CN102558309A

CN102558309A - Transcription activator-like effector nucleases, and encoding genes and application thereof

Info

Publication number: CN102558309A
Application number: CN2012100297659A
Authority: CN
Inventors: 肖磊; 赵金龙
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2012-02-10
Filing date: 2012-02-10
Publication date: 2012-07-11
Anticipated expiration: 2032-02-10
Also published as: CN102558309B

Abstract

The invention discloses a pair of transcription activator-like effector nucleases, and encoding genes and application thereof. The pair of transcription activator-like effector nucleases (TALEN) is obtained by fusing a pair of deoxyribonucleic acid (DNA) recognition proteins and two heterologous subunits of a Flavobacterium okeanokoites 1(Fok 1) DNA incision enzyme and can recognize two adjacent loci on goat or sheep beta lactoglobulin gene (BLG) exon2 specifically. When the transcription activator-like effector nucleases are transferred to a host cell simultaneously, the exon2 loci of the BLG gene of the host cell can be targeted by the transcription activator-like effector nucleases, and the targeted loci are subjected to genetic mutation, so that the targeted modification of the goat or sheet BLG gene is realized, and the transcription activator-like effector nucleases have the advantages of high specificity, high targeting efficiency, high accuracy and the like.

Description

Pair of transcription activator-like effector nucleases, coding gene and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a pair of transcription activator-like effector nucleases, and a coding gene and application thereof.

Background

Targeted modification of genomes at the discretion of humans has been a dream for many scientists. The specific deletion or addition of the required sequence on the endogenous genome can construct various animal models for basic research of biology and disease mechanism research on one hand, and can produce animal reactors for cheap production of biological components which are required by us and are difficult to obtain from other ways on the other hand.

The method for producing the protein for medical use or health care by utilizing the mammary gland of the large animal has great economic value and social value of reducing the overall medical cost to a great extent. The principle of the large animal mammary gland apparatus is that after target protein is knocked into a protein promoter with high efficiency and specific expression in mammary gland through a genome targeted modification technology, the high-value target protein is expressed in a large amount and specifically in the mammary gland of a large animal through the promoter. The beta lactoglobulin gene is a main target gene for manufacturing a mammary gland reactor, has high expression quantity and strong space-time specificity, and can be expressed only in mammary glands of adult female animals. The expressed product is secreted only into the mammary gland for collection and purification.

However, people have not found a simple and efficient method for carrying out genome-targeted modification on a genome. The traditional gene targeting technology relies on the random exchange of homologous chromosomes naturally occurring in cells, the targeting efficiency is very low, usually only 10 < -6 > -10 < -8 >, and the targeting method is widely applied only in mice, but not widely applied in other model animals and large mammals due to too low efficiency.

Recently developed sequence-specific nucleases can be used for precise genome-targeted modification. Generally, sequence-specific nucleases consist of a DNA recognition domain and a non-specific endonuclease domain. The principle is that firstly, a nuclease is positioned to a genome region needing editing by a DNA recognition domain, then a non-specific endonuclease cuts double-stranded DNA so as to cause double-strand break (DSB), and the introduced DSB activated DNA self-repair can cause mutation of a gene and promote homologous recombination of the DNA at the site. Zinc Finger Nucleases (ZFNs) are the most clearly and widely used sequence-specific nucleases in current research. The principle is that two zinc finger proteins specifically recognize two DNA sequences which are separated by 5-7bp, two monomers of a non-specific DNA cutting protein Fok1 which is expressed by fusion with the zinc finger proteins are positioned together, and double-stranded DNA at the position can be cut off when the DNA cutting proteins form a dimer, so that DSB is generated. The emergence of ZFN advances the genome targeted modification technology forward, however, the ZFN technology has the problems of uncertainty in targeting, low efficiency, high mop rate and the like, and researchers are difficult to design zinc finger nucleases of specific and efficient target genome target sequences by themselves, which still is the bottleneck restricting the wide application of ZFN. The cost of purchasing high-efficiency specific zinc finger nucleases is high (20 ten thousand RMB/gene), and the ordinary researchers or commercial companies cannot bear the cost at all.

In 2009, two research groups found that one transcription activator-like effector (TALE) of plant pathogens xanthomas, which can regulate plant gene expression, shows DNA binding specificity, and the recognition code of TALE has the characteristics of modularization and simplification, which brings new hopes for scientists to develop simpler novel genome targeted modification technology.

The TALE is fused to Fok1 to form transcription activator-like effector nucleases (TALENs). TALENs target on the same principle as ZFNs, but recognize different proteins for specific DNA. TALEs consist of tens of tandem "protein modules" that specifically recognize DNA and flanking N-and C-terminal sequences. Each "protein module" comprises 34 amino acids, with

residues

12 and 13 being key sites for targeted recognition, referred to as repeat variable di-Residues (RVDs) sites. However, unlike each zinc finger protein recognizing a specific triplet of bases, each RVDs on TALEs can recognize only one base.

Two research groups of Sangamo BioSciences and Harvard university, respectively, performed genome-targeted modification-related studies using TALEs technology, and both papers were published in the same journal of Natural Biotechnology (Nature Biotechnology).

Edward Rebar led group ligated truncated fragments with different C-terminal TALEs to the catalytic domain of the nuclease FokI. When researchers targeted constructed TALENs to endogenous human NTF3 and CCR5 genes, it was demonstrated that TALENs were able to specifically cleave these gene fragments. The harvard university research group developed a strategy based on hierarchical ligation to construct TALEs containing 12 repeat modules. They reduced the DNA sequence of each module while retaining RVDs while minimizing the reproducibility of the remaining sequences. Monomers with specific linker sequences were then obtained by 12-fold PCR and cloned into a backbone vector containing TALE N-terminal and C-terminal sequences. To construct TALE transcription factors, researchers in turn fuse TALEs to the activation domain of one transcription factor. In the next targeting assay, researchers have demonstrated that they specifically upregulate the expression of two of the four endogenous genes tested.

The Rudolf janisch group at MIT in this 7 th month also demonstrated the targeting effect of TALENs in human embryonic stem cells and human ipscs. It was further verified that TALENs are very good genome editing tools by comparing the targeting effect of TALENs at five loci with their ZFNs at the same positions before, which resulted in that five groups of TALENs were similar in targeting efficiency and accuracy to ZFNs purchased from santamo BioSciences, inc.

Disclosure of Invention

The invention provides a pair of short peptides, and a pair of transcription activator-like effector (TALE) obtained by constructing the pair of short peptides can specifically recognize two adjacent nucleotides on a goat or sheep Beta Lactoglobulin Gene (BLG) exon 2; the pair of transcription activator-like effector nucleases (TALEN) constructed by the pair of transcription activator-like effector can accurately and efficiently target the beta lactoglobulin genes of goats or sheep.

A pair of short peptides, wherein the pair of short peptides respectively have amino acid sequences shown as SEQ ID NO.1 and SEQ ID NO. 2.

The present invention provides a pair of polynucleotides encoding the pair of short peptides described above, respectively.

Preferably, the pair of polynucleotides have base sequences shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

Wherein the polynucleotide consists of a nucleotide sequence SEQ ID NO: 16 and SEQ ID NO: 20, wherein the TALENs recognition modules for recognizing the base A are NI-A (shown as SEQ ID NO: 22), the TALENs recognition module for recognizing the base T are NG-T (shown as SEQ ID NO: 23), the TALENs recognition module for recognizing the base C are HD-C (shown as SEQ ID NO: 24), and the TALENs recognition module for recognizing the base G are NK-G (shown as SEQ ID NO: 25).

The invention also provides a pair of proteins, wherein the pair of proteins consists of the N end and the C end of the amino acid sequence framework of the transcriptional activator-like effector which are respectively added at the two ends of the pair of short peptides; wherein, the N end and the C end of the amino acid sequence frame of the transcription activator-like effector are natural or artificially modified sequences.

The pair of proteins can specifically recognize two nucleotide sequences on a goat or sheep Beta Lactoglobulin Gene (BLG) exon2 respectively, wherein the two nucleotide sequences are respectively selected from the following two nucleotide sequences:

(1) SEQ ID NO: 16 or SEQ ID NO: 16 sequence, wherein one or two nucleotides of the sequence are substituted to form a nucleotide sequence;

(2) SEQ ID NO: 20 or SEQ ID NO: 20 sequence derived from the substitution of one or two nucleotides of the sequence.

Preferably, the pair of proteins have amino acid sequences as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. The proteins are transcription activator-like effectors designated BLG-TALE-L1 and BLG-TALE-R2.

The present invention also provides a pair of polynucleotides encoding the pair of proteins described above, respectively.

Preferably, the pair of polynucleotides have base sequences shown as SEQ ID NO.7 and SEQ ID NO.8, respectively.

The invention also provides a pair of fusion proteins, which are formed by fusing the pair of proteins with the DNA cutting protein respectively.

Preferably, the DNA cleavage protein is an endonuclease.

Preferably, the pair of proteins is fused to two subunits of the DNA cleavage protein, respectively.

More preferably, the DNA cleavage protein is a natural or artificially modified Fok1DNA endonuclease.

Most preferably, the pair of fusion proteins have amino acid sequences shown as SEQ ID NO.9 and SEQ ID NO.10, respectively. The fusion protein is transcription activator-like effector nuclease and is named as BLG-TALEN-L1 and BLG-TALEN-R2.

The present invention also provides a pair of polynucleotides encoding the pair of fusion proteins described above, respectively.

Preferably, the pair of polynucleotides have base sequences shown as SEQ ID NO.11 and SEQ ID NO.12, respectively.

The present invention also provides a vector comprising any one of the above-mentioned pair of polynucleotides.

Preferably, a plasmid vector containing a gene encoding a transcription activator-like effector nuclease capable of expressing the transcription activator-like effector nuclease can be constructed by ligating a polynucleotide capable of specifically recognizing the base sequence shown in SEQ ID NO.16 or SEQ ID NO.20 to an intermediate vector pCMV-NLS-TALEbackyne-Fok 1(R) -intercediate, ligating the intermediate vector to a final vector pEF1a-NLS-TALE backyne-Fok 1(R) -pA (shown in SEQ ID NO: 48) or a final vector pEF1a-NLS-TALE backyne-Fok 1(L) -IRES-PURO-pA (shown in SEQ ID NO: 49).

The invention also provides a host cell transformed with the vector.

Preferably, the host cell is a goat or sheep cell; more preferably, the host cell is a goat or sheep IPS cell.

The invention also provides an application of the pair of fusion proteins or the pair of polynucleotides in targeted modification of goat or sheep beta lactoglobulin genes.

Preferably, the pair of fusion proteins have amino acid sequences shown as SEQ ID NO.9 and SEQ ID NO.10 respectively; the pair of polynucleotides have base sequences shown as SEQ ID NO.11 and SEQ ID NO.12 respectively.

The invention also provides a method for targeting the goat or sheep beta lactoglobulin gene, which comprises the following steps: transferring the pair of fusion proteins or the pair of polynucleotides or the vector containing the pair of polynucleotides into goat or sheep IPS cells, and carrying out amplification culture at 30-37 ℃ for 3-7 days to obtain the cells with the beta lactoglobulin gene subjected to targeted modification.

Preferably, the goat or sheep IPS cell is also transferred with anti-puro protein or plasmid capable of expressing the anti-puro protein, so that the screening is facilitated.

Preferably, the amplification culture is carried out at 30 ℃ for at least 1 day, so that a better targeting effect can be obtained.

The invention designs a pair of transcription activator-like effector nucleases (BLG-TALEN-L1 and BLG-TALEN-R2) aiming at one site of a goat or sheep BLG gene, wherein the pair of TALENs are respectively obtained by fusing a DNA recognition structural domain capable of recognizing a section of nucleotide on the BLG gene exon2 and two heterologous subunits of a Fok1DNA endonuclease. When the pair of transcription activator-like effector nucleases is transferred into a host cell, the pair of transcription activator-like effector nucleases can target the exon2 locus of the BLG gene of the host cell and enable the targeted locus to generate gene mutation including base deletion, base insertion and the like, thereby realizing targeted modification of the goat or sheep BLG gene and having the advantages of strong specificity, high targeting efficiency, high accuracy and the like.

Drawings

FIG. 1 is an artificially designed DNA sequence and site recognized by a transcription activator-like effector nuclease;

FIG. 2 is a schematic diagram of a 18 identification module connection strategy; wherein,

a: PCR adds enzyme cutting recognition sequence and connection joint process schematic diagram for each recognition module;

b: PCR is used for adding enzyme digestion recognition sequences and connecting joints for each recognition module;

c: PCR amplifying 6 module segment and intermediate carrier schematic diagram;

d: schematic diagram of the finally constructed TALEN plasmid;

FIG. 3 is a schematic diagram of the intermediate vector pCMV-NLS-TALE backbone-Fok1(R) -intercediate;

FIG. 4 is a schematic representation of the final vector pEF1a-NLS-TALE backbone-Fok1(R) -pA;

FIG. 5 is a schematic representation of the final vector pEF1a-NLS-TALE backbone-Fok1(L) -IRES-PURO-pA;

FIG. 6 is a schematic representation of the final TALEN plasmid pEF1a-NLS-TALE backbone N tertiary-BLG-TALEN-L1-TALE backbone C tertiary-Fok 1(L) -IRES-PURO-pA;

FIG. 7 shows the genotype change of BLG gene at the targeting site; wherein, -represents a deletion of a base, and + represents an insertion of a base.

Detailed Description

The techniques used in the following examples, including molecular biology techniques such as PCR amplification and detection, cell transfection, etc., and cell culture, detection techniques, etc., are conventional techniques known to those skilled in the art, unless otherwise specified; the instruments, reagents, cell lines, etc. used are generally available to those of ordinary skill in the art, unless otherwise indicated by the specification.

Example 1 design of TALENs target sequences

1. Goat and sheep BLG genomic sequences were downloaded from NCBI (GenBank accession No.: goat is Z33881.1, sheep is X12817.1) and exon2 was selected as the targeting target;

2. designing primers, carrying out PCR amplification on the targeted site fragments on the genome, and sequencing, wherein the PCR primers and the sequencing primers are shown in table 1;

TABLE 1

3. Designing TALENs recognition sequences:

determining the TALENs recognition sequence according to the sequence obtained by sequencing and the following principle:

(1) the 0 th base is T (the base before the first base in the recognition sequence is the 0 th base)

(2) The last base is T

(3) The recognition sequence length is between 14 and 19

(4) The length of the Spacer (Spacer) between the two recognition sequences is controlled between 14 and 21 (12 or 13 is also possible, but the efficiency may be lower)

The positions of the designed target sequences are shown in FIG. 1, and the specific sequences are shown in Table 2.

TABLE 2

TALE name	Target sequence
		BLG-TALE-L1(SEQ ID NO：16)	gtctcagccctccact
BLG-TALE-L2(SEQ ID NO：17)	cagccctccactccct
		BLG-TALE-L3(SEQ ID NO：18)	gtctcagccct
BLG-TALE-R1(SEQ ID NO：19)	gcagctggggtcgtgctt
		BLG-TALE-R2(SEQ ID NO：20)	gcagctggggtcgtgct
BLG-TALE-R3(SEQ ID NO：21)	ggctgcagctggggt

Example 2 ligation between TALENs recognition modules and construction of recombinant vectors

1. Acquisition of TALENs recognition Module (modulator)

(1) Four recognition modules, NI, NG, HD, NK, were synthesized that recognize base A, T, C, G, respectively, with the sequences shown in Table 3.

TABLE 3

(2) Four fragments were ligated into pEASY-B vector (purchased from Beijing Quanyujin Co.) by:

taking 3 mul of PCR product;

② adding 1 mul pEASY-B vector;

③25℃，7min；

transforming DH5a competent cells, and coating kanamycin plates;

fifthly, selecting cloning, extracting plasmid in small quantity, carrying out enzyme digestion and sequencing to finally obtain the identification modules NI, NG, HD and NK connected to the vector pEASY-B.

2. Identifying connections between modules

Connection strategy: taking the connection of 19 identification modules as an example, the connection strategy is explained. Since the last half module that can recognize base T is already on the vector, only 18 modules need to be ligated, as shown in FIG. 2.

(1) The recognition sequence (target sequence) is divided into three parts (the original sequence SEQ ID NO: 16-21 is removed the last base, as shown below), that is, each recognition sequence is divided into three segments, each segment contains 3-6 bases, each segment corresponds to 3-6 recognition modules, and each segment is taken as a unit, and the 3-6 recognition modules of the segment are connected.

BLG-TALE-L1- gtctcagccctccac

BLG-TALE-L2- cagccctccactccc

BLG-TALE-L3- gtctcagccc

BLG-TALE-R1- gcagctggggtcgtgct

BLG-TALE-R2- gcagctggggtcgtgc

BLG-TALE-R3- ggctgcagctgggg

(2) Connection method between 3-6 identification modules

PCR amplification adding enzyme digestion recognition sequence and connecting joint

Take the connection between 6 identification modules as an example: FIG. 2(A) is a schematic diagram of the process of adding restriction enzyme recognition sequences and connecting linkers in 6 modular PCR, wherein the primers F1, F7, F8, R6, R7 and R8 have Bbsl restriction enzyme recognition sequences, and the primers F2, F3, F4, F5, R1, R2, R3, R4 and R5 have Bsal restriction enzyme recognition sequences. The Bbsl recognition sequence (SEQ ID NO: 26) is GAAGACNN 'NNNNNN, the Bsal recognition sequence (SEQ ID NO: 27) is GGTCTCN' NNNNNN, the two enzymes belong to type IIs enzymes, the same enzyme digestion recognition sequence can generate a plurality of sticky recognition ends, and theoretically can generate 4⁴The 24 linkers can be generated using a type IIs enzyme, with sticky recognition ends, and restrictions of 4, 6 for the first Gly and Leu codons for each module. We chose 16 of them to design primers, except F1 and R8, F_nCan be reacted with R_n+1Is not ligated to the cohesive ends on the other primers.

Similarly, if 5 modules, 4 modules and 3 modules are connected, F4R6, F3R6 and F2R6 primers are added to the 4 th, 3 rd and 2 nd connecting modules respectively, the primers added to the corresponding front module and the last module are kept unchanged, and the number of connected fragments is correspondingly reduced by 1, 2 and 3 module fragments.

The respective primer sequences (SEQ ID NOS: 28-43) are shown in Table 4.

TABLE 4

Note: recognition sequence with lower case bold letter as enzyme cutting site

The PCR amplification system (50. mu.l) was:

DNA Template (Template): 0.5. mu.l (about 50ng)

Primers (Primer): each 1 μ l (50 μ M)

LA Taq enzyme (Takara): 0.3. mu.l

10 × buffer (buffer): 5 μ l

dNTP：2.5μl(2.5μM)

ddH₂O：40.7μl

PCR procedure: 2min at 95 ℃; 15s at 95 ℃, 30s at 55.8 ℃, 11s at 72 ℃ and 36 cycles; prolonging the temperature at 72 ℃ for 10 min.

After PCR, the fragment shown in FIG. 2(B) is obtained, and each module is added with a different endonuclease recognition sequence and a different linker according to the target binding sequence, and two linkers in the same color indicate that the sticky ends generated by the two linkers can be connected.

② purification

The resulting PCR product was subjected to agarose gel electrophoresis to determine the concentration. The PCR fragment was purified using a general-purpose DNA purification recovery kit (centrifugal column type) from Tiangen corporation, and the concentration of each product was calibrated by agarose gel electrophoresis after purification.

Thirdly enzyme digestion connection

The adjacent modules can not be cut by Bsa1 any more after being connected, so the connection can be carried out simultaneously by enzyme cutting connection, and the enzyme cutting connection system is as follows:

a module: 100 ng/module (3-6)

Bsa1(NEB)：1μl

T4 ligase (fermentas): 1 μ l

T4 ligase buffer (NEB): 2 μ l

ddH₂O: make up to 20 μ l

PCR digestion ligation procedure: 5min at 37 ℃, 5min at 20 ℃ and 35-45 cycles; 10min at 80 ℃.

(3) The fragments of the three 3-6 modules were ligated to the intermediate vector pCMV-NLS-TALEbackne-Fok 1(R) -intercediate

Amplification of 3-6 Modular fragments

And (3) carrying out agarose gel electrophoresis on all 20 mu l of products subjected to the enzyme digestion and connection in the previous step, wherein a plurality of bands with gradient appear in the size of a module length multiple, and the uppermost band is recovered by gel cutting. Carefully placing the cut gel pieces into the tip of a 200. mu.l pipette with a filter membrane, and placing the tip into a 1.5ml centrifuge tube (EP tube); centrifuging at the maximum rotating speed for 5min, and blowing all liquid which is not thrown into the centrifuge tube in the gun head into the centrifuge tube by using a 200-microliter pipette gun after centrifuging; the centrifuged liquid can be used as a template for PCR amplification of the multi-module fragment. The primers for PCR amplification were F-assom and R-assom, and the sequences are shown in Table 5.

TABLE 5

Primer name	Primer sequences
		F-assem(SEQ ID NO：44)	CGGGAGCCGACGTCGACAG
R-assem(SEQ ID NO：45)	CGCTCGAGCGACACGCAGG

PCR System (50. mu.l):

template: 2 μ l

Primer: each 0.5. mu.l (50. mu.M)

Accuprime pfx：0.3μl

10 × buffer: 5 μ l

ddH₂O：42.2μl

PCR procedure: 2min at 95 ℃; 35 cycles of 95 ℃ for 15s, 64 ℃ for 30s, 68 ℃ for 50 s; extending the temperature for 10min at 68 ℃.

② purification of PCR products

The PCR product was subjected to agarose gel electrophoresis to confirm the presence or absence of a foreign band and the concentration of the desired band. If no bands or low proportion of bands relative to the target band, the PCR product was purified using direct Kit; if the amount of the impurity band is large, the gel needs to be recovered and purified. After purification, the band concentration was determined by electrophoresis.

③ enzyme digestion of the intermediate vector pCMV-NLS-TALE backbone-Fok1(R) -intercediate

When 3-6 modular fragments are ligated due to the bbs1 cleavage site in the final vector, ligation is performed simultaneously with bbs1 cleavage. Therefore, it cannot be directly linked to the final vector but first linked to an intermediate vector without the bbs1 cleavage site. A schematic of the intermediate vector is shown in FIG. 3.

Intermediate vector enzyme digestion system:

plasmid: 5 μ g

BsmB1：2μl

DTT(100mM)：1μl

ddH₂O: make up to 100. mu.l

The digestion was carried out overnight at 37 ℃ with 0.5. mu.l of BsmB1 added every two hours and mixed well, preferably by changing the tube, to eliminate the uncut circular plasmid pinned to the wall. After the enzyme had been cut, electrophoresis was carried out to determine whether the plasmid had been completely linearized. After the determination is finished, Kit purifies the enzyme digestion product, and electrophoresis is carried out to calibrate the concentration of the carrier.

(iv) ligation of the three fragments to an intermediate vector

Like Bsa1, Bbs1 is also a type IIs enzyme, which is not cleaved after the ligation of the cohesive ends produced by the cleavage, so that this ligation can also be carried out simultaneously with the cleavage.

Carrier: 100ng

A module: 200 ng/module

Bbs1(fermentas)：1μl

T4 ligase (fermentas): 1 μ l

T4 ligase buffer (NEB): 2 μ l

ddH₂O: make up to 20 μ l

And (3) enzyme digestion connection program: PCR procedure: 5min at 37 ℃, 5min at 20 ℃ and 35-45 cycles; 10min at 80 ℃.

Fifthly, transfection, selection and cloning, small amount plasmid extraction, enzyme digestion identification and sequencing identification

After ligation, 10. mu.l of the transformed DH5a was competent, and the remaining 10. mu.l was frozen at-20 ℃. A certain number of monoclonals (more than 10 monoclonals per plate) are picked up on the next day, a small amount of plasmids are extracted on the third day, the obtained plasmids are identified by digestion with BamH1 and Pst1, a band of about 2kb is correctly connected, and a band of about 550bp is generated when the plasmids are self-connected. Sequencing after the enzyme digestion is correct, and obtaining 14-19 fragments which are successfully connected and cloned after the sequencing is correct. The sequencing primers are shown in Table 6, wherein the amino acid sequences of the successfully connected BLG-TALE-L1 and BLG-TALE-R2 are respectively shown as SEQ ID NO: 5, SEQ ID NO: 6 is shown in the specification; the amino acid sequence of 15.5 modules in the BLG-TALE-L1 is shown in SEQ ID NO: 1, the amino acid sequence of 16.5 modules in the BLG-TALE-L1 is shown as SEQ ID NO: 2, respectively.

TABLE 6

Primer name	Primer sequences
		TALE-Forward sequencing (SEQ ID NO: 46)	CTCCCCTTCAGCTGGACAC
TALE-reverse sequencing (SEQ ID NO: 47)	AGCTGGGCCACGATTGAC

(4) The fragments ligated into the intermediate vector and sequenced correctly were ligated into the final vector pEF1a-NLS-TALE backbone-Fok1(R) -pA and pEF1a-NLS-TALEbackbone-Fok1(L) -1RES-PURO-pA

The schematic diagrams of the final vector pEF1a-NLS-TALE backbone-Fok1(R) -pA and pEF1a-NLS-TALE backbone-Fok1(L) -IRES-PURO-pA are shown in FIGS. 4 and 5, and the base sequences are shown in SEQ ID NO: 48, SEQ ID NO: shown at 49.

The intermediate vector connected with the correct fragment and two final vectors are subjected to double enzyme digestion by BamH1 and Pst1 at the same time, and the corresponding fragments are recovered by cutting gel. TALEs containing modulators were ligated to the two vectors on the left and right of the final vector in the order of left and right at the time of design. Connecting, transfecting, selecting and cloning, extracting plasmids in a small amount, carrying out double enzyme digestion identification on BamH1 and Pst1, and sequencing and identifying. And identifying the correct clone as the final TALENs plasmid required by us. A schematic of the final TALEN plasmid BLG-TALEN-L1 is shown in FIG. 6.

EXAMPLE 3 transfection of plasmids

1. Adding 100 μ l matrigel into each well of 6-well plate, shaking to make it fully spread at the bottom of the whole well, placing in 5% CO₂The incubator is 30 min.

2. Sucking out the culture medium from the bottle for culturing IPS cells T25, sucking PBS once, adding 1ml of 0.25% pancreatin, shaking back and forth to uniformly cover the bottom of the bottle, and placing in 5% CO₂The incubator is 5 min.

3. After digestion, 1ml of 10% DMEM was added to neutralize the pancreatin, and the digested cells were transferred to a 15ml centrifuge tube, counted, centrifuged, at 1200rpm, for 5 min.

4. The cells were resuspended in the appropriate amount of 4 × Dox ES0, 200 ten thousand IPS cells were placed in 6-well plates with matrigel, and 2ml of fresh 4 × Dox ES0 was added.

5. The transfection was performed simultaneously with passage.

6. The constructed BLG-TALEN-L1, BLG-TALEN-L2, BLG-TALEN-L3, BLG-TALEN-R1, BLG-TALEN-R2 and BLG-TALEN-R3 are paired and combined to transfect cells according to Table 7, and 9 combinations are combined in total.

TABLE 7

	BLG-TALEN-R1	BLG-TALEN-R2	BLG-TALEN-R3
				BLG-TALEN-L1	L1+R1	L1+R2	L1+R3
BLG-TALEN-L2	L2+R1	L2+R2	L2+R3
				BLG-TALEN-L3	L3+R1	L3+R2	L3+R3

Plasmids, transfection reagents and medium solutions were mixed according to the following protocol:

the proportion of each component in the system is as follows: TALEN-L: TALEN-R: Lv-EF1a-Mcherry ═ 5: 2

Total DNA opti MEM 2. mu.g 100. mu.l

Total DNA/. mu.gene ═ 2. mu.g/. mu.l

7. The next day after transfection, the Mcherry fluorescence intensity and transfection efficiency were observed under a fluorescence microscope. If the transfection was successful, the medium in the 6 wells was aspirated off and 2ml of fresh 10% DMEM with 2.5. mu.g/ml puro was added.

8. Placing in 5% CO₂The culture was carried out in an incubator at 37 ℃ for two days, and 2ml of fresh 10% DMEM culture solution containing 2.5. mu.g/ml puro per day was replaced.

9. Removing pesticide, and transferring to 30 deg.C 5% CO₂The culture was carried out in an incubator for two days, and the culture medium was changed to 2ml of 10% DMEM.

10. 5% CO shifted to 37 deg.C₂Culturing in an incubator until the cell amount is enough for gene extraction and identification.

Example 4 cell targeting assay

1. 300 mul of 0.25% pancreatin is added into the 6-well plate after the medicine is killed, and the mixture is shaken back and forth evenly. The cells were left at 37 ℃ for 5min and blown with a gun to digest all cells.

2. Mu.l of the liquid was pipetted into a 1.5ml EP tube and the 6-well plate was washed twice with 400. mu.l of PBS and also added to the EP tube.

3. Centrifuging at 13200rpm/min for 5min, and discarding the supernatant.

4. The genome was extracted with a direct PCR Kit (thermo cat # F-140), and the DNA fragment of the targeting region was PCR-amplified.

5. Identification of genotype and targeting efficiency of targeted cells

And adding A into the PCR fragment of the genome of the IPS cell treated by the BLG-TALEN-L1/BLG-TALEN-R2 combination, then connecting the PCR fragment into a PMD18-T vector, monoclonalizing the DNA fragment, and sequencing to obtain the genotype of the target site of the BLG gene. A total of 30 samples were sent and 7 clones were sequenced and mutated, as shown in FIG. 7.

The system A is as follows:

DNA：10μl

rTaq：0.5μl

10xbuffer：1.5

dNTP：0.5μl

ddH2O：2.5μl

then mixing evenly, and placing at 72 ℃ for 20 min.

The results show that: in 7 mutated clones, 5 were deleted and 2 were inserted. The number of bases deleted varies from 6 to 59, and the inserted bases are cc, g and acaa, respectively.

The probability that the BLG-TALEN-L1/BLG-TALEN-R2 combination mutates the BLG gene is (7X 2)/30, i.e., 46.7%, assuming no double knock-outs in the cell. The research only designs TALENs molecules at one site of the BLG gene to obtain a pair of TALENs capable of modifying the gene at fixed points, and the efficiency is very high. The superiority of TALENs technology compared to ZFN technology can be seen. The pair of polynucleotides may recognize SEQ ID NO: 16 and SEQ ID NO: 20, and can recognize a nucleotide sequence derived by substituting one or two nucleotides of the two sequences. The pair of polynucleotides or the fusion protein expressed by the polynucleotides are TALENs capable of efficiently targeting goat and sheep genes, and provide a very efficient tool for manufacturing goat or sheep mammary gland reactors through homologous recombination. By transfecting goat or sheep cells with the polynucleotide of the present invention or injecting the fusion protein of the present invention into goat or sheep cells, homologous recombination can be promoted, a target gene can be inserted, and a protein with high economic value can be obtained, so that production can be performed in mammary gland.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A pair of short peptides, which are characterized in that the pair of short peptides respectively have amino acid sequences shown as SEQ ID NO.1 and SEQ ID NO. 2.

2. A pair of polynucleotides, wherein said pair of polynucleotides encode a pair of short peptides according to claim 1, respectively.

3. The pair of polynucleotides of claim 2, wherein said pair of polynucleotides have base sequences as set forth in SEQ ID No.3 and SEQ ID No.4, respectively.

4. A pair of proteins, wherein said pair of proteins consists of a pair of short peptides according to claim 1, each of which has an N-terminus and a C-terminus framed by an amino acid sequence of a transcriptional activator-like effector; wherein, the N end and the C end of the amino acid sequence frame of the transcription activator-like effector are natural or artificially modified sequences.

5. The pair of proteins of claim 4, wherein said pair of proteins have amino acid sequences as set forth in SEQ ID No.5 and SEQ ID No.6, respectively.

6. A pair of polynucleotides encoding a pair of proteins according to claim 4 or 5, respectively.

7. The pair of polynucleotides of claim 6, wherein said pair of polynucleotides have base sequences as set forth in SEQ ID No.7 and SEQ ID No.8, respectively.

8. A pair of fusion proteins, wherein the pair of fusion proteins is obtained by fusing the pair of proteins according to claim 4 to DNA cleavage proteins, respectively.

9. The pair of fusion proteins of claim 8, wherein said pair of proteins are fused to two subunits of a DNA cleavage protein, respectively.

10. The pair of fusion proteins of claim 9, wherein the DNA cleavage protein is a native or engineered Fok1 endonuclease DNA.

11. The pair of fusion proteins of any one of claims 8 to 10, wherein the pair of fusion proteins have amino acid sequences as shown in SEQ ID No.9 and SEQ ID No.10, respectively.

12. A pair of polynucleotides encoding a pair of fusion proteins according to any one of claims 8 to 10, respectively.

13. A pair of polynucleotides encoding a pair of fusion proteins according to claim 11, respectively.

14. The pair of polynucleotides according to claim 13, wherein said pair of polynucleotides have base sequences shown as SEQ ID No.11 and SEQ ID No.12, respectively.

15. A vector comprising any one of a pair of polynucleotides according to any one of claims 2 to 3, 6 to 7, 13 or 14.

16. A host cell transformed with the vector of claim 15.

17. A vector comprising any one of the pair of polynucleotides of claim 12.

18. A host cell transformed with the vector of claim 17.

19. The host cell of claim 16 or 18, wherein the host cell is a goat or sheep IPS cell.

20. Use of a pair of fusion proteins according to claim 11 or a pair of polynucleotides according to claim 14 for the targeted modification of the goat or sheep beta lactoglobulin gene.

21. A method of targeting a goat or sheep beta lactoglobulin gene comprising: transferring the pair of fusion proteins as claimed in claim 11 or the pair of polynucleotides as claimed in claim 13 or 14 or the vector containing the pair of polynucleotides as claimed in claim 13 or 14 into goat or sheep IPS cells, and performing amplification culture at 30-37 ℃ for 3-7 days to obtain the cells with the targeted modified beta lactoglobulin gene.

22. The method of claim 21, wherein the goat or sheep IPS cells are also transfected with or express a plasmid resistant to puro protein.

23. The method of claim 21 or 22, wherein said amplifying is performed at 30 ℃ for at least 1 day.