminals and corresponding universal primers and the gene to be inserted to the pLHG-4. The recombinant cells can be obtained by transfect the above targeting vector cells into cells, when using the site-directed gene mutation library contrasted by the said method, we only need to insert the interest gene between the homology arms then the site-directed insertion of the genes to be completed.

[0070] The targeting vector obtained by using said method contains the universal primers, greatly reducing the difficulty and workload of the screening test. And there are not the promoters starting a positive screening gene expression on the inside of the two homologous arms, and there are also negative screening genes on the outside of the homologous arms. The said targeting vector transfect cells, then the positive clones are screened by the culture medium containing corresponding drugs with positive screening genes, the positive clones are enriched with high efficiency, the method of cell screening is simple and does not need a lot of manpower and material resources, greatly reducing the cost of gene targeting, at the same time, the foreign gene can be stably expressed at the H11 locus, and a stable platform for the transgene is built.

[0071] The beneficial effects of the present invention are described combined with specific examples in detail below.

Example 1: Construction of Site-Directed Cutting System of Three Porcine H11 Sites

[0072] One. Construction of TALEN Site-Directed and Targeted Cutting System

[0073] 1. Construction of Target Sequence

[0074] Find the sequences of porcine H11 site in gene library. The present invention first according to the gene sequence of porcine H11 locus, as follows:

[0075] 5-TACTGAAATGTGACCTACTTTCTTATGTTCCTGGAAGTTTAGATCAGGGT GGGCAGCTCTGGG-3

[0076] 2. Design of the TALEN Site

[0077] At present, the TALEN system uses FokI incision enzyme activity to cut the target gene, because the FokI can play the activity by forming a dipolymer, in the actual operation we should select two adjacent (interval 14-18 base) target sequences (generally more than a dozen bases) to construct respectively TAL identification modules.

[0078] The site of TALEN cutting system is designed according to the target, schematic diagram shown in FIG. 1, the specific sequences are as follows:

[0079] L1: 5-TTCTTATGTTCCTGGAAG-3 T carrier: L15, the structure of the carrier is: cmv-sp6-NLS-TAL-T-IRES-puro-pA, this carrier is purchased from Shanghai SiDanSai Biotechnology Co., Ltd;

[0080] L2: 5-TCTTATGTTCCTGGAAGT-3 T carrier: L15, the structure of the carrier is: cmv-sp6-NLS-TAL-T-IRES-puro-pA, this carrier is purchased from Shanghai SiDanSai Biotechnology Co., Ltd;

[0081] L3: 5-CTTATGTTCCTGGAAGTT-3 T carrier: L15, the structure of the carrier is: cmv-sp6-NLS-TAL-T-IRES-puro-pA, this carrier is purchased from Shanghai SiDanSai Biotechnology Co., Ltd;

[0082] R1: 3-GTAGCCTATAAAACCCAG-5 A carrier: R10, the structure of the carrier is: cmv-sp6-NLS-TAL-A-pA, this carrier is purchased from Shanghai SiDanSai Biotechnology Co., Ltd;

[0083] R2: 3-AGCCTATAAAACCCAGAG-5 C carrier: R12, the structure of the carrier is: cmv-sp6-NLS-TAL-C-pA, this carrier is purchased from Shanghai SiDanSai Biotechnology Co., Ltd;

[0084] 3. TALEN is constructed by using FastTALE TALEN rapid construction kit (Cat. No. 1802-030) of Shanghai SiDanSai Biotechnology Co., Ltd, the procedure of construction is:

(One) Design, Select the Appropriate Module According to the Selected Site, the Design Results are as Follows:

[0085]

TABLE-US-00001 L1: 5-TTCTTATGTTCCTGGAAG-3 Tcarrier:L15 Selectedmodule: TT1CT2TA3TG4TT5CC6TG7GA8AG9 L2: 5-TCTTATGTTCCTGGAAGT-3 Tcarrier:L15 Selectedmodule: TC1TT2AT3GT4TC5CT6GG7AA8GT9 L3: 5-CTTATGTTCCTGGAAGTT-3 Tcarrier:L15 Selectedmodule: CT1TA2TG3TT4CC5TG6GA7AG8TT9 R1: 5-GTAGCCTATAAAACCCAG-3 Acarrier:R10 Selectedmodule: GT1AG2CC3TA4TA5AA6AC7CC8AG9 R2: 5-AGCCTATAAAACCCAGAG-3 Ccarrier:R12 Selectedmodule: AG1CC2TA3TA4AA5AC6CC7AG8AG9

(Two) Adding Modules

[0086] Add the required modules (a total of 5 tubes) respectively in turn into the 200 ul PCR tube in accordance with the selected modules in the first step.

TABLE-US-00002 TABLE 1 module 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

TABLE-US-00003 TABLE 2 module 2 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

(Three) Adding Sample

[0087] Add other solutions respectively into the reagent kit in accordance with the following system, the system is as follows:

TABLE-US-00004 TABLE 3 reaction system System Module 1.5 L 9 Solution1 1 L Solution2 1 L Solution3 2 L Carrier 1.5 L ddH2O 1 L Total volume 20 L

(Four) Connect

[0088] 1) The above mixture is respectively placed on the PCR instrument to complete the connection, the reaction procedure is as follows:

TABLE-US-00005 37 C. 5 min {close oversize brace} 15 cycle 16 C. 10 min 80 C. 10 min 12 C. 2 min

[0089] 2) Take out the reaction solution in the previous step, respectively add 1 L solution 4, 0.5 L solution 5 (total volume 21.5 L), then incubation for 60 minutes at 37 C.

(Five) Transform

[0090] 1) Take out the competence in the kit, and put it on the ice for 10 min to melt.

[0091] 2) Take 10 L of the final connection product in step 4 to join in, mixing them.

[0092] 3) Lay them on the ice for 20 min

[0093] 4) Heat Shock at 42 C. for 60 s.

[0094] 5) Ice-bath for 3 min.

[0095] 6) Add 500 L SOC, recovery on the shaking table at 37 C. for 30 min.

[0096] 7) 4000 rpm, centrifugal for 5 min, pour the most supernatant (leave about 150 u L).

[0097] 8) Resuspend the cells, uniformly coat them on the LB plates resisting kna.

[0098] 9) Culture at 37 C. for 16 h.

(Six) Select the Clones

[0099] 10 clones are selected on the culture plate, cultured in the shaking table at 37 C. overnight (more than 16 h). The primer 305 (5-CTCCCCTTCAGCTGGACAC-3) and 306 (5-AGCTGGGCCACGATTGAC-3) are sent to the company (Beijing TIANYI HUIYUAN Ltd.) for sequencing, select the correct clones to obtain TALEN: TALEN-H11-L1, TALEN-H11-L2, TALEN-H11-L3, TALEN-H11-R1 and TALEN-H11-R2, extract the plasmid to complete the next experiment.

[0100] Two. Construction of CRISPR/Cas9 Targeted Cutting System

[0101] 1. Find the sequences of porcine H11 site in gene library, select the sgRNA target for gene knockout according to PAM sequence, as follows: 5-TACTGAAATGTGACCTACTTTCTTATGTTCCTGGAAGTTTAGATCAGGGTGG GCAGCTCTGGG-3,

[0102] Location 1 of sgRNA target site (named as H11-sg1): 5-GTTCCTGGAAGTTTAGATCAGGG-3, the nucleotide sequences identifying the target site in the corresponding sgRNA sequences are shown in SEQ ID NO:15, the DNA sequences encoding the above sequences are shown in SEQ ID NO:24.

[0103] Location 2 of sgRNA target site (named as H11-sg2): 5-AGATCAGGGTGGGCAGCTCTGGG-3, the nucleotide sequences identifying the target site in the corresponding sgRNA sequences are shown in SEQ ID NO:16, the DNA sequences encoding the above sequences are shown in SEQ ID NO:25.

[0104] 2. Construction of the sgRNA Expression Plasmid

[0105] Use the cas9/gRNA construction kit (Catalog. No. VK001-01) of ViewSolid Biotech company to complete the construction, the construction process is as follows:

[0106] (1) According to the two target sequences mentioned above, the corresponding primer sequences are designed, synthesized by Beijing TIANYI HUIYUAN Ltd., the specific sequences are shown in Table 4:

TABLE-US-00006 TABLE4 PrimersequencesofthetwosgRNAtargets Nameofthe nucleotide Sequences(5-3) H11-sg1-F AAACACCGGTTCCTGGAAGTTTAGATCA H11-sg1-R CTCTAAAACTGATCTAAACTTCCAGGAAC H11-sg2-F AAACACCGAGATCAGGGTGGGCAGCTCT H11-sg2-R CTCTAAAACAGAGCTGCCCACCCTGATCT

[0107] (2) Formation of Oligonucleotide Dipolymer (Oligoduplex)

[0108] The synthetic oligo is diluted to 10 M, mixed in the following proportions

TABLE-US-00007 H11-sg1-F 1 L H11-sg1-R 1 L Solution1 5 L H2O 3 L Final system 10 L

[0109] After mixing respectively, processing in accordance with the following program: 95 C. 3 min; the sample tube is placed in the 95 C. water to cool the above mixture from 95 C. to 25 C.; and then to deal with 5 min at 16 C., finally get the oligonucleotide dipolymer-1.

TABLE-US-00008 H11-sg2-F 1 L H11-sg2-R 1 L Solution1 5 L H2O 3 L Final system 10 L

[0110] After mixing respectively, processing in accordance with the following program: 95 C. 3 min; the sample tube is placed in the 95 C. water to cool the above mixture from 95 C. to 25 C.; and then to deal with 5 min at 16 C., finally get the oligonucleotide dipolymer-2.

[0111] (3) The Oligonucleotide Dipolymers are Inserted into the Carrier Respectively

[0112] Reaction in the following reaction system:

TABLE-US-00009 Cas9/gRNA Vector 1 L oligoduplex-1 2 L H2O 7 L Final system 10 L

[0113] After full mixing, standing at room temperature (25 C.). for 5 min, get the carrier Cas9/gRNA-H11-sg1.

TABLE-US-00010 Cas9/gRNA Vector 1 L oligoduplex-2 2 L H2O 7 L Final system 10 L

[0114] After full mixing, standing at room temperature (25 C.). for 5 min, get the carrier Cas9/gRNA-H11-sg2.

[0115] (4) Transform

[0116] The final products (carrier Cas9/gRNA-H11-sg1, Cas9/gRNA-H11-sg2) of the step (3) are respectively added into the 50 L DH5a competent cells which had just thawed, mixing gently, ice bath for 30 min, then heat shock at 42 C. for 90 s, standing on the ice for 2 min, apply directly on the ampicillin resistance plate.

[0117] (5) Test and Verify

[0118] Pick five white colonies to shake bacteria, and extract the DNA of plasmid for sequencing. The primer for sequencing is 5-TGAGCGTCGATTTTTGTGATGCTCGTCAG-3, the sequencing results of Cas9/gRNA-H11-sg2 and Cas9/gRNA-H11-sg1 were obtained, the sequencing results are shown in SEQ ID NO:39 and SEQ ID NO:40. The results indicate that the DNA sequence encoding sgRNA (the sequences of target site 1 and target site 2) can be successfully inserted into the Cas9/gRNA vector backbone by the above operation.

[0119] Three. Construction of CRISPR/Cas9n Targeted Cutting System

[0120] 1. Design the Target

[0121] According to the H11 locus of the mouse, find the Eif4 and Drg genes (the site of the mouse is located in the middle of the two genes) of the pig, bring up the middle area in NCBI to find out the H11 site of pig, select the sgRNA target for knocking out the genes according to the PAM sequence (PAM sequence is NGG), as follows:

TABLE-US-00011 5-TACTGAAATGTGACCTACTTTCTTATGTTCCTGGAAGTTTAGATCAG GGTGGGCAGCTCTGGG-3

[0122] Design the sgRNA target for knocking out the genes: location 1 of SgRNA-L target site (named H11-sgL2): 5-AGATCAGGGTGGGCAGCTCTGGG-3, the nucleotide sequences identifying the target in the corresponding sgRNA-L sequence are shown in SEQ ID NO:17; the DNA sequence encoding the above sequences are shown in SEQ ID NO:26.

[0123] Location 2 of sgRNA-R target site (named as H11-sgR1): 5-TTCCAGGAACATAAGAAAGTAGG-3, the nucleotide sequences identifying the target site in the corresponding sgRNA sequences are shown in SEQ ID NO:18, the DNA sequences encoding the above sequences are shown in SEQ ID NO:27. The two target sequences was arrangement of head to head, they are 4 bp apart from each other, that is 4 bp interval.

[0124] 2. Construction of sgRNA Expression Plasmids

[0125] First design the primer sequences according to the target sequence, then send them to Beijing TIANYI HUIYUAN Ltd. to synthetise single-stranded oligonucleotides, specific sequences are as follows:

TABLE-US-00012 (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

[0126] Wherein H11-sgL2-F and H11-sgL2-R were annealed to obtain a double stranded DNA fragment H11-sgL2 with a viscous end, the pX335 (addgene, Plasmid 42335) vector (its nucleotide sequence is as shown in SEQ ID NO:41) is digested by Bbs I enzyme to recover fragment, H11-sgL2 is 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-gR1 with a viscous end, the pX335 vector is digested by Bbs I enzyme to recover fragment, H11-gR1 is connected to the fragment to obtain pX335-sgRNA-H11-R vector. The two plasmids were sent to Beijing TIANYI HUIYUAN Ltd. to carry out sequencing and verification, the sequence of sequencing primers bbsR is: 5 -GACTATCATATGCTTACCGT-3, the results of sequencing are respectively show in SEQ ID NO:42 and SEQ ID NO:43. The results show that the sgRNA encoding sequence of the sgRNA target site 1 and the target site 2 of can be inserted into the pX335 vector backbone through the above operation.

Example 2: Verify the Efficiency of Three Methods for Site-Directed Cutting System of Porcine H11 Sites

[0127] 1. Separate the Porcine Fetal Fibroblast Cells

[0128] PEF cells are isolated from the aborted porcine fetus (methods of separation in reference: Li Hong, Wei Hongjiang, Xu Chengsheng, Wangxia, Qing Yubo, Zeng Yangzhi; Establishment of the fetal fibroblast cell lines of Banna Mini-Pig Inbred and their biological characteristics; Journal of Hunan Agricultural University (natural science ed); Vol. 36, issue 6; in December 2010; 678-682).

[0129] 2. Eukaryotic Transfection

[0130] The recombinant plasmids TALEN-H11-L1 and TALEN-H11-R1, TALEN-H11-L2 and TALEN-H11-R1, TALEN-H11-L3 and TALEN-H11-R1, TALEN-H11-L1 and TALEN-H11-R2, TALEN-H11-L2 and TALEN-H11-R2, TALEN-H11-L3 and TALEN-H11-R2 in example 1, are cotransfected into PEF cells by electroporation in 2.5 g respectively, to obtain five kinds of recombinant cells. The recombinant plasmids Cas9/gRNA-H11-sg1 and Cas9/gRNA-H11-sg2 obtained in example 1 (Two) are cotransfected into PEF cells by electroporation in 4 g respectively, to obtain the recombinant cells. The recombinant plasmids pX335-sgRNA-H11-L and pX335-sgRNA-H11-R obtained in example 1 (Three) are cotransfected into PEF cells by electroporation in 2 g respectively, to obtain a kind of recombinant cell. The specific steps of transfection are: the nuclear transfer instrument (Amaxa, types: AAD-1001S) and a set of transfection kit of mammalian fibroblast cells (Amaxa, No.: VPI-1002) are used to transfect. First use 0.1% trypsin (Gibco, No.: 610-5300AG) to digest adherent cells, use the fetal bovine serum (Gibco, No.: 16000-044) to terminate the digestion, use the phosphate buffer (Gibco, No.: 10010-023) to wash the cells two times, add the transfection reagents, use the procedure T-016 to transfect cells.

[0131] 3. Extraction of DNA

[0132] Eight kinds of recombinant cells could be obtained by step 2, wherein five kinds of recombinant cells obtained in TALEN targeted and site-directed cutting system, two kinds of recombinant cells obtained in CRISPR/Cas9 targeted and site-directed cutting system, a kind of recombinant cell obtained in CRISPR/Cas9n targeted and site-directed cutting system, The above eight kinds of recombinant cells are cultured for 48 hours at 37 C., then collect the cells. The specific steps are: First use 0.1% trypsin (Gibco, No.: 610-5300AG) to digest adherent cells, use the fetal bovine serum (Gibco, No.: 16000-044) to terminate the digestion, use the phosphate buffer (Gibco, No.: 10010-023) to wash the cells two times, add 200 microliters of cell lysate GA (component of DNA extraction kit DP304 in TIANGEN company). Respectively extract the genomic DNA of the above eight kinds of recombinant cells reference the steps of kit manual.

[0133] 4. Validation of PCR Enzyme Digestion Efficiency

[0134] (1) Using the primer H11-F (5-GCGAGAATTCTAAACTGGAG-3) and the primer H11-R (5-GATCTGAGGTGACAGTCTCAA-3) the PCR amplification is carried out by using five kinds of recombinant cells DNA as template, which are obtained from the TALEN target cutting system in step 3, recovered 387 bp fragment; using the primer H11-F (5-GCGAGAATTCTAAACTGGAG-3) and the primer H11-R (5-GATCTGAGGTGACAGTCTCAA-3) the PCR amplification was carried out by using two kinds of recombinant cells DNA as template, which were collected from the CRISPR/Cas9 target cutting system in step 3, recovered PCR amplification products of about 370 bp; using the primer H11-F: 5-GCGAGAATTCTAAACTGGAG-3 and the primer H11-R: 5-GATCTGAGGTGACAGTCTCAA-3 to compose the primer pair, the PCR amplification is carried out by using genomic DNA of recombinant cells as template, which are collected from the CRISPR/Cas9 target cutting system, recovered 387 bp fragment.

[0135] The PCR results of recombinant cells of said TALEN target cutting system and CRISPR/Cas9 target cutting system are identified with enzyme cutting by using T7 endonuclease I (T7 endonuclease I, T7E1) (NO: #E001L) of VIewSolid Biotech. Specific steps are:

[0136] (2) The PCR products of mutant DNA and wild type DNA are mixed with the following system, and the heat denaturation and annealing treatment are carried out (95 C. 5 min, naturally cooled to room temperature).

TABLE-US-00013 TABLE 5 PCR amplification reaction system Number 1 2 PCR products in the 5 ul 0 experimental group PCR products in the 0 5 ul control group Buffer2 (NEB) 1.1 ul 1.1 ul ddH2O 4.4 ul 4.4 ul Total 10.5 ul

[0137] (3) The 0.5 ul T7E1 enzyme is added to the above reaction system, after reaction at 37 C. for 30 min, enzyme digestion results are detected by 2% agarose gel electrophoresis, the electrophoretogram of the recombinant cells enzyme digestion results of the TALEN target cutting system is shown in FIG. 2, the electrophoretogram of the recombinant cells enzyme digestion results of the CRISPR/Cas9n target cutting system is shown in FIG. 3. Wherein, the Lane 1 in FIG. 2 is TALEN-H11-L1 and TALEN-H11-R1, the Lane 2 is TALEN-H11-L2 and TALEN-H11-R1, the Lane 3 is TALEN-H11-L3 and TALEN-H11-R1, the Lane 4 is TALEN-H11-L1 and TALEN-H11-R2, the Lane 5 is TALEN-H11-L2 and TALEN-H11-R2, the Lane 6 is TALEN-H11-L3 and TALEN-H11-R2, the Lane P is positive transfection Cas9n, the Lane N is control cell. If the TALEN is effective, the target will be cutted out of the 160 bp+230 bp band, target 2 will be cutted out of the 170 bp+220 bp band, the restriction fragment after cutting can be seen from the above figure, and the bands of 3, 4, 5, 6 combination are brighter, the cutting efficiency is higher than 1, 2 groups. Figure of T7EI enzyme digestion: the Lane 1 is TALEN-H11-L1 and TALEN-H11-R1, the Lane 2 is TALEN-H11-L2 and TALEN-H11-R1, the Lane 3 is TALEN-H11-L3 and TALEN-H11-R1, the Lane 4 is TALEN-H11-L1 and TALEN-H11-R2, the Lane 5 is TALEN-H11-L2 and TALEN-H11-R2, the Lane 6 is TALEN-H11-L3 and TALEN-H11-R2, the Lane P is positive transfection Cas9n (introduced in another patent), the Lane N is control cell. If the TALEN is effective, the target will be cutted out of the 160 bp+230 bp band, target 2 will be cutted out of the 170 bp+220 bp band, the restriction fragment after cutting can be seen from the above figure, and the bands of 3, 4, 5, 6 combination are brighter, the efficiency is estimated at about 2%-3%.

[0138] From the results of FIG. 3, if the sgRNA is effective, the target position 1 will cutted out the 160 bp+230 bp band, the target position 2 will cutted out the 170 bp+220 bp band, the fuzzy restriction fragment can be seen from the FIG. 3, so the pair of gRNA have certain activity. The specificity of the pair of sgRNA in the cleavage of H11 target site is very strong, which can effectively reduce the miss phenomenon existing in the CRISPR/Cas9 system, greatly increase the efficiency of the fixed point insertion of exogenous gene, and then reduce the impact of the mutation on the non target site of genome caused by nonspecific cleavage.

[0139] The identification procedures of cutting results of recombinant cells of CRISPR/Cas9 targeted cutting system are as follows: the PCR amplification product is connected with PMD-18T vector (Takara, No.: D101A), to obtain the connected products, the details of the operation procedures see the description of kit.

[0140] The obtained products are transformed into Escherichia coli. DH5a competent cells, and then coated on the LB solid medium plate containing 500 mg/ml ampicillin to culture, 40 clones are randomly selected from two groups respectively and sequenced, proportion of mutant clones in the total number of clones is calculated, so the efficiency of the recombinant plasmid Cas9/gRNA-H11-sg1 and Cas9/gRNA-H11-sg2 plasmid is calculated.

[0141] Experimental results are shown in FIG. 4, the results show that: the efficiency of Cas9/gRNA-H11-sg1 is 63% (7 mutants occurred in 11 clones), the efficiency of the Cas9/gRNA-H11-sg2 plasmid is 58% (23 mutants occurred in 40 clones). The results show that the sgRNA could identify the porcine H11 sites efficiently, and carry out fixed point cutting on this site efficiently with the aid of Cas9 enzyme. We can see from the mutation rate of the H11 site of the genomic DNA, for Cas9/gRNA-H11-sg1, its efficiency is 63%, it shows that there are H11 sites of 63 chromosomes in the H11 sites of the 100 chromosomes of the genome identified by the sgRNA, and cutted. In the same way, the efficiency of Cas9/gRNA-H11-sg2 is also very high. It has laid a solid foundation for high efficiency and fixed-point integration experiment to the porcine H11 site.

Example 3: Method of Fixed-Point Insertion of Green Fluorescent Protein Gene

[0142] Method of fixed-point insertion of green fluorescent protein gene to the porcine H11 site with the aid of the CRISPR/Cas9 targeted cutting system constructed by the said target site 1 in the Example 1(Two), comprises the following steps:

[0143] 1. Construction of Targeting Vector

[0144] (1) Synthetic Fragment

[0145] According to the DNA sequence of porcine H11 site, design the 3-terminal homology arm (shown as SEQ ID NO:30), corresponding universal primer (shown as SEQ ID NO:31) and plus the restriction site respectively on two ends: MluI (ACGCGT) and FseI (GGCCGGCC) to join, synthetic fragments are as follows:

TABLE-US-00014 5-ACGCGTttcccgaggctGagttagttgGtccagccagtgattgagt tgcgtgcggagggcttcttatcttagTTTTATAGGCTACACTGTTAACA CTCAGGCTGTTTTCTACCGTTTAGTCAAAATATAGTCACCTTGCCTGCT TCACCTGTCCATCAGAGAATGGCCTCATTAATTGACTCTCTAGTATGAA GTCAAAGTAGCTTTGGTGGCCCTAAATGGACAAGTATCAAGAGACTGGG TGAATTGAGGAGCTTGAGACTGTCACCTCAGATCGAAAAGACTGAAAAA TCACCTCAGATCAAAAAGACTGAAAAATCTTCAGTCTGGAAAGGGGACT CAAAACCATAATTAGAGTATTCTGGTAGAATCCTTTTCTCCACTGTTAT TCATACAGTTAAGGTGAATAACTAAAAGTAATTGTGAGCTGAGGAGTAA GATACAACACACAAGGAATCAGTTAACAGAGTCTCGAGTGAAATTATAA ATGGAAAGAATTATGACTTGAATCATAACTCTGAGGCCCCATTTTCCCT AACAACTTTTGTCCCAATAAACGTGGGTATTTGTTTGGGAGAAACTATC ATATACATGATTACCCAGTAAACAGACTGTTTACTAAGTGGGTTTAATT TTAGAAATTGCGCGCTGCAATCTGGTATTAACCATACAACTACCTACCT ATAGGGTCAGCCCAGCCTGAACTATCCCATTGGGGTCTTTATTAAGGCT CAAGAAACGGCCATAGCTTCTTCCTTTAAAATGAGTGTTTATTTCTATG AGCTTTAAAGAAAAAAACAGATAATTTCCCTCAACCTACTGAAGAGGAA GGGATTCAGGAAGAAATAAACACAACAATGCCATTCACTTCAGGCCGGC C-3

[0146] (2) The DNA fragments obtained in the previous step are cutted into the vector pLHG-4 by MluI (ACGCGT) and FseI (GGCCGGCC) (recovering the fragment of the about 9 KB size, pLHG-4 sequences are shown in SEQ ID NO:44) (PLHG-4 construction steps see Dr. Li Hegang's thesis), the vector named pLHG-H11-AR, the sequences are as follows:

TABLE-US-00015 5-CTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAA CGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAG CACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGA TCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCC TGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCC TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGG GGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCA AAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA GACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGA CTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTT TTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGA GCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTT ACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAA CCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTC AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCC TGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGAT CAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTA AGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCAC TTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTG AGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAG AGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAAC TTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGC ACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCT GAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCA ATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAG CTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGC CAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCA GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCA CTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTT AGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGAT CCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTC CACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATC CTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCT ACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAG TGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGAT AAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGG CGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAA AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGC CTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGT CGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCA GCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACC GCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCA GCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCC TCTCCCCGCGCGTTGGCCGATTCATTAATCAGCTGGCACGACAGGTTTC CCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCT CACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATG TTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATG ACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAA GCTGGAGCTACTTAAGGGCGCGCCATGAGATGAACTGCTCTGGGATGCC TAGGTAAATTTCTCTGCATTTCAGTTTCTTTTTAGGAAAGTCAGAACTG TTCCTTGCAAGATGAGTTCTGAGAACAGAATGTGTTGCAGAAAGTACTG GAGTCTTTCTAAAAATTTATCCTATGATATTTCCAAGAGACATGGTCAC CCTTAAGCAAAGTTATACAAGTATTCATGGTCAATTAATACCATTTGGG GGGGTGTCTTTTTTCTAGGGCTGCACCCATAGCATAAGGAGGTTCCCAG GAGGTGTGGCCGTCAGCTTATGCCACAACCACAGAAACACCAGATCCAA GCGGCATCTGTGACCTATACCACAGCTCATAGCAACGCCAGATCCTTAG CCCCCTTGATTAAAGCCAGGGATCAAACCTGCCTCCTCAAGGATGCTAG TCAGACTCGTTTACTCTGAGCCACGACAGGAACTCCAAGTAATACCATT TTTAATCTGGAAAAAAATCTAAATATCATTAAATCCAACCTTGTTATTA TAAAAGAAGGTACCCCATAGCAAAGGTAGCTAATTCATTCAACTAATGT GCAGCTCATTAAGGGTGGAGCTGGGAAGTGAGATCTCCTACTTAGCGTC ACATGCCACCTTGCCTAATAATGATGTATTTGTCTATCAAATGCCTACA AAGACATACAGAGTCTCTCCCTGGACAGTTTTCATTTTATTATGTGATC GTTACTACCCCAAAGATTTCTTTCTTGATTTTATTTTGTCCCTCATATT CTGTCTGTCATCCCTACATTCAGATATCAGAGGTGGGGGTATTGGGGAG GGGGAGATGAGGAGAGGAAAAGGATTGGTTGGTGCATGGCCAGTCAAGT TGAAGATGACTGCAACAATCACGAGAAATCTCTGCAAAACTATAAAAGC TTCCTGGGGTGCCTTCTGAAAAAGTCTGATCCAAGTTGCTTTATTAGGG CCTGGACCATTTCTAGAAGTAGATGAATGCATTCCTTTCATTGGCTAGG AGGTGGGGATGGGGCAGAGAGCATACTTCTGTTTCTGCAGCTGAGACCT GGACATGGTGAACCTGGAGTAGCTACCCATATGGCATGGACAGGTCCAA CTGCTGCCCCCTCCTTTGTCCCCCAAGAAGCCAGCAGGGGCAGGATGAA GGCCACCTTGGGGCTGCCCTGAGCCTCCTGCAGTATGCCTGGCAACTAC TTTCTTAGCCATCTTTAAGGCCCAATCTTGGGTAAAATACTACTCAACC CATTCTTTAGCCACCTTCTCCAAATGCTTCTAGAAAGCGGCCCCCACAA GTAGGTTCTCTGCAGCAGCACAGTGCAAATGGAGGAACACGACCTCAGT AATTATTTTGTCACTGCAAAGTATCTACAACCTTTGCTATAAAAATTAA CACCTTGCTTTCCCTGAAAAATAGCCCAGTCATATCCAGCATTTTCCAG CATCCAGGGCAGAGTGCTTGCTCCTCCCCCAGTCAACAGGACTGTTCAT ACCGAGGAAATGATTTGAGGGTTCTTTAAGCATTTACGCTGTTAATGCT AAAGCTTTCACGACTTCTACCTGAGGGGGGCTTGAGGGAGGGGGGAGGT TTATGTCCCTGCACCGCCAGGAGCCTGGTCTTTGGTAGGAACGCAGAGG CAGCCGGCGACCTTCCACCCTCAGTGTGTCCTTCCCCAGGAGTTTAGGG AAGTGAATCCCTAGATCCAGCCAACATTTCCACTCCCATTTTCAAGAGA TTAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAAGCATCGGCAGGTCAGC AAACCAGCAGTTCTCCATCCTTGGGATCTTAGCAGCCGACGACCTTAAT TAAACGCGGTGGCGGCCGCATTACCCTGTTATCCCTAGAATTCGATGCT GAAGTTCCTATAGTTTCTAGAGTATAGGAACTTCGGTCATAACTTCGTA TAGCATACATTATACGAAGTTATTCCGGATAAGATACATTGATGAGTTT GGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAA TTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACA AGTTGGGGTGGGCGAAGAACTCCAGCATGAGATCCCCGCGCTGGAGGAT CATCCAGCCGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAG AAGGCGGCGGTGGAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTT GGTCGGTCATTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGA AGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAA GCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATC ACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGG CCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCG GCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCAT GCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGC TCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTAC GTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGC CGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACT TTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTT CGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCAC AGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCC TCGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAA GAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCA GCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAA GCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACG ATCCTCATGCTAGCTTATCATCGTGTTTTTCAAAGGAAAACCACGTCCC CGTGGTTCGGGGGGCCTAGACGTTTTTTTAACCTCGACTAAACACATGT AAAGCATGTGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACC TTCTGGGCATCCTTCAGCCCCTTGTTGAATACGCTTGAGGAGAGCCATT TGACTCTTTCCACAACTATCCAACTCACAACGTGGCACTGGGGTTGTGC CGCCTTTGCAGGTGTATCTTATACACGTGGCTTTTGGCCGCAGAGGCAC CTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTCGCTACAGACG TTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCACGACATT CAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATG CTCGTCAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAG ACGGCAATATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTA TTCCAAGCGGCTTCGGCCAGTAACGTTAGGGGGGGGGGGGGAGAGGGGC GGAATTGGATCCGATATCTTACTTGTACAGCTCGTCCATGCCGAGAGTG ATCCCGGCGGCGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCT CGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTC GGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGG TCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGT TCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTG GCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCC TTGAAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCT CGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAA GATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAG TCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGT AGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGT GGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCG CCCTCGCCGGACACGCTGAACTTGTGGCCGTTTACGTCGCCGTCCAGCT CGACCAGGATGGGCACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCAC CATCTTAAGGATCTGACGGTTCACTAAACCAGCTCTGCTTATATAGACC TCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGT TACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCC ATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGC TATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAG CGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGT CATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCA ATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGG CAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTAT TGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGG TCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGC GGAACTCCATATATGGGCTATGAACTAATGACCCCGTAATTGAGATCTG AAGTTCCTATAGTTTCTAGAGTATAGGAACTTCGGTCATAACTTCGTAT AGCATACATTATACGAAGTTATACGCGTttcccgaggctGagttagttg GtccagccagtgattgagttgcgtgcggagggcttcttatcttagTTTT ATAGGCTACACTGTTAACACTCAGGCTGTTTTCTACCGTTTAGTCAAAA TATAGTCACCTTGCCTGCTTCACCTGTCCATCAGAGAATGGCCTCATTA ATTGACTCTCTAGTATGAAGTCAAAGTAGCTTTGGTGGCCCTAAATGGA CAAGTATCAAGAGACTGGGTGAATTGAGGAGCTTGAGACTGTCACCTCA GATCGAAAAGACTGAAAAATCACCTCAGATCAAAAAGACTGAAAAATCT TCAGTCTGGAAAGGGGACTCAAAACCATAATTAGAGTATTCTGGTAGAA TCCTTTTCTCCACTGTTATTCATACAGTTAAGGTGAATAACTAAAAGTA ATTGTGAGCTGAGGAGTAAGATACAACACACAAGGAATCAGTTAACAGA GTCTCGAGTGAAATTATAAATGGAAAGAATTATGACTTGAATCATAACT CTGAGGCCCCATTTTCCCTAACAACTTTTGTCCCAATAAACGTGGGTAT TTGTTTGGGAGAAACTATCATATACATGATTACCCAGTAAACAGACTGT TTACTAAGTGGGTTTAATTTTAGAAATTGCGCGCTGCAATCTGGTATTA ACCATACAACTACCTACCTATAGGGTCAGCCCAGCCTGAACTATCCCAT TGGGGTCTTTATTAAGGCTCAAGAAACGGCCATAGCTTCTTCCTTTAAA ATGAGTGTTTATTTCTATGAGCTTTAAAGAAAAAAACAGATAATTTCCC TCAACCTACTGAAGAGGAAGGGATTCAGGAAGAAATAAACACAACAATG CCATTCACTTCAGGCCGGCCTCTAGAATGCATGTTTAAACAGGCCGCGG GAATTCGATTATCGAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGG CAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACA CAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCA ACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCC CTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGT GACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCAC CGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAG CTTTGCTCCTTCGCTTTCTGGCTCAGAGGCTGGGAAGGGGTGGGTCCGG GGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCC TCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGC TGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGGTCCTCGCC ATGGATCCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAA ACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCA AAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGAT GATTGGAAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGAT ACTCTGTAGATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGT CAAAGTGACGTATCCAGGACTGACGAAGGTTCTCGCACTAAAAGTGGAT AATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAACCGT TGATGGAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGG TGCTTCGCGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGC GTTGAATATATTAATAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAAC TTGAGATTAATTTTGAAACCCGTGGAAAACGTGGCCAAGATGCGATGTA TGAGTATATGGCTCAAGCCTGTGCAGGAAATCGTGTCAGGCGATCTCTT TGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCT ACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGTATAAT GTGTTAAACTACTGATTCTAATTGTTTGTGTATTTTAGATTCCAACCTA TGGAACTGATGAATGGGAGCAGTGGTGGAATGCAGATCCTAGAGCTCGC TGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTATTGTTTGCC CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCT TTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCAT TCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGG AAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGA GGCGGAAAGAACCAGCTGGGGCTCGAGGGGGGGCCCGGTACCCAATTCG CC-3

[0147] (3) Synthetic Fragment

[0148] According to the DNA sequence of porcine H11 site, design the 5-terminal homology arm (shown as SEQ ID NO:28), corresponding universal primer (shown as SEQ ID NO:29) and plus RFP encoding sequence, polyA sequence and plus the restriction site respectively on two ends: Asc I (GGCGCGCC), Pac I (TTAATTAA), synthetic fragments are as follows:

TABLE-US-00016 5-GGCGCGCCCATTGAGCCACGAACAGAACTCCCTCTTACCAACTTAT TACTACTAACTTCCCAAGTACTGGCTGCTCAGCTGCTTCCTTGGGCATG GGGGAGGGAGCACTATTTTTTCCTCTCCTGACTTCATCCTCTTCCTTTT AATTTCCATAAGGTTCCCTGTGGCCCTGTGCTTTTTTATTTTGAGGCCT TGCACATCCTTCTGGCCCTGATTGCTTCTCAACTCATCTTGTGCCTGCT GGACTTCCACCGTTGTTTCATGTATCTCGTTAGCTGAGATAGCACTTCC TCCTGCCCTTACCCTTTATCTGGCTCTTAGCTCCTGAAAACTGCATTAT TAGCTTCCTCTTTTGCCTCTACTCTTACTCAACCAAAATTGTTTTAAGA TCTGTGGATCTAGCTTCTGCTGTGCTATTCTTAGGAACACTTTTATTTC CTCTTAGCTCCATCTCACCAGTTATTGGCTAATGGCTTTGCTTGGTACC TACATCTGTACATTTCTTTCGTACTAGCTTCTAGACTGAAAAAGGACTG TTGGTTCAACATGAAAGGGAAGGAGGTAAAAGAGGACACACAGGAAAGA TGGATTGGGATTCAGGTCTCTGCTGTTGTTACTTGAGATTGCTTTCTAG ATTCTACTTGTGGAAACAAAAAGCCTTTGCGAGAATTCTAAACTGGAGT ATTTCTGTAATTGAGGAGTCTTGCTCAGCAAATCCCACTTAGGGGACTA ATGAAGTACCAGGAAGAGACAGACCATGCTCAATCCACAAAGCCAGGTT TTACTGAAATGTGACCTACTTTCTTATGCGATCGCCTgccgaaagagta atgTtggCCgagataggagaagacGatgatatcacgctacgacggaaac AGTACTATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCA AGGTGCGCATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGG CGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAG GTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTC AGTTCCAGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCC CGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTG ATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCC TGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTT CCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCC TCCACCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCA AGATGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTCAA GACCACCTACATGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAAG ACCGACATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCG TGGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGCCTAAGA ATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAA ATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTG CATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA TTAATTAA-3

[0149] (4) Asc I (GGCGCGCC), Pac I (TTAATTAA) double-enzyme digest vector pLHG-H11-AR (recovering of 8 KB size fragments), connected with the DNA fragment obtained from the last step, to obtain the final carrier pLHG-H11, shown as SEQ ID NO:45.

[0150] 2. Verification of the Vector Efficiency

[0151] (1) Separate the Porcine Fetal Fibroblast Cells

[0152] PEF cells are isolated from the aborted porcine fetus, the specific separation method see the literature: Li Hong, Wei Hongjiang, Xu Chengsheng, Wangxia, Qing Yubo, Zeng Yangzhi; Establishment of the fetal fibroblast cell lines of Banna Mini-Pig Inbred and their biological characteristics.

[0153] (2) Linearization

[0154] The pLHG-H11 are linearized using BclI (NEB, R0160S), using the agarose gel extraction kit (DP209) of TIANGEN BIOTECH (BEIJING) CO., LTD, recycling fragments for the next experiment, specific operation method see the kit instructions.

[0155] (3) Eukaryotic Transfection

[0156] The recombinant plasmids Cas9/gRNA-H11-sg1 and the linearized pLHG-H11 are cotransfected into PEF cells by electroporation in 2.5 g respectively, to obtain the recombinant cells. The specific steps of transfection are: transfection is carried out by using nuclear instrument (Amaxa and types: AAD-10015) and a set of mammalian fibroblast cells transfection Kit (Amaxa, No.: VPI-1002). First use 0.1% trypsin (Gibco, No.: 610-5300AG) to digest adherent cells, use the fetal bovine serum (Gibco, No.: 16000-044) to terminate the digestion, use the phosphate buffer (Gibco, No.: 10010-023) to wash the cells two times, add the transfection reagents, use the procedure T-016 to transfect cells.

[0157] (4) Cell Selection

[0158] After the electrotransformation, the recombinant cells are cultured for 72 hours at 30 C., and then the cells are collected. The cells are diluted, a certain number of cells in each of the 10 cm culture dishes, change the culture medium every 2-3 days. FIG. 2 is the clone of planking for 6 days.

[0159] After planking for 10 days, the cells begin to form monoclone, the half of cells in each of the monoclonal cells are collected to use for genome extraction, the rest of the cells continue to be cultured. A total of 132 clones are collected.

[0160] 5) Cell Positive Identification

[0161] PCR amplification is performed using the following general primers, and the ampliconic sequences are:

TABLE-US-00017 TABLE6 TheprimersusingforPCRamplification Primer name Sequences(5-3) Remarks H11-L-F1 CTCAGTCCCAGGCTTTACATC Amplification H11-L-R1 CCAACATTACTCTTTCGGCAG oftheleftarm H11-L-F2 ACTGGCTTTCTGAGTTAGGG Amplification H11-L-R2 GTTTCCGTCGTAGCGTGATA oftheleftarm H11-R-F3 CGGAGGGCTTCTTATCTTAG Amplification H11-R-R3 GTGTGGAGCTGTTTAGGGAC oftheright arm

[0162] Please add the steps of electrophoresis, the electrophoresis results are shown in FIG. 5, the P1 indicate the amplified fragments by the primer H11-L-F1 and H11-L-R1, the size of 1.2 kb, the P2 indicate the amplified fragments by the primer H11-L-F2 and H11-L-R2, the P3 indicate the amplified fragments by the primer H11-R-F3 and H11-R-R3.

[0163] It can be drawn by the PCR identification, 31 positive clones are obtained from 132 clones (all 3 pairs of primer are amplificated), the positive rate is 23%, the screened positive clones are excited under ultraviolet light (blue light), the results are shown in FIG. 6A and FIG. 6B, the screened positive clones can stimulate the green fluorescence from FIGS. 6A and 6B, this shows that the vector can be used well for fixed-point insertion of H11 sites.