CN102850444B - A pair of transcription activator like effector nucleases of L3 and R1 and a coding gene and an application thereof - Google Patents

Abstract

The invention discloses a pair of transcription activator like effector nucleases, a coding gene and an application thereof. The pair of transcription activator like effector nucleases (TALEN) is obtained by respective fusion of a pair of DNA recognins with two heterogenous subunits of a Fok1 DNA endonuclease, and can specifically recognize two adjacent loci on human RHD or RHCE gene exon1. When the pair of transcription activator like effector nucleases is simultaneously transferred to a host cell, targeting of the exon1 loci of the host cell gene can be realized; the targeted loci are subject to gene mutation; therefore, targeting modification of the human RHCE or RHD gene is realized, and advantages of strong specificity, high targeting efficiency, high accuracy and the like are provided.

Description

A pair transcriptional activation increment effector nuclease L3 and R1 and encoding gene thereof and application

Technical field

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

Background technology

Common blood group system, except abo blood group, also has Rh blood group.Rh blood group is divided into Rh+ and Rh-.RH positive can accept the blood of RH negative patient, but RH negative patient can not accept RH positive blood, because the antigen in RH positive blood will stimulate RH, negative human body will produce RH antibody.If again input RH positive blood, hemolytic blood transfusion reaction can be caused.And according to pertinent data introduction, Rh is positive, and blood group accounts for 99.7% in China Han and most of national people, so cause in the blood group of China Rh-very rare, a lot of patient is had to lose one's life because not having the blood of suitable distribution type.Therefore Rh-blood also has the title of panda blood.

The antigen of the Rh blood group system identified has kind more than 50, wherein antigen D, C, c, E, and e wants most.Wherein antigen D is by RHD genetic expression, antigens c, and c, E, e are by RHCE genetic expression, if can these two gene knockouts, then the blood of the RH positive then becomes RH feminine gender.

Because of RHD gene and RHCE genetic homology higher, the present invention is directed to one section of identical sequence in RHD gene and RHCE gene, design TALEN, reached the object of gene knockout

According to the wish of the mankind, the dream that directed targeting modification is many scientists is always carried out to genome.Endogenous genome is deleted or added the sequence of our needs specifically, various animal model can be constructed like this for Basic of Biology research and pathogenic mechanism research

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, usually only has 10 ^-6-10 ^-8, this shooting method has only obtained extensively application in mouse, and all can not get widespread use because efficiency is too low in other model animals and large mammal.

The very fast sequence-specific nuclease of developed recently may be used for accurate genome targeting modification.Generally be made up of a DNA recognition structure territory and a non-specific nucleic acid restriction endonuclease structural domain sequence-specific nuclease.Principle first nuclease is navigated to by DNA differential threshold the genome area needing editor, then non-specific nucleic acid restriction endonuclease cuts off double-stranded DNA thus 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 research is the clearest now is also most widely used sequence-specific nuclease.Its principle is the DNA sequence dna of two zinc finger protein specific recognition, two sections of 5-7bp of being separated by, and two of the non-specific DNA scinderin Fok1 of amalgamation and expression with it single aggressiveness location together, DNA scinderin can cut off the double-stranded DNA of this position when forming dimer, thus 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 is high 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 restricting ZFN widespread use.And business buys efficiently 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 to find in phytopathogen Xanthomonas a kind of can transcriptional activation increment effector (the transcription activator-like effector of regulating plant genetic expression, TALE) DNA binding specificity is shown, and its recognition code has the feature of modularization and simplification, develop more easy novel gene group targeting modification technology for scientists and bring new hope.

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

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

Truncated segment with different C-end TALE is connected on the catalyst structure domain of nuclease FokI by the research group that Edward Rebar leads.When researchist is by the endogenic mankind NTF3 of TALENs target of structure and CCR5 gene, confirm that TALENs can shear these gene fragments specifically.Research group of Harvard University develops a kind of strategy connected based on layering and builds the TALEs comprising 12 replicated blocks.They decrease the DNA sequence dna of each module on the basis retaining RVDs, drop to minimum by the repeatability of residue sequence simultaneously.And then obtain the monomer with specificity catenation sequence by 12 heavy PCR, and be cloned in the skeleton carrier comprising TALE N-end and C-end sequence.In order to build TALE transcription factor, TALE is fused to again the activation domain of a transcription factor by researchist.In ensuing targeting detects, researchist confirms that it can make four two genetic expressions detected in native gene raise specifically.

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

Summary of the invention

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

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

The invention provides one-to-many Nucleotide, a pair small peptide that described one-to-many Nucleotide is encoded above-mentioned respectively.

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

Wherein, described polynucleotide are by identifying that the TALENs identification module of corresponding base in nucleotide sequence on people RHCE gene or RHD gene is sequentially formed by connecting respectively, wherein, the TALENs identification module of identification base A is NIA, the TALENs identification module of identification base T is NG-T, the TALENs identification module of identification base C is HD-C, identifies that the TALENs identification module of bases G is NK-G.

Present invention also offers a pair protein, by a pair above-mentioned small peptide two ends, described a pair protein adds that the N of transcriptional activation increment effector aminoacid sequence framework holds and C end forms respectively; Wherein, the N end of described transcriptional activation increment effector aminoacid sequence framework and C end are natural or through engineered sequence.

This can distinguish the two sections of nucleotide sequences identified specifically on people's RHD gene or RHCE gene to protein, described two sections of nucleotide sequences are selected from following two nucleotide sequences respectively:

(1) ctcattctcctct(SEQ ID NO:17 sequence) or one of this sequence or two Nucleotide through replacement the nucleotide sequence that derives;

(2) ctaaggaagcgtcatagt(SEQ ID NO:18 sequence) or one of this sequence or two Nucleotide through replacement the nucleotide sequence that derives.

Preferably, described this has aminoacid sequence as shown in SEQ ID NO.5 and SEQ ID NO.6 respectively to protein.This protein is transcriptional activation increment effector, called after RH-TALE-L3 and RH-TALE-R1.

Present invention also offers one-to-many Nucleotide, a pair protein that described one-to-many Nucleotide is encoded above-mentioned respectively.

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

Present invention also offers a pair fusion rotein, described a pair fusion rotein is merged with DNA scinderin respectively by a pair above-mentioned protein and forms.

Preferably, described DNA scinderin is DNA restriction endonuclease.

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

More preferably, described DNA scinderin is natural or through engineered Fok1 DNA restriction endonuclease.

Most preferably, described a pair fusion rotein has the aminoacid sequence as shown in SEQ ID NO.9 and SEQ ID NO.10 respectively.This fusion rotein is transcriptional activation increment effector nuclease, called after RH-TALEN-L3 and RH-TALEN-R1.

Present invention also offers one-to-many Nucleotide, a pair fused protein that described one-to-many Nucleotide is encoded above-mentioned respectively.

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

Present invention also offers a kind of recombinant vectors of any polynucleotide comprised 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-TALE backbone-Fok1 (R)-intermediate, again this intermediate carrier is connected on final carrier pEF1a-NLS-TALE backbone-Fok1 (R)-pA or final carrier pEF1a-NLS-TALE backbone-Fok1 (L)-IRES-PUROpA, build to obtain and comprise the plasmid vector that encoding transcription activates increment effector nuclease gene, transcriptional activation increment effector nuclease can be expressed.

Present invention also offers a kind of host cell transformed with above-mentioned recombinant vectors.

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

Present invention also offers a kind of above-mentioned a pair fusion rotein or the application of above-mentioned one-to-many Nucleotide in people RHD gene or RHCE gene target are modified.

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

Present invention also offers a kind of method of people RHD gene or the target practice of RHCE gene, comprise: by above-mentioned a pair fusion rotein or above-mentioned one-to-many Nucleotide or containing this, people's isolated cells is proceeded to the carrier of polynucleotide, in 30-37 DEG C of amplification cultivation 1-4 days, obtain the cell that prion protein gene is targeted modification.

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

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

The site that the present invention is directed to people RHD gene or RHCE gene devises a pair transcriptional activation increment effector nuclease (RH-TALEN-L3 and RH-TALEN-R1), and this is merged by two allos subunits in the DNA recognition structure territory and a Fok1 DNA restriction endonuclease that can identify RHD or RHCE gene the preceding paragraph Nucleotide respectively TALENs and obtains.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 RHD gene or RHCE gene, and make the producer sudden change of target practice site, comprise base deletion, base insertion etc., thus the targeting modification realized people RHD gene or RHCE gene, have that high specificity, target practice efficiency are high, accuracy advantages of higher.

Accompanying drawing explanation

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

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;

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

C:PCR increases 6 modular segments and intermediate carrier schematic diagram;

D: the final TALEN plasmid schematic diagram built;

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

Fig. 4 is final carrier pEF1a-NLS-TALE backboneFok1 (R) pA schematic diagram;

Fig. 5 is final carrier pEF1a-NLS-TALE backboneFok1 (L)-IRESPURO-pA schematic diagram;

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

Embodiment

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

The design of embodiment 1TALENs target sequence

1, from the upper and lower manned RHCE gene (GENE ID:6006) of NCBI and RHD genome sequence (GENE ID:6007)

2, design primer site fragment that pcr amplification genome is practiced shooting, and check order, wherein, PCR primer and sequencing primer are in table 1;

Table 1

3, TALENs recognition sequence (target sequence) is designed:

According to the sequence obtained that checks order, and determine TALENs recognition sequence according to following principle:

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

(2) last bit base is T

(3) recognition sequence length is between 13-19

Intervening sequence (Spacer) cut to lengthen between (4) two recognition sequences between 14-21 (12,13 also can, but efficiency may be lower)

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

Table 2

TALE title	Target sequence
		RH-Tale-L1（SEQ ID NO:13）	ctcattctcctcttct
RH-Tale-L2（SEQ ID NO:14）	ctcattctcctctt
		RH-Tale-L3（SEQ ID NO:1）	ctcattctcctct
RH-Tale-R1（SEQ ID NO:15）	ctaaggaagcgtcatagt
		RH-Tale-R2（SEQ ID NO:16）	ctaaggaagcgtcat
RH-Tale-R3（SEQ ID NO:2）	ctaaggaagcgt

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

1, the acquisition of TALENs identification module (modular)

(1) synthesis identifies four identification modules NI, NG, HD, NK of base A, T, C, G respectively, and 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. PCR primer 3 μ l is got; 2. 1 μ l pEASY-B carrier is added; 3. 25 DEG C, 7min; 4. transform DH5a competent cell, mycin flat board received by coating card; 5. picked clones, a small amount of extracts plasmid, enzyme is cut, check order, and finally obtains identification module NI, NG, HD, NK of being connected in carrier pEASY-B.

2, the connection between identification module

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

(1) three parts are divided into by recognition sequence (target sequence) (to be respectively table 2 Central Plains sequence S and to remove last base, as follows), namely every bar recognition sequence is first divided into three sections, every section containing 3-6 base, correspondingly every section of corresponding 3-6 identification module, first in units of every section, the 3-6 of this section identification module is connected.

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

1. pcr amplification adds restriction endonuclease recognition sequence and jointing

Connection between 6 identification modules: Fig. 2 (A) is that 6 module PCR add restriction endonuclease recognition sequence and jointing process schematic, 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 is the recognition sequence of GAAGACNN ' NNNN, Bsa1 is GGTCTCN ' NNNN, and these two enzymes all belong to typeIIsenzymes, and same restriction endonuclease recognition sequence can produce multiple viscosity identification end, can produce 4 in theory ⁴individual viscosity identification end, add ending and the beginning Gly codon 4 kinds of each module, the restriction that Leu codon is 6 kinds, utilizes a typeIIs enzyme can produce 24 kinds of joints.We devise primer at 16 kinds of choosing wherein, except F1 and R8, and F _ncan with R _n+1sticky end be connected, and can not to be connected with the sticky end on other primers.

Similarly, if 5 modules, 4 modules, 3 model calling, then respectively the 4th, 3rd, 2nd link block adds F4R6, F3R6, F2R6 primer, the primer that module before corresponding and last module add remains unchanged, and the segments of connection then reduces by 1,2,3 modular segments accordingly.

Each primer sequence is in table 4.

Table 4

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

PCR amplification system (50 μ l) is: DNA profiling (Template): 0.5 μ l(is about 50ng); Primer (Primer): each 1 μ l(50 μM); LA Taq enzyme (Takara): 0.3 μ l; 10 × damping fluid (buffer): 5 μ l; DNTP:2.5 μ l(2.5 μM); 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.

Can obtain as the fragment in Fig. 2 (B) after PCR, each module is coupled with different endonuclease recognition sequence 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 primer of gained is carried out agarose gel electrophoresis, determines concentration.Use the universal DNA purifying of Tian Gen company to reclaim test kit (centrifugal column type) purified PCR fragments, after purifying, carry out agarose gel electrophoresis and demarcate the concentration of each product.

3. enzyme cuts connection

Because no longer being cut by Bsa1 after adjacent model calling so this connection can be cut connection and carry out by enzyme simultaneously, 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 cuts linker: 37 DEG C of 5min, 20 DEG C of 5min, 3545 circulations; 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. increase 3-6 modular segments

20 μ l products previous step enzyme being cut 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 200 μ l liquid-transfering guns of band filter membrane, rifle head is placed in 1.5ml centrifuge tube (EP pipe); With the centrifugal 5min of maximum speed of revolution, the liquid be not all thrown in centrifuge tube in rifle head all blows in centrifuge tube by the liquid-transfering gun of centrifugal rear use 200 μ l; The centrifugal liquid got 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	Primer sequence
		F-assem	CGGGAGCCGACGTCGACAG
R-assem	CGCTCGAGCGACACGCAGG

PCR system (50 μ l): template: 2 μ l; Primer: each0.5 μ l(50 μM); Accuprime pfx: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 primer is carried out agarose gel electrophoresis, confirms the concentration with or without assorted band and object band.If without assorted band or assorted band very low relative to object band ratio; use direct Kit purified pcr product; If assorted band needs glue to reclaim purifying more at most.Purifying rear electrophoresis demarcates band concentration.

3. enzyme cuts intermediate carrier pCMV-NLS-TALE backbone-Fok1 (R)-intermediate

Because final carrier has bbs1 restriction enzyme site, and when being connected with 3 3-6 modular segments, connection is cut with the enzyme of bbs1 and is carried out simultaneously.So can not directly link on final carrier, and can only first link there is no bbs1 restriction enzyme site intermediate carrier on.Fig. 3 is shown in by the schematic diagram of intermediate carrier.

Intermediate carrier enzyme cuts 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 cut through night, and enzyme hits and often crosses two hours benefit 0.5 μ l BsmB1, and mixes, and preferably changes a pipe, to eliminate nail circular plasmids not digested on tube wall.Enzyme cuts the whether all linearizings of rear electrophoresis determination plasmid.After determining, Kit purifying digestion products, electrophoresis demarcates carrier concn.

4. the connection of three fragments and intermediate carrier

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

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 cuts linker: PCR program: 37 DEG C of 5min, 20 DEG C of 5min, 3545 circulations; 80 DEG C of 10min.

5. transfection, selected clone, extract plasmid in a small amount, enzyme cuts qualification, order-checking qualification

Get 10 μ l after having connected 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 BamH1 obtained and Pst1 enzyme cut qualification, connect the correct band that should have about 2kb, and the meeting certainly connected is had an appointment 550bp band.Enzyme is cut correctly and is sent order-checking, and order-checking correctly can obtain 14-19 fragment successful connection and clone.Sequencing primer in table 6, wherein, the aminoacid sequence of RH-TALE-L3 and RH-TALE-R1 of successful connection respectively as SEQ ID NO:5, shown in SEQ IDNO:6; The aminoacid sequence of 12.5 modules in RH-TALE-L3 is as shown in SEQ ID NO:1, and the aminoacid sequence of 17.5 modules in RH-TALE-R1 is as shown in SEQ ID NO:2.

Table 6

Primer	Primer sequence
		TALE-forward checks order	CTCCCCTTCAGCTGGACAC
TALE-backward sequencing	AGCTGGGCCACGATTGAC

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

Final carrier pEF1a-NLS-TALEbackbone-Fok1 (R)-pA and pEF1a-NLS-TALEbackbone-Fok1 (L)-IRES-PURO-pA are on the basis of ZFN carrier (being purchased from Sigma company), obtain after adding the N-terminal of TALEN and C-terminal with BamHI+KpnI enzyme after cutting.Fig. 4 and Fig. 5 is shown in by the schematic diagram of final carrier.

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

The transfected with human 293T cell of embodiment 3 plasmid

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

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

3, add in 1ml10%DMEM after having digested and pancreatin, the cell digested 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 completing matrigel, add the 10%DMEM that 2ml is fresh.

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

6, the RH-TALEN-L1 will built, RH-TALEN-L2, RH-TALEN--L3, RH-TALEN-R1, RH-TALEN-R2, RH-TALEN-R3 press table 7 combinations of pairs transfectional cell between two, totally 12 kinds of combinations.

Table 7

	RHCE-TALEN-R1	RHCE-TALEN-R2	RHCE-TALEN-R3
				RH-TALEN-L1	L1+R1	L1+R2	L1+R3
RH-TALEN-L2	L2+R1	L2+R2	L2+R3
				RH-TALEN-L3	L3+R1	L3+R2	L3+R3

According to following scheme mixing plasmid, transfection reagent and medium solution:

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

STb gene: opti MEM=2 μ g:100 μ l

STb gene: F μ gene=2 μ g:5 μ l

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

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

9, remove medicine to kill, the 5%CO of 37 DEG C ₂be cultured to the use that cell concentration enough takes out gene identification in incubator, nutrient solution is changed to 2ml10%DMEM.

Embodiment 4 cell targeting is identified

1, with adding 300 μ l0.25% pancreatin in 6 orifice plates after medicine being killed, shake even back and forth.Place 5min for 37 DEG C, make that all cells is all digested to get off with rifle piping and druming.

2,300 μ l liquid are sucked in 1.5ml EP pipe, 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 region DNA fragment.

5, genotype and the target practice efficiency of target practice cell is identified

Be connected in PMD18-T carrier after the genomic PCR fragment of the 293T cell of above-mentioned RH-TALEN-L3/RH-TALEN-R1 combined treatment is added A, mono-clonal DNA fragmentation, after sending order-checking, obtain the genotype in the target practice site of RHD or RHCE gene.

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: send 30 sample order-checkings to have 4 clones to there occurs sudden change altogether, see Fig. 6.Wherein three clones there occurs base deletion, and a clone there occurs base and inserts.Because 293T is triploid, in hypothesis cell, there is no two to knock out or under three prerequisites knocked out, it is 4/30*3 that RH-TALEN-L3/RH-TALEN-R1 combines the probability making RH gene there occurs sudden change, namely 40%.This research only devises in a site of RH gene that TALENs molecule just obtains can a pair TALENs of this gene of pointed decoration, 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:18 to polynucleotide, and can identify that one of these two sequences or two Nucleotide are substituted rear derived nucleotide sequence.This is the TALENs that efficiently can practice shooting to people's gene to polynucleotide or its fusion rotein of expressing, they for by homologous recombination modified R HD or RHCE gene sudden change or other genetic modifications provide an instrument very efficiently.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 homologous recombination be promoted, inserting goal gene, obtaining the albumen of high economic worth.

The all documents mentioned in the present invention are quoted as a reference all 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 (12)

1. a pair fusion rotein, is characterized in that, described a pair fusion rotein is transcriptional activation increment effector nuclease, is merged respectively form by a pair protein with two subunits of DNA scinderin; Described DNA scinderin is for the aminoacid sequence of a pair protein described in natural Fok1 DNA restriction endonuclease is respectively as shown in SEQ ID NO.5 and SEQ IDNO.6.

2. a pair fusion rotein described in claim 1, is characterized in that, the aminoacid sequence of described a pair fusion rotein is respectively as shown in SEQ ID NO.9 and SEQ ID NO.10.

3. one-to-many Nucleotide, is characterized in that, described one-to-many Nucleotide is encoded a pair fusion rotein as claimed in claim 1 respectively.

4. one-to-many Nucleotide, is characterized in that, described one-to-many Nucleotide is encoded a pair fusion rotein as claimed in claim 2 respectively.

5. one-to-many Nucleotide according to claim 4, 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.

6. a recombinant vectors, is characterized in that, comprises one-to-many Nucleotide described in the arbitrary claim of claim 3,4 or 5.

7. a host cell, is characterized in that, containing recombinant vectors described in claim 6.

8. host cell according to claim 7, is characterized in that, described host cell behaviour isolated cells.

9. a pair application of fusion rotein in people RHD gene or RHCE gene target are modified described in claim 2.

10. the application of one-to-many Nucleotide described in claim 5 in people RHD gene or RHCE gene target are modified.

The method of 11. 1 kinds of people RHD genes or RHCE gene targeting, it is characterized in that, comprise: proceed to people's isolated cells by containing the recombinant vectors of one-to-many Nucleotide described in claim 4 or 5, in 30-37 DEG C of amplification cultivation 3-7 days, obtain the cell that RHD gene or RHCE gene are targeted modification.

12. methods according to claim 11, is characterized in that, also proceed to the plasmid that anti-puro albumen maybe can express anti-puro albumen in described people's isolated cells; Add puro medicine and kill the cell screening and successfully proceed to after albumen or plasmid proceed to cell, kill 3 days in 37 DEG C of medicines, 30-37 DEG C of amplification cultivation 1-4 days, obtain the cell that RHD gene or RHCE gene are targeted modification.