CN102702331B - Pair of transcription activator-like effector nucleases (TALEN), encoding gene and application thereof - Google Patents

Abstract

The invention discloses a pair of transcription activator-like effector nucleases (TALEN), encoding gene and application thereof. The pair of transcription activator-like effector nucleases are obtained from fusing a pair of DNA recognition proteins with two heterogenous subunits of Fok1 DNA endonuclease respectively and can recognize specifically two adjacent sites of human RPE65 gene exon1. When the pair of transcription activator-like effector nucleases are transferred into host cells, the RPE65 gene exon1 site of the host cells can be targeted and then genic mutation occurs at the targeted locus, which achieves the target modification of RPE65 gene; in addition, the TALEN has the advantages of high specificity, high targeting efficiency, high accuracy, and on the like.

Description

A pair of transcriptional activation increment effector nuclease and encoding gene and application

Technical field

The present invention relates to genetically engineered field, relate in particular to a pair of transcriptional activation increment effector nuclease and encoding gene and application.

Background technology

People RPE65 gene is called retinal pigment epithelium gene, and a key protein that molecular weight is 65kDa in coding retinal pigment epithelium, participates in the committed steps such as material cycle, the regeneration of visual pigment Visual purple such as retinene.If people lacks the protein of this genes encoding, 11-cis retinene disappearance, rod photoreceptor cell is to light stimulation by Fails To Respond, and the sudden change of this gene can cause Lay uncle congenital amaurosis (LCA2) and retinitis pigmentosa.

According to the mankind's wish, genome is carried out to the dream that directed targeting modification is many scientists always.On endogenous genome, delete specifically or add the sequence of our needs, can construct on the one hand various animal models for Basic of Biology research and pathogenic mechanism research, can produce on the other hand reaction of animals device and produce in order to cheapness the very difficult biological components obtaining from other approach again that we need.

People never find simple method efficiently to carry out genome targeting modification to genome.Traditional gene targeting depends on abiogenous homologous chromosomes in cell and exchanges at random, and its target practice efficiency is very low, conventionally only has 10 ^-6-10 ^-8, this shooting method has only obtained application extensive in mouse, and in other model animals and large mammal, all can not get widespread use because efficiency is too low.

The very fast sequence-specific nuclease of developed recently can be for accurate genome targeting modification.Generally formed by a DNA recognition structure territory and a non-specific endonuclease structural domain by sequence-specific nuclease.Principle is first by DNA differential threshold, nuclease to be navigated to the genome area that needs editor, then thereby non-specific endonuclease cuts off double-stranded DNA and causes DNA double splitting of chain (double-strand break, DSB) the DNA self-regeneration that, the DSB of introducing activates can cause the sudden change of gene and promote this site DNA homology restructuring.Zinc finger nuclease (Zinc-finger nucleases, ZFN) is that studying the clearest is now also most widely used sequence-specific nuclease.Its principle is the DNA sequence dna of two sections of 5-7bp of being separated by of two zinc finger protein specific recognition, and two single aggressiveness of the non-specific DNA scinderin Fok1 of amalgamation and expression are with it located together, DNA scinderin can cut off the double-stranded DNA of this position while forming dimer, thereby causes DSB.The appearance of ZFN makes genome targeting modification technology stride forward major step, but, also there is the problems such as target uncertainty, efficiency are low, mop rate height in ZFN technology, investigator is difficult to the Zinc finger nuclease that designed, designed goes out special and efficient target gene group aim sequence and remains the bottleneck that restricts ZFN widespread use.And business is bought efficient special Zinc finger nuclease expensive (200,000 Renminbi/gene), this expense cannot be born at all by general Study person or commercial company.

2009 Nian Liangge study group find a kind of transcriptional activation increment effector (the transcription activator-likeeffector that can regulating plant genetic expression in phytopathogen Xanthomonas, TALE) show DNA binding specificity, and its recognition code has the feature of modularization and simplification, develop more easy novel gene group targeting modification technology for scientists and brought new hope.

TALE and Fok1 form transcriptional activation increment effector nuclease (transcription activator-like effector nucleases, TALEN) after merging.The target practice principle of TALEN is identical with ZFN, just identifies the albumen difference of specific DNA.TALEs is made up of the series connection " module " of dozens of specific recognition DNA and N-end and the C-end sequence of both sides.Each " module " comprises 34 amino acid, the 12nd and 13 residues are critical sites of target identification, be known as and repeat variable di-residues(RVDs) site.But being different from the triplet base of each zinc finger protein identification specificity, the each RVDs on TALEs only can identify a base.

The Liang Ge research group of Sangamo BioSciences company and Harvard University utilizes respectively TALEs technology to carry out the correlative study of genome targeting modification, and two sections of research papers are published on " Nature Biotechnol " (Nature Biotechnology) magazine of same first phase.

Edward Rebar leader's research group will be connected on the catalyst structure domain of nuclease FokI with the truncated segment of different C-end TALE.When researchist is by the endogenic mankind NTF3 of TALENs target building with when CCR5 gene, confirm that TALENs can shear these gene fragments specifically.Research group of Harvard University has developed a kind of strategy connecting based on layering and has built the TALEs that comprises 12 replicated blocks.They have reduced the DNA sequence dna of each module on the basis that retains RVDs, the repeatability of residue sequence are dropped to minimum simultaneously.And then obtained the monomer with specificity catenation sequence by 12 heavy PCR, and be cloned in the skeleton carrier that comprises TALE N-end and C-end sequence.In order to build TALE transcription factor, researchist is fused to TALE again the activation domain of a transcription factor.In ensuing targeting detects, researchist confirms that it can make four two genetic expressions that detect in native gene raise specifically.

July in this year, the Rudolf Jaenisch group of MIT also verified the target practice effect of TALEN in human embryo stem cell and people iPSC.Its by the TALENs in five sites with before its target practice effect at the ZFNs of same position compare, show that five groups of TALENs are similar to the ZFNs buying from Sangamo BioSciences company with in tolerance range at target practice efficiency, further verified that TALENs is extraordinary genome edit tool.

Summary of the invention

The invention provides a pair of small peptide, utilize this to build to small peptide a pair of transcriptional activation increment effector (TALE) obtaining and can identify specifically two sections of adjacent nucleotides on people RPE65 genome; Utilize this transcriptional activation increment effector to be built to a pair of transcriptional activation increment effector nuclease (TALEN) obtaining, can practice shooting accurately and efficiently to people RPE65 gene.

A pair of small peptide, described a pair of small peptide has respectively the aminoacid sequence as shown in SEQ ID NO.1 and SEQ ID NO.2.

The invention provides one-to-many Nucleotide, the described one-to-many Nucleotide above-mentioned a pair of small peptide of encoding respectively.

Preferably, described one-to-many Nucleotide has respectively the base sequence as shown in SEQ ID NO.3 and SEQ ID NO.4.

Wherein, described polynucleotide are sequentially formed by connecting by identifying respectively on people RPE65 gene the TALENs identification module of corresponding base in nucleotide sequence SEQID NO:17 and SEQ ID NO:22, wherein, the TALENs identification module of identification base A is that NI-A(is as shown in SEQ ID NO:23), the TALENs identification module of identification base T is that NG-T(is as shown in SEQ ID NO:24), the TALENs identification module of identification base C is that HD-C(is as shown in SEQ ID NO:25), the TALENs identification module of identification bases G is that NK-G(is as shown in SEQ ID NO:26).

The present invention also provides a pair of protein, and described a pair of protein adds by above-mentioned a pair of small peptide two ends that respectively the N end of transcriptional activation increment effector aminoacid sequence framework and C end form; Wherein, the N of described transcriptional activation increment effector aminoacid sequence framework end and C end are for natural or pass through engineered sequence.

This can identify respectively two sections of nucleotide sequences on people RPE65 gene specifically to protein, and described two sections of nucleotide sequences are selected from respectively following two nucleotide sequences:

(1) one of SEQ ID NO:17 sequence or SEQ ID NO:17 sequence or two derivative nucleotide sequences of Nucleotide process replacement;

(2) one of SEQ ID NO:22 sequence or SEQ ID NO:22 sequence or two derivative nucleotide sequences of Nucleotide process replacement.

Preferably, described this has respectively the aminoacid sequence as shown in SEQ ID NO.5 and SEQ ID NO.6 to protein.This protein is transcriptional activation increment effector, called after RPE65-TALE-L2 and RPE65-TALE-R3.

The present invention also provides one-to-many Nucleotide, the described one-to-many Nucleotide above-mentioned a pair of protein of encoding respectively.

Preferably, described one-to-many Nucleotide has respectively the base sequence as shown in SEQ ID NO.7 and SEQ ID NO.8.

The present invention also provides a pair of fusion rotein, and described a pair of fusion rotein is merged and forms with DNA scinderin respectively by above-mentioned a pair of protein.

Preferably, described DNA scinderin is DNA restriction endonuclease.

Preferably, described a pair of protein merges with two subunits of DNA scinderin respectively.

More preferably, described DNA scinderin is natural or through engineered Fok1DNA restriction endonuclease.

Most preferably, described a pair of fusion rotein has respectively the aminoacid sequence as shown in SEQ ID NO.9 and SEQ IDNO.10.This fusion rotein is transcriptional activation increment effector nuclease, called after RPE65-TALEN-L2 and RPE65-TALEN-R3.

The present invention also provides one-to-many Nucleotide, the described one-to-many Nucleotide above-mentioned a pair of fused protein of encoding respectively.

Preferably, described one-to-many Nucleotide has respectively the base sequence as shown in SEQ ID NO.11 and SEQ IDNO.12.

The present invention also provides a kind of carrier that comprises any polynucleotide in above-mentioned one-to-many Nucleotide.

Preferably, can first the polynucleotide of base sequence shown in energy specific recognition SEQ ID NO.17 or SEQ ID NO.22 be connected on intermediate carrier pCMV-NLS-TALEbackbone-Fok1 (R)-intermediate, again this intermediate carrier is connected on final carrier pEF1a-NLS-TALE backbone-Fok1 (R)-pA or final carrier pEF1a-NLS-TALEbackbone-Fok1 (L)-IRES-PURO-pA, build and obtain the plasmid vector that comprises encoding transcription activation increment effector nuclease gene, can express transcriptional activation increment effector nuclease.

The present invention also provides a kind of use host cell that above-mentioned carrier transforms.

Preferably, described host cell behaviour isolated cells; More preferably, described host cell behaviour 293T cell.

The present invention also provides a kind of above-mentioned a pair of fusion rotein or the application of above-mentioned one-to-many Nucleotide in people RPE65 gene target is modified.

Preferably, described a pair of fusion rotein has respectively the aminoacid sequence as shown in SEQ ID NO.9 and SEQ ID NO.10; Described one-to-many Nucleotide has respectively the base sequence as shown in SEQ ID NO.11 and SEQID NO.12.

The present invention also provides a kind of method of people RPE65 gene targeting, comprise: by above-mentioned a pair of fusion rotein or above-mentioned one-to-many Nucleotide or contain this carrier to polynucleotide and proceed to people's isolated cells, in 30-37 DEG C of amplification cultivation 1-4 days, obtain prion protein gene by the cell of targeting modification.

Preferably, described a pair of fusion rotein has respectively the aminoacid sequence as shown in SEQ ID NO.9 and SEQ ID NO.10; Described one-to-many Nucleotide has respectively the base sequence as shown in SEQ ID NO.11 and SEQID NO.12.

Preferably, in described people's isolated cells, also proceed to anti-puro albumen and maybe can express the plasmid of anti-puro albumen, be convenient to screening.

A pair of transcriptional activation increment effector nuclease (RPE65-TALEN-L2 and RPE65-TALEN-R3) has been designed in a site that the present invention is directed to people RPE65 gene, and this is merged and obtained by two allos subunits can identifying the DNA recognition structure territory of RPE65 gene the preceding paragraph Nucleotide and a Fok1DNA restriction endonuclease respectively TALENs.When this is proceeded to host cell to transcriptional activation increment effector nuclease simultaneously, it can be practiced shooting to the site of host cell RPE65 gene, and the site producer sudden change that makes to practice shooting, comprise base deletion, base insertion etc., thereby realize targeting modification to people RPE65 gene, have that high specificity, target practice efficiency are high, accuracy advantages of higher.

Brief description of the drawings

Fig. 1 is DNA sequence dna and the site of the transcriptional activation increment effector nuclease identification of artificial design;

Fig. 2 is 18 identification module connection strategy schematic diagram; Wherein,

A:PCR is that each identification module adds restriction endonuclease recognition sequence and jointing process schematic diagram;

B:PCR is schematic diagram after each identification module interpolation restriction endonuclease recognition sequence and jointing;

C:PCR increase 6 module fragments and intermediate carrier schematic diagram;

D: the final TALEN plasmid schematic diagram building;

Fig. 3 is intermediate carrier pCMV-NLS-TALE backbone-Fok1 (R)-intermediate schematic diagram;

Fig. 4 is final carrier pEF1a-NLS-TALE backbone-Fok1 (R)-pA schematic diagram;

Fig. 5 is final carrier pEF1a-NLS-TALE backbone-Fok1 (L)-IRES-PURO-pA schematic diagram;

Fig. 6 is the Genotypic variation of RPE65 gene in target practice site; Wherein ,-representing base deletion ,+expression base is inserted.

Embodiment

The technology using in following examples, comprises pcr amplification and detection, cell transfecting equimolecular biology techniques, and cell cultures, detection technique etc., unless stated otherwise, is the known routine techniques of those skilled in the art; Plant and instrument, reagent and the clone etc. that use, only this specification sheets is dated especially, is that the research of general this area and technician can obtain by public approach.

The design of embodiment 1 TALENs target sequence

1, from the upper and lower manned RPE65 genome sequence of NCBI (NC 000001.10)

2, the site fragment of practicing shooting on design primer pcr amplification genome, and order-checking, wherein, PCR primer and sequencing primer are in table 1;

Table 1

3, design TALENs recognition sequence (target sequence):

The sequence obtaining according to order-checking, and determine TALENs recognition sequence according to following principle:

(1) the 0th bit base is that the base before first of T(recognition sequence is the 0th)

(2) last bit base is T

(3) recognition sequence length is between 13-19

Intervening sequence (Spacer) length between (4) two recognition sequences be controlled between 13-21 (12 also can, but efficiency may be lower)

As shown in Figure 1, concrete sequence is in table 2 in the target sequence position that design obtains.

Table 2

TALE title	Target sequence
		RPE65-TALE-L1（SEQ ID NO:16）	tccttcttcattct
RPE65-TALE-L2（SEQ ID NO:17）	gagaacttccttcttcatt
		RPE65-TALE-L3（SEQ ID NO:18）	gagaacttccttcttcat
RPE65-TALE-L4（SEQ ID NO:19）	gagaacttccttctt
		RPE65-TALE-R1（SEQ ID NO:20）	tcaggatccagagttct
RPE65-TALE-R2（SEQ ID NO:21）	tcaggatccagagtt
		RPE65-TALE-R3（SEQ ID NO:22）	tcaggatccagagt

Connection between embodiment 2 TALENs identification modules and the structure of recombinant vectors

1, the acquisition of TALENs identification module (modular)

(1) synthetic four identification module NI, NG, HD, the NK that identifies respectively base A, T, C, G, sequence is in table 3.

Table 3

(2) four fragments are connected into pEASY-B carrier (purchased from Beijing Quan Shi King Company), method of attachment is:

1. get PCR product 3 μ l; 2. add 1 μ l pEASY-B carrier; 3. 25 DEG C, 7min; 4. transform DH5a competent cell, coating card is received mycin flat board; 5. picking is cloned, a small amount of is extracted plasmid, enzyme is cut, checked order, and finally obtains being connected to identification module NI, NG, HD, NK in carrier pEASY-B.

2, the connection between identification module

Connection strategy: with the example that is connected to of 19 identification modules, connection strategy is described.Because last half that can identify base T is on carrier, so as long as connect 18 modules, connection diagram is shown in Fig. 2.

(1) recognition sequence (target sequence) being divided into three parts (is respectively former sequence SEQ IDNO:16-22 and removes last base, as follows), be that every recognition sequence is first divided into three sections, every section contains 3-6 base, correspondingly every section of corresponding 3-6 identification module, first, taking every section as unit, the 3-6 of this section identification module connected.

(2) method of attachment between 3-6 identification module

1. pcr amplification adds restriction endonuclease recognition sequence and jointing

Add restriction endonuclease recognition sequences and jointing process schematic diagram taking example: the Fig. 2 (A) that is connected between 6 identification modules as 6 module PCR, wherein primers F 1, F7, F8, R6, R7, R8 are with Bbs1 restriction endonuclease recognition sequence, and primers F 2, F3, F4, F5, R1, R2, R3, R4, R5 are with Bsa1 restriction endonuclease recognition sequence.Bbs1 recognition sequence (SEQ ID NO:27) is GAAGACNN ' NNNN, the recognition sequence (SEQ ID NO:28) of Bsa1 is GGTCTCN ' NNNN, these two enzymes all belong to type IIs enzymes, same restriction endonuclease recognition sequence can produce multiple viscosity identification ends, can produce in theory 44 viscosity identification ends, add the ending of each module and start 4 kinds of Gly codons, the restriction that Leu codon is 6 kinds, utilizes a type IIs enzyme can produce 24 kinds of joints.We have designed primer at 16 kinds of choosing wherein, except F1 and R8, and F _ncan with R _n+1sticky end be connected, and can not be connected with the sticky end on other primers.

Similarly, if 5 modules, 4 modules, 3 modules connect, respectively the 4th, the 3rd, the 2nd link block added F4R6, F3R6, F2R6 primer, corresponding module above remains unchanged with the primer that last module adds, and the segments of connection reduces by 1,2,3 module fragment accordingly.

Each primer sequence (SEQ ID NO:29-44) is in table 4.

Table 4

Note: small letter overstriking letter is the recognition sequence of restriction enzyme site

Pcr amplification system (50 μ are l): DNA profiling (Template): the about 50ng of 0.5 μ l(); Primer (Primer): each 1 μ l(50 μ M); LA Taq enzyme (Takara): 0.3 μ l; 10 × damping fluid (buffer): 5 μ l; DNTP:2.5 μ l(2.5 μ M); ddH ₂o:40.7 μ l.

PCR program: 95 DEG C of 2min; 95 DEG C of 15s, 55.8 DEG C of 30s, 72 DEG C of 11s, 36 circulations; 72 DEG C extend 10min.

After PCR, can obtain as the fragment in Fig. 2 (B), each module is coupled with different restriction endonuclease recognition sequences and different joints according to object binding sequence, and two joints of same color represent that the sticky end that both produce can be connected.

2. purifying

The PCR product of gained is carried out to agarose gel electrophoresis, determine concentration.Use the universal DNA purifying of Tian Gen company to reclaim test kit (centrifugal column type) purifying PCR fragment, the concentration of carrying out agarose gel electrophoresis and demarcate each product after purifying.

3. enzyme is cut connection

Can enzyme cut connection and carry out simultaneously so no longer can be cut this connection by Bsa1 because of adjacent module after connecting, enzyme is cut linked system and is: module: 100ng/ module (3-6); Bsa1 (NEB): 1 μ l; T4 ligase enzyme (fermentas): 1 μ l; T4 ligase enzyme damping fluid (NEB): 2 μ l; ddH ₂o: mend to 20 μ l.

PCR enzyme is cut linker: 37 DEG C of 5min, 20 DEG C of 5min, 35-45 circulation; 80 DEG C of 10min.

(3) fragment of three sections of 3-6 modules is connected on intermediate carrier pCMV-NLS-TALEbackbone-Fok1 (R)-intermediate

1. the 3-6 module that increases fragment

The 20 μ l products of previous step enzyme being cut to connection all carry out agarose gel electrophoresis, there will be one of a block length multiple size several have the band of gradient, cut glue and reclaim uppermost band.The fritter gel of switchback is placed on carefully in the rifle head of the 200 μ l liquid-transfering guns with filter membrane, rifle head is placed in 1.5ml centrifuge tube (EP pipe); With the centrifugal 5min of maximum speed of revolution, the liquid-transfering gun of centrifugal rear use 200 μ l all blows to the liquid not all being thrown in centrifuge tube in rifle head in centrifuge tube; The centrifugal liquid getting off both can be used as the use of the template of pcr amplification multimode fragment.The primer of pcr amplification is F-assem and R-assem, and sequence is in table 5.

Table 5

Primer title	Primer sequence
		F-assem（SEQ ID NO:45）	CGGGAGCCGACGTCGACAG
R-assem（SEQ ID NO:46）	CGCTCGAGCGACACGCAGG

PCR system (50 μ l): template: 2 μ l; Primer: each 0.5 μ l(50 μ M); Accuprimepfx:0.3 μ l; 10 × damping fluid: 5 μ l; ddH ₂o:42.2 μ l.

PCR program: 95 DEG C of 2min; 95 DEG C of 15s, 64 DEG C of 30s, 68 DEG C of 50s, 35 circulations; 68 DEG C extend 10min.

2. purified pcr product

PCR product is carried out to agarose gel electrophoresis, confirm to have or not the concentration of assorted band and object band.If very low with respect to object band ratio without assorted band or assorted band, use direct Kit purified pcr product; If assorted band needs glue to reclaim purifying more at most.Purifying rear electrophoresis is demarcated band concentration.

3. enzyme is cut intermediate carrier pCMVNLS-TALE backbone-Fok1 (R)-intermediate

Because having bbs1 restriction enzyme site on final carrier, and while being connected with 3 3-6 module fragments, connect and the enzyme of bbs1 is cut simultaneously and carried out.So can not directly link on final carrier, and can only first link on the intermediate carrier that there is no bbs1 restriction enzyme site.Fig. 3 is shown in by the schematic diagram of intermediate carrier.

Intermediate carrier enzyme is cut system: plasmid: 5 μ g; BsmB1:2 μ l; DTT(100mM): 1 μ l; ddH ₂o: supply 100 μ l.

37 DEG C of enzymes are cut and are spent the night, and hit every mistake two hours of enzyme is mended 0.5 μ l BsmB1, and mixes, and preferably changes a pipe, to eliminate nail not digested circular plasmids on tube wall.Enzyme cuts rear electrophoresis and determines whether all linearizings of plasmid.After determining, Kit purifying enzyme is cut product, and electrophoresis is demarcated carrier concn.

4. being connected of three fragments and intermediate carrier

Identical with Bsa1, Bbs1 is also type IIs enzyme, can not be cut by this enzyme, so this connects and that it(?) also can enzyme cuts that connection carries out simultaneously after the sticky end that enzyme is cut generation connects.

Carrier: 100ng; Module: 200ng/ module; Bbs1 (fermentas): 1 μ l; T4 ligase enzyme (fermentas): 1 μ l; T4 ligase enzyme damping fluid (NEB): 2 μ l; ddH ₂o: mend to 20 μ l.

Enzyme is cut linker: PCR program: 37 DEG C of 5min, 20 DEG C of 5min, 35-45 circulation; 80 DEG C of 10min.

5. transfection, selected clone, extracts plasmid in a small amount, and enzyme is cut qualification, order-checking qualification

After having connected, get 10 μ l and transform DH5a competence, 10 remaining μ l are frozen in-20 DEG C.Within second day, choose the mono-clonal (being greater than 10/plate) of some amount, within the 3rd day, in a small amount extract plasmid, the plasmid obtaining is cut qualification with BamH1 and Pst1 enzyme, and connecting correct should have the band about 2kb, and the meeting certainly the connecting 550bp band of having an appointment.Enzyme send order-checking after cutting correctly, and order-checking correctly can obtain 14-19 fragment successful connection and clone.Sequencing primer is in table 6, wherein, the RPE65-TALE-L2 of successful connection, the aminoacid sequence of RPE65-TALE-R3 is respectively as SEQ ID NO:5, shown in SEQ ID NO:6; The aminoacid sequence of 18.5 modules in RPE65-TALE-L2 is as shown in SEQID NO:1, and the aminoacid sequence of 13.5 modules in RPE65-TALE-R3 is as shown in SEQ IDNO:2.

Table 6

Primer title	Primer sequence
		TALE-forward order-checking (SEQ ID NO:47)	CTCCCCTTCAGCTGGACAC
TALE-backward sequencing (SEQ IDNO:48)	AGCTGGGCCACGATTGAC

(4) will connect into intermediate carrier and the correct fragment that checks order is connected into final carrier pEF1a-NLS-TALE backbone-Fok1 (R)-pA and pEF1a-NLS-TALEbackbone-Fok1 (L)-IRES-PURO-pA

Final carrier pEF1a-NLS-TALE backbone-Fok1 (R)-pA and pEF1a-NLS-TALE backbone-Fok1 (L)-IRES-PURO-pA are on the basis of ZFN carrier (being purchased from Sigma company), add to obtain after the N-terminal of TALEN and C-terminal with BamHI+KpnI enzyme after cutting.The N-terminal of TALEN and the nucleotide sequence of C-terminal are as shown in SEQ IDNO:49; The schematic diagram of final carrier is shown in Fig. 4 and Fig. 5.

The intermediate carrier and two the final carriers that are connected with correct fragment are used to BamH1 and Pst1 double digestion simultaneously, cut glue and reclaim corresponding fragment.Left and right order during according to design is connected into the TALE that contains Modulars on two of the left and right carrier of final carrier.Connection, transfection, picking clone, a small amount of extract plasmid, BamH1 and the qualification of Pst1 double digestion, order-checking qualification.Identify that correct clone is the final TALENs plasmid that we need.

The transfected with human 293T cell of embodiment 3 plasmids

1, in the 6 each holes of orifice plate, add 100 μ l matrigels, rock back and forth, make it to be paved with the bottom in whole hole, complete and be placed on 5%CO ₂30min in incubator.

2, by the substratum sucking-off of cultivating in IPS cell T25 bottle, PBS inhales one time, adds 1mL0.25% pancreatin, rocks back and forth, at the bottom of making its uniform fold bottle, is placed in 5%CO ₂5min in incubator.

3, add in 1ml 10%DMEM and pancreatin after having digested, the cell digesting is transferred in 15ml centrifuge tube, cell counting, centrifugal, 1200rpm, 5min.

4, with appropriate 10%DMEM re-suspended cell, get 2,000,000 293T cells and be placed in 6 orifice plates of completing matrigel, add the 10%DMEM that 2ml is fresh.

5, go down to posterity and carry out transfection simultaneously.

6, by the RPE65-TALEN-L1 building, RPE65-TALEN-L2, RPE65-TALEN-L3, RPE65-TALEN-L4, RPE65-TALEN-R1, RPE65-TALEN-R2, RPE65-TALEN-R3 presses table 7 combinations of pairs transfectional cell between two, totally 12 kinds of combinations.

Table 7

	RPE65-TALEN-R1	RPE65-TALEN-R2	RPE65-TALEN-R3
				RPE65-TALEN-L1	L1+R1	L1+R2	L1+R3
RPE65-TALEN-L2	L2+R1	L2+R2	L2+R3
				RPE65-TALEN-L3	L3+R1	L3+R2	L3+R3
RPE65-TALEN-L4	L4+R1	L4+R2	L4+R3

Mix plasmid, transfection reagent and medium solution according to following scheme:

The ratio of each composition: TALEN-L:TALEN-R:Lv-EF1a-Mcherry=5:5:2 in system

Total DNA:opti MEM=2 μ g:100 μ l

Total DNA:F μ gene=2 μ g:5 μ l

7, after transfection second day, can be at fluorescence microscopy Microscopic observation Mcherry fluorescent brightness and transfection efficiency.If transfection success, sops up the substratum in 6 holes, add the fresh 10%DMEM of 2ml of 2.0 μ g/ml puro.

8, be placed in 5%CO ₂in incubator, cultivate two days for 37 DEG C, change the fresh 10%DMEM nutrient solution of 2ml of 2.0 μ g/ml puro every day.

9, remove medicine and kill, the 5%CO of 37 DEG C ₂in incubator, be cultured to cell concentration and enough take out the use of gene identification, nutrient solution is changed to 2ml 10%DMEM.

Embodiment 4 cell targeting qualifications

1, in 6 orifice plates after medicine is killed with adding 300 μ l 0.25% pancreatin, shake back and forth even.Place 5min for 37 DEG C, make with rifle piping and druming that all cells is all digested to get off.

2,300 μ l liquid are sucked in 1.5ml EP pipes, wash 6 orifice plates twice with the PBS of 400 μ l, also add in EP pipe.

3, the centrifugal 5min of 13200rpm/min, abandoning supernatant.

4, with Direct PCR Kit(thermo article No.: F-140) extracting genome, and pcr amplification target practice regional DNA fragment.

5, genotype and the target practice efficiency of qualification target practice cell

After the genomic PCR fragment of the 293T cell of above-mentioned RPE65-TALEN-L2/RPE65-TALEN-R3 combined treatment is added to A, be connected in PMD18-T carrier, mono-clonal DNA fragmentation, send the genotype that obtains the target practice site of RPE65 gene after order-checking.Send altogether 21 sample order-checkings to have 3 clones that sudden change has occurred, see Fig. 6.

Adding A system is: DNA:10 μ l

rTaq:0.5μl

10xbuffer：1.5

dNTP：0.5μl

ddH2O：2.5μl

Then mix, be placed in 72 DEG C of 20min.

Result shows: in the clone of 4 sudden changes, one of them lacks 8 bases, and two other has inserted respectively tggt and gttg.Do not have in cell under two prerequisites that knock out in hypothesis, the probability that RPE65-TALEN-L2/RPE65-TALEN-R3 combination makes RPE65 gene that sudden change occur is (3 × 2)/21,28.6%.This research only designed in a site of RPE65 gene TALENs molecule just obtain can this gene of pointed decoration a pair of TALENs, and efficiency is very high.The superiority compared to ZFN technology of visible TALENs technology.This can identify the nucleotide sequence of SEQ ID NO:17 and SEQ ID NO:22 to polynucleotide, and can identify one of these two sequences or two Nucleotide and be substituted rear derivative nucleotide sequence.This fusion rotein to polynucleotide or its expression is can be to the efficient TALENs practicing shooting of people's gene, they for by homologous recombination modified R PE65 gene sudden change or other genetic modifications an instrument very is efficiently provided.By injecting people's isolated cells by polynucleotide transfected with human isolated cells of the present invention or by fusion rotein of the present invention, can promote homologous recombination, insert goal gene, obtain the albumen of high economic worth.

All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (17)

1. a pair of protein, is characterized in that, described a pair of protein amino acid sequence is respectively as shown in SEQID NO.5 and SEQ ID NO.6.

2. one-to-many Nucleotide, is characterized in that, the described one-to-many Nucleotide a pair of protein as claimed in claim 1 of encoding respectively.

3. one-to-many Nucleotide as claimed in claim 2, is characterized in that, the base sequence of described one-to-many Nucleotide is respectively as shown in SEQ ID NO.7 and SEQ ID NO.8.

4. a pair of fusion rotein, is characterized in that, described a pair of fusion rotein is merged and forms with DNA scinderin respectively by a pair of protein claimed in claim 1, and described DNA scinderin is natural Fok1DNA restriction endonuclease.

5. a pair of fusion rotein as claimed in claim 4, is characterized in that, described a pair of protein merges with two subunits of DNA scinderin respectively.

6. a pair of fusion rotein as described in claim as arbitrary in claim 4 or 5, is characterized in that, the aminoacid sequence of described a pair of fusion rotein is respectively as shown in SEQ ID NO.9 and SEQ ID NO.10.

7. one-to-many Nucleotide, is characterized in that, the described one-to-many Nucleotide a pair of fusion rotein as described in claim as arbitrary in claim 4-5 of encoding respectively.

8. one-to-many Nucleotide, is characterized in that, the described one-to-many Nucleotide a pair of fusion rotein as claimed in claim 6 of encoding respectively.

9. one-to-many Nucleotide as claimed in claim 8, is characterized in that, the base sequence of described one-to-many Nucleotide is respectively as shown in SEQ ID NO.11 and SEQ ID NO.12.

10. a carrier that comprises one-to-many Nucleotide described in the arbitrary claim of claim 2-3,8-9.

11. 1 kinds of host cells that transform with carrier described in claim 10.

12. 1 kinds of carriers that comprise one-to-many Nucleotide described in claim 7.

13. 1 kinds of host cells that transform with carrier described in claim 12.

14. host cell as described in claim 11 or 13, is characterized in that, described host cell behaviour isolated cells.

15. 1 kinds of a pair of fusion rotein or one-to-many Nucleotide application in the people RPE65 of non-diagnosis or therapeutic purpose gene target is modified as claimed in claim 8 as claimed in claim 6.

The method of the people RPE65 gene targeting of 16. 1 kinds of non-diagnosis or therapeutic purpose, it is characterized in that, comprise: by one-to-many Nucleotide described in a pair of fusion rotein or claim 8 or 9 described in claim 6 or contain the carrier of one-to-many Nucleotide described in claim 8 or 9 and proceed to people's isolated cells, in 30-37 DEG C of amplification cultivation 1-4 days, obtain RPE65 gene by the cell of targeting modification.

17. methods as claimed in claim 16, is characterized in that, also proceed to anti-puro albumen and maybe can express the plasmid of anti-puro albumen in described people's isolated cells.