CN104844696A - Design, synthesis and application of transcription activator like effector function protein - Google Patents

Abstract

A "transcription activator like effector (TALE)" functional protein, the repeat variable double amino acid residue RVD (repeat variable di-residues) of the "transcription activator like effector" functional protein Arginine R and valine V respectively, or valine V and isoleucine I respectively, or glycine G and aspartic acid D respectively, or arginine R and tryptophan W respectively , or respectively threonine T and glycine G, or respectively cysteine C and glycine G, or respectively glutamic acid E and cysteine C.

Description

Design, synthesis and application of a transcriptional activator-like effector functional protein

technical field

The present invention relates to the design and synthesis of a "transcription activator like effector (TALE)" functional protein and the application of the above functional protein, in particular to the above functional protein. the

Background technique

In the post-genome era, we urgently need new bioengineering technologies such as efficient gene manipulation and synthesis. For example, we often need to suppress or silence the expression of a gene. Traditional gene knockout (Gene Knockout) technology relies on homologous recombination naturally occurring in cells, and its efficiency is very low, usually at the level of 10 ^-6 ; RNAi technology is simple and easy to implement, but it is difficult to obtain 100% inhibition effect. In addition to suppressing or silencing specific genes, we often need to modify a few bases or even a certain sequence of specific genes. Also only relying on homologous recombination technology, it is difficult to obtain the desired effect.

TALE (Transcription Activator Like Effectors) was first discovered on the plant pathogen Xanthomonas, which specifically binds to DNA and regulates plant genes during the pathogen infection process. TALE proteins can pass through the nuclear membrane into the nucleus and bind to specific DNA binding domains to regulate the expression of genes related to disease and resistance in the plant genome. Simply put, TALE is composed of 4 or more tandem "protein modules" that specifically recognize DNA and the N-terminal and C-terminal sequences on both sides, and each "protein module" contains 34 amino acids, of which the 12th and The 13th amino acid is the key site for target recognition, which is called repeat variable diresidue (or RVDs). The mechanism of TALE recognition of DNA is that a nucleotide on the DNA target is recognized by an RVD on a repeat sequence. the

Theoretically, for any of the bases A, T, G, and C, RVDs that specifically bind to it can be found. Therefore, for any DNA sequence, we can easily design and synthesize TALEs composed of corresponding protein modules. The problems that need to be pointed out are: 1) Although the corresponding RVD can be designed for A, T, G, and C, the corresponding relationship between them needs to be further optimized; 2) Can the designed and synthesized TALE effectively target The expected location on the genome is also determined by many other factors; 3) The composition of the protein module also has room for further optimization. the

Although TALE still has many problems to be studied and solved, this does not hinder its application. One of the biggest application prospects is TALEN. TALEN is a fusion protein, which is fused with a DNA sequence that specifically recognizes TALE and an endonuclease (Nuclease) that can generate a double strand break (DSB) on the DNA sequence. TALEN is a heterodimeric molecule (that is, two units of TALE-Nuclease act together), which can cut DNA between two specific recognition sequences that are relatively close together. the

The DSB produced by TALEN can be repaired in the following two ways: 1) Non Homologous End Joining (NHEJ): NHEJ is a natural repair mechanism that can be used to introduce nucleotide deletions for inactivation or knockout A specific target gene; 2) Homologous recombination (Homologous Recombination, HR): DSB promotes homologous recombination, in the presence of a DNA template, can produce specific DNA sequence changes, and can also integrate transgenes into the DNA sequence. The NHEJ pathway can be used for gene silencing, while HR can be used for gene editing (Gene Editing), or gene knock-in (Gene Knock-in). Regardless of the approach, compared with purely relying on homologous recombination, the repair produced by TALEN can greatly improve the efficiency of gene recombination, which provides a convenient technical means for us to engage in genome customization and develop more Facile novel genome-targeted modification technology offers new hope. the

Another big application prospect of TALE is TALEA (transcription activator-like (TAL) effector activator). TALEA is a fusion protein that fuses TALE that recognizes a specific DNA sequence with the transcription factor activation domain VP64 (VP64 Activation Domain) to construct a transcriptional activator TALEA that recognizes a specific DNA sequence on the promoter. The fusion protein will bind to the specific DNA sequence near the gene promoter, and bind to Polymerase II through the VP64 activation region, thereby activating the transcription of the gene and increasing the expression of the endogenous target gene. In practice, it is necessary to select a target sequence (generally 12-18 bases) upstream of the promoter of the target gene to construct a TALE recognition module. the

TALE technology has begun to emerge in the field of life sciences. In 2011, two groups from France and the United States cooperated to use TALEN technology to knock out and inactivate IgM function in rats, with an efficiency as high as 60%. In 2011, several groups, including China, used TALEN technology to knock out and inactivate genes such as hey2 in zebrafish, and the efficiency reached more than 30%. the

TALEs have special structural features, including a nitrogen-terminal (N-terminal) secretion signal, a central DNA-binding domain, a nuclear localization signal (NLS) and a carbon-terminal (C-terminal) activation domain. In the DNA binding domain, almost all TALE proteins that have been discovered are composed of highly conserved repeating units with different numbers (12-30). Among these repeating units (generally containing 33-35 amino acids), except for the 12th and 13th Amino acids are not the same, other components are very conservative. The variable amino acids at the 12th and 13th positions are called repeat variable di-residues RVD (repeat variable di-residues). TALE mainly recognizes DNA sequences through the RVD in the repeat unit. A total of 14 types of RVD have been reported so far. NI (asparagine and isoleucine) specifically recognizes A, HD (histidine and aspartic acid) specifically recognizes C, NN (asparagine and asparagine) can recognize G and A, NK ( Asparagine and lysine) specifically recognize G, NS (asparagine and serine) can recognize G, A, C and T, NG (asparagine and glycine) specifically recognize T, NH (asparagine and Histidine) specifically recognizes G, N* (asparagine, 13th vacancy) can recognize T, C, G and A, NP (asparagine and proline) can recognize T, A and C, HN (histidine and asparagine) can recognize G and A, NT (asparagine and threonine) can recognize G and A, SN (serine and asparagine) can specifically recognize G, SH (serine and histamine) Acid) specifically recognizes G. the

Problems existing in the prior art: some TALEs are not specific enough, and some TALEs are not efficient enough in recognition. Therefore, the development of new high-efficiency TALE sequences (ie, new RVDs) has become a technical problem that needs to be solved urgently in this field. the

Contents of the invention

The purpose of the present invention is to solve the above-mentioned technical problems. It is hoped that some improvements can be made to the TALE protein by design to realize the optimization of the RVD sequence of the TALE protein, so as to achieve a target for any base of A, T, G, and C. Specific binding RVD sequence. the

RV specifically recognizes A bases; VI specifically recognizes C bases; GD specifically recognizes C bases; RW specifically recognizes C bases; TG recognizes T bases, and can also weakly recognize C and G bases; CG It can recognize G bases, and can also weakly recognize T and C bases; EC can strongly recognize A, G, and C bases, and can also weakly recognize T bases. the

The technical scheme adopted in the present invention is as follows. the

In the present invention, TALEN is designed and synthesized for the HHB gene sequence, and the experimental method adopted is the luciferase single-strand annealing recombination assay (luciferase single-strand annealing recombination assay, SSA), which is commonly used in the world, and is used to detect endogenous gene Homologous recombination detection efficiency method - SURVEYOR experiment. The experimental principle is shown in Figures 1 and 2. the

Although there is a general demand for new TALEs with better recognition specificity and higher recognition efficiency, there are also methods for testing the recognition efficiency of TALEs in the prior art (SSA, SURVEYOR, see below for details), but since TALEs were discovered, new Structural TALE is rarely reported. Among many reasons, the screening workload is too large to be tolerated, which is one of the most important reasons. the

As we all know, since there are 20 kinds of basic amino acids, there are 20 ² or 400 permutations and combinations of RVDs containing two amino acids, and the amount of screening experiments is quite large. In the present invention, the property of plasmid incompatibility in Escherichia coli is used to screen useful RVDs from a large number of permutations and combinations in Escherichia coli. The specific steps are shown in Figure 1. In this method, a screening system is first established, and then a TALEN library is established according to 400 situations in which 20 basic amino acids are arranged in pairs. The TALEN in the TALEN library and the reporter plasmid are co-transformed into E. coli, and positive clones are selected and amplified. Co-transform again with the reporter plasmid for verification. The above-mentioned new method we designed greatly reduces the amount of screening experiments, making it feasible to select effective new TALEs from a large number of possible combinations.

Using the above method, we selected effective TALEs from hundreds of amino acid combinations, which are as follows: The repeat variable diamino acid residue RVD (repeat variable di- residues) are arginine R and valine V respectively, or valine V and isoleucine I respectively, or glycine G and aspartic acid D respectively, or arginine R and tryptophan respectively Acid W, or threonine T and glycine G, respectively, or cysteine C and glycine G, respectively, or glutamic acid E and cysteine C, respectively. Moreover, we have also confirmed the bases specifically recognized by the above-mentioned new TALEs, and these new TALEs can be applied to the specific recognition of these bases, respectively as follows:

The repeat variable di-amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively arginine R and valine V, which are used for the specificity of adenine (A). sex recognition; the repeat variable di-amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively valine V and isoleucine I, which are used for cytosine ( C) specific recognition; the repeated variable di-amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are glycine G and aspartic acid D, respectively, for the The specific recognition of pyrimidine (C); the repeat variable di-amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively arginine R and tryptophan W, with For the specific recognition of cytosine (C); the repeat variable di-amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively threonine T and glycine G, It is used for strong recognition of thymine (T), and weak recognition of cytosine (C) and guanine (G) bases; the repeat of the "transcription activator-like effector" functional protein is variable The double amino acid residues RVD (repeat variable di-residues) are cysteine C and glycine G, which are used for strong recognition of guanine (G), and can also recognize thymine (T) and cytosine (C ) for weak recognition; the repeat variable di-amino acid residue RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein is glutamic acid E and cysteine C, respectively, for the Stronger recognition of adenine (A), guanine (G) and cytosine (C) and weaker recognition of thymine (T). the

Description of drawings

Figure 1 Schematic diagram of the screening experiment process

Figure 2 Schematic diagram of luciferase single-strand annealing recombination assay (SSA): first, the luciferase gene was modified. A stop codon and target gene sequence were added in the middle of the luciferase gene; then another piece of luciferase gene sequence was followed by molecular biological means, and the sequence in the light gray area (about 800bp) on the way was the same. When TALEN is effective, TALEN will cut the double-stranded DNA at the target sequence position to form a double-stranded DNA break, due to the two light gray region sequences (that is, a gene on the left side of "stop codon" in Figure 1 and " The gene on the right side of "target sequence" is the same, homologous recombination will occur at this time, and an active luciferase reporter gene will be formed. In the experiment, the expression of luciferase can be measured to detect the strength of the effect of TALEN. the

Figure 3 SURVEYOR principle diagram: Firstly, PCR method is used to amplify the target gene fragment from the genome. These include deletions and sequences that do not produce changes. Then, the amplified fragments are re-annealed in vitro, and at this time, the missing fragments and the unchanged sequences will anneal to form a partially unpaired double-stranded structure. Next, use SURVEYOR enzyme to carry out enzyme digestion experiments, this enzyme can recognize unpaired regions for digestion. We can detect the efficiency of the deletion by the content of a, b, and c in the gel detection graph, so as to obtain the effect of TALEN. the

Figure 4 SSA experimental results of RVD-RV

Figure 5 SSA experimental results of RVD-VI

Figure 6 SSA experimental results of RVD-GD

Figure 7 SSA experimental results of RVD-RW

Figure 8 SSA experimental results of RVD-TG

Figure 9 SSA experimental results of RVD-CG

Figure 10 SSA experimental results of RVD-EC

Figure 11 SURVEYOR experimental results

Detailed ways

Example 1

The repeat variable di-residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively arginine R and valine V, referred to as RVD-RV. The corresponding SSA experimental results are shown in Figure 4, where the positive control is a TALE designed and synthesized according to existing rules in the field: NI (asparagine and isoleucine) recognizes A, HD (histidine and aspartic acid) C is recognized by NN (asparagine and asparagine), G is recognized by NG (asparagine and glycine), and T is recognized by NG (asparagine and glycine). Compared with the positive control, the recognition rate of RVD-RV for adenine (A) is higher, and compared with the positive control, the relative efficiency reaches 150%. Therefore, this TALE can be used for the specific recognition of adenine (A). the

Example 2

The repeat variable di-amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively valine V and isoleucine I, referred to as RVD-VI. The corresponding SSA experimental results are shown in Figure 5. Compared with the positive control, RVD-VI has a high recognition rate for cytosine (C), and compared with the positive control, the relative efficiency reaches 198%. Therefore, this TALE can be used for the specific recognition of cytosine (C). the

Example 3

The repeat variable di-residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively glycine G and aspartic acid D, referred to as RVD-GD. The corresponding SSA experimental results are shown in Figure 6. Compared with the positive control, RVD-GD has a high recognition rate for cytosine (C), and compared with the positive control, the relative efficiency reaches 103%. Therefore, this TALE can be used for the specific recognition of cytosine (C). the

Example 4

The repeat variable di-residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively arginine R and tryptophan W, referred to as RVD-RW. The corresponding SSA experimental results are shown in Figure 7. Compared with the positive control, the recognition rate of cytosine (C) by RVD-RW is higher, and compared with the positive control, the relative efficiency reaches 68.5%. Therefore, this TALE can be used for the specific recognition of cytosine (C). the

Example 5

The repeat variable di-residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively threonine T and glycine G, referred to as RVD-TG. The corresponding SSA experimental results are shown in Figure 8. Compared with the positive control, RVD-TG has a higher recognition rate for thymine (T), and compared with the positive control, the relative efficiency has reached 132%; for cytosine (C) and guanine (G) bases Weaker recognition, with relative efficiencies of 61% and 36%, respectively, compared to the positive control. Therefore, the TALE can be used for stronger recognition of thymine (T), and weaker recognition of cytosine (C) and guanine (G) bases. the

Example 6

The repeat variable di-amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively cysteine C and glycine G, referred to as RVD-CG. The corresponding SSA experimental results are shown in Figure 9. Compared with the positive control, RVD-CG has a higher recognition rate for guanine (G), and the relative efficiency reaches 88.8% compared with the positive control; it has a weaker recognition for thymine (T) and cytosine (C) , compared with the positive control, the relative efficiencies were 1% and 34%, respectively. Therefore, the TALE can be used for stronger recognition of guanine (G), and weaker recognition of thymine (T) and cytosine (C). the

Example 7

The repeat variable di-amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are glutamic acid E and cysteine C, respectively, referred to as RVD-EC. The corresponding SSA experimental results are shown in Figure 10. Compared with the positive control, RVD-EC has a higher recognition rate for adenine (A), guanine (G) and cytosine (C), and the relative efficiencies are 211%, 421 and 200% respectively compared with the positive control %; Thymine (T) was weakly recognized, compared with the positive control, the relative efficiency was 13%. Therefore, the TALE can be used for strong recognition of adenine (A), guanine (G) and cytosine (C), and weak recognition of thymine (T). the

Appendix:

(1)

In Example 1, the RVD-RV amino acid sequence:

MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEKI

KPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVG

KQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNL

TPEQVVAIASRVGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLP

VLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASRVGGKQAL

ETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASH

DGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPD

QVVAIASRVGGKQALETVQRLLPVLCQAHGLTPDQVVAIASRVGGKQALETVQRLLPVLC

QAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIAASNGGGKQALETV

QRLLPVLCQAHGLTPAQVVAIASRVGGKQALETVQRLLPVLCQAHGLTPDQVVAIASRVGG

GKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQV

VAIASRVGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQA

HGLTPAQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGL

PHAPALIKRTNRRIPERTSHRVA

(2)

In embodiment 2, RVD-VI amino acid sequence:

MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEKI

KPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVG

KQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNL

TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPV

LCQAHGLTPAQVVAIAASVIGGKQALETVQRLLPVLCQAHGLTPAQVVAIAASNIGGKQALETV

QRLLPVLCQAHGLTPDQVVAIAASVIGGKQALETVQRLLPVLCQAHGLTPAQVVAIAASVIGGK

QALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAI

ASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIAASNIGGKQALETVQRLLPVLCQAHGLT

PDQVVAIAASVIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVL

CQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIAASVIGGKQALETV

QRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGG

KQALETVQRLLPVLCQAHGLTPDQVVAIAASVIGGKQALETVQRLLPVLCQAHGLTPAQVVA

IASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRT

NRRIPERTS HRVA

(3)

In embodiment 3, RVD-GD amino acid sequence:

MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEK

IKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGV

GKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPL

NLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRL

LPVLCQAHGLTPDQVVAIASGDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGK

QALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVA

IASGDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAH

GLTPAQVVAIASGDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASGDGGKQALETVQRL

LPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASGDGGK

QALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVA

IASGDGGKQALETVQRLLPVLCQAHGLTPAQVVAIAASNIGGKQALETVQRLLPVLCQAHG

LTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGRPALESIVAQLS

RPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVA 

(4)

In embodiment 4, RVD-RW amino acid sequence:

MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEK

IKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGV

GKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPL

NLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASRWGGKQALETVQRL

LPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASRWGGK

QALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVA

IASRWGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAH

GLTPAQVVAIASRWGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRL

LPVLCQAHGLTPDQVVAIASRWGGKQALETVQRLLPVLCQAHGLTPAQVVAIAASNIGGKQ

ALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAI

ASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHG

LTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIAASNIGGKQALETVQRLLP

VLCQAHGLTPDQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAL

DAVKKGLPHAPALIKRTNRRIPERTSHRVA 

(5)

In Example 5, the RVD-TG amino acid sequence:

MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEKI

KPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVG

KQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNL

TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASTGGGKQALETVQRLLPV

LCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALET

VQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHD

GGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQ

VVAIAASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIAASNIGGKQALETVQRLLPVLCQA

HGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASTGGGKQALETVQR

LLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQ

ALETVQRLLPVLCQAHGLTPAQVVAIASTGGGKQALETVQRLLPVLCQAHGLTPAQVVAIA

SNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLT

PAQVVAIASTGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAP

ALIKRTNRRIPERTSHRVA

(6)

In Example 6, the RVD-CG amino acid sequence:

MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEK

IKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGV

GKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPL

NLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLL

PVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIAASHDGGKQ

ALETVQRLLPVLCQAHGLTPAQVVAIASCGGGKQALETVQRLLPVLCQAHGLTPDQVVAI

ASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHG

LTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLP

VLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQAL

ETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIAS

NGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLT

PAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIAASNIGGKQALETVQRLLPVL

CQAHGLTPDQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDA

VKKGLPHAPALIKRTNRRIPERTSHRVA 

(7)

In Example 7, the amino acid sequence of RVD-EC:

MAPKKKRKVYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHGTVDLRTLGYSQQQQEK

IKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGV

GKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPL

NLTPEQVVAIASECGGKQALETVQRLLPVLCQAHGLTPDQVVAIASECGGKQALETVQRL

LPVLCQAHGLTPDQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGK

QALETVQRLLPVLCQAHGLTPAQVVAIASECGGKQALETVQRLLPVLCQAHGLTPDQVVA

IASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHG

LTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLL

PVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIAASHDGGKQ

ALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAI

ASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIAASNIGGKQALETVQRLLPVLCQAHGL

TPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGRPALESIVAQLSR

PDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVA 

Claims (8)

1. A "transcription activator like effector (Transcription Activator Like Effectors, TALE)" functional protein, characterized in that the repeated variable double amino acid residue RVD (repeat) of the "transcription activator like effector" functional protein variable di-residues) are respectively arginine R and valine V, or are respectively valine V and isoleucine I, or are respectively glycine G and aspartic acid D, or are respectively arginine R and tryptophan W, or threonine T and glycine G, respectively, or cysteine C and glycine G, respectively, or glutamic acid E and cysteine C, respectively. the 2. The application of the functional protein according to claim 1, characterized in that the repeated variable double amino acid residue RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein is arginine respectively Acid R and valine V are used for the specific recognition of adenine (A) in DNA. the 3. The application of the functional protein according to claim 1, characterized in that the repeated variable double amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively valamine Acid V and Isoleucine I are used for the specific recognition of cytosine (C) in DNA. the 4. The application of the functional protein according to claim 1, characterized in that the repeated variable double amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively glycine G and aspartic acid D for the specific recognition of cytosine (C) in DNA. the 5. The application of the functional protein according to claim 1, characterized in that the repeated variable double amino acid residue RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein is respectively arginine Acid R and tryptophan W for the specific recognition of cytosine (C) in DNA. the 6. The application of the functional protein according to claim 1, characterized in that the repeated variable double amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively threonine Acid T and glycine G are used for strong recognition of thymine (T) in DNA, and weak recognition of cytosine (C) and guanine (G) bases in DNA. the 7. The application of the functional protein according to claim 1, characterized in that the repeated variable double amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively cysteine Amino acid C and glycine G are used for strong recognition of guanine (G) in DNA, and weak recognition of thymine (T) and cytosine (C) in DNA. the 8. The application of the functional protein according to claim 1, characterized in that the repeated variable double amino acid residues RVD (repeat variable di-residues) of the "transcription activator-like effector" functional protein are respectively glutamine Acid E and cysteine C, for strong recognition of adenine (A), guanine (G) and cytosine (C) in DNA, and weak recognition of thymine (T) in DNA identify. the