CN103724410B - The fusion protein and method of one class regulation and control CCR5 and CXCR4 genes - Google Patents

Abstract

One species activating transcription factor (Transcription Activator Like Effectors, TALE), it is characterized in that the class activating transcription factor (TALE) can recognize and combine with the DNA fragmentation in mankind CCR5 gene activities region or CXCR4 gene activities region, the mankind CCR5 gene activities region one has 3, and the CXCR4 gene activities region one has 2.TALE is connected to form fusion protein TALEN with endonuclease, and the specific site of CCR5 genes or CXCR4 genes is sheared using the TALEN, can allow to carry out the cell after gene regulation and possess the ability for resisting inhibition of HIV.

Description

The fusion protein and method of one class regulation and control CCR5 and CXCR4 genes

Technical field

The present invention relates to the method to CCR5 and CXCR4 gene alterations, and the fusion protein used in this method, especially It is related to including class activating transcription factor (Transcription Activator Like Effectors, TALE) and endonuclease The fusion protein of enzyme/partial nucleic acid restriction endonuclease, and change with the fusion protein method of CCR5 and CXCR4 genes.

Background technology

The joint assessment carried out according to ministry of Health of China, UNAIDS and WHO in 2011 to Chinese AIDS Epidemic, by The end of the year 2011, estimation China survival patients infected hiv and the people of AIDS patients 780,000 (62~940,000 people), wherein new hair The people of HIV person 4.8 ten thousand (4.1~5.4 ten thousand people), AIDS has turned into the serious public health problem of China.

In AIDS evolution, trial of strength a moment of HIV and human autoimmune's system did not all stop, but so far Also HIV can be spontaneously removed without a infected.Because causing its gene order as viral persistence is replicated It is continually changing, makes HIV biological property and its sensitiveness of immune response is changed.For example, by entering thin to it The change of the Co receptor selectivity of born of the same parents can accelerate disease development, by producing substantial amounts of virus mutant, to reach escape The monitoring of self immune system.In addition, inhibition of HIV can also be to be incorporated into the genome of host cell, dexterously in the form of DNA It is stored at not clear cell place or anatomy place, referred to as virus repository storehouse.The foundation and removing in virus repository storehouse are one Individual dynamic process, from start to infect first day, virus repository storehouse began to set up.Virus repository storehouse is to effect a radical cure AIDS most One of big obstacle.These features of inhibition of HIV so that exploitation can effectively control HIV, duplications, mutation, release, hide In terms of medicine become certainty.

The system research carried out by more than 30 years to HIV biology and human body AntiHIV1 RT activity immune response, develops more than 30 Plant inverase.These medicines are broadly divided into 1) viral entry inhibitor；2) efabirenz；3) non-nucleosides Class RTI；4) integrase inhibitor；5) protease inhibitors.Current clinical widely used AntiHIV1 RT activity/AIDS is controlled Treatment scheme, is the combination of at least three kinds medicines, and a conventional line suggested design is that two kinds of efabirenzs are added A kind of non-nucleoside reverse transcriptase inhibitor or the protease inhibitors or integrase inhibitor for having Ritonavir excitement.These HIV can be suppressed to the level that can't detect in blood plasma, seminal fluid etc. by therapeutic scheme, maintain or recover impaired human immunity work( Can, extend patient's life-span, improve the quality of living, reduce HIV spread.The HAART that nineteen ninety-five proposes (HighlyActive Antiretroviral Therapy, HAART) scheme, by the optimum organization to these medicines, makes Chinese mugwort Grow morbidity and mortality and persistently reduce 70% so that it is a kind of pronounce that the disease for death penalty switchs to one kind by early stage medical science can Treatment and controllable chronic disease.

But antiviral therapy can not effect a radical cure AIDS at present, main cause is that do not have to suppress and clear to hiding HIV Except effect.Once treatment is interrupted, viral load will quick rebound.Its main cause is the dyeing that HIV is incorporated into permissive cell On body, hide in the intracellular of human body, dexterously escaped the identification of human immune system and inverase；Meanwhile, these quilts The cell of infection can hide for a long time, form HIV virus repository storehouse.So existing medicine at all can not be HIV from internal It is basic to remove, do not reach " basic healing ".Today is found to from AIDS and HIV, although the preventing and treating to AIDS is achieved Very great achievement, but AIDS still can not effect a radical cure, and effective AIDS vaccine succeeds in developing, AIDS still not within the foreseeable future from us The prevention and treatment research of disease once enters bottleneck period.

But the appearance of " Berlin patient ", pointing out people " modification " HIV co-receptor gene C CR5 can reach to AIDS " the feature healing " of disease, efficiently controls hiding HIV for us and provides a new treatment method.HIV " feature Cure " although referring to that the inhibition of HIV integrated can not be removed, can be by increasing being difficult to inhibition of HIV for CCR5 receptor defectses The cell of sense, to reach the resistant function to HIV, so as to fundamentally recover the immunologic function of the infected, reaches function and curing Purpose." Berlin patient " is exactly a HIV person, and he is by receiving the donor bones of HIV Co receptor gene C CR5 defects Marrow, cell therein then replace and felt by HIV because normal replication and can not be functioned by HIV destruction gradually The T lymphocytes of dye, including the HIV permissive cells for having HIV genomes are hidden, so as to reach " feature healing ".This phenomenon Us are pointed out to simulate the cell of natural CCR5 acceptors missing, the immune T that CCR5/CXCR4 is lacked by manual method Cell is fed back to patient, so as to reach the purpose of AIDS " feature healing ".

There are breakthrough progress, wherein zinc finger protein nuclease on chromosome and its modification technique in the past few years (ZincFinger Nuclease, ZFN) represents to be therein.The research group in the U.S. carries out CCR5 knockouts etc. using ZFN Research, and clinical research is carried out in HIV person, very gratifying progress is achieved, related research work has been enter into The second stage of clinical optimization ZFN, which modifies CCR5 allele efficiency and improved, feeds back CCR5-modified CD4 T cell in cell The conceptual phase of ratio.TALEN (the transcription activator-like effector being recently developed Nuclease, class activating transcription factor nuclease) there is obvious advantage in terms of specificity, design synthesis than ZFN, be TALEN is applied to clinical there is provided more solid foundation.

TALE (Transcription Activator Like Effectors) is phytopathogen Xanthomonas campestris first (Xanthomonas) found on, be specifically binding to DNA, plant gene is adjusted during the pathogenic bacterial infection Control.TALE albumen can enter through nuclear membrane to be combined in nucleus with specific DNA binding domain, with disease in regulation and control Plant Genome The expression of disease and resistance related gene.Briefly, TALE is series connection " the albumen mould by 4 or more specific recognition DNA Block " and the N- ends of both sides and C- end sequences composition, and each " module " includes 34 amino acid, wherein the 12nd and 13 Amino acids are the critical sites of targets identification, referred to as the variable bis-amino acid residue (repeat of repetition Variablediresidue, or RVDs).TALE recognizes that DNA mechanism is a nucleotides on DNA target point by one RVD identifications on repetitive sequence.

In theory, for any one base of A, T, G, C, the RVDs of particular combination therewith can be found.Therefore, to any Section of DNA sequence, we can easily design, synthesize the TALE of correspondence module composition.It may be noted that the problem of be, Although 1) corresponding RVD can be designed for A, T, G, C, the corresponding relation between them needs further Optimization；2) can the TALE of design synthesis be efficiently targetted with reference to the desired location on genome, further depend on it is many other because Element；3) composition of module also has the space further optimized.

Solved although TALE also has many problems to await further investigation, this simultaneously its application of without prejudice to.One Individual maximum application prospect is TALEN.TALEN is a fusion protein, by recognizing TALE and energy with certain segment DNA sequence-specific On DNA sequence dna produce double-strand break (double strand break, DSB) endonuclease (Nuclease) fusion and Into.TALEN is heterodimer molecule (i.e. the TALE-Nuclease collective effects of two units), can be separated by two nearer Specific recognition sequence between cutting DNA.

The DSB that TALEN is produced can be repaired by following two approach：1) non-homologous end joining (Non HomologousEnd Joining, NHEJ)：NHEJ is, with natural repair mechanism, to be used to introduce nucleotide deletion to lose Living or one specific target gene of knockout；2) homologous recombination (Homologous Recombination, HR)：DSB promotes same Source is recombinated, in the presence of a DNA masterplate, can produce specific DNA sequences change, also can be by integrated transgene to DNA In sequence.NHEJ approach can be used for gene silencing, and HR can be used for modification gene (Gene Editing), or gene knock-in (GeneKnock-in).Either which kind of approach, the reparation that TALEN is produced with merely dependent on homologous recombination compared with, gene hair The efficiency of raw restructuring is greatly improved, and this is engaged in genome customizing (genome customization) for us and provides convenient Technological means, new hope is brought to develop more easy novel gene group targeting modification technology.

TALE technologies have begun to show up prominently in life science.2011, French and two, U.S. group cooperation, Using TALEN technologies, inactivation IgM functions are knocked out in rats, efficiency high is up to 60%.It is 2011, several including China Group, using TALEN technologies, in zebra fish, knocks out the genes such as inactivation hey2, efficiency has also reached more than 30%.

TALE has special architectural feature, including nitrogen end (N-terminal) secretion signal, the DNA binding domain in center and nuclear location The activation domain of sequence (Nuclear localization signal, NLS) and carbon teminal (C-terminal).It is nearly all in DNA binding domain It has been found that TALE albumen be all to be made up of quantity different (12~30), highly conserved repeat unit, these repeat units In (typically containing 33~35 amino acid), except the 12nd and 13 amino acids it is not near it is identical in addition to, other components all ten Code insurance is kept.Wherein the 12nd and 13 variable amino acid is referred to as the variable bis-amino acid residue RVD (repeat of repetitive sequence variable di-residues).TALE is mainly the RVD passed through in repeat unit and recognizes DNA sequence dna.At present it has been reported that RVD has 14 kinds.NT (amino acid name) specific recognition A, HD (amino acid name) specific recognitions C, NN (amino acid name Claim) G and A can be recognized, NK (amino acid name) specific recognition G, NS (amino acid name) can recognize G, A, C and T, NG (amino acid name) specific recognition T, NH (amino acid name) specific recognition G, N* (amino acid name) can recognize T, C, G and A, NP (amino acid name) can recognize T, A and C, and HN (amino acid name) can recognize G and A, and NT (amino acid name) can To recognize G and A, SN (amino acid name) specific recognition G, SH (amino acid name) specific recognitions G.

The major part of TALE technologies is to enter functional molecular, such as activating transcription factor in the fusion of TALE albumen carbon teminal (Activation Domain, AD) nuclease, methylase, demethylase, recombinase etc..It is special in TALE fusion proteins After opposite sex identification DNA sequence dna and combination, the function group of its carbon teminal plays a role, so as to reach the effect regulated and controled to functional protein Really.Now about being typically all to be believed by plasmid transfection, virus infection or injection in the report of TALE correlation technique application The RNA made method directly expresses TALE fusion proteins, allows it to play a role.

Due to TALEN (transcription activator-like effector nuclease, class transcriptional activation because Sub- nuclease) there is obvious advantage in terms of specificity, design synthesis than ZFN, this patent is carried out using TALEN to gene Fixed point is knocked out, and is screened and is optimized TALEN of the specificity for HIV CCR5/CXCR4 acceptor genes, reaches safe efficient, standard True fixed point knocks out the purpose of the CCR5/CXCR4 acceptor genes of patient, so as to suppress invasion of the virus to human immune system.

The content of the invention

Definition：

For disambiguation, the following term in the present invention is defined.

Shearing is cut：Refer to the fracture of DNA molecular covalent backbone (covalent backbone) under given conditions. Shearing or cutting can be carried out by (but not limited to) following methods：The enzymolysis or chemical hydrolysis of phosphodiester bond.DNA's is single-stranded What fracture and double-strand break were all possible to, double-strand break, which is likely due to two single chain breaks, to be caused.

Polynucleotide：Large biological molecule material, including DNA (DNA) and the major class of ribonucleic acid (RNA) two.

Nuclease (English：Nuclease)：It is the class of enzymes of catalytic phosphatase diester linkage hydrolysis using nucleic acid as substrate.To make It is divided into endonuclease and exonuclease with position.

Endonuclease：It is a kind of ferment, the phosphate diester key inside cleavable polynucleotide chain, with exonuclease phase Correspondence.From the point of view of the specificity to substrate, the enzyme that DNase I, DNase II etc. decompose DNA can be divided into；RNase, RNaseTl etc. Decompose RNA enzyme.In general, do not have base specific mostly, but also have spleen RNase, RNaseTl etc. or restricted Restriction endonuclease that can recognize and cut off the enzyme of specific base or base sequence.

Partial nuclease：Partial nuclease is one section of polypeptide, when it is combined with another section of polypeptide (identical or different), shape Into the dimer complex (i.e. nuclease) with nucleic acid shear active (particularly double-strand shear active).

Nuclease shearing function domain (cleavage domain)：It is the knot with DNA or RNA shear actions in nuclease Structure domain (domain).

TALEN (transcription activator-like effector nuclease, class activating transcription factor core Sour enzyme)：TALEN is a fusion protein, can be produced by the TALE that is recognized with certain segment DNA sequence-specific and on DNA sequence dna Double-strand break (double strand break, DSB) endonuclease (Nuclease) or partial nucleic acid restriction endonuclease fusion and Into, the present invention in " TALE-1-L "-FokI, " TALE-1 '-L "-FokI, " TALE-1-L "-endonuclease, " TALE-1 '- L "-endonuclease belongs to TALEN.

It is more concise in order to state, below will the present invention will be described in detail since most preferred technical scheme. It is surprisingly found by the inventors that, there are some active regions in CCR5 and CXCR4 genes, if to some of these active regions DNA fragmentation is cut off, then after treating gene repair, inhibition of HIV by the cell after can not attacking these gene repair again, from And reach the purpose of function and curing AIDS.These active regions one have 5, wherein have 3 in CCR5 genes, its nucleotides Sequence is respectively：Active region 1)：

tgcacagggtggaacaagatggattatcaagtgtcaagtccaatctatgacatcaattattatacatcggagccctg Ccaaaaaatcaatgtgaagcaaatcgcag,

Active region 2)：

Caacatgctggtcatcctcatcctgataaactgcaaaaggctgaagagcatgactg acatctacctg,

Active region 3)：

tcttcattacacctgcagctctcattttccatacagtcagtatcaattctggaagaatttccagacattaaagatag ；

Active region one has 2 in CXCR4 genes, and its nucleotide sequence is respectively：

Active region 1 ')：

TAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCCATGAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATT TCAATAAAA,

Active region 2 ')：

TAAAATCTTCCTGCCCACCATCTACTCCATCATCTTCTTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCA TGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCA。

In practical operation, inventor devises a kind of fusion protein-TALEN including TALE and endonuclease to realize Cutting to specific site in above-mentioned active region.Further to improve the accuracy of cleavage site, inventor devises one Each in cutting to the common completions of TALEN to target site, this pair of TALEN include respectively TALE albumen and One single-stranded a certain section in one partial nucleic acid restriction endonuclease, one of TALE identifications DNA double chain, another TALE is then A certain section during another of identification is single-stranded, after two TALEN are recognized respectively and combine its target DNA fragments, two TALEN In partial nucleic acid restriction endonuclease will be in spatially close proximity, the two partial nucleic acid restriction endonucleases, which will be combined to form, to be had The dimer of nucleic acid shear active, i.e., the complete endonuclease with nucleic acid shear active, so as to be carried out to target site Cutting.For example, for the active region 1 from CCR5 genes：

tgcacagggtggaacaagatggattatcaagtgtcaagtccaatctatgacatcaattattatacatcggagccctg ccaaaaaatcaatgtgaagcaAatcgcag,

Inventor devises a pair of TALEN, is respectively " TALE-1-L "-FokI and " TALE-1-R "-FokI, wherein TALE-1-L RVD sequences are NI NG NN NI HD NI NG HD NI NI NG NG NI NG NG NI NG, TALE-1- R RVD sequences are NN HD NG NG HD NI HD NI NG NG NN NI NG NG NG NG NG NG, wherein TALE-1- That L is recognized and combined is-the TATGACATCAATTATTAT-3 ' of part 5 ' that runic is marked in above-mentioned active region 1, and TALE- 1-R identifications are then the single-stranded anti-sense strand complementaries in above-mentioned active region, its DNA fragmentation for recognize and combining for 5 '- The part that italic is marked in TGCTTCACATTGATTTTTT-3 ', the above-mentioned active region 1 of correspondence.When two TALEN fusion proteins Recognize respectively and with reference to after its target DNA fragments, the partial nucleic acid restriction endonuclease FokI in two TALEN will be in active region 1 The part that Wave line is marked is combined into the dimer with cleavage activity, and the DNA fragmentation at this is cut.Cut Cheng Hou, DNA can typically carry out self-regeneration (this reparation can also be carried out under manual intervention), and reparation finishes CCR5 genes still Possess normal function, but experiment finds that inhibition of HIV can not attack such cell again.

Similar, inventor have also been devised other paired TALEN, including " TALE-2-L "-FokI and " TALE-2-R "- FokI；" TALE-3-L "-FokI and " TALE-3-R "-FokI；" TALE-4-L "-FokI and " TALE-4-R "-FokI；“TALE- 5-L "-FokI and " TALE-5-R "-FokI；" TALE-6-L "-FokI and " TALE-4-R "-FokI；" TALE-7-L "-FokI and “TALE-7-R”-FokI；" TALE-1 '-L "-FokI and " TALE-1 '-R "-FokI；" TALE-2 '-L "-FokI and " TALE- 2’-R”-FokI；" TALE-3 '-L "-FokI and " TALE-3 '-R "-FokI；" TALE-4 '-L "-FokI and " TALE-4 '-R "- FokI；" TALE-5 '-L "-FokI and " TALE-5 '-R "-FokI；" TALE-6 '-L "-FokI and " TALE-6 '-R "-FokI.Its Corresponding RVD sequences and the DNA sequence dna of identification see attached list 1-3 and 1-2, or 1 ' -3 ' and 1 ' -2 '.

Because TALE albumen inherently possesses specific to the identification of DNA sequence dna, therefore, those skilled in the art are not Hardly possible is it is contemplated that independent one in above-mentioned paired TALEN can just complete the cutting to DNA specific sites, it is only necessary to part Endonuclease replaces with the endonuclease with cleavage activity, and simply recognition accuracy is not paired as described above TALEN.The TALEN that therefore, it can be used alone includes " TALE-1-L "-endonuclease, " TALE-1-R "-endonuclease Enzyme, " TALE-2-L "-endonuclease, " TALE-2-R "-endonuclease, " TALE-3-L "-endonuclease, " TALE-3- R "-endonuclease, " TALE-4-L "-endonuclease, " TALE-4-R "-endonuclease, " TALE-5-L "-endonuclease Enzyme, " TALE-5-R "-endonuclease, " TALE-6-L "-endonuclease, " TALE-7-L "-endonuclease, " TALE- 7-R "-endonuclease, " TALE-1 '-L "-endonuclease, " TALE-1 '-R "-endonuclease, " TALE-2 '-L "-core Sour restriction endonuclease, " TALE-2 '-R "-endonuclease, " TALE-3 '-L "-endonuclease, " TALE-3 '-R "-endonuclease Enzyme, " TALE-4 '-L "-endonuclease and " TALE-4 '-R "-endonuclease, " TALE-5 '-L "-endonuclease, " TALE-5 '-R "-endonuclease, " TALE-6 '-L "-endonuclease and " TALE-6 '-R "-endonuclease.

Further, inventor is due to the limitation of objective condition, and the above-mentioned TALEN listed does not have limit and is possible to, Due to the present invention provide 25 TALEN identification DNA fragmentation throughout above-mentioned 5 active regions, and can reach resist HIV disease The effect of poison, those skilled in the art are it is contemplated that arrive, in addition to above-mentioned 25 TALEN enumerated, in above-mentioned 5 active regions The other DNA fragmentations of selection recognize target sequence as TALEN, and correspondence designs different TALEN to CCR5 or CXCR4 genes Regulated and controled, be all feasible so as to reach the effect for resisting inhibition of HIV.

The present invention also provides the method regulated and controled with above-mentioned TALEN to CCR5 and CXCR4 genes.Including：A) thin First paragraph nucleotide sequence, the first paragraph nucleic acid sequence encoding first paragraph polypeptide (i.e. the first TALEN), so that when first are introduced in born of the same parents Section nucleotide sequence is expressed as in cell after first paragraph polypeptide, class activating transcription factor (TALE) identification in first paragraph polypeptide And the DNA fragmentation in mankind's CCR5 or CXCR4 gene activity region (being referred to as the first DNA fragmentation) is combined, first paragraph polypeptide has Shear the function of CCR5 or CXCR4 genes.

This method be may further include and second segment nucleotide sequence b) is introduced in cell, and the second segment nucleotide sequence is compiled Code second segment polypeptide (i.e. the 2nd TALEN), above-mentioned 2nd TALEN is different from the first TALEN, so that when second segment nucleotide sequence exists It is expressed as in cell after second segment polypeptide, the class activating transcription factor (TALE) in second segment polypeptide recognizes and combines the mankind DNA fragmentation (being referred to as the second DNA fragmentation) in CCR5 or CXCR4 gene activities region, above-mentioned second DNA fragmentation is different from first DNA fragmentation, second segment polypeptide and first paragraph polypeptide can together with shear CCR5 or CXCR4 genes.

When this method only includes step a) without including step b), the albumen combined in the first TALEN with TALE For the endonuclease with shear active, and when the method comprising the steps of a) and when step b), the first TALEN, second The albumen combined in TALEN with TALE can be the partial nucleic acid restriction endonuclease for not having shear active individually, such as FokI.

Above-mentioned first paragraph polypeptide and second segment polypeptide can be encoded by same nucleic acid, thus above-mentioned first paragraph nucleotide sequence and Second segment nucleotide sequence is on same nucleic acid, disposable to introduce cell；Above-mentioned first paragraph polypeptide and second segment polypeptide Thus first paragraph nucleotide sequence and second segment nucleotide sequence can be introduced into cell respectively in two times by different IPs acid encoding.

This method may further include step c) and one section of polynucleotide, the polynucleotide bag introduced in cell Include first area and second area, one section of gene of the first area and CCR5 or CXCR4 gene double-strand regions of fracture upstream Sequence is identical, and the second area is identical with one section of gene order in CCR5 or CXCR4 gene double-strand regions of fracture downstream.Pass through Above-mentioned steps c) can accelerate the reparation of the CCR5 or CXCR4 genes after shearing, so as to ensure after being regulated and controled by the above method CCR5 or CXCR4 genes can not only resist HIV attack, and can recover its normal function as early as possible.

Further, first paragraph nucleic acid, second segment nucleic acid and polynucleotide are supported on genophore, such as conventional gland Viral vector Ad5/F35 etc..Above-mentioned cell can then be selected from candidate stem cell, T cell, macrophage, dendritic cells and antigen In delivery cell.

Brief description of the drawings：

Fig. 1：Flow cytometer detection CCR5-TALEN knocks out CCR5 results.FACS detections TALEN-1-L/TALEN-1-R is knocked out Cell surface CCR5 expressions (left figure) after ghost cell, wherein FACS detections TALEN-1-L/TALEN-1-R is knocked out Cell surface CXCR4 expressions are used as control (right figure) after ghost cell.

Fig. 2：Flow cytometer detection CCR5-TALEN knocks out CCR5 results.FACS detections TALEN-2-L/TALEN-2-R is knocked out Cell surface CCR5 expressions (left figure) after ghost cell, wherein FACS detections TALEN-2-L/TALEN-2-R is knocked out Cell surface CXCR4 expressions are used as control (right figure) after ghost cell.

Fig. 3：Flow cytometer detection CCR5-TALEN knocks out CCR5 results.FACS detections TALEN-3-L/TALEN-3-R is knocked out Cell surface CCR5 expressions (left figure) after ghost cell, wherein FACS detections TALEN-3-L/TALEN-3-R is knocked out Cell surface CXCR4 expressions are used as control (right figure) after ghost cell.

Fig. 4：Flow cytometer detection CCR5-TALEN knocks out CCR5 results.FACS detections TALEN-4-L/TALEN-4-R is knocked out Cell surface CCR5 expressions (left figure) after ghost cell, wherein FACS detections TALEN-4-L/TALEN-4-R is knocked out Cell surface CXCR4 expressions are used as control (right figure) after ghost cell.

Fig. 5：Flow cytometer detection CCR5-TALEN knocks out CCR5 results.FACS detections TALEN-5-L/TALEN-5-R is knocked out Cell surface CCR5 expressions (left figure) after ghost cell, wherein FACS detections TALEN-5-L/TALEN-5-R is knocked out Cell surface CXCR4 expressions are used as control (right figure) after ghost cell.

Fig. 6：Flow cytometer detection CCR5-TALEN knocks out CCR5 results.FACS detections TALEN-6-L/TALEN-6-R is knocked out Cell surface CCR5 expressions (left figure) after ghost cell, wherein FACS detections TALEN-6-L/TALEN-6-R is knocked out Cell surface CXCR4 expressions are used as control (right figure) after ghost cell.

Fig. 7：Flow cytometer detection CCR5-TALEN knocks out CCR5 results.FACS detections TALEN-7-L/TALEN-7-R is knocked out Cell surface CCR5 expressions (left figure) after ghost cell, wherein FACS detections TALEN-7-L/TALEN-7-R is knocked out Cell surface CXCR4 expressions are used as control (right figure) after ghost cell.

Fig. 8：7 couples of TALEN knock out the summary of CCR5 gene efficiency.

Fig. 9：7 couples of TALEN knock out the survival rate of cell after CCR5 genes, including MTT methods and method for cell count.

Figure 10：CCR5 expresses negative Ghost cell after airflow classification 7 is knocked out to TALEN.

Figure 11：The inhibiting rate that the negative Ghost cell of CCR5 expression are infected pseudovirus after 7 couples of TALEN are knocked out.

Figure 12：Flow cytometer detection CXCR4-TALEN knocks out CCR5 results.FACS detections TALEN-1 '-L/TALEN-1 '-R strike Except cell surface CXCR4 expressions (right figure) after ghost cell, wherein FACS detections TALEN-1 '-L/TALEN-1 '-R strike Except cell surface CCCR5 expressions are used as control (left figure) after ghost cell.

Figure 13：Flow cytometer detection CXCR4-TALEN knocks out CCR5 results.FACS detections TALEN-2 '-L/TALEN-2 '-R strike Except cell surface CXCR4 expressions (right figure) after ghost cell, wherein FACS detections TALEN-2 '-L/TALEN-2 '-R strike Except cell surface CCR5 expressions are used as control (left figure) after ghost cell.

Figure 14：Flow cytometer detection CXCR4-TALEN knocks out CCR5 results.FACS detections TALEN-3 '-L/TALEN-3 '-R strike Except cell surface CXCR4 expressions (right figure) after ghost cell, wherein FACS detections TALEN-3 '-L/TALEN-3 '-R strike Except cell surface CCR5 expressions are used as control (left figure) after ghost cell.

Figure 15：Flow cytometer detection CXCR4-TALEN knocks out CCR5 results.FACS detections TALEN-4 '-L/TALEN-4 '-R strike Except cell surface CXCR4 expressions (right figure) after ghost cell, wherein FACS detections TALEN-4 '-L/TALEN-4 '-R strike Except cell surface CCR5 expressions are used as control (left figure) after ghost cell.

Figure 16：Flow cytometer detection CXCR4-TALEN knocks out CCR5 results.FACS detections TALEN-5 '-L/TALEN-5 '-R strike Except cell surface CXCR4 expressions (right figure) after ghost cell, wherein FACS detections TALEN-5 '-L/TALEN-5 '-R strike Except cell surface CCR5 expressions are used as control (left figure) after ghost cell.

Figure 17：Flow cytometer detection CXCR4-TALEN knocks out CCR5 results.FACS detections TALEN-6 '-L/TALEN-6 '-R strike Except cell surface CXCR4 expressions (right figure) after ghost cell, wherein FACS detections TALEN-6 '-L/TALEN-6 '-R strike Except cell surface CCR5 expressions are used as control (left figure) after ghost cell.

Figure 18：6 couples of TALEN knock out the summary of CXCR4 gene efficiency.

Figure 19：6 couples of TALEN knock out the survival rate of cell after CXCR4 genes.

Figure 20：CXCR4 expresses negative Ghost cell after airflow classification 6 is knocked out to TALEN.

Figure 21：The inhibiting rate that the negative Ghost cell of CXCR4 expression are infected pseudovirus after 6 couples of TALEN are knocked out.

Embodiment

The present invention will be illustrated by specific embodiment below, but these specific embodiments are not construed as to this The limitation of invention, some details, which are modified, to be still fallen within protection scope of the present invention.

Example 1TALEN plasmid amplifications；

50 μ l competent cell DH5 α (TIANGEN) are taken from -80 DEG C of refrigerators first, as the about 10min that thaws on ice.So Adding TALEN DNAs in the backward competent cell suspension, (50ul competent cell can be by 500nG super coiled DNAs institute Saturation), gently rotating centrifugal pipe stands 30min to mix content in ice bath.And then the thermal shock 90 in 42 DEG C of water-baths Second, it is immediately placed in cooled on ice 3-5 minutes after thermal shock, then addition 500ul LB fluid nutrient mediums (being free of antibiotic) into pipe, Take 100 μ l to be coated in the screening flat board containing Amp after shaking up, face up placement half an hour, treat that bacterium solution is cultured base suction completely Culture dish is inverted after receipts, 37 DEG C are cultivated 16 hours.

Picking monoclonal is cultivated in 3ml Amp LB culture mediums in 37 DEG C of shaking tables of bacterium on culture plate；16h 3ml bacterium solutions are inoculated into 100ml Amp LB afterwards, continue to cultivate 12h in 37 DEG C of shaking tables.

Example 2TALEN plasmid extractions (are usedPlasmid Plus Maxi)

First carry out following preparation.RNase A enzymes, mixing are added into P1 buffer solutions, and is stored in 2-8 DEG C；Then Adding proportion is 1: 1000 theretoCheck solution P2 whether containing SDS precipitations, if any 37 DEG C can be placed in A little time is to treat that it is completely dissolved；Before use, adding ethanol (96-100%) (see a bottle label) into solution PE.

Largely extracted followed by plasmid.With 4 DEG C of centrifuge, 6000 × G centrifugations 15min is collected shakes bacterium overnight 100mL Escherichia coli bacteria liquids.Then thalline is resuspended with 5ml solution P1, is repeated to mix with oscillator.Then 5ml solution P2 are added, Gently by bottle turned upside down 4-6 times until bacterium cracks completely, in grume.Solution after cracking is placed in room temperature 3min again. If having added LyseBlue reagents, cell suspension will become au bleu.New QIAfilter DNA adsorption columns are put into totally In suitable centrifuge tube, it is ensured that enough volume solubilization liquid BB.5mlS3 lysates are added in cellular lysate liquid forwardly, on Lower be inverted 4-6 times has been sufficiently mixed.If having added LyseBlue, mixed solution is known completely colorless.Then lysate is turned Move in QIAfilter pillars, and be incubated 10 minutes at room temperature.During incubation, by GIAGEN Plasmid Plus from Stem is put into the Plus of QIAvac 24；And then gently insertion plunger into QIAfilter pipes and filtration cell lysate, is stayed Go out sufficient space solubilization liquid BB.Then 5ml solution B B are added into the cell pyrolysis liquid of clarification, simultaneously turned upside down 4-6 times is mixed Transfer cell pyrolysis liquid is into GIAGEN Plasmid Plus centrifugal columns afterwards.With about -300mbar vavuum pump suction filtration lysates, Until lysate is all taken out down from adsorption column.Add 0.7ml solution Es TR and use vavuum pump suction filtration, until lysate is whole Taken out down from adsorption column.Equally, also add 0.7ml solution PE and use vavuum pump suction filtration, until lysate is all from adsorption column Take out down.By adsorption column, 10000G (9700rpm) centrifuges 1min with the complete washing for removing residual in desk centrifuge remittance afterwards Buffer solution.GIAGEN Plasmid Plus adsorption columns are put into a clean 2ml centrifuge tube again.To adsorption column interposition Put addition 400ul solution ddH₂O, about 1min is placed in room temperature, is then centrifuged for eluting target DNA.Finally surveyed with Nanodrop Determine plasmid concentration.Plasmid concentration, can be diluted to 1ug/ul by cell transfecting for convenience.

Example 3Ghost cell cell culture

Before passage, cell is observed under the microscope and covers with culture dish bottom.Then cell culture medium (DMEM), PBS, pancreatin are placed in room temperature half an hour, while after ultraviolet irradiating cell super-clean bench 30min, can be tested on super-clean bench.

First nutrient solution original in the individual layer Ghost cell covered with blake bottle is siphoned away with suction pipe, added 1ml0.25% trypsin solutions vitellophag is (depending on the volume of addition is with specific culture dish size.For example, 10cm culture dishes are trained Foster cell can use the trypsin solutions of 5ml 0.25%), it is placed in vitellophag about 5 minutes in 37 DEG C of incubators；In under inversion mirror The change of cell attachment and form is observed, when former adherent cell gradually tends to be circular, hikes up and starts to be separated into a small amount of aggregation Cell mass when, add 10ml nutrient solutions and terminate digestion.Then by this postdigestive cell in centrifuge, normal temperature 800rpm from The heart 5 minutes.Cell is resuspended with a certain amount of DMEM culture mediums again, and cell is counted under the microscope with blood counting instrument.Count Afterwards, appropriate Ghost cell are taken (to add 3 × 10 in such as one 10cm culture dish⁶Individual cell) in culture dish or blake bottle In, put in 37 DEG C of cell culture incubators and cultivate.In order to ensure that cell state is good, the passage in every two days of control cell is once.Cell is trained In supporting, culture medium DMEM additions 10%FBS.In order to prevent cell contamination, 1% antibiosis is added in cell culture medium after sizing Plain (penicillin＆ streptomycin).

Example 4Ghost cell electricity turns conditional FP tree

Electricity turns forward pass for Ghost cell., with 3*10⁴cells/cm²Cell density be layered on a diameter of 10cm culture dish In, in cell culture incubator, with 37 DEG C, 5%CO₂Under the conditions of cultivate.After passage 24h, observation cell state is good, covers with During 90% area of culture dish, vitellophag, room temperature centrifugation 100G*10min.Then counted.Turn anti-according to each 20ul electricity System is answered to take 5*10⁵Cell, takes the cell suspension of proper volume to centrifuge, and turning buffer solution SE (SF or SG) with 20ul electricity is resuspended carefully Born of the same parents, fully dispel after cell, add the Pmax plasmid 0.4ul that concentration is 1uG/ul, fully mix.Electricity in 16 holes turns reaction bar In the electric revolving cup of neck, the reaction system of 20ul previous steps is added.LONZA electroporations are opened, setting electricity turns parameter, starts electricity Hit.After electric shock terminates, room temperature is placed after 1min, then the addition 80ul culture mediums in each electric revolving cup, and room temperature places 10min, together When, the preheating of 500ul culture mediums is added in 24 orifice plates.And then 100ul electricity swivels system is added in 24 orifice plates, notes using up cell Amount is uniformly layered in hole.

12h after electric shock, observes cell attachment situation, and change liquid；36h after electricity turns, is turned with the different electricity of fluorescence microscope Buffer solution and different electricity turn cell GFP expressions under parameter.Different electricity are turned with buffer solution and electricity turns to carry out cell under parameter Count, to compare cell survival rate under different condition.Turn GFP expression rates under parameter with the different electricity of flow cytometer detection simultaneously.It is determined that thin Born of the same parents' survival rate and GFP expression efficiencies higher electricity turn condition, and the electricity for being defined as Ghost cell cell lines turns parameter.

The difference of example 5 TALEN knocks out Ghost cell efficiency and survival rate compares

Electricity turns forward pass for Ghost cell, with 3*10⁴cells/cm²Cell density be layered on a diameter of 10cm culture dish In, in cell culture incubator, with 37 DEG C, 5%CO₂Under the conditions of cultivate.After passage 24h, observation cell state is good, covers with During 90% area of culture dish, vitellophag is simultaneously counted, and turning reaction system according to each 100ul electricity takes 3.5*10⁶Cell.After digestion Cell, room temperature centrifugation 100G*10min.Turn buffer solution SF respin cells with 100ul electricity, fully dispel after cell, add dense The TALEN-L/TALEN-R plasmids difference 3ul that Pmax plasmids 2ul that degree is 1uG/ul, concentration are 1uG/ul, and fully mix. The reaction system of mixing is added in electric revolving cup.LONZA electroporations are opened, the electricity that determination is explored before setting turns parameter EN- 138, start electric shock.After electric shock terminates, room temperature places 1min.500ul culture mediums are added in each electric revolving cup again, room temperature is placed 10min, meanwhile, the preheating of 2.5ml culture mediums is added in 6 orifice plates.Take 200ul in 600ul electricity swivels system to add in 6 orifice plates, note Make cell try one's best uniformly to be layered in hole.12h after electric shock, observes cell attachment situation, and change liquid；36h after electricity turns, uses fluorescence microscopy Sem observation difference electricity turns buffer solution and different electricity turn cell GFP expressions under parameter.Different electricity are turned with buffer solution and electricity turns Cell count is carried out under parameter, to compare cell survival rate under different condition, while turning GFP under parameter with the different electricity of flow cytometer detection Expression rate simultaneously detects that Ghost cell surface Cs XCR4/CCR5 knocks out situation.Determine that cell survival rate and CXCR4/CCR5 knock out effect Rate higher TALEN, and the cell after the preferable TALEN of efficiency is knocked out will be knocked out and be transferred in culture dish expand culture.

Example 6FACS detection TALEN plasmids knock out efficiency

Digestion Ghost cell are simultaneously counted, and take 3*10⁶Cell is homogeneously disposed in 3 1.5ml centrifuge tubes respectively, is encoded to 1., 2., 3., in addition, taking transfection Pmax Ghost cell 1*10⁶In 1.5ml centrifuge tubes, it is encoded to 4..Through 4 DEG C, After 850rpm*3min centrifugation, supernatant is abandoned, is washed with 1ml2%FBS PBS 2 times.Diluted respectively with 100ul2%FBS PBS 10ul CXCR4-APC and CCR5-PE antibody, are added separately to 2., 3. in cell, 1., be 4. separately added into 100ul l2%FBS PBS, sample is incubated 10min in 4 DEG C of lucifuges.Washed with 1ml2%FBS PBS 2 times；Finally use flow cytometer detection：Adjust respectively Compensation between Ghost cell streamings parameters and FITC/PE/APC.

Digest the Ghost cell after different TALEN transfections and count, take 1*10⁶In 1.5ml centrifuge tubes；Through 4 DEG C, After 850rpm*3min centrifugations, supernatant discarding is washed 2 times with 1ml2%FBS PBS；In each cell sample centrifuge tube, add and use 100ul2%FBS PBS dilution 10ul CXCR4-APC and 10ul CCR5-PE antibody, sample is incubated in 4 DEG C of lucifuges 10min.Washed with 1ml2%FBS PBS after 2 times, carry out flow cytometer detection：Determine different cell CCR5 and CXCR4 after transfection TALEN Expression, as a result represented with FITC/PE and FITC/APC double fluorescence scatter diagrams.

CCR5/CXCR4-neGative Ghost cell after example 7FACS sortings TALEN is knocked out

Digestion Ghost cell are simultaneously counted, and take 3*10⁶Cell is homogeneously disposed in 3 1.5ml centrifuge tubes respectively, is encoded to ①、②、③.After 4 DEG C, 850rpm*3min centrifugations, supernatant discarding is washed 2 times with 1ml2%FBS PBS.Use respectively 100ul2%FBS PBS dilutions 10ul CXCR4-APC and CCR5-PE antibody, is added separately to 2., 3. in cell, 1. add 100ul 12%FBS PBS, sample is incubated 10min in 4 DEG C of lucifuges.Washed with 1ml2%FBS PBS after 2 times, carry out streaming inspection Survey：Compensation between adjustment Ghost cell streamings parameters and PE/APC respectively.

Digestion knocks out the Ghost cell after the higher several groups of difference TALEN transfections of efficiency and counted, and is placed in 1.5ml centrifugations Guan Zhong；After 4 DEG C, 850rpm*3min centrifugations, supernatant discarding is washed 2 times with 1ml2%FBS PBS.Each cell sample centrifugation Guan Zhong, according to cell number：Antibody volume=10⁶：10ul ratio adds the PBS dilutions CXCR4-APC with 100ul2%FBS With 10ul CCR5-PE antibody, sample is incubated 10min in 4 DEG C of lucifuges.Washed with 1ml2%FBS PBS after 2 times, carry out streaming Sorting：During sorting, transfection CCR5-TALEN Ghost cell select APC-positive/PE-neGative cell mass, turn The Ghost cell for contaminating CXCR4-TALEN select APC-neGative/PE-positive cell mass, expand culture after sorting. After cell expansion culture, cell mass CCR5/CXCR4 expression after being sorted by flow cytometer detection, to ensure the efficiency of separation.

The pseudovirus of example 8 is coated with

1. pseudovirus is coated with the previous day, by 293T cells with 10⁶The density in cells/ holes is layered in 6 orifice plates, second day generation When cell covers with 80% or so, start transfection procedure；

2. take 2ug PNL4-3 plasmids, 2ug HIV Env plasmids fully to be mixed in EP pipes；

3. adding the culture medium that 200ul is free of serum in well mixed plasmid, fully mix；

4. room temperature places 10min；

5. the transfection mixture prepared point is added in 6 orifice bores, make rotaring redyeing system uniformly dispersing in hole；

6. the cell after transfection is placed in 37 DEG C, 5%CO₂Condition of culture under cultivate；

7. cell changes liquid after transfection 4-8h；

8. transfection two time points of 48h and 72h harvest pseudovirus respectively；

9. harvest pseudovirus：4500rpm centrifuges 10min, removes cell fragment；The hyclone of general addition 10% (FBS), make FBS final concentration of 20% in pseudovirus, be distributed into after 300-500ul/ pipes to freeze and spend refrigerator in -80.

Ghost cell after the pseudovirus of example 9 infection TALEN knockouts

CXCR4-neGative and CCR5-neGative Ghost cell bed boards after 1.TALEN is knocked out

CXCR4-positive/CCR5-neGative's and CXCR4-neGative/CCR5-positive after sorting Ghostcell groups expand culture, detect that the co-receptor expression rate after its sorting knocks out efficiency and reaches more than 90%, and count.Press According to 2*10⁴The density in cells/ holes spreads 96 orifice plates, and per hole culture volume 100ul, the pseudovirus of each type lays 2 Cell mass after multiple holes, every kind of knockout all sets the cell controls that two holes are not added with pseudovirus.Do not knocked out by TALEN Counted after Ghost cell digestion, according to 2*10⁴The density in cells/ holes spreads 96 orifice plates, each per hole culture volume 100ul The pseudovirus of type lays 2 multiple holes, infects pseudovirus the positive ginseng suppressed after TALEN is knocked out as detection According to.Meanwhile, two holes of setting are not added with the cell controls of pseudovirus.96 orifice plates completed cultivate 12h in cell culture incubator, culture Condition is 37 DEG C, 5%CO₂, make cell completely adherent.

2. select the infection of different type pseudovirus

Several typical pseudovirus and type strain for selecting co-receptor single.By CXCR4-neGative and CCR5- 1h after neGative Ghostcell bed boards, pseudovirus 50ul is added per hole, is then cultivated in incubator and poison is detected after 48h Value.

3. pseudovirus infection poison value is read in detection

Add after pseudovirus 48h, suck cell conditioned medium with vavuum pump, note the hole of different cell types and different pseudovirus Between can not pollute mutually.100ulPBS is added in each hole again, 50ul cell pyrolysis liquids and Luciferase substrates is added Mixture, room temperature lucifuge cracking 4min.Then each hole takes 100ul reaction supernatant in blank, by determining each sample The fluorescence readings of product reacts the situation of pseudovirus infection cell.Meanwhile, by glimmering with the corresponding pseudovirus of Ghost cell infection Light readings compares, and calculates the inhibiting rate that the Ghost cell after CCR5/CXCR4 is knocked out are infected pseudovirus.

Table 1：The CCR5 high-activity areas that 3 TALEN are combined

TALEN high-activity areas 1：

tgcAcagggtggaacaagatggattatcaagtgtcaagtccaatctatgacatcaattattatacatcggagccctg ccaaaaaatcaatgtgaagcaaatcgcag

TALEN high-activity areas 2：

Caacatgctggtcatcctcatcctgataaactgcaaaaggctgaagagcatgactgacatctacctg

TALEN high-activity areas 3：

Tcttcattacacctgcagctctcattttccatacagtcagtatcaattctggaagaatttccagacattaaagatag

Table 2：The CCR5 high activity sequences that 7 couples of TALEN are combined

Sequence 1.

It is left：5 '-TATGACATCAATTATTAT-3 ' (are combined) with table 3TALEN-1-L

It is right：5 '-TGCTTCACATTGATTTTTT-3 ' (are combined) with table 3TALEN-1-R

Sequence 2.

It is left：5 '-TGGAACAAGATGGATTAT-3 ' (are combined) with table 3TALEN-2-L

It is right：5 '-TAATTGATGTCATAGAT-3 ' (are combined) with table 3TALEN-2-R

Sequence 3.

It is left：5 '-TATCAAGTGTCAAGTCCAAT-3 ' (are combined) with table 3TALEN-3-L

It is right：5 '-TGGCAGGGCTCCGATGTATAAT-3 ' (are combined) with table 3TALEN-3-R

Sequence 4.

It is left：5 '-TGCTGGTCATCCTCAT-3 ' (are combined) with table 3TALEN-4-L

It is right：5 '-TCATGCTCTTCAGCCTT-3 ' (are combined) with table 3TALEN-4-R

Sequence 5.

It is left：5 '-TGGTCATCCTCATCCT-3 ' (are combined) with table 3TALEN-5-L

It is right：5 '-TCAGTCATGCTCTTCAGC-3 ' (are combined) with table 3TALEN-5-R

Sequence 6.

It is left：5 '-TGCTGGTCATCCTCATC-3 ' (are combined) with table 3TALEN-6-L

It is right：5 '-TCATGCTCTTCAGCCTT-3 ' (are combined) with table 3TALEN-6-R

Sequence 7.

It is left：5 '-TACACCTGCAGCTCTCAT-3 ' (are combined) with table 3TALEN-7-L

It is right：5 '-TGGAAATTCTTCCAGAAT-3 ' (are combined) with table 3TALEN-7-R

Table 3：The TALEN RVD sequences combined with table 2CCR5 sequences

TALEN-1RVD sequences：

TALEN-1-L：NI NG NN NI HD NI NG HD NI NI NG NG NI NG NG NI NG

TALEN-1-R：NN HD NG NG HD NI HD NI NG NG NN NI NG NG NG NG NG NG

TALEN-2RVD sequences：

TALEN-2-L：NN NN NI NI HD NI NI NN NI NG NN NN NI NG NG NI NG

TALEN-2-R：NI NI NG NG NN NI NG NN NG HD NI NG NI NN NI NG

TALEN-3RVD sequences：

TALEN-3-L：NI NG HD NI NI NN NG NN NG HD NI NI NN NG HD HD NI NI NG

TALEN-3-R：NN NN HD NI NN NN NN HD NG HD HD NN NI NG NN NG NI NG NI NI NG

TALEN-4RVD sequences：

TALEN-4-L：NN HD NG NN NN NG HD NI NG HD HD NG HD NI NG

TALEN-4-R：HD NI NG NN HD NG HD NG NG HD NI NN HD HD NG NG

TALEN-5RVD sequences：

TALEN-5-L：NN NN NG HD NI NG HD HD NG HD NI NG HD HD NG

TALEN-5-R：HD NI NN NG HD NI NG NN HD NG HD NG NG HD NI NN HD

TALEN-6RVD sequences：

TALEN-6-L：NN HD NG NN NN NG HD NI NG HD HD NG HD NI NG HD

TALEN-6-R：HD NI NG NN HD NG HD NG NG HD NI NN HD HD NG NG

TALEN-7RVD sequences：

TALEN-7-L：NI HD NI HD HD NG NN HD NI NN HD NG HD NG HD NI NG

TALEN-7-R：NN NN NI NI NI NG NG HD NG NG HD HD NI NN NI NI NG

Table 4：TALEN is knocked out after CCR5, cloning and sequencing analysis.

NO.1TALEN-1 knocks out the sequencing result of CCR5 target sequences 1

Wild type CCR5 target sequences 1 (WT)：

GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGAACAAG

ATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGGAGCCCTGCCAAAAA

ATCAATGT

NO1：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGAA

CAAGATGGATTATCAAGTGTCAAGTCCAATCTATG:::TCAATTATTATACATCGGAGCCCTGCCA

AAAAATCAATGT

NO2：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGAA

CAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGGAGCC::::CA

AAAAATCAATGT

NO3：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGAA

CAAGATGGATTATCAAGTGTCAAGTCCAAT:::::ACATCAATTATTATACATCGGAGCCCTGCCA

AAAAATCAATGT

NO4：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGAA

CAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGGAGCC:::::A

AAAAATCAATGT

NO5：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGAA

CAAGATGGATTATCAAGTGTCAAGTCCAATCT:::::ATCAATTATTATACATCGGAGCCCTGCCA

AAAAATCAATGT

NO6：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGAA

CAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGGAGCC::::::

AAAAATCAATGT

NO7：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGAA

CAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGG:::::::CCA

AAAAATCAATGT

NO8：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGAA

CAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGG:::::::CCA

AAAAATCAATGT

NO9：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGAA

CAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCG::::::::CCA

AAAAAT CAAT GT

NO10：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGA

ACAAGATGGATTATCAAGTGTCAAGTCCAATC:::::::::AATTATTATACATCGGAGCCCTGCC

AAAAAATCAATGT

NO11:GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGA

ACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTAT::::::::::AGCCCTGCC

AAAAAATCAATGT

NO12：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGA

ACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATA::::::::::::::C

AAAAAATCAATGT

NO13：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGA

ACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACA:::::::::::::

：AAAAATCAATGT

NO14：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGA

ACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGG:::::::::

::::::::::TGT

NO15：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGA

ACAAGATGGATTATCAAGTGTCAAGTCCAATC::::::::::::::::::::::::::::::::::

:::::ATCAATGT

NO16：GGCAA::::::::::::::::::::::::::::::::::::::::::::::::::::::::

::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

::AAAATCAATGT

NO17：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGA

ACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGEAGCCCT

GCCAAAAAATCAA

NO18：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGA

ACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGGAGCCCTGC

CAAAAAATCAATG

NO19：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGA

ACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGGAGCCCT

GCCAAAAAATCAA

NO20：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGA

ACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGGAGCCCT

GCCAAAAAATCAA

NO21：GGCAACTAAATACATTCTAGGACTTTATAAAAGATCACTTTTTATTTATGCACAGGGTGGA

ACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATACATCGGAGCC

CTGCCAAAAAATC

NO.2 TALEN-2 knock out the sequencing result of CCR5 target sequences 2

Wild type CCR5 target sequences 2 (WT)：

GATCACTTTTTATTTATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGA

CATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO1：GATCACTTTTTATTTATGCACAGGGTGGAACAAGATGGATTAT:AAGTGTCAAGTCCAATCT

ATGACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO2：GATCACTTTTTATTTATGCACAGGGTGGAACAAGATGGATTATCA:GTGTCAAGTCCAATCT

ATGACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO3：GATCACTTTTTATTTATGCACAGGGTGGAACAAGATGGAT:::CAAGTGTCAAGTCCAATCT

ATGACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO4：GATCACTTTTTATTTATGCACAGGGTGGAACAAGATGGATTA::::::GTCAAGTCCAATCT

ATGACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO5：GATCACTTTTTATTTATGCACAGG:::::::::::TGGATTATCAAGTGTCAAGTCCAATCT

ATGACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO6：GATCACTTTTTATTTATGCACAGGGTGGAACAAG:::::::::::::TGTCAAGTCCAATCT

ATGACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO7：GATCACTTTTTATTTAT:::::::::::::CAAGATGGATTATCAAGTGTCAAGTCCAATCT

ATGACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO8：GATCACTTTTTATTTATGCACAGGGTGGAACAAGATG:::::::::::::::::TCCAATCT

ATGACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO9：GATCACTTTTTATTTATGCACAGGGTGG:::::::::::::::::::::TCAAGTCCAATCT

ATGACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO10：GATCACTTTTTA:::::::::::::::::::::::::::TTATCAAGTGTCAAGTCCAATC

TATGACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO11：GATCACTTTTTATTTATGCACAGGGTGGAACAAGATGGA::::::::::::::::::::::

::::::ATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO12：GATCACTTTTTATTTATGCACAG::::::::::::::::::::::::::::::::::::::

::::::::::::::TTATACATCGGAGCCCTGCCAAAAAATCAATGTGAA

NO13：GATCACTTTTTATTTATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAA

TCTATGACATCAATTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGA

NO.3TALEN-3 knocks out the sequencing result of CCR5 target sequences 3

Wild type CCR5 target sequences 3 (WT)：

ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAATTATTATAC

ATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO1：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACA:CAATTATT

ATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO2：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTATG::ATCAATTATT

ATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO3：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAAT:::T

ATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO4：ATGCACAGGGTGGAACAAGATGGAT:::::AGTGTCAAGTCCAATCTATGACATCAATTATT

ATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO5：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAA:::::::CATCAATTATT

ATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO6：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAA:::::::CATCAATTATT

ATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO7：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTC:::::::::ACATCAATTATT

ATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO8：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTC:::::::::ACATCAATTATT

ATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO9：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCC::::::::::ATCAATTATT

ATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO10：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTC:::::::::::::CAATTAT

TATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO11：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACAT:::::::

:::::::CGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO12：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAATC:::::::::::::::

::TACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO13：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCC:::::::GA::TC::::::

::TACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO14：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAA:::::::::::::::::::TTAT

TATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO15：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCA::::::::::::::::::

::::::TCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGCCTCC

NO16：ATGCACAGGGTGGAACAAGATGGATTATCAAGTGTCAAGTCCAATCTATGACATCAA

TTATTATACATCGGAGCCCTGCCAAAAAATCAATGTGAAGCAAATCGCAGCCCGCCTCCTGC

NO.4TALEN-4 knocks out the sequencing result of CCR5 target sequences 4

Wild type CCR5 target sequences 4 (WT)：

GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAACTGCAAAAGGCTGAAGAGCATGACT

GACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCTGGGCT

CACTAT

NO1：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGAT:AACTGCAAAAGGCTGAAGAGCAT

GACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCTG

GGCTCACTAT

NO2：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTG::AAACTGCAAAAGGCTGAAGAGCAT

GACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCTG

GGCTCACTAT

NO3：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTG:::AACTGCAAAAGGCTGAAGAGCAT

GACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCTG

GGCTCACTAT

NO4：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTG::::ACTGCGTAAGGATGAAGAACAT

GATTGACATGTACCTGCTCAACCTGTCCGTCTCTGATTTGTTTTTCCTTGTTACTGTCCGGGTCTG

GGATCCTTAT

NO5：GGTTTTGTGGGCAACACGCTGGTCATCCTCATCCTGATAAA:::::AAAGGCTGAAGAGCAT

GACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCTG

GGCTCACTAT

NO6：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAA:::::::AGGCTGAAGAGCAT

GACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCTG

GGCTCACTAT

NO7：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAA:::::::AGGCTGAAGAGCAT

GACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCTG

GGCTCACTAT

NO8：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTG::::::::CAAAAGGCTGAAGAGCAT

GACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCTG

GGCTCACTAT

NO9：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGA::::::::AAAAGGCTGAAGAGCAT

GACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCCTACTGTCCCCTTCTG

GGCTCACTAT

NO10：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGA::::::::AAAAGGCTGAAGAGGA

TGACTGACTTCTTGCTGCTCAACCTGATCATCTCTGAGCTGTTTTTCCTTCTTACTGTCCCCTTGT

GGCCTCATGAT

NO11：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTG::::::::CAAAAGGCTGAAGAGCA

TGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO12：GGTTTTGTGGGCAACATGCTGGTCATCC::::::::::AAACTGCAAAAGGCTGAAGAGCA

TGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO13：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGA::::::::::AAGGCTGAAGAGCA

TGACTGACATCTACCTGCTCAACCTGGCCATCACTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO14：GGTTTTGTGGGCAACATGCTGGTCATCC:::::::::::AACTGCAAAAGGCTGAAGAGCA

TGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO15：GGTTTTGTGGGCAACATGCTGGTCATCCTC::::::::::::TGCAAAAGGCTGAAGAGCA

TGACTAACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO16：GGTTTTGTGGGCAACATGCTGGTCATCCT::::::::::::::GCAAAAGGCTGAAGAGCA

TGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO17：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGA::::::::::::::::::AGAGCA

TGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO18：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGA:::::::::::::::::::GAGCA

TGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO19：GGTTTTGTGGGCAACATGCT:::::::::::::::::::AACTGCAAAAGGCTGAAGAGCA

TGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

：GGCTCACTAT

NO20：GGTCTTGTGGGCAACATGCTGGTCATCCTC:::::::::::::::::::::::TGAAGAGC

ATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTC

TGGGCTCACTAT

NO21：GGTTTTGTGGGCAACATGCTGGTCATCCT::::::::::::::::::::::::GAAGAGCA

TGACTGACATCTACCTGCCCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO22：GGTTTTGTGGGCAACATGCTGGTCAT::::::::::::::::::::::::GCTGAAGAGCA

TGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO23：GGTTTTGTGGGCAACATGCTG:::::::::::::::::::::::::::::::::AAGAGCA

TGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO24：GGTTTTGTGGGCAACATGCTG::::::::::::::::::::::::::::::::::AAGAGC

ATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTC

TGGGCTCACTAT

NO25：GGTTTTGTGGGCA:::::::::::::::::::::::::::::::::::::::::::::GCA

TGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTCT

GGGCTCACTAT

NO26：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCT::::::::::::::::::::::::::

:::::::::::::::::::::::::::::::::::::::TGTTTTTCCTTCTTACTGTCCCATTCT

GGGCTCACTAT

NO27：GGTTTTGTGGGCAACATGCTGGTCATCC:::::::::::::::::::::::::::::::::

::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::CCTTCT

GGGCTCACTAT

NO28：GGTTTTGTGGGCAACATGCTGGTCATCC:::::::::::::::::::::::::::::::::

::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::CCTTCT

GGGCTCACTAT

NO29：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAACTGCGAAAGGCTGAAGA

GCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCT

TCTGGGCTCACT

NO30：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGA

CAAAAGGCTGAAG

NO31：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAACTGGAAAATGCTGAAC

AGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCC

TTCTGGGGATCCT

NO32：GGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAACTGCAAAAGGCTGAAGAGC

ATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTTCCTTCTTACTGTCCCCTTC

TGGGCTCACTAT

NO.5TALEN-5 knocks out the sequencing result of CCR5 target sequences 5

Wild type CCR5 target sequences 5 (WT)：

GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAACTGCAAAAGGCTGAA

GAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO1：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCT:ATAAACTGCAAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO2：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGA:::ACTGCAAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO3：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAA:::::AAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO4：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCT:::::ACTGCAAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO5：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATA::::::AAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO6：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATC::::::::CTGCAAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO7：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGAT::::::::AAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO8：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATC:::::::::::::AAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO9：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATC::::::::::::::AAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO10：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTC::::::::::::::AAACTGCAAAAGG

CTGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO11：GTTCATCTTTGGTTTTGTGGGCAACATGCTG::::::::::::::::TAAACTGCAAAAGG

CTGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO12：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTG:::::::::::::::

:TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO13：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCA::::::::::::::::::::

CTGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO14：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCT::::::::::::::::::::::

:::::::GCATGACTGACATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO15：GTTCATCTTTGGTTTTG::::::::::::::::::::::::::::::::::::::::::::

::::::::::::::::::ATCTACCTGCTCAACCTGGCCATCTCTGACCTGTTTTT

NO.6TALEN-6 knocks out the sequencing result of CCR5 target sequences 6

Wild type CCR5 target sequences 6 (WT)：

GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAACTGCAAAAGGCTGAA

GAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTC

NO1：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAA:TGCAAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTC

NO2：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATAAA::::AAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTC

NO3：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGAT:::::::AAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTC

NO4：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATC::::::::CTGCAAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTC

NO5：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTG::::::::CAAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTC

NO6：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTG::::::::CAAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTC

NO7：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTG::::::::CAAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTC

NO8：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTG:::::::::AAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTC

NO9：GTTCATCTTTGGT:::::::::::::::::::::::::::::::::::::::::CAAAAGGC

TGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTC

NO10：GTTCATCTTTGGTTTTGTGGGCAACATGCTG::::::::::::::::::::::::::::::

::::::::::::ACTGACATCTACCTGCTCAACCTGGCCATCTC

NO11：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGATCAAAAGG

CTGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCATCTC

NO12：GTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCTGACTGCAA

AAGGCTGAAGAGCATGACTGACATCTACCTGCTCAACCTGGCCA

NO.7TALEN-7 knocks out the sequencing result of CCR5 target sequences 7

Wild type CCR5 target sequences 7 (WT)：

TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGTCAGTAT

CAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO1：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGT:A

GTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO2：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATA::::CA

GTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO3：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAG:::

::ATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO4：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCC::::::::A

GTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO5：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCA::::::::

GTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO6：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATA::::::

::ATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO7：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTT::::::::::CA

GTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO8：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATT:::::::::::CA

GTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO9：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCT:::::::::::::::CA

GTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO10：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAG::::::::::::::::::::

::::::::::::::::::::::::::ACATTAAAGATAGTCATCT

NO11：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAG

TCAGTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO12：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGT

CAGTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO13：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGT

CAGTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO14：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGT

CAGTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

NO15：TTTACCAGATCTCAAAAAGAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACAGT

CAGTATCAATTCTGGAAGAATTTCCAGACATTAAAGATAGTCATCT

Table 1 '：The CXCR4 high-activity areas that 2 TALEN are combined

TALEN high-activity areas 1 '：

TAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCCATGAAGGAACCCTGTTTCCGTGAAGAAAA TGCTAATTTCAAIAAAA

TALEN high-activity areas 2 '：

TAAAATCTTCCTGCCCACCATCTACTCCATCATCTTCTTAACTGGCATTGTGGGCAATGGATTGGTCAT CCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCA

Table 2 '：The CXCR4 high activity sequences that 6 couples of TALEN are combined

The of sequence 1 '

It is left：CTTCCTGCCCACCATCT (is combined) with the TALEN-1 ' of table 3 '-L

It is right：GCCCACAATGCCAGTT (is combined) with the TALEN-1 ' of table 3 '-R

The of sequence 2 '

It is left：ACACCGAGGAAATGGGCTCA (is combined) with the TALEN-2 ' of table 3 '-L

It is right：CACGGAAACAGGGTTCCTT (is combined) with the TALEN-2 ' of table 3 '-R

The of sequence 3 '

It is left：ATGACTCCATGAAGGAAC (is combined) with the TALEN-3 ' of table 3 '-L

It is right：ATTGAAATTAGCATTTT (is combined) with the TALEN-3 ' of table 3 '-R

The of sequence 4 '

It is left：CCTGCCCACCATCTACT (is combined) with the TALEN-4 ' of table 3 '-L

It is right：GCCCACAATGCCAGT (is combined) with the TALEN-4 ' of table 3 '-R

The of sequence 5 '

It is left：CTACTCCATCATCTT (is combined) with the TALEN-5 ' of table 3 '-L

It is right：GACCAATCCATTGCCC (is combined) with the TALEN-5 ' of table 3 '-R

The of sequence 6 '

It is left：ATCCTGGTCATGGGTT (is combined) with the TALEN-6 ' of table 3 '-L

It is right：ACTTGTCCGTCATGCTT (is combined) with the TALEN-6 ' of table 3 '-R

Table 3 '：The TALEN RVD sequences combined with the CXCR4 sequences of table 2 '

TALEN-1 ' RVD sequences：

TALEN-1’-L：HD NG NG HD HD NG NN HD HD HD NI HD HD NI NG HD NG

TALEN-1’-R：NN HD HD HD NI HD NI NI NG NN HD HD NI NN NG NG

TALEN-2 ' RVD sequences：

TALEN-2’-L：NI HD NI HD HD NN NI NN NN NI NI NI NG NN NN NN HD NG HD NI

TALEN-2’-R：HD NI HD NN NN NI NI NI HD NI NN NN NN NG NG HD HD NG NG

TALEN-3 ' RVD sequences：

TALEN-3’-L：NI NG NN NI HD NG HD HD NI NG NN NI NI NN NN NI NI HD

TALEN-3’-R：NI NG NG NN NI NI NI NG NG NI NN HD NI NG NG NG NG

TALEN-4 ' RVD sequences：

TALEN-4’-L：HD HD NG NN HD HD HD NI HD HD NI NG HD NG NI HD NG

TALEN-4’-R：NN HD HD HD NI HD NI NI NG NN HD HD NI NN NG

TALEN-5 ' RVD sequences：

TALEN-5’-L：HD NG NI HD NG HD HD NI NG HD NI NG HD NG NG

TALEN-5’-R：NN NI HD HD NI NI NG HD HD NI NG NG NN HD HD HD

TALEN-6 ' RVD sequences：

TALEN-6’-L：NI NG HD HD NG NN NN NG HD NI NG NN NN NN NG NG

TALEN-6’-R：NI HD NG NG NN NG HD HD NN NG HD NI NG NN HD NG NG

Table 4 '：TALEN is knocked out after CXCR4, cloning and sequencing analysis.

NO.1TALEN-1 ' knocks out the sequencing result of CXCR4 target sequences 1

Wild type CXCR4 target sequences 1 (WT)：

TCTTCCTGCCCACCATCTACTCCATCATCTTCTTAACTGGCATTGTGGGCAATGGATTGGTCATCC

TGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTCAGTGG

CCGACC

NO1：TCTTCCTGCCCACCATCTACTCCATC::CTTCTTAACTGGCATTGTGGGCAATGGATTGGTC

ATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTCA

GTGGCCGACC

NO2：TCTTCCTGCCCACCATCTACTCCA:::::TTCTTAACTGGCATTGTGGGCAATGGATTGGTC

ATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTCA

GTGGCCGACC

NO3：TCTTCCTGCCCACCATCTACT:::::::CTTCTTAACTGGCATTGTGGGCAATGGATTGGTC

ATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTCA

GTGGCCGACC

NO4：TCTTCCTGCCCACCATCTACTCCATC:::::::::ACTGGCATTGTGGGCAATGGATTGGTC

ATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTCA

GTGGCCGACC

NO5：TCTTCCTGCCCACCATCTAC::::::::CTTCTTAACTGGCATTGTGGGCAATGGATTGGTC

ATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTCA

GTGGCCGACC

NO6：TCTTCCTGCCCACCATCTACTCCATCAT:::::::::TGGCATTGTGGGCAATGGATTGGTC

ATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTCA

GTGGCCGACC

NO7：TCTTCCTGCCCACCATCTACT::::::::::CTTAACTGGCATTGTGGGCAATGGATTGGTC

ATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTCA

GTGGCCGACC

NO8：TCTTCCTGCCCACCATCTACTC::::::::::TTAACTGGCATTGTGGGCAATGGATTGGTC

ATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTCA

GTGGCCGACC

NO9：TCTTCCTGCCCACCATCTACT::::::::::::TAACTGGCATTGTGGGCAATGGATTGGTC

ATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTCA

GTGGCCGACC

NO10：TCTTCCTGCCCAC:::::::::::::::::::::::::GGCATTGTGGGCAATGGATTGGT

CATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTC

AGTGGCCGACC

NO11：TCTTCCTGCC:::::::::::::::CATCTTCTTAACTGGCATTGTGGGCAATGGATTGGT

CATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTC

AGTGGCCGACC

NO12：TCT:::::::::::::::::::::::::::::::::::GGCATTGTGGGCAATGGATTGGT

CATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCTGTC

AGTGGCCGACC

NO13：TCTTCCTGCCCACCATCTAC:::::::::::::::::::::::::::::::::::::::::

::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

::::::::::C

NO14：TCTTCCTGCCCACCATCTACTCCATCATCTTCTTAACTGGCATTGTGGGCAATGGATT

GGTCATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCACCT

GTCAGTGGCCG

NO.2TALEN-2 ' knocks out the sequencing result of CXCR4 target sequences 2

Wild type CXCR4 target sequences 2 (WT)：

CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCC

ATGAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTAC

TCCATCA

NO1：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATG：

CTCCATGAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCAT

CTACTCCATCA

NO2：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGG::TATGA

CTCCATGAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCAT

CTACTCCATCA

NO3：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGA

CTCCATGAAGGAAC::::TTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCAT

CTACTCCATCA

NO4：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGG:::::ATGA

CTCCATGAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCAT

CTACTCCATCA

NO5：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGA

:::::::::GGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCAT

CTACTCCATCA

NO6：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTAT::

:::::::AAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCAT

CTACTCCATCA

NO7：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTA:::

:::::::AAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCAT

CTACTCCATCA

NO8：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAA::::::::::GGGACTATGA

CTCCATGAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCAT

CTACTCCATCA

NO9：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGG:::::::::

:::CATGAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCAT

CTACTCCATCA

NO10：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATG

ACTCCAT::::::::::::TTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCA

TCTACTCCATCA

NO11：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAA:::::::::::::::TATG

ACTCCATGAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCA

TCTACTCCATCA

NO12：CCTCTTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGC:::::::::::::

:::::::::AGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCA

TCTACTCCATCA

NO.3TALEN-3 ' knocks out the sequencing result of CXCR4 target sequences 3

Wild type CXCR4 target sequences 3 (WT)：

TTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCCATGAA

GGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCAT

CATC

NO1：TTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCC

ATGAAGGAACCCTGTTTC:TGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACT

CCATCATC

NO2：TTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCC

ATGAAGGAACC:GTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACT

CCATCATC

NO3：TTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCCA

TGAAGGAACCCTGT::CCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACT

CCATCATC

NO4：TTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCCA

TGAAGGAACCCTGTTT::GTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACT

CCATCATC

NO5：TTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCC

ATGAA::::::TGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACT

CCATCATC

NO6：TTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCCA

TGAAGGAACCCTGTTTCCG:::::::::TGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACT

CCATCATC

NO7：TTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTC:

::::::::::CTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACT

CCATCATC

NO8：TTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCC

ATGAAGGAACCCTGTTTCCGTGA:::::::::::::TCAATAAAATCTTCCTGCCCACCATCTACT

CCATCATC

NO9：TTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCCA

TG:::::::::::::TCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACT

CCATCATC

NO10：TTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTC

CATGAAGGAACCCTGTTTCCGTGAA::::::::::::::AATAAAATCTTCCTGCCCACCATCTAC

TCCATCATC

NO11：TTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTC

CATGAAGGAACCCTGTTT:::::::::::::::::TTTCAATAAAATCTTCCTGCCCACCATCTAC

TCCATCATC

NO12：TTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTC

CATGAAG::::::::::::::::::::::::::AATTTCAATAAAATCTTCCTGCCCACCATCTAC

TCCATCATC

NO13：TTTTGCAGATATACACTTCAGATAACTACACCGAGGAAATGGG::::::::::::::::::

:::::::::::::::::::::::::::::::::::::::::TAAAATCTTCCTGCCCACCATCTAC

TCCATCATC

NO.4TALEN-4 ' knocks out the sequencing result of CXCR4 target sequences 4

Wild type CXCR4 target sequences 4 (WT)：

GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTC

CATCATCTTCTTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAA

ACTGAGAAGC

NO1：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCT

ACTCCATCA:CTTCTTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGA

AGAAACTGAGAAGC

NO2：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCT

ACTCCATCATCT:CTTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGA

AGAAACTGAGAAGC

NO3：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCT

ACTCCATCATCTTCTTA:CTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGA

AGAAACTGAGAAGC

NO4：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCT

ACTCCATCATCTTCTTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGT:ACCAGA

AGAAACTGAGAAGC

NO5：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCT

ACTCCATCATCTTCT::ACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGA

AGAAACTGAGAAGC

NO6：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCT

ACTCCATCATCT:::TAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGA

AGAAACTGAGAAGC

NO7：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCT

ACTCCATCATCTTCTTAAC:::CATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGA

AGAAACTGAGAAGC

NO8：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCT

ACTCCATCATCTTCTT:::::GCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGA

AGAAACTGAGAAGC

NO9：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCT

ACTC::::::::TCTTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGA

AGAAACTGAGAAGC

NO10：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATC

TACTCCA::::::::TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAG

AAGAAACTGAGAAGC

NO11：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATC

TACTCCATCATCTT:::::::::CATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAG

AAGAAACTGAGAAGC

NO12：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATC

TACTCCATCATCTTCTTAAC::::::::::GGCAATGGATTGGTCATCCTGGTCATGGGTTACCAG

AAGAAACTGAGAAGC

NO13：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATC

TACTCCATCATC:::::::::::::TTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAG

AAGAAACTGAGAAGC

NO14：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATC

T:::::::::::::::TAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAG

AAGAAACTGAGAAGC

NO15：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATC

TACTCCAT::::::::::::::::::TGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAG

AAGAAACTGAGAAGC

NO16：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATC

TACTCCAT:::::::::::::::::::TGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAG

AAGAAACTGAGAAGC

NO17：GAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCA:::::::::::::::::::::::

::::::::::::::::::::::::::::::::::::TGGATTGGTCATCCTGGTCATGGGTTACCAG

AAGAAACTGAGAAGC

NO.5TALEN-5 ' knocks out the sequencing result of CXCR4 target sequences 5

Wild type CXCR4 target sequences 5 (WT)：

AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATCATCTTCTTAACTGGCAT

TGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTG

NO1：AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATCATCTTCTTAAC::

::::TGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTG

NO2：AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATCATCT::::::::G

GCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTG

NO3：AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATC:::::::::::TG

GCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTG

NO4：AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATCATCTT::::::::

:::::GTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTG

NO5：AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATCATCTTCTTAA:::

::::::::::::ATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTG

NO6：AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATCAT:::::::::::

::::TGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTG

NO7：AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCAT::::::::::::::

::::TGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTG

NO8：AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATCATCTTCTT:::::

:::::::::::::::GATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTG

NO9：AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATCAT:::::::::::

::::::::::::::::::TGGTCATCCTGGTCATGGGTTACCAGAAGAAACTG

NO10：AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATCATCTTCTTAACT

GGCATTGTGGGCAATG::::::::::::::::::::::::::::::::::ACTG

NO11：AGAAAATGCTAATTTCAATAAAATCTTCCTGCCCACCATCTACTCCATCATC:::::::::

:::::::::::::::::::::::::::CTGGTCATGGGTTACCAGAAGAAACTG

NO.6TALEN-6 ' knocks out the sequencing result of CXCR4 target sequences 6

Wild type CXCR4 target sequences 6 (WT)：

TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTGAGAAGC

ATGACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO1：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAG:AGAAACTGAG

AAGCATGACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO2：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAA::AACTGAG

AAGCATGACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO3：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGA:::AACTGAG

AAGCATGACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO4：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTAC::::AGAAACTGAG

AAGCATGACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO5：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAG:::::::TGAG

AAGCATGACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO6：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGA::::::G

AAGCATGACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO7：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGA:::::::::G

AAGCATGACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO8：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTAC::::::::::TGAG

AAGCATGACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO9：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTAC::::::::::TGAG

AAGCATGACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO10：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAACTG:

::::::::::GGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO11：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAA::::

:::::::GACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO12：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAA::::

:::::::::::GACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO13：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAAA::::

::::::::::::ACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO14：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCAGAAGAA:::::

:::::::::::GACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO15：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCATGGGTTACCA:::::::::::

::::::::ACGGACAAGTACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC

NO16：TTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGG::::::::::::::::::::::::

:::::::::::::::::TACAGGCTGCACCTGTCAGTGGCCGACCTCCTCTTTGTCATCAC。

Claims (23)

1. a species activating transcription factor (Transcription Activator Like Effectors, TALE), its feature Be the class activating transcription factor (TALE) can with mankind CCR5 gene activities region or CXCR4 gene activities region DNA fragmentation recognize and combine that the mankind CCR5 gene activities region one has 3, and its nucleotide sequence is respectively：Activity Region 1)：tgcacagggtggaacaagatggattatcaagtgtcaagtccaatctatgacatcaattattatacatcgg Agccctgccaaaaaatcaatgtgaagcaaatcgcag, active region 2)：caacatgctggtcatcctcatcctgataa Actgcaaaaggctgaagagcatgactgacatctacctg, active region 3)：tcttcattacacctgcagctctcattt tccatacagtcagtatcaattctggaagaatttccagacattaaagatag；The CXCR4 gene activities region has altogether There are 2, its nucleotide sequence is respectively：Active region 1 ')：TAACTACACCGAGGAAATGGGCTCAGGGGACTATGACTCC ATGAAGGAACCCTGTTTCCGTGAAGAAAATGCTAATTTCAATAAAA, active region 2 ')： TAAAATCTTCCTGCCCACCATCTACTCCATCATCTTCTTAACTGGCATTGTGGGCAATGGATTGGTCATCCTGGTCA TGGGTTACCAGAAGAAACTGAGAAGCATGACGGACAAGTACAGGCTGCA, if to the above-mentioned active regions of CCR5 or CXCR4 DNA fragmentation in domain is cut off, then is treated after gene repair, inhibition of HIV can not will be attacked after these gene repair again Cell；

The DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 1), its nucleotides sequence is classified as 5 '-TATGACATCAATTATTAT-3 ', the class activating transcription factor (TALE) and the single-stranded identification of above-mentioned nucleotide sequence are simultaneously tied Close, its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal-NI NG NN NI HD NI NG HD NI NI NG NG NI NG NG NI NG-C ends, above-mentioned class activating transcription factor (TALE) is designated as “TALE-1-L”；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 1) antisense strand, its Nucleotides sequence is classified as 5 '-TGCTTCACATTGATTTTTT-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotides sequence List chain recognizes and combined that its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is： N-terminal-NN HD NG NG HD NI HD NI NG NG NN NI NG NG NG NG NG NG-C ends, above-mentioned class transcriptional activation The factor (TALE) is designated as " TALE-1-R "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 1), its nucleotides sequence It is classified as 5 '-TGGAACAAGATGGATTAT-3 ', the class activating transcription factor (TALE) and the single-stranded identification of above-mentioned nucleotide sequence And combine, its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal-NN NN NI NI HD NI NI NN NI NG NN NN NI NG NG NI NG-C ends, above-mentioned class activating transcription factor (TALE) is designated as “TALE-2-L”；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 1) antisense strand, its Nucleotides sequence is classified as 5 '-TAATTGATGTCATAGAT-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotide sequence Single-stranded identification is simultaneously combined, and its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N End-NI NI NG NG NN NI NG NN NG HD NI NG NI NN NI NG-C ends, above-mentioned class activating transcription factor (TALE) it is designated as " TALE-2-R "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 1), its nucleotides sequence It is classified as 5 '-TATCAAGTGTCAAGTCCAAT-3 ', the class activating transcription factor (TALE) and the single-stranded knowledge of above-mentioned nucleotide sequence Not and combine, it repeats variable bis-amino acid residue (repeat variable diresidue, RVD) sequence and is：N-terminal-NI NG HD NI NI NN NG NN NG HD NI NI NN NG HD HD NI NI NG-C ends, above-mentioned class activating transcription factor (TALE) it is designated as " TALE-3-L "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 1) antisense strand, its Nucleotides sequence is classified as 5 '-TGGCAGGGCTCCGATGTATAAT-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotides Sequence ss recognizes and combined that it repeats variable bis-amino acid residue (repeat variable diresidue, RVD) sequence For：N-terminal-NN NN HD NI NN NNNN HD NG HD HD NN NI NG NN NG NI NG NI NI NG-C ends, it is above-mentioned Class activating transcription factor (TALE) is designated as " TALE-3-R "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2), its nucleotides sequence 5 '-TGCTGGTCATCCTCAT-3 ' are classified as, the class activating transcription factor (TALE) and the single-stranded identification of above-mentioned nucleotide sequence are simultaneously With reference to its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal-NN HD NG NN NN NG HD NI NG HD HD NG HD NI NG-C ends, above-mentioned class activating transcription factor (TALE) is designated as " TALE-4- L”；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2) antisense strand, its Nucleotides sequence is classified as 5 '-TCATGCTCTTCAGCCTT-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotide sequence Single-stranded identification is simultaneously combined, and its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N End-HD NI NG NN HD NG HD NG NG HD NI NN HD HD NG NG-C ends, above-mentioned class activating transcription factor (TALE) it is designated as " TALE-4-R "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2), its nucleotides sequence 5 '-TGGTCATCCTCATCCT-3 ' are classified as, the class activating transcription factor (TALE) and the single-stranded identification of above-mentioned nucleotide sequence are simultaneously With reference to its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal-NN NN NG HD NI NG HD HD NG HD NI NG HD HD NG-C ends, above-mentioned class activating transcription factor (TALE) is designated as " TALE-5- L”；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2) antisense strand, its Nucleotides sequence is classified as 5 '-TCAGTCATGCTCTTCAGC-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotide sequence Single-stranded identification is simultaneously combined, and its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N End-HD NI NN NG HD NI NG NN HD NG HD NG NG HD NI NN HD-C ends, above-mentioned class activating transcription factor (TALE) it is designated as " TALE-5-R "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2), its nucleotides sequence It is classified as 5 '-TGCTGGTCATCCTCATC-3 ', the class activating transcription factor (TALE) and the single-stranded identification of above-mentioned nucleotide sequence And combine, its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal-NN HD NG NN NN NG HD NI NG HD HD NG HD NI NG HD-C ends, above-mentioned class activating transcription factor (TALE) is designated as “TALE-6-L”；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 3), its nucleotides sequence It is classified as 5 '-TACACCTGCAGCTCTCAT-3 ', the class activating transcription factor (TALE) and the single-stranded identification of above-mentioned nucleotide sequence And combine, its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal-NI HD NI HD HD NG NN HD NI NN HD NG HD NG HD NI NG-C ends, above-mentioned class activating transcription factor (TALE) is designated as “TALE-7-L”；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 3) antisense strand, its Nucleotides sequence is classified as 5 '-TGGAAATTCTTCCAGAAT-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotide sequence Single-stranded identification is simultaneously combined, and its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N End-NN NN NI NINI NG NG HD NG NG HD HD NI NN NI NI NG-C ends, above-mentioned class activating transcription factor (TALE) it is designated as " TALE-7-R "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2 '), its nucleotides sequence It is classified as 5 '-CTTCCTGCCCACCATCT-3 ', the class activating transcription factor (TALE) and the single-stranded identification of above-mentioned nucleotide sequence And combine, its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal-HD NG NG HD HD NG NN HD HD HD NI HD HD NI NG HD NG-C ends, above-mentioned class activating transcription factor (TALE) is designated as “TALE-1’-L”；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2 ') antisense strand, its Nucleotides sequence is classified as 5 '-GCCCACAATGCCAGTT-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotides sequence list Chain recognizes and combined that its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal- NN HD HD HD NI HD NI NI NG NN HD HD NI NN NG NG-C ends, above-mentioned class activating transcription factor (TALE) It is designated as " TALE-1 '-R "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 1 '), its nucleotides sequence It is classified as 5 '-ACACCGAGGAAATGGGCTCA-3 ', the class activating transcription factor (TALE) and the single-stranded knowledge of above-mentioned nucleotide sequence Not and combine, it repeats variable bis-amino acid residue (repeat variable diresidue, RVD) sequence and is：N-terminal-NI HD NI HD HD NN NI NN NN NI NI NI NG NN NN NN HD NG HD NI-C ends, above-mentioned class transcriptional activation because Sub (TALE) is designated as " TALE-2 '-L "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 1 ') antisense strand, its Nucleotides sequence is classified as 5 '-CACGGAAACAGGGTTCCTT-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotides sequence List chain recognizes and combined that its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is： N-terminal-HD NI HD NN NN NI NI NI HD NI NN NN NN NG NG HD HD NG NG-C ends, above-mentioned class transcription swashs The factor (TALE) living is designated as " TALE-2 '-R "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2 '), its nucleotides sequence It is classified as 5 '-ATGACTCCATGAAGGAAC-3 ', the class activating transcription factor (TALE) and the single-stranded identification of above-mentioned nucleotide sequence And combine, its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal-NI NG NN NI HD NG HD HD NI NG NN NI NI NN NN NI NI HD-C ends, above-mentioned class activating transcription factor (TALE) It is designated as " TALE-3 '-L "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2 ') antisense strand, its Nucleotides sequence is classified as 5 '-ATTGAAATTAGCATTTT-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotide sequence Single-stranded identification is simultaneously combined, and its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N End-NI NG NG NN NI NI NI NG NG NI NN HD NI NG NG NG NG-C ends, above-mentioned class activating transcription factor (TALE) it is designated as " TALE-3 '-R "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2 '), its nucleotides sequence It is classified as 5 '-CCTGCCCACCATCTACT-3 ', the class activating transcription factor (TALE) and the single-stranded identification of above-mentioned nucleotide sequence And combine, its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal-HD HD NG NN HD HD HD NI HD HD NI NG HD NG NI HD NG-C ends, above-mentioned class activating transcription factor (TALE) is designated as “TALE-4’-L”；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2 ') antisense strand, its Nucleotides sequence is classified as 5 '-GCCCACAATGCCAGT-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotides sequence list Chain recognizes and combined that its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal- NN HD HD HD NI HD NI NI NG NN HD HD NI NN NG-C ends, above-mentioned class activating transcription factor (TALE) is designated as “TALE-4’-R”；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2 '), its nucleotides sequence 5 '-CTACTCCATCATCTT-3 ' are classified as, the class activating transcription factor (TALE) and the single-stranded identification of above-mentioned nucleotide sequence are simultaneously With reference to its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal-HD NG NI HD NG HD HD NI NG HD NI NG HD NG NG-C ends, above-mentioned class activating transcription factor (TALE) be designated as " TALE-5 '- L”；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2 ') antisense strand, its Nucleotides sequence is classified as 5 '-GACCAATCCATTGCCC-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotides sequence list Chain recognizes and combined that its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal- NN NI HD HD NI NI NG HD HD NI NG NG NN HD HD HD-C ends, above-mentioned class activating transcription factor (TALE) It is designated as " TALE-5 '-R "；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2 '), its nucleotides sequence 5 '-ATCCTGGTCATGGGTT-3 ' are classified as, the class activating transcription factor (TALE) and the single-stranded identification of above-mentioned nucleotide sequence are simultaneously With reference to its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N-terminal-NI NG HD HD NG NN NN NG HD NI NG NN NN NN NG NG-C ends, above-mentioned class activating transcription factor (TALE) is designated as " TALE- 6’-L”；

Or the DNA fragmentation that the class activating transcription factor (TALE) recognizes and combined comes from active region 2 ') antisense strand, its Nucleotides sequence is classified as 5 '-ACTTGTCCGTCATGCTT-3 ', the class activating transcription factor (TALE) and above-mentioned nucleotide sequence Single-stranded identification is simultaneously combined, and its variable bis-amino acid residue (repeat variable diresidue, RVD) sequence of repetition is：N End-NI HD NG NG NN NG HD HD NN NG HD NI NG NN HD NG NG-C ends, above-mentioned class activating transcription factor (TALE) it is designated as " TALE-6 '-R ".

2. a kind of fusion protein, it is characterised in that the fusion protein includes the class activating transcription factor described in claim 1 And at least one Complete Nucleotide enzyme shearing function domain (cleavage domain) or partial nuclease shearing function (TALE) Domain.

3. fusion protein according to claim 2, it is characterised in that the partial nuclease shearing function domain is wild type Restriction enzyme FokI has the half of shearing function domain.

4. fusion protein according to claim 2, it is characterised in that the partial nuclease shearing function domain is manually to change Moulding restriction enzyme FokI has the half of shearing function domain.

5. a kind of kit, it is characterised in that the kit includes the fusion protein 2 as described in claim 3 or 4, described After 2 fusion proteins are recognized and combined with Target Nucleotide Sequence respectively, the partial nuclease on 2 fusion proteins, which is in, to be connect Near position, forms an endonuclease with shearing DNA functions.

6. kit according to claim 5, it is characterised in that 2 fusion proteins be respectively " TALE-1-L "- FokI and " TALE-1-R "-FokI；Or be respectively " TALE-2-L "-FokI and " TALE-2-R "-FokI；Or be respectively " TALE-3-L "-FokI and " TALE-3-R "-FokI；Or be respectively " TALE-4-L "-FokI and " TALE-4-R "-FokI； Or be respectively " TALE-5-L "-FokI and " TALE-5-R "-FokI；Or be respectively " TALE-6-L "-FokI and " TALE- 4-R”-FokI；Or be respectively " TALE-7-L "-FokI and " TALE-7-R "-FokI；Or be respectively " TALE-1 '-L "- FokI and " TALE-1 '-R "-FokI；Or be respectively " TALE-2 '-L "-FokI and " TALE-2 '-R "-FokI；Or respectively For " TALE-3 '-L "-FokI and " TALE-3 '-R "-FokI；Or be respectively " TALE-4 '-L "-FokI and " TALE-4 '-R "- FokI；Or be respectively " TALE-5 '-L "-FokI and " TALE-5 '-R "-FokI；Or be respectively " TALE-6 '-L "-FokI " TALE-6 '-R "-FokI.

7. a kind of kit, it is characterised in that the kit includes 1 fusion protein as claimed in claim 2, described to melt After hop protein can be recognized and combined with Target Nucleotide Sequence, endonuclease can exercise DNA shearing functions in specific site.

8. kit according to claim 7, it is characterised in that the fusion protein is " TALE-1-L "-endonuclease Enzyme；Or be " TALE-1-R "-endonuclease；Or be " TALE-2-L "-endonuclease；Or for " TALE-2-R "- Endonuclease；Or be " TALE-3-L "-endonuclease；Or be " TALE-3-R "-endonuclease；Or be " TALE-4-L "-endonuclease；Or be " TALE-4-R "-endonuclease；Or be " TALE-5-L "-endonuclease Enzyme；Or be " TALE-5-R "-endonuclease；Or be " TALE-6-L "-endonuclease；Or for " TALE-7-L "- Endonuclease；Or be " TALE-7-R "-endonuclease；Or be " TALE-1 '-L "-endonuclease；Or be " TALE-1 '-R "-endonuclease；Or be " TALE-2 '-L "-endonuclease；Or in " TALE-2 '-R "-nucleic acid Enzyme cutting；Or be " TALE-3 '-L "-endonuclease；Or be " TALE-3 '-R "-endonuclease；Or be " TALE- 4 '-L "-endonuclease；Or be " TALE-4 '-R "-endonuclease；Or be " TALE-5 '-L "-endonuclease；Or Person is " TALE-5 '-R "-endonuclease；Or be " TALE-6 '-L "-endonuclease；Or be " TALE-6 '-R "-core Sour restriction endonuclease.

9. a kind of polynucleotide, it is characterised in that the TALE albumen or coding of the polymerized nucleoside acid encoding claim 1 Fusion protein included by the kit of one of one of claim 2-4 fusion protein or coding claim 5-8.

10. a kind of genophore, it is characterised in that the genophore includes the polynucleotide described in claim 9.

11. genophore according to claim 10, it is characterised in that the genophore is adenovirus vector.

12. a kind of free cell, it is characterised in that the TALE or right that the free cell was imported into claim 1 will Ask fusion protein included by one of fusion protein or claim 5-8 described in any one of the 2-4 kit or One of polynucleotide or claim the 10-11 genophore described in person's claim 9.

13. the method for CCR5 or CXCR4 genes in the regulating cell of non-diagnostic and therapeutic purposes, it is characterised in that the side Method includes：A) first paragraph nucleotide sequence, the first paragraph nucleic acid sequence encoding first paragraph polypeptide, described first are introduced in cell Section polypeptide includes the fusion protein described in any one of claim 2-4, referred to as the first fusion protein, so that when first paragraph nucleic acid Sequence is expressed as in cell after first paragraph polypeptide, and the class activating transcription factor (TALE) in first paragraph polypeptide is recognized and combined DNA fragmentation in mankind's CCR5 or CXCR4 gene activity region, referred to as the first DNA fragmentation, first paragraph polypeptide has shearing CCR5 Or the function of CXCR4 genes.

14. method according to claim 13, it is characterised in that further comprise：B) second segment nucleic acid is introduced in cell Sequence, the second segment nucleic acid sequence encoding second segment polypeptide, the second segment polypeptide includes claim 2-4 any one institute The fusion protein stated, referred to as the second fusion protein, above-mentioned second fusion protein are different from the first fusion protein, so as to work as second segment Nucleotide sequence is expressed as in cell after second segment polypeptide, and class activating transcription factor (TALE) identification in second segment polypeptide is simultaneously With reference to the DNA fragmentation in mankind's CCR5 or CXCR4 gene activity region, referred to as the second DNA fragmentation, above-mentioned second DNA fragmentation is not Be same as the first DNA fragmentation, second segment polypeptide and first paragraph polypeptide can together with shear CCR5 or CXCR4 genes.

15. method according to claim 13, wherein first polypeptide is " TALE-1-L "-endonuclease；Or For " TALE-1-R "-endonuclease；Or be " TALE-2-L "-endonuclease；Or be " TALE-2-R "-endonuclease Enzyme；Or be " TALE-3-L "-endonuclease；Or be " TALE-3-R "-endonuclease；Or for " TALE-4-L "- Endonuclease；Or be " TALE-4-R "-endonuclease；Or be " TALE-5-L "-endonuclease；Or be " TALE-5-R "-endonuclease；Or be " TALE-6-L "-endonuclease；Or be " TALE-7-L "-endonuclease Enzyme；Or be " TALE-7-R "-endonuclease；Or be " TALE-1 '-L "-endonuclease；Or for " TALE-1 '- R "-endonuclease；Or be " TALE-2 '-L "-endonuclease；Or be " TALE-2 '-R "-endonuclease；Or For " TALE-3 '-L "-endonuclease；Or be " TALE-3 '-R "-endonuclease；Or be " TALE-4 '-L "-nucleic acid Restriction endonuclease；Or be " TALE-4 '-R "-endonuclease；Or be " TALE-5 '-L "-endonuclease；Or be " TALE- 5 '-R "-endonuclease；Or be " TALE-6 '-L "-endonuclease；Or be " TALE-6 '-R "-endonuclease.

16. method according to claim 14, wherein the first polypeptide is " TALE-1-L "-FokI, the second polypeptide is “TALE-1-R”-FokI；Or first polypeptide be " TALE-2-L "-FokI, the second polypeptide be " TALE-2-R "-FokI I；Or First polypeptide is " TALE-3-L "-FokI, and the second polypeptide is " TALE-3-R "-FokI；Or first polypeptide be " TALE-4-L "- FokI, the second polypeptide is " TALE-4-R "-FokI；Or first polypeptide be " TALE-5-L "-FokI, the second polypeptide be " TALE- 5-R”-FokI；Or first polypeptide be " TALE-6-L "-FokI, the second polypeptide be " TALE-4-R "-FokI；Or more than first Peptide is " TALE-7-L "-FokI, and the second polypeptide is " TALE-7-R "-FokI；Or first polypeptide be " TALE-1 '-L "-FokI, Second polypeptide is " TALE-1 '-R "-FokI；Or first polypeptide be " TALE-2 '-L "-FokI, the second polypeptide for " TALE-2 '- R”-FokI；Or first polypeptide be " TALE-3 '-L "-FokI, the second polypeptide be " TALE-3 '-R "-FokI；Or more than first Peptide is " TALE-4 '-L "-FokI, and the second polypeptide is " TALE-4 '-R "-FokI；Or first polypeptide be " TALE-5 '-L "- FokI, the second polypeptide is " TALE-5 '-R "-FokI；Or first polypeptide be " TALE-6 '-L "-FokI, the second polypeptide is “TALE-6’-R”-FokI。

17. method according to claim 14, wherein first paragraph polypeptide and second segment polypeptide are encoded by same nucleic acid carrier Produce.

18. method according to claim 14, wherein first paragraph polypeptide are with second segment polypeptide by two different nucleic acid carriers It is separately encoded generation.

19. the method according to claim 13 or 14, further comprises c) introducing one section of polynucleotide, institute in cell Stating polynucleotide includes first area and second area, the first area and CCR5 the or CXCR4 gene double-strand regions of fracture One section of gene order of upstream is identical, the second area and one section of base in CCR5 or CXCR4 gene double-strand regions of fracture downstream Because sequence is identical.

20. method according to claim 14, wherein first paragraph nucleic acid, second segment nucleic acid and polynucleotide are supported on base Because on carrier.

21. method according to claim 20, wherein the genophore is adenovirus vector.

22. method according to claim 21, wherein the adenovirus vector is Ad5/F35 carriers.

23. the method according to claim 13 or 14, wherein the cell is thin selected from candidate stem cell, T cell, macrophage Born of the same parents, dendritic cells, antigen presenting cell, mescenchymal stem cell or ghost (3) X4/R5 cell lines.