WO2014169810A1

WO2014169810A1 - Isolated oligonucleotide and use thereof

Info

Publication number: WO2014169810A1
Application number: PCT/CN2014/075403
Authority: WO
Inventors: 吴璐; 许奇武; 王磊; 原辉
Original assignee: 深圳华大基因科技服务有限公司
Priority date: 2013-04-16
Filing date: 2014-04-15
Publication date: 2014-10-23
Also published as: CN105008536A; HK1214302A1

Abstract

Disclosed in the present invention are an isolated oligonucleotide and use thereof. The isolated oligonucleotide comprises: a first nucleic acid molecule, the first nucleic acid molecule encoding a double-base recognition module, wherein the double-base recognition module consists of a first single-base recognition module and a second single-base identification module in series, and the first single-base recognition module and the second recognition module both contain repeat variable di-residue amino acids; a first amplification sequence, wherein the first amplification sequence is located at the 5' side of the first nucleic acid molecule, and the first amplification sequence comprises a lis-type endoenzyme recognition sequence; and a second amplification sequence, wherein the second amplification sequence is located at the 3' side of the first nucleic acid molecule, and the second amplification sequence comprises a lis-type endoenzyme recognition sequence. The oligonucleotide can quickly obtain various combinations of RVD, thereby being capable of identifying any target nucleic acid sequence.

Description

Isolated oligonucleotides and uses thereof

Technical field

The invention relates to the field of biotechnology. In particular, the invention relates to isolated oligonucleotides and uses thereof. More specifically, the present invention relates to an isolated oligonucleotide, an oligonucleotide library, a method of constructing a nucleic acid expressing a TALE repeat, and a method of altering a cellular genome. Background technique

TALEN (Transcription activator-like effectors nucleases) The directed gene modification technology consists of two parts. One part of the TALEN directed cutting target sequence forms a DNA double-stranded incision (DSB), and the other part of the Doner vector provides the sequence to be edited, completing the genome-directed editing work. . TALEN is composed of more than 12 tandem protein modules and Fokl endonuclease. Each protein module contains 34 amino acids. The 12th and 13th amino acid residues are key sites for base recognition and are called double amino acid residues. RVD). The target gene sequence can be identified by changing the sequence of the RVD module. TALEN binds to the target sequence and cleaves the DNA double strand to form DSB. Under the mechanism of intracellular non-homologous end repair, a frameshift mutation occurs at the DBS to form a stop codon. Target gene knockout is accomplished due to early termination of target gene transcription. TALEN technology has the characteristics of high knockout efficiency and strong specificity. TALEN can select targets in any part of the genome, knock out genes, and change target gene sequences, so TALEN technology is the preferred method for studying gene function.

However, Talen vectors make it difficult to directly synthesize RVD module sets due to the sequence repeatability in their recognition sequences. Therefore, the current methods and means for constructing TALE repeat sequences still need to be improved. Summary of the invention

The present invention is directed to solving at least some of the above technical problems or at least providing a useful commercial option. To this end, it is an object of the present invention to provide an oligonucleotide which can be effectively used to construct a TALE repeat and its use.

In a first aspect of the invention, the invention proposes an isolated oligonucleotide. According to an embodiment of the invention, the isolated oligonucleotide comprises: a first nucleic acid molecule encoding a double base recognition module, the double base recognition module being identified by a first single base in tandem a module and a second single base recognition module, wherein the first single base recognition module and the second single base recognition module each comprise a repeating variable biamino acid residue; a first amplification sequence, the first amplification a sequence is located on the 5' side of the first nucleic acid molecule, and the first amplified sequence comprises a lis type endonuclease recognition sequence; and a second amplified sequence, the second amplified sequence is located in the first nucleic acid The 3' side of the molecule, and the second nucleic acid sequence comprises a lis type endonuclease recognition sequence. Since the oligonucleotide molecule comprises a nucleic acid sequence encoding two single base recognition modules, such oligonucleotide molecules encoding different RVDs can be used as starting materials by cutting with restriction enzymes. The cohesive ends are formed on both sides of the oligonucleotide molecule, and the ligation can be directly performed, whereby various different combinations of RVDs can be obtained quickly, so that any target nucleic acid sequence can be recognized. Can greatly reduce the enzyme digestion The number of times, and the inventors have surprisingly found that the mismatch rate that enables module unit connections is greatly reduced. Generally, for longer RVD combinations, the more the number of cleavage connections, the more difficult the correct connection.

According to an embodiment of the invention, the oligonucleotide may also have the following additional technical features:

In one embodiment of the invention, the first single base recognition module and the second single base recognition module each have the following amino acid sequence:

LTPDQVVAIAS*RVD*GGKQALETVQRLLPVLCQDHG, where *RVD* at positions 12 and 13 represent the RVD sequence, a repeating variable diamino acid, which determines the different recognition bases. Wherein, the RVD corresponding to base A is NI; the RVD corresponding to base C is HD; the RVD corresponding to base T is NG; and the RVD corresponding to base G is N. Thus, the first single base recognition module and the second single base recognition module each have an amino acid sequence selected from one of the following:

Identification base A: LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG;

Identification base C: LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG;

Identify base T: LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG and

Identify the base G: LTPDQWAIASNNGGKQALETVQRLLPVLCQDHG.

The inventors have surprisingly found that by using the single base recognition module of the single base recognition module described above, it is possible to effectively exert a function of specifically recognizing a base in a plurality of cells including animal cells and plant cells.

And the inventors have surprisingly found that the recognition efficiency of the obtained TALEN for the target nucleotide can be further improved by shortening the lengths of the C-terminus and the N-terminus of both sides of the RVD and optimizing the partial amino acids as compared with the wild-type TALEN.

In an embodiment of the present invention, the RVD in the first single base identification module and the second single base identification module meets one of the following conditions: the RVD of the first single base identification module is NI, The RVD of the second single base identification module is I; the RVD of the first single base identification module is NI, and the RVD of the second single base identification module is NG; the first single base identification module The RVD of the second single base identification module is HD; the RVD of the first single base identification module is NI, and the RVD of the second single base identification module is NN; The RVD of the single base identification module is NG, the RVD of the second single base identification module is NI; the RVD of the first single base identification module is NG, and the RVD of the second single base identification module Is the NG; the RVD of the first single base identification module is NG, the RVD of the second single base identification module is HD; the RVD of the first single base identification module is NG, the second single The RVD of the base identification module is N; the RVD of the first single base identification module is HD, and the RVD of the second single base identification module is NI; The RVD of the base identification module is HD, the RVD of the second single base identification module is NG; the RVD of the first single base identification module is HD, and the RVD of the second single base identification module is HD; The RVD of the first single base identification module is HD, the RVD of the second single base identification module is N; the RVD of the first single base identification module is NN, and the second single base identification The RVD of the module is NI; the RVD of the first single base identification module is NN, the RVD of the second single base identification module is NG; and the RVD of the first single base identification module is N, The RVD of the second single base identification module is HD; or the RVD of the first single base identification module is NN, and the RVD of the second single base identification module is NN. Thus, the oligonucleotide can encode one that recognizes AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CG, CC, GA, GT, GC, GG. Thus, by taking all of the above possible first bases The combination of the recognition module and the second base recognition module, respectively, after constructing the oligonucleotide molecule, can efficiently obtain a library of oligonucleotide molecules that can be used to construct any RVD combination, thereby requiring only the corresponding oligonucleoside The acid molecules are ligated to obtain the desired RVD combination that recognizes the predetermined target nucleotide sequence.

According to an embodiment of the present invention, the lis-type endonuclease is at least one selected from the group consisting of Bsal, Bbsl and BsmBI. Preferably, the lis-type endonuclease is Bsal, and the lis-type endonuclease recognition sequence is GGTCTCNNNN, wherein ^^ is , T, G or C. Thereby, the efficiency of cleavage by endonuclease and ligation of the cleavage products to obtain different RVD combinations can be further improved.

According to an embodiment of the present invention, the first amplification sequence further comprises an Xbal cleavage site, and the first amplification sequence may have the general formula (M) ₁₍₎ . ₂₍₎ TCTAGA(;M;) ₂ . _{_{_{8 GGTCTC (H;. 8 25}}} , wherein M is a, T, C or G;. H is a, T, C or G, and consistent with the coding sequence of the first single-base recognition module optionally, The second amplified sequence further comprises a Xhol cleavage site, and the second amplified sequence has the general formula (M') io-2oCTCGAG(M') 2-8GGTCTC(H')i _8- 25, wherein M' is A, T, C or G; 11' is A, T, C or G and matches the coding sequence of the second single base recognition module. The sequences of M and M' are variable, in the context of the present invention In one embodiment, the first nucleic acid molecule or the single base recognition module coding sequence is ligated to the vector as an amplification template, and the sequences of M and M' can be respectively set to coincide with the 5' end of the vector and with the vector 3 'End sequence matching. Thus, the corresponding oligonucleotide can be conveniently saved or amplified, thereby facilitating the efficiency of subsequent construction of talen. According to an embodiment of the invention, the vector or plasmid can be Containing the selection marker, such as a drug resistance gene, which can easily be amplified or screening vector or plasmid.

According to an embodiment of the present invention, the first amplified sequence and the second amplified sequence satisfy the following condition: the first amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 1, The second amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 10; the first amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 2, and the second amplified sequence has a nucleotide sequence of SEQ ID NO: 11; the first amplified sequence has a nucleotide sequence as set forth in SEQ ID NO: 3, and the second amplified sequence has SEQ ID NO: 12 a nucleotide sequence shown; the first amplification sequence has a nucleotide sequence as shown in SEQ ID NO: 4, and the second amplification sequence has a nucleotide sequence as shown in SEQ ID NO: The first amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 5, and the second amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 14; The amplified sequence has the nucleotide sequence set forth in SEQ ID NO: 6, and the second amplified sequence has the nucleotide sequence set forth in SEQ ID NO: 15. The first amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 7, and the second amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 16; The sequence has the nucleotide sequence set forth in SEQ ID NO: 8, the second amplified sequence has the nucleotide sequence set forth in SEQ ID NO: 17; or the first amplified sequence has the SEQ ID NO: The nucleotide sequence shown by 9, which has the nucleotide sequence shown as SEQ ID NO: 18.

The sequences for SEQ ID NOs: 1-18 are summarized in Table 1, below.

0Ϊ869Ϊ/ 0Ζ OAV The inventors have surprisingly found that by using the combination of the first amplification sequence and the second amplification sequence described above, the efficiency of amplification of the oligonucleotide molecule can be effectively improved, and the inventors have found that, based on the first amplification sequence described above, And the second amplification sequence, by using the above-mentioned first amplification sequence and the second amplification sequence for each of the two-base recognition modules, respectively, for each double-base recognition module, constructing for connection Starting material, so that when the larger RVD combination is connected, only the first amplification sequence and the second amplification sequence of each oligonucleotide molecule need to be selected, and the final RVD combination can be completed by one enzyme-cutting connection. The construction does not require the replacement and amplification of the intermediate vector, and only needs to be transformed once after transformation. In theory, it takes only 6 hours to obtain the correct plasmid, which can be downstream after one transformation and amplification. experiment of. The whole experiment process is very simple, and the experiment is really experimental, which makes the experiment controllable.

In a second aspect of the invention, the invention provides a library of oligonucleotides, comprising: a plurality of isolated oligonucleotides, wherein the isolated oligonucleotides are as described above Oligonucleotides. Thus, the oligonucleotide library can be effectively used to construct different RVD combinations. According to an embodiment of the invention, the different oligonucleotides are each arranged in a different container. Thereby, the construction of the RVD combination can be conveniently performed. In addition, according to an embodiment of the present invention, a large number of oligonucleotide types are preset in the oligonucleotide library. First, for each combination of the first base recognition module and the second base recognition module, respectively, respectively The oligonucleotide molecule, and then, for each of the two-base recognition modules, the respective first oligonucleotide sequence and the second amplification sequence are used to construct respective oligonucleotide molecules. Thus, by simply ligating the corresponding oligonucleotide molecules, it is possible to obtain a desired RVD combination that can recognize a predetermined target nucleotide sequence. Moreover, when the RVD combination is connected, only the first amplification sequence and the second amplification sequence of each oligonucleotide molecule need to be selected, and the construction of the final RVD combination can be completed by one enzyme-cutting connection. For the replacement and amplification of the intermediate vector, it is only necessary to carry out the transformation after one connection. In theory, it takes only 6 hours to obtain the correct plasmid, and then the downstream experiment can be carried out after one transformation and amplification identification. The whole experiment process is very simple, and the experiment is really experimental, which makes the experiment controllable. In addition, PCR amplification is not required in the experiment to obtain individual single-double unit modules, but each single-double unit module is directly constructed on the plasmid, and a large number of fragment libraries are obtained by amplification of the plasmid, thereby reducing The mutation introduced by PCR, and the experimental controllability is better. The final vector is obtained directly by restriction enzyme ligation of the plasmid and the plasmid, and different vectors can be used to screen for the correct vector.

According to a third aspect of the invention, the invention proposes a method of constructing a nucleic acid expressing a TALE repeat. The method comprises: providing a first oligonucleotide and a second oligonucleotide, the first oligonucleotide and the second oligonucleotide being the oligonucleotides described above; using a lis type inscribed The first oligonucleotide and the second oligonucleotide are cleaved by an enzyme to obtain a first oligonucleotide cleavage product and a second oligonucleotide cleavage product, wherein the first oligonucleotide The cleavage product forms a cohesive end at the first amplified sequence and the second amplified sequence, and the second oligonucleotide cleavage product forms a sticky end at the first amplified sequence and the second amplified sequence, and The sticky end formed by the second amplified sequence of the first oligonucleotide cleavage product matches the sticky end formed by the first amplified sequence of the second oligonucleotide cleavage product; and the first oligo A nucleotide cleavage product is ligated to the second oligonucleotide cleavage product to obtain an oligonucleotide that expresses a TALE repeat. With this method, different RVD combinations can be obtained efficiently by connecting different oligonucleotides. In the actual operation, only the first amplification sequence and the second amplification sequence on both sides of the oligonucleotide molecule need to be selected, and a large RVD combination can be obtained by one connection. It should be noted, The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining "first" and "second" may include one or more of the features, either explicitly or implicitly. In the description of the present invention, the meaning of "plurality" is two or more, unless specifically defined otherwise. Thus, according to an embodiment of the present invention, any number of RVD combinations can be accomplished by one enzymatic ligation reaction in one enzymatic ligation system at a time. It is not necessary to carry out the replacement and amplification of the intermediate vector, and only needs to perform the transformation after one connection. In theory, it takes only 6 hours to obtain the correct plasmid, and then the downstream experiment can be carried out after one transformation and amplification identification. The whole experiment process is very simple, and the experiment is really experimental, which makes the experiment controllable. According to the current conventional method, only the construction process takes 2 to 5 days. In addition, PCR amplification is not required in the experiment to obtain individual single-double unit modules, but each single-double unit module is directly constructed on the plasmid, and a large number of fragment libraries are obtained by amplification of the plasmid, thereby reducing The mutation introduced by PCR, and the experimental controllability is better. The final vector is obtained directly by restriction enzyme ligation of the plasmid and the plasmid, and different vectors can be used to screen for the correct vector.

According to an embodiment of the invention, the above method may further have the following additional technical features:

In one embodiment of the invention, the RVD sequence of the first oligonucleotide and the second oligonucleotide molecule is determined based on a predetermined target nucleic acid sequence. Preferably, the RVD sequence of the first oligonucleotide and the second oligonucleotide molecule is determined based on the following relationship:

The RVD corresponding to base A is I;

The RVD corresponding to base C is HD;

The RVD corresponding to base T is NG;

The RVD corresponding to base G is NN. Thereby, the efficiency of the designed RVD combination to recognize a predetermined target nucleotide sequence can be effectively improved.

Further, according to an embodiment of the present invention, at least one of the first oligonucleotide and the second oligonucleotide comprises a plurality of different oligonucleotides, wherein at least one sticky end of each oligonucleotide is One sticky end of the other oligonucleotide matches. For example, for multiple oligonucleotides, the forward primer cohesive terminus _{Fn of} each oligonucleotide is only linked to the reverse primer sticky end Rn _{+1 of the} other oligonucleotide, but not to other primers. The sticky ends are connected, whereby a plurality of oligonucleotides can be ligated at one time, thereby improving the construction efficiency.

In a fourth aspect of the invention, the invention provides a method for altering a cellular genome, comprising: determining an RVD sequence of a TALE for a predetermined target nucleic acid sequence in a genome of a cell; constructing according to the method described above An oligonucleotide expressing a TALE repeat sequence capable of specifically recognizing the target nucleic acid sequence; and introducing an oligonucleotide expressing a TALE repeat sequence and a nucleic acid encoding a TALE DNA modifying enzyme into the cell. Thus, according to an embodiment of the present invention, a specific RVD combination can be efficiently constructed for a predetermined target nucleic acid sequence, thereby effectively improving the efficiency of changing the genome of the cell. According to an embodiment of the invention, the target nucleic acid sequence has a length of 4 to 20 nt, preferably 12 to 20 nt. According to an embodiment of the invention, the TALE DNA modifying enzyme is Fok I. Preferably, the oligonucleotide expressing the TALE repeat sequence and the nucleic acid encoding the TALE DNA modifying enzyme are constructed on the same vector. Therefore, it is possible to effectively improve the efficiency of changing the genome of the cell.

In a fifth aspect of the invention, the invention provides an isolated polypeptide, characterized in that the polypeptide is The oligonucleotides described are encoded. Thus, the obtained polypeptide is capable of specifically recognizing the base sequence.

In a fifth aspect of the invention, the invention proposes a carrier. According to an embodiment of the invention, the vector comprises a nucleic acid molecule encoding the oligonucleotide described above. Among them, it is preferred that the vector is a plasmid. Thereby, the corresponding oligonucleotide can be conveniently stored or amplified, thereby facilitating the efficiency of subsequent construction of talen. According to an embodiment of the present invention, a vector or a plasmid may contain a selection marker, such as a drug resistance gene, so that amplification or screening of the vector or plasmid can be conveniently performed. In addition, according to an embodiment of the present invention, a screening marker of a vector containing an oligonucleotide, such as a drug resistance gene, may be different from a screening marker of a finally constructed expression vector, thereby facilitating screening by more convenient one-time ligation reaction. A combination of identification modules required.

In a sixth aspect of the invention, the invention proposes a plasmid library. According to an embodiment of the present invention, the plasmid library comprises: a plurality of plasmids, wherein the plasmid is the vector described above. Preferably, the different plasmids are each placed in separate containers.

In a seventh aspect of the invention, the invention proposes a cell. According to an embodiment of the present invention, the cells are obtained by altering the genome by the method of changing the genome as described above. Thus, the resulting cells carry mutations, such as silencing of gene expression, in the genome as compared to the original cells.

In an eighth aspect of the invention, the invention proposes a method of modifying a gene. According to an embodiment of the present invention, the method comprises: selecting at least two corresponding plasmids from the plasmid library described above based on the target sequence of the gene; and cutting the at least two plasmids with a lis endonuclease , in order to obtain a plurality of plasmid cleavage products, wherein the plasmid cleavage products are respectively formed with cohesive ends, and one cohesive end of each plasmid matches at most one cohesive end of the other plasmid; the plurality of plasmids are cleaved The product is ligated to obtain an oligonucleotide expressing a TALE repeat, the TALE repeat identifying a target sequence of the gene; identifying a target sequence of the gene based on the TALE repeat, by means of a TALE DNA modifying enzyme, The gene changes. Thereby, it is possible to efficiently modify a predetermined gene, such as gene silencing, to achieve the purpose of gene knockout. According to an embodiment of the invention, of the plurality of plasmids, one cohesive end of each plasmid matches at most one sticky end of the other plasmid. For example, for multiple plasmids, the forward primer sticky end F _{n of} each plasmid is only linked to the reverse primer sticky end R _{n+1 of the} other plasmid, and cannot be linked to the sticky end of the other primers, thereby Multiple plasmids are ligated at one time, thereby increasing construction efficiency.

According to an embodiment of the invention, the method may also have the following additional technical features:

In one embodiment of the invention, the corresponding plasmid is determined based on the following relationship:

The RVD corresponding to base A is I;

The RVD corresponding to base C is HD;

The RVD corresponding to base T is NG;

The RVD corresponding to base G is N.

In one embodiment of the invention, the gene is ΡΡΑΛγ, at least one of the target sequences TGACACAGAGATGCCATT and GAATCAGCTCTGTGG. The additional aspects and advantages of the invention will be set forth in part in the description which follows. detailed description

The invention will now be described by way of specific examples. It should be noted that the embodiments described below are merely exemplary and do not limit the scope of the invention, and the reagents used in the following examples are commercially available, and, if not, In particular, the methods of the steps not explicitly illustrated in the following examples are all carried out by conventional methods known to those skilled in the art. Example 1 Construction of an oligonucleotide library

1, determine the identification module

The inventor can choose AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC,

16 recognition modules for CG, GA, GT, GC, GG (16 double bases) NI-NI, NI-NG, NI-HD, NI-Shun, NG-NK NG-NG NG-HD, NG-Shun HD-NK HD-NG, HD-HD, HD-Li, cis-NI, NN-NG, NN-HD, NN-NN sequences are sequentially connected to the carrier p-FUS-B2, respectively, and identified as correct Increased template.

The base sequence of a single module is as follows:

NI(A)

CTGACCCCGG ACCAAGTGGT GGCTATCGCC AGCAACATTG GCGGCAAGCA AGCGCTCGAA HD(C)

CTGACTCCGG ACCAAGTGGT GGCTATCGCC AGCCACGATG GCGGCAAGCA AGCGCTCGAA

NG(T)

CTGACCCCGG ACCAAGTGGT GGCTATCGCC AGCAACGGTG GCGGCAAGCA

AGCGCTCGAA Li (G)

CTGACCCCGG ACCAAGTGGT GGCTATCGCC AGCAACAATG GCGGCAAGCA AGCGCTCGAA where p-FUS-B2 was purchased from addgene, using p-FUS-B2 as a carrier, and two single modules were joined together to form the basic amplification template used by the inventors. Sequencing is correct and ready for use.

2. Amplification recognition module

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5'-ctagacgtctcgatagcctcgagTGGTTggtctcTcAGTCCATGGTCCTGGCAC In the above 18 primers, both have the Bsal restriction recognition sequence GGTCTCN'NNNN. In this type lis enzymes, with recognition sequence a cleavage recognition sticky end may generate a plurality, theoretically cause An adhesive identification terminal ^44. Since the amino acids at the end and beginning of each module are Gly and Leu, 24 types of linkers can be generated using a type lis enzyme, taking into account the limitations of degenerate codons (4 Gly codons, 6 Leu codons). We selected 18 of them to design primers. Except for F 1 and R9, F _n can be attached to the sticky end of R _n+1 and not to the sticky end of other primers. It should be noted that in the above amplification primers, Xhol and Xbal I restriction sites were respectively introduced at both ends of the amplification product: C'TCGAG and T'CTAGA.

The procedure for PCR amplification is as follows:

The PCR reaction system includes:

Single and double template plasmid (50ng/ul) lul

Phusion High-Fidelity PCR Master Mix 25ul

Positive and negative primers (lOuM) 5ul

Double steamed water 19ul

Total volume 50ul

Reaction conditions:

35 cycles

After PCR, the PCR product was recovered and purified for the single-module product; the PCR product of the two-module was subjected to tapping recovery due to the interference of single module, and the product with a size of about 280 bp was selected for recovery and purification.

3. Construct a plasmid library of the recognition module

Due to the presence of the Xhol and Xbal cleavage sites on the PNN4-plasmid, the inventors selected the PNN4 plasmid resistant to tetracycline resistance as the background plasmid for the plasmid library and the single- and double-modulus linkage due to the resistance of PNN4 and the final vector. Kanamycin resistance is different, all can achieve a one-step connection more efficiently. PNN4 was purchased from Addgene.

The PNN-4 module vector plasmid and the previously obtained recognition module amplification product were digested with Xhol and Xbal, and the target fragment was recovered, and the digested products were ligated to obtain the plasmid library of each recognition module. 180 plasmids. According to the serial number of the primer, it is named aN (where a represents the primer code of 1-9, N represents the base recognized by the module, and can be single base A, T, C, G, or 16 double base AA , AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, GG) The conditions for the digestion reaction are as follows:

Enzyme digestion system:

Xbal, lO U /μΙ 0.75ul

Xhol lO U /μΙ 0.75ul

NEB buffer 4 5ul

PNN-4 or single and dual modules 500ng

Double distilled water to make up the volume to 50ul

Total volume 50ul

Reaction conditions: 37 ° C 4h

The reaction conditions are as follows:

Connection system:

T4 liagase 0.4ul

T4 buffer lul

PNN-4 20ng obtained after enzyme digestion

Single and double modules recovered after enzyme digestion 200ng

Double steamed water to make up the volume to lOul

Total volume lOul Reaction conditions: 16 ° C 2 h. After the ligation product was transformed into the competent bacteria TransTl, the plasmid was extracted, and the plasmid was subjected to restriction enzyme digestion (Xhol and Xbal) to obtain a plasmid of the size corresponding to the single- and double-module size, and Sanger was sequenced to confirm the sequence.

Finally, all plasmids connected to the single and double modules on PNN-4 were used.

4. Construction of a plasmid expressing a semi-repeat unit

In the Talen protein, the last half of the repeat unit module actually contains half of the RVD sequence and also the C-terminus of the partial talen protein. In this embodiment, the two parts are combined into one modular unit, simultaneously with other modules. The connection is made, which simplifies the connection process and simplifies the final vector into a final vector that does not need to select different corresponding identification ATCGs according to different end recognition sequences ATCG.

Since the last module is a half unit and contains a partial N-terminal sequence, the inventors used synthetic and PCR methods to obtain a corresponding sequence. The present invention uses a synthetic C terminal seq as a module, and uses a forward fragment primer and a half-R for PCR amplification.

The PCR amplification method is as follows:

PCR amplification system:

Synthetic C terminal seq 10ng

Phusion High-Fidelity PCR Master Mix 25ul

half-R ( lOuM) 2ul

Double steamed water to make up to 50ul Total volume 50ul

The reaction conditions are as follows:

98 °C 30s

98 °C 10s

60 °C 20s J 20 cycles

72 °C 20s

72 °C 5min

After the first step of PCR amplification, 10 ul was taken from the PCR system, lOul of the forward fragment primer was added, mixed and heated to 95 ° 5 min, and then left to cool at room temperature. After annealing, lul 1 Ox Klenow fragment Buffer and lul klenow fragment were added and treated at 37 ° C for 30 min.

3 ul of the product of the above procedure was taken out, and as a module, PCR reaction was carried out using the amplification primer F/R under the following conditions:

PCR amplification system:

Klenow enzyme treated product 3ul

Phusion High-Fidelity PCR Master Mix 25ul

Amplification primer F\R ( lOuM) 2ul

Double steamed water to make up to 50ul

Total volume 50ul

The reaction conditions are as follows:

98 °C 30s

98 °C 10s

60 °C 20s 35 cycles

72 °C 20s

72 °C 5min

The PCR product recovers a fragment of about 200 bp in length, and the gel is recovered and purified, and then ligated to the P-easy vector. The reaction conditions are as follows:

P-easy blunt vector lul

PCR product 05.ul-4ul (20ng)

After mixing for 15 min at room temperature, the competent cells were directly transformed, and then the monoclonal was picked for Manger using M13F for Sanger sequencing. Save the correct plasmid for use.

Using the amplification primer F/R for PCR amplification, a half module and a partial C-terminal module unit relative to the last bit of the recognition sequence can be obtained, and the module unit is connected to the PNN-4 like other single and dual modules. The method is the same as other single and double modules, and the correct sequencing plasmid is obtained. Since half of the modules are at the 10th position of the connection, the bases identified by them are named 10A, 10T, 10C, 10G, respectively.

Its full length sequence is as follows:

HD:

gcgcTCTAGACCTTAAACCGGCCAACATACCggtctcCactgaccCCGGACCAAGTGG

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The identification of the talen target site starts from 5' to 3' and the previous digit identified is T. You can use the online tool htt s: / bogl ab .pip.iastate. edii/node/add/taien3⁄4fi talen site selection. To construct a 20bp target, N = A, G, T or C), the middle 18bp is the TALEN carrier target sequence we want to construct as needed.

1, select the target sequence

In this example, the inventors selected the gene PPAR _Y 2 (NCBI request number ACCESSION: NG_011749 REGION: 5001..151507), and the target sequence of the present invention is located on exon 2 of the ΡΡΑΙγ2 gene, and the sequence is TGACACAGGAGTGCCATTctggcccaccaacttcgGAATCAGCTCTGTGGA . Thus, the left arm of the constructed Talen recognizes the 17 base sequence TGACACAGATGGCCCATT, and the right arm recognizes the 15 base sequence GAATCAGCTCTGTGG with an interval of 17 bases. In the present invention, it is suggested that all the selection intervals in the design include the enzyme digestion. The talen of the site is beneficial for subsequent target efficiency verification. The talen design is based on the fact that the 5' end of the gene sequence of the target site starts with T, ie the sequence starts with T and ends with A. This T is very important to improve the efficiency of the talen.

2. Determine the RVD module

Based on the target sequence determined above, the sequence of the RVD module is determined as follows:

Talen left arm RVD sequence:

Talen right arm RVD sequence:

HD HD NI HD NI to NI NH HD NG to NI NG NG HD

After determining the RVD recognized by the target Talen of ΡΡΑΙ2, we selected the plasmid from the plasmid library: 1TG, 2AC, 3AC, 4AG, 5AG, 6AT, 7GC, 8CA, 9T, 10T for a total of 10 plasmids and the final vector plasmid of the present invention. Enzyme-cleavage ligation, and finally the right-handed Talen left arm was obtained. The plasmids were selected: 1CC, 2AC, 3AG, 4AG, 5CT, 6G, 7A, 8T, 9T, 10C - a total of 10 plasmids and the final vector plasmid of the present invention were ligated and ligated to obtain the right Talen right arm.

3, module connection

The plasmid concentration of each plasmid library was uniformly diluted to 100 ng/ul. Since the restriction site on the vector was Esp3I, the final vector was first digested, and the vector with a fragment size of 4K was recovered by tapping:

The volume of the enzyme digestion component is as follows:

Esp3I (BsmBI), lO U /μΙ 0.75ul

T4 buffer lul

Double distilled water 14.85ul

Final carrier lmg Total volume 20ul

The reaction conditions were rrc 4h.

The reaction system for the final talen left and right arms is obtained by enzyme digestion as follows:

Component volume

Restriction recovery of purified vector (100 ι^μΐ- ¹ ) lul

10 module plasmids (100 ng μ plant ¹ ) 0.5ul each

Bsal 0.75ul

BSA(IOX) lul

ATP (lOmM) lul

T7 ligase 0.25ul

Double distilled water to make up the volume to 20ul total volume 20ul

Reaction conditions:

37 ° C 5 min

20 °C lOmin J 35 cycles

After the end of the reaction, the ligation product was transformed and the resistance was Ka. The clones were screened for Sanger and used immediately.

4, verification

After sequencing, the assembly and alignment of the above-mentioned lenγ2 Talen left arm RVD sequence: The RVD sequence of Talen's left arm is: The corresponding RVD amino acid sequence is:

ETVQRLLPVLCQDHG (SEQIDNO: 37)

TVQRLLPVLCQDHG (SEQIDNO: 38)

TVQRLLPVLCQDHG (SEQIDNO: 39) TVQRLLPVLCQDHG (SEQIDNO: 40)

TVQRLLPVLCQDHG (SEQIDNO: 41)

TVQRLLPVLCQDHG (SEQIDNO: 42)

ETVQRLLPVLCQDHG (SEQIDNO: 43) TVQRLLPVLCQDHG ( SEQ ID NO: 44)

LTPDQVVAIAS

E ( SEQ ID NO: 45 )

RVD consists of a total of 598 amino acids, including 34* 17+20=598 ^

The primer sequences for Sanger sequencing are:

TAL-F1 5'-ttggcgtcggcaaacagtgg ( SEQ ID NO: 35 )

TAL-R2 5'-ggcgacgaggtggtcgttgg (SEQ ID NO: 36)

Since the recognition length of the entire Talen RVDs is about 2k, in order to ensure the correctness of each RVD sequence, we sequence the entire sequence RVDs into three parts. The principle of sequence alignment is to ensure that the three sequences can overlap. Starting with the amino acid LTPE, the QAHG cutoff to the last half of the module ensures complete alignment of each amino acid.

The vector matched by the detection sequence can be used for the detection of downstream target efficiency.

Example 3 Gene knockdown The targeting vector constructed in the previous Example 2 was transfected into 293T cells:

Cell transfection

Adjust the cells to a good state before transfection (cell morphology is clear, full and translucent). On the day before the official transfection, the cells are passaged into a 6-well plate, so that the density of the cells at the time of transfection is 80-90%. In addition to the experimental group, one well was reserved as a negative control and one well was a positive control.

In this experiment, X-tremeGENE HP DNA Transfection Reagent was used for cell transfection (taking 6孑L plate as an example). In short, the steps are as follows:

Prepare the transfection complex: a) 200 μΐ serum-free medium or Opti-MEM I medium. b) Add 2 μ _§ DNA (experimental group: Talen plasmid 1 μ _{§ ;} positive control group: EGFP-N1 plasmid 2 μ _§ ; negative control group: none). c) A brief gentle vortex (no more than 10s). d) Add 6μ1 X-tremeGENE HP DNA DNA Transfection Reagent. e) A brief gentle whirlpool. f) Incubate for 15-30 minutes (15 to 25 ° C) at room temperature.

The transfection complex was evenly added dropwise to the cells prepared in advance, and the dish was gently shaken by the cross method. The cells were cultured at 37 ° C in a 5% CO ₂ incubator.

At 24 h after transfection, the cell transfection efficiency was estimated by observing the green fluorescence ratio of the positive control group (CHO cells were normally over 80%). Discard the medium and replace with new complete medium.

After transfection for 24h, the medium can be changed to a selection medium containing puromycin. After about 7 days of culture, the medium is replaced with normal complete medium. At this time, the negative control cells are dead, and the experimental group still has viable cells (antibiotic concentration and The screening time should be pre-tested in advance)

During the sieving period, a large amount of cell death occurs after suspension in the culture medium, and the liquid can be changed every 1-2 days, and the concentration of puromycin is always maintained.

After changing the normal complete medium, the cells in the experimental group grow to more than 80%, that is, the genomic DNA can be extracted. Subsequent identification discarded the medium, and each well was washed once with PBS, and then digested with 0.25% trypsin. After the cells were digested into a round shape, the whole medium was added to terminate the digestion, and the cell suspension was collected, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.

Cellular genomic DNA was extracted using the Blood Tissue Cell Genome Extraction Kit (TIANGEN).

Using genomic DNA as a template, PCR was performed using primers designed according to the target site in advance, and the size of the target fragment was determined by agarose gel electrophoresis, and the PCR product was sent for sequencing.

If there is a set of peaks at the target site in the sequencing result, it indicates that the target site has a knockout, and the Talen effect is effective. The sequenced correct PCR product was subjected to a large number of enzyme digestion to remove the non-knockout gene, and the restriction enzyme product was recovered and subjected to TA cloning. After colony PCR, the enzyme was digested, and the correct clone was sampled and sequenced. The sequencing result verified the correct experiment. Sex.

Preparation of monoclonal cells

Put 0.1% gelatin on a 10cm culture dish in advance and place it in a 37°C incubator. The cells that were initially identified to be knocked out were trypsinized into cell suspensions and counted by cell counting plates to 2000 per dish. The cells were added to the culture dish, and cultured at 37 ° C in a 5% CO 2 incubator for one week to form a cell population with a clear island edge, which is a cell monoclonal.

Detection of monoclonal cells

The single-cell clones that have been extended after the dilution are transferred to a 96-well plate by a pipette and transferred to a 96-well plate. After the cell density reaches 90%, they are passaged into a 48-well plate, and a small amount of cells are isolated. Using fl40 phire animal The tissue direct per kit was subjected to PCR amplification, and agarose gel electrophoresis showed that 93 of the 96 samples were banded at 530 bp. The PCR product was digested with a Phol restriction enzyme at 75 °C. The electrophoresis revealed that the original 530 bp band was not cut into positive clones, and the PCR results were sent for sequencing. Subsequently, the sequencing results were analyzed, and the corresponding clonal cells with knockout at the Phol site (GGCC) were single cell lines after TALEN knockout. The preliminary sequencing results showed that there were 23 sequencing samples, of which there were 6 non-knockout cells, 2 knockouts but 2 single cells, 9 knockouts (9 sequencing results are not obvious), and 2 knockouts (27). , 64).

Further, PCR products of No.27 and No.64 were ligated to PMD18-T for T-cloning sequencing. The analysis results are as follows:

Thus, it was verified that a plurality of pure and ppair2 knockout cell clones were successfully obtained by the above experiments, and two strains were pure and double knockout.

In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Moreover, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any embodiment or example. Although the embodiments of the present invention have been shown and described, it is understood that the foregoing embodiments are illustrative and not restrictive Variations, modifications, alterations and variations of the above-described embodiments are possible within the scope of the invention.

Claims

Claim

An isolated oligonucleotide, comprising:

a first nucleic acid molecule, the first nucleic acid molecule encoding a double base recognition module, wherein the double base identification module is composed of a first single base identification module and a second single base identification module connected in series, the first single The base recognition module and the second single base recognition module each comprise a repeating variable diamino acid residue;

a first amplification sequence, the first amplification sequence is located on the 5' side of the first nucleic acid molecule, and the first amplification sequence comprises a lis endonuclease recognition sequence;

A second amplification sequence, the second amplification sequence is located on the 3' side of the first nucleic acid molecule, and the second nucleic acid sequence comprises a lis type endonuclease recognition sequence.

The oligonucleotide according to claim 1, wherein the first single base recognition module and the second single base recognition module each have an amino acid sequence:

LTPDQVVAIAS*RVD*GGKQALETVQRLLPVLCQDHG,

Wherein *RVD* indicates repeated variable diamino acid residues.

The oligonucleotide according to claim 2, wherein the RVD in the first single base identification module and the second single base identification module satisfy one of the following conditions:

The RVD of the first single base identification module is L; the RVD of the second "single base identification module is NI; and the RVD of the first - single base identification module is L the second "single base" The RVD of the recognition module is NG, and the RVD of the first-single base identification module is L. The RVD of the second "single base identification module is HD. The RVD of the first-single base identification module is L. The RVD of the second "single base identification module" is NN, and the RVD of the first-single base identification module is NG, and the RVD of the second base identification module is the first-single base identification of the NI The RVD of the module is NG, the RVD of the second single base identification module is NG, the RVD of the first-base identification module is NG, and the RVD of the second single base identification module is HD. - the RVD of the single base identification module is NG, the RVD of the second single base identification module is NN, the RVD of the first-single base identification module is HD, and the second single base identification module The RVD is NI; the RVD of the first-single base recognition module is HD, and the RVD of the second single base identification module is NG, the first-single base identification The RVD of the block is HD, the RVD of the second single base identification module is HD, the RVD of the first-base identification module is HD, and the RVD of the second single base identification module is NN. - the RVD of the single base identification module is N, the RVD of the second single base identification module is NI; the RVD of the first - single base identification module is N, and the second single base identification module The RVD of the first-base identification module is N, the RVD of the second single-base identification module is HD, or the RVD of the first single-base identification module is N, The RVD of the second single base identification module is NN.

The oligonucleotide according to claim 1, wherein the lis-type endonuclease is at least one selected from the group consisting of Bsal, Bbsl, and BsmBI.

The oligonucleotide according to claim 1, wherein the lis endonuclease recognition sequence is GGTCTCNNNN, where N is A, T, G or C.

The oligonucleotide according to claim 1, wherein the first amplification sequence further comprises an Xbal cleavage site, and the first amplification sequence has the general formula CM; > _1Q . _2Q TCTAGA (M _{_{;.) 2 8 GGTCTC (;}} H;. 8 25, wherein M is a, T, C or G; !! is, T, C or G, and consistent with the first single-base recognition sequence coding module .

The oligonucleotide according to claim 1, wherein the second amplified sequence further comprises a Xhol cleavage site, and the second amplified sequence has the general formula (M>. _2Q CTCGAG (M'_{_{;) 2 8 GGTCTC (;.}} H ';. 8 25, wherein M' is a, T, C or G; 11 is, T, C or G, and the second single-base recognition sequence coding module match.

The oligonucleotide according to claim 1, wherein the first amplified sequence and the second amplified sequence satisfy the following conditions:

The first amplification sequence has a nucleotide sequence as shown in SEQ ID NO: 1, and the second amplification sequence has a nucleotide sequence as shown in SEQ ID NO: 10;

The first amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 2, and the second amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 11;

The first amplification sequence has a nucleotide sequence as shown in SEQ ID NO: 3, and the second amplification sequence has a nucleotide sequence as shown in SEQ ID NO: 12;

The first amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 4, and the second amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 13;

The first amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 5, and the second amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 14;

The first amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 6, and the second amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 15;

The first amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 7, and the second amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 16;

The first amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 8, and the second amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 17;

The first amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 9, and the second amplified sequence has a nucleotide sequence as shown in SEQ ID NO: 18.

9. An oligonucleotide library, comprising:

A plurality of isolated oligonucleotides, wherein the isolated oligonucleotide is the oligonucleotide according to any one of claims 1 to 8.

10. The oligonucleotide library according to claim 9, wherein the different oligonucleotides are respectively disposed in different containers.

11. A method of constructing a nucleic acid expressing a TALE repeat, characterized in that it comprises:

Providing a first oligonucleotide and a second oligonucleotide, the first oligonucleotide and the second oligonucleotide are the oligonucleotides according to any one of claims 1 to 8;

The first oligonucleotide and the second oligonucleotide are cleaved using a lis-type endonuclease to obtain a first oligonucleoside An acid cleavage product and a second oligonucleotide cleavage product, wherein the first oligonucleotide cleavage product forms a cohesive end at the first amplification sequence and the second amplification sequence, and the second oligonucleoside The acid cleavage product forms a cohesive end at the first amplified sequence and the second amplified sequence, and the sticky end formed by the second amplified sequence of the first oligonucleotide cleavage product and the second oligonucleoside The sticky end of the first amplified sequence of the acid cleavage product is matched;

The first oligonucleotide cleavage product is ligated to the second oligonucleotide cleavage product to obtain an oligonucleotide that expresses a TALE repeat.

12. The method according to claim 11, wherein the RVD sequence of the first oligonucleotide and the second oligonucleotide molecule is determined based on the following relationship:

The RVD corresponding to base A is I;

The RVD corresponding to base C is HD;

The RVD corresponding to base T is NG;

The RVD corresponding to base G is N.

13. The method according to claim 11, wherein at least one of the first oligonucleotide and the second oligonucleotide comprises a plurality of different oligonucleotides, wherein each oligonucleotide One sticky end matches at most one sticky end of the other oligonucleotide.

14. A method of altering a cellular genome, the method comprising:

Determining the RVD sequence of TALE for a predetermined target nucleic acid sequence in the genome of the cell;

The method according to any one of claims 11 to 13, constructing an oligonucleotide expressing a TALE repeat, the TALE repeat capable of specifically recognizing the target nucleic acid sequence;

An oligonucleotide expressing a TALE repeat and a nucleic acid encoding a TALE DNA modifying enzyme are introduced into the cell.

The method according to claim 14, wherein the target nucleic acid sequence has a length of 4 to 20 nt, preferably 12 to 20 nt.

16. The method according to claim 14, wherein the TALE DNA modifying enzyme is Fok I.

17. The method according to claim 14, wherein the oligonucleotide expressing the TALE repeat sequence and the nucleic acid encoding the TALE DNA modifying enzyme are constructed on the same vector.

18. An isolated polypeptide, characterized in that the polypeptide is encoded by the oligonucleotide according to any one of claims 1-8.

A vector comprising a nucleic acid molecule encoding the oligonucleotide of any one of claims 1 to 8.

20. The vector according to claim 19, wherein the vector is a plasmid.

21. A plasmid library, comprising:

A plurality of plasmids, wherein the plasmid is the vector of claim 19 or 20.

22. The plasmid library according to claim 21, wherein the different plasmids are respectively disposed in different containers.

A cell, which is obtained by modifying a genome by the method according to any one of claims 14 to 17.

24. A method of modifying a gene, comprising:

Selecting at least two corresponding plasmids from the plasmid library of claim 21 or 22 based on the target sequence of the gene;

The at least two plasmids are cleaved using a lis-type endonuclease to obtain a plurality of plasmid cleavage products, wherein the plasmid cleavage products are respectively formed with cohesive ends, and one viscous end of each plasmid is at most Matching one sticky end of the other plasmid;

Ligating the plurality of plasmid cleavage products to obtain an oligonucleotide expressing a TALE repeat sequence, the TALE repeat sequence identifying a target sequence of the gene;

The target sequence of the gene is identified based on the TALE repeat, and the gene is altered by means of TALE DNA modifying enzyme.

The method according to claim 24, wherein the corresponding plasmid is determined based on the following relationship: the RVD corresponding to the base A is I;

The RVD corresponding to base C is HD;

The RVD corresponding to base T is NG;

The RVD corresponding to base G is N.

The method according to claim 24, wherein the gene is ΡΡΑΙγ, at least one of the target sequences TGACACAGAGATGCCATT and GAATCAGCTCTGTGG.