WO2015165274A1 - Taler protein having a transcription inhibiting effect by means of steric hindrance, and application thereof - Google Patents

Abstract

Provided are a TALER protein having a transcription inhibiting effect by means of steric hindrance, and application thereof, comprising the use of a TALER protein in the inactivation of a target promoter; the upstream and downstream of the promoter of interest have at least one TALER protein target, or the upstream of the promoter of interest does not have a TALER protein target but its downstream has at least one TALER protein target. Also provided is the use of a TALER protein in the inactivation of a protein of interest-encoding gene; the protein of interest-encoding gene is expressed by the promoter; the upstream and downstream of the promoter of interest have at least one TALER protein target, or the upstream of the promoter of interest does not have a TALER protein target but its downstream has at least one TALER protein target.

Description

[Name of invention established by ISA according to Rule 37.2] TALER protein which exerts transcriptional inhibition by steric hindrance and its application Technical field

The present invention relates to the TASTAT protein exerting transcriptional inhibition through steric hindrance and its use.

Background technique

The synthetic gene lines have been carefully designed to assemble gene regulatory devices according to their functions, to sense, integrate, and process molecular information in cells and to perform certain functions. Various synthetic gene lines have been developed to enable customizable, programmable functions within cells, including dynamic behavior, switching and memory, intercellular communication, adaptability, cellular polarization, digital and analog computation, and complex biosynthetic pathways. Most of these genetic lines are constructed using limited genetic elements and expensive, inefficient "try-and-error" methods. Therefore, in order to simplify the design and optimize the complex operation of living cells, it is necessary to develop a large-scale, well-defined synthetic gene component library and corresponding computational models and simulation methods.

In the field of synthetic biology research for mammals, engineered synthetic transcriptional activators and repressors are an important goal in supporting scalable gene line design. Currently, a common strategy for constructing mammalian/eukaryotic transcriptional repressors is to fuse a transcriptional repression domain and an engineered DNA binding protein domain, such as zinc finger proteins, transcriptional activator-like factor (TALE) proteins, and inactivation. The Cas9 (dCas9) nuclease is in an RNA-directed CRISPR (cluster-like periodic interval short palindromic repeat) system. However, transcriptional repressor domains, such as the Krüppel-associated cassette (KRAB) transcriptional repressor domain and the mSin interacting domain (SID4), typically result in epigenetic modifications near the target promoter and thus have a very slow response time. Therefore, this transcriptional inhibition is not suitable for constructing a gene line that responds quickly and reversibly.

Another mode of transcriptional repression that is ubiquitous in prokaryotes is through steric hindrance of non-functional domains, which are not common in eukaryotes, such as the Lac inhibitor (LacI) and the tetracycline repressor (TetR). The DNA is formed into a loop by oligomerization to bind to a specific DNA sequence near the promoter, thereby preventing binding of the transcription initiation core element in the promoter region). Studies have shown that in the context of mammalian gene regulation, placement of the LacI binding site downstream of the cytomegalovirus promoter (CMV) or CAG promoter in synthetic gene lines can inhibit gene expression, although inhibition efficiency is in lactation. The expression system is lower than the prokaryotic expression system. Similarly, the dCas9 protein does not fuse into any transcriptional repressor domain in mammalian systems and also exhibits a weak transcriptional repressor function.

The TALER protein consists of several tandem "protein modules" that specifically recognize DNA and the N-terminal and C-terminal sequences flanking the sides. Each "protein module" contains 33-35 amino acid residues, and the 12th and 13th amino acid residues are key sites for targeted recognition and are referred to as repetitive variable double-linked amino acid residue (RVDs) sites. Each RVD on the TALER protein recognizes only one base. TALEN (Transcription Activator-Like Effector Nuclease) is an artificially modified restriction endonuclease that uses the TALER protein (as a DNA binding domain) and the restriction endonuclease FokI (as a DNA cleavage domain, also known as a suppression domain). The TALEN fusion protein obtained by fusion. TALEN binds to the target site of the genome in the cell, forming a dimer that exerts endonuclease activity, resulting in double-stranded DNA breaks (DSB, Double-Strand Breaks) in the spacer region of the left and right TALEN, thereby inducing a DNA damage repair mechanism. Cells can pass DNA was repaired by non-homologous end joining (NHEJ). The NHEJ repair mechanism is not accurate and is prone to errors (missing/insertion), resulting in a frameshift mutation, so that gene knockout can be achieved.

Invention disclosure

It is an object of the present invention to provide a TAREL protein which exerts transcriptional repression by steric hindrance and its use.

The invention protects the use of a TALER protein for inactivating a promoter of interest; each of the upstream and downstream of the promoter of interest has at least one target of the TALER protein, or the upstream of the promoter of interest does not have the The target of the TALER protein but downstream of it has at least one target for the TALER protein.

Specifically, the upstream of the promoter of interest has one target of the TALER protein and has 1-3 targets of the TALER protein downstream thereof.

More specifically, the distance between the target of the TALER protein upstream of the promoter of interest and the target of the nearest TALER protein downstream of the promoter of interest is 72-100 bp.

The promoter of interest may specifically be a CMVmini promoter.

The invention also protects the use of a TALER protein for inactivating a gene encoding a protein of interest; the gene encoding the protein of interest is initiated by a promoter of interest; each of the upstream and downstream of the promoter of interest has at least one of the TALER proteins The target, or the target upstream of the promoter of interest, does not have a target for the TALER protein but has at least one target for the TALER protein downstream thereof.

Specifically, the upstream of the promoter of interest has one target of the TALER protein and has 1-3 targets of the TALER protein downstream thereof.

More specifically, the distance between the target of the TALER protein upstream of the promoter of interest and the target of the nearest TALER protein downstream of the promoter of interest is 72-100 bp.

The promoter of interest may specifically be a CMVmini promoter.

The invention also protects the use of a TALER protein for inactivating a functional fragment of interest; the functional fragment of interest is a DNA fragment; each of the upstream and downstream of the functional fragment of interest has at least one target of the TALER protein, or The upstream of the functional fragment of interest does not have a target for the TALER protein but has at least one target for the TALER protein downstream thereof.

Specifically, the target functional fragment has a target of one of the TALER proteins upstream and has 1-3 targets of the TALER protein downstream thereof.

More specifically, the distance between the target of the TALER protein upstream of the functional fragment of interest and the target of the TAREL protein nearest the downstream of the functional fragment of interest is 72-100 bp.

The invention also protects a DNA molecule combination comprising a DNA molecule A, a DNA molecule B and a DNA molecule C; the DNA molecule A comprises a promoter and a Gal4/vp16 encoding gene from upstream to downstream; the DNA molecule The upstream to downstream sequence includes the following elements: a 5×UAS sequence, a target of a TALER protein, a CMVmini promoter, 1-3 targets of the TALER protein, and a gene encoding a protein of interest; the DNA molecule C is from upstream to downstream. The promoter and the gene encoding the TALER protein are in turn included.

The promoter in the DNA molecule A can specifically be the pEF1a promoter. The DNA molecule can also have a gene encoding a fluorescent protein (specifically, a gene encoding TagBFP), and the fluorescent protein The coding gene is located upstream or downstream of the gene encoding the Gal4/vp16, and both are linked by the coding gene of the 2A-ligation peptide.

The protein of interest in the DNA molecule B may specifically be a fluorescent protein such as mKate2.

In the DNA molecule C, the promoter may specifically be a CMV promoter. The DNA molecule may further have a gene encoding a fluorescent protein (specifically, a gene encoding EYFP), and the gene encoding the fluorescent protein is located upstream or downstream of the gene encoding the TALER protein, and the two are connected by 2A. The gene encoding the peptide is linked.

The invention also protects a plasmid combination comprising a plasmid A, a plasmid B and a plasmid C; the plasmid A having the DNA molecule A according to any one of the above; the plasmid B having the DNA molecule B according to any of the above; The plasmid C has the DNA molecule C of any of the above.

The present invention also protects a method for regulating expression of a protein of interest, comprising the steps of: expressing said protein of interest by transfecting said DNA molecule A and said DNA molecule B into a host cell; The DNA molecule M and the DNA molecule B are transfected into the DNA molecule C to inhibit expression of the protein of interest.

The present invention also protects a method for regulating expression of a protein of interest, comprising the steps of: expressing said protein of interest by transfecting said plasmid A and said plasmid B into a host cell; by transfecting said plasmid The plasmid C is transfected into cells of A and the plasmid B to inhibit expression of the protein of interest.

The CMV promoter can be the CMV promoter in the plasmid of any of Examples 1-2.

The coding gene for the EYFP may be the coding gene for EYFP in the plasmid of any of Examples 1-2.

The gene encoding the 2A linker peptide may be the gene encoding the 2A linker peptide in the plasmid of any of Examples 1-2.

The gene encoding the TALER protein may be a gene encoding a TALER protein in a plasmid according to any one of Examples 1-2 (e.g., a gene encoding TALER1, a gene encoding TALER2, a gene encoding TALER4, and the like).

The 5 x UAS sequence can be the 5 x UAS sequence in the plasmid of any of Examples 1-2.

The target of the TALER protein may be a target sequence of a TALER protein (such as a T1 sequence, a T2 sequence, a T4 sequence, etc.) in the plasmid of any of Examples 1-2.

The CMVmini promoter can be the CMVmini promoter in the plasmid of any of Examples 1-2.

The coding gene of mKate2 may be the coding gene of mKate2 in the plasmid of any of Examples 1-2.

The pEF1a promoter may be the pEF1a promoter in the plasmid of any of embodiments 1-2.

The gene encoding the TagBFP may be the gene encoding TagBFP in the plasmid of any of Examples 1-2.

The gene encoding Gal4/vp16 may be the gene encoding Gal4/vp16 in the plasmid of any of Examples 1-2.

The plasmid A may specifically be a pEF1a-TagBFP-2A plasmid.

The plasmid B may specifically be a plasmid carrying a target of the TALER protein in any of the examples (e.g., pT1+T1+ plasmid, pT2+T2+ plasmid, pT4+T4+ plasmid, etc.). The plasmid B may specifically be pCMV-TALER1 plasmid, pCMV-TALER9 plasmid, pCMV-TALER10 plasmid, pCMV-TALER12 plasmid, pCMV-TALER13 plasmid, pCMV-TALER14 plasmid, pCMV-TALER16 plasmid, pCMV-TALER21 plasmid, pCMV-TALER29 Plasmid or pCMV-TALER35 plasmid.

The plasmid C may specifically be a plasmid carrying the gene encoding the TALER protein in any of the examples (e.g., pCMV-TALER1 plasmid, pCMV-TALER2 plasmid, pCMV-TALER4 plasmid, etc.). The plasmid C can specifically be pT1+T1+ plasmid, pT9+T9+ plasmid, pT10+T10+ plasmid, pT12+T12+ plasmid, pT13+T13+ plasmid, pT14+T14+ plasmid, pT16+T16+ plasmid, pT21+T21+ plasmid, pT29+T29+ Plasmid or pT35+T35+ plasmid.

DRAWINGS

Figure 1 is a schematic diagram showing the mechanism of action of the TALER protein.

Figure 2 is a schematic representation of the mechanism of action of the pCMV-TALERx plasmid, pTx+Tx+ plasmid and pEF1a-TagBFP-2A plasmid.

3 is a result of the first step of the first embodiment.

4 is a result of the second step of the second embodiment.

Figure 5 is the result of step one of the second embodiment.

Figure 6 is the result of step two of the second embodiment.

Figure 7 is the result of step three of the second embodiment.

Figure 8 is the result of step four of the second embodiment.

The best way to implement the invention

The following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. In the quantitative tests in the following examples, three repetitions were set, and the results were averaged. HEK293 cells: Invitrogen. A schematic diagram of the mechanism of action of TALER protein is shown in Figure 1.

In the examples, the cells were transfected with plasmids: 24 well plates were used, and 0.5 mL of HEK293 cell suspension (containing 6×10 ⁴ HEK293 cells) was inoculated into each well. After 24 hours of culture, the new DMEM medium was replaced, and then proceeded. Plasmid transfection.

Example 1. Functional verification and specificity analysis of TALER proteins

A schematic diagram of the mechanism of action of pCMV-TALERx plasmid, pTx+Tx+ plasmid and pEF1a-TagBFP-2A plasmid is shown in Fig. 2. Under the action of the pEF1a promoter, TagBFP and Gal4/vp16 are expressed (the 2A-linked peptide between TagBFP and Gal4/vp16 is a self-splicing peptide, so TagBFP can represent a table of Gal4/vp16) Amount). Gal4/vp16 activates the 5xUAS sequence, thereby activating the transcriptional initiation of the CMVmini promoter, and mKate2 is expressed. Under the action of the CMV promoter, EYFP and TALER1 proteins are expressed (the 2A linker peptide between EYFP and TALER1 proteins is a self-splicing peptide, so EYFP can represent the expression level of TALER1 protein). The TALER1 protein binds to the T1 sequence and exerts transcriptional repression through steric hindrance. The CMVmini promoter between the two T1 sequences is inactivated, and mKate2 is inhibited.

The pCMV-TALER1 plasmid is shown in SEQ ID NO: 1. In SEQ ID NO: 1, the 1-549th nucleotide from the 5' end is the CMV promoter, and the nucleotides 6013-1319 are the coding gene of EYFP (enhanced yellow fluorescent protein), and the nucleotides 1326-1379 The gene encoding the 2A-linked peptide, the nucleotides 1389-4220 are the genes encoding the TALER1 protein, and the nucleotides 4227-4259 are the genes encoding the nuclear localization signal SV40NLS.

The pT1+T1+ plasmid is shown in SEQ ID NO:27. In SEQ ID NO:27, the nucleotides 4275-4367 from the 5' end are 5×UAS sequences, and the nucleotides 4383-4396 are T1 sequences (target sequences of TALER1 protein), nucleotides 4403-4462 For the CMVmini promoter, nucleotides 4469-4482 are T1 sequences, and nucleotides 4532-5237 are genes encoding mKate2 (far red fluorescent protein).

The pEF1a-TagBFP-2A plasmid is shown in SEQ ID NO:53. In sequence 53, the nucleotides 4250-5423 from the 5' end are pEF1a (promoter), the nucleotides 5488-6177 are the genes encoding TagBFP (blue fluorescent protein), and the nucleotides 6178-6243 The acid is a gene encoding a 2A-ligation peptide, and the nucleotide at positions 6250-6933 is a gene encoding Gal4/vp16 (fusion transcription factor).

The pCMV-TALER2 plasmid is shown in SEQ ID NO: 2. In sequence 2, nucleotides 1-549 are CMV promoters, nucleotides 603-1319 are EYFP encoding genes, and nucleotides 1326-1379 are 2A connecting peptide encoding genes, 1389- The 4220 nucleotide is the gene encoding the TALER2 protein, and the nucleotides 4227-4259 are the genes encoding the nuclear localization signal SV40NLS.

The pCMV-TALER4 plasmid is shown in SEQ ID NO:3. In SEQ ID NO: 3, nucleotides 1713 to 2301 are CMV promoters, nucleotides 2315 to 3031 are EYFP encoding genes, and nucleotides 3038 to 3091 are encoding genes of 2A linked peptides, and The 5932 nucleotide is the coding gene of the TALER4 protein, and the nucleotide 5939-5971 is the coding gene of the nuclear localization signal SV40NLS.

The pCMV-TALER5 plasmid is shown in SEQ ID NO: 4. In SEQ ID NO: 4, nucleotides 5842-6430 are CMV promoters, nucleotides 6444-7160 are encoding genes of EYFP, and nucleotides 7167-7220 are encoding genes of 2A linking peptides, 7230- The 2387 nucleotide is the coding gene of the TALER5 protein, and the nucleotide 2394-2426 is the coding gene of the nuclear localization signal SV40NLS.

The pCMV-TALER9 plasmid is shown in SEQ ID NO: 5. In SEQ ID NO: 5, nucleotides 6148-6736 are the CMV promoter, nucleotides 6750-7466 are the coding gene of EYFP, and nucleotides 7473-7526 are the coding genes of 2A junction peptide, 7536- The 2693 nucleotide is the coding gene of the TALER9 protein, and the 2700-2732 nucleotide is the coding gene of the nuclear localization signal SV40NLS.

The pCMV-TALER10 plasmid is shown in SEQ ID NO: 6. In SEQ ID NO: 6, the 1792-2380 nucleotide is the CMV promoter, the 2394-3110 nucleotide is the EYFP encoding gene, and the 3117-3170 nucleotide is the 2A linkage. The coding gene of the peptide, the nucleotides 3180-6623 are the genes encoding the TALER10 protein, and the nucleotides 6630-6662 are the genes encoding the nuclear localization signal SV40NLS.

The pCMV-TALER11 plasmid is shown in SEQ ID NO: 7. In SEQ ID NO: 7, the 1766-2354 nucleotide is the CMV promoter, the 2368-3084 nucleotide is the EYFP coding gene, the 3091-3144 nucleotide is the 2A junction peptide coding gene, and the 3154- 4 The 6597 nucleotide is the coding gene of the TALER11 protein, and the nucleotide 6604-6636 is the coding gene of the nuclear localization signal SV40NLS.

The pCMV-TALER12 plasmid is shown in SEQ ID NO: 8. In SEQ ID NO: 8, the nucleotides 1705-2293 are the CMV promoter, the nucleotides 2307-3023 are the coding genes of EYFP, and the nucleotides 3030-3083 are the coding genes of the 2A junction peptide, The 6332 nucleotide is the coding gene of the TALER12 protein, and the nucleotides 6339-6371 are the coding genes of the nuclear localization signal SV40NLS.

The pCMV-TALER13 plasmid is shown in SEQ ID NO: 9. In SEQ ID NO: 9, the nucleotides 1687-2275 are the CMV promoter, the nucleotides 2289-3005 are the coding genes of EYFP, and the nucleotides 301-32-365 are the coding genes of the 2A junction peptide, pp. 3075- The nucleotide at position 6212 is the gene encoding the TALER13 protein, and the nucleotide at positions 6219-6251 is the gene encoding the nuclear localization signal SV40NLS.

The pCMV-TALER14 plasmid is shown in SEQ ID NO: 10. In SEQ ID NO: 10, the 1764-2352 nucleotide is the CMV promoter, the 2366-3082 nucleotide is the EYFP coding gene, the 3089-3142 nucleotide is the 2A junction peptide coding gene, and the 3152- The 6289 nucleotide is the coding gene of the TALER14 protein, and the nucleotides 6296-6328 are the coding genes of the nuclear localization signal SV40NLS.

The pCMV-TALER15 plasmid is shown in SEQ ID NO: 11. In SEQ ID NO:11, nucleotides 1-549 are CMV promoters, nucleotides 603-1319 are EYFP encoding genes, and nucleotides 1326-1379 are 2A linking peptide encoding genes, 1394- The 4597 nucleotide is the coding gene of the TALER15 protein, and the nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.

The pCMV-TALER16 plasmid is shown in SEQ ID NO: 12. In SEQ ID NO: 12, nucleotides 1-549 are CMV promoters, nucleotides 603-1319 are EYFP encoding genes, and nucleotides 1326-1379 are 2A linking peptide encoding genes, 1394- The 4597 nucleotide is the coding gene of the TALER16 protein, and the nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.

The pCMV-TALER17 plasmid is shown in SEQ ID NO: 13. In SEQ ID NO: 13, the nucleotides 1-549 are the CMV promoter, the nucleotides 6013-1319 are the coding genes of EYFP, and the nucleotides 1326-1379 are the coding genes of the 2A junction peptide, 1394- The 4597 nucleotide is the coding gene of the TALER17 protein, and the nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.

The pCMV-TALER18 plasmid is shown in SEQ ID NO: 14. In SEQ ID NO: 14, nucleotides 1-549 are CMV promoters, nucleotides 603-13-119 are EYFP encoding genes, and nucleotides 1326-1379 are 2A linking peptide encoding genes, 1394- The 4597 nucleotide is the coding gene of the TALER18 protein, and the nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.

The pCMV-TALER19 plasmid is shown in SEQ ID NO: 15. In SEQ ID NO:15, the nucleotides 1711-2299 are CMV promoters, the nucleotides 2313-3029 are EYFP encoding genes, and the nucleotides 3036-3089 are 2A. The gene encoding the linker peptide, the nucleotides 3099-6440 are the genes encoding the TALER19 protein, and the nucleotides 6447-6479 are the genes encoding the nuclear localization signal SV40NLS.

The pCMV-TALER20 plasmid is shown in SEQ ID NO: 16. In SEQ ID NO:16, nucleotides 1-549 are CMV promoters, nucleotides 603-1319 are EYFP encoding genes, and nucleotides 1326-1379 are 2A linking peptide encoding genes, 1394- The 4597 nucleotide is the coding gene of the TALER20 protein, and the nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.

The pCMV-TALER21 plasmid is shown in SEQ ID NO: 17. In SEQ ID NO:17, nucleotides 1616-2204 are CMV promoters, nucleotides 2218-2934 are EYFP encoding genes, nucleotides 2941-2994 are 2A linking peptide encoding genes, and 3004- The 6345 nucleotide is the coding gene of the TALER21 protein, and the nucleotide number 6352-6384 is the coding gene of the nuclear localization signal SV40NLS.

The pCMV-TALER22 plasmid is shown in SEQ ID NO: 18. In SEQ ID NO: 18, the nucleotides 1-549 are the CMV promoter, the nucleotides 6013-1319 are the coding genes of EYFP, and the nucleotides 1326-1379 are the coding genes of the 2A junction peptide, 1394- The 3985 nucleotide is the coding gene of the TALER22 protein, and the nucleotides 3993-4025 are the coding genes of the nuclear localization signal SV40NLS.

The pCMV-TALER23 plasmid is shown in SEQ ID NO: 19. In SEQ ID NO: 19, nucleotides 1-549 are CMV promoters, nucleotides 603-13-1 are EVFP encoding genes, and nucleotides 1326-1379 are 2A linking peptide encoding genes, 1394- The 3985 nucleotide is the coding gene of the TALER23 protein, and the nucleotides 3993-4025 are the genes encoding the nuclear localization signal SV40NLS.

The pCMV-TALER24 plasmid is shown in SEQ ID NO: 20. In SEQ ID NO: 20, nucleotides 1-549 are CMV promoters, nucleotides 603-13-119 are EYFP encoding genes, and nucleotides 1326-1379 are 2A connecting peptide encoding genes, 1394- The 3985 nucleotide is the coding gene of the TALER24 protein, and the nucleotides 3993-4025 are the genes encoding the nuclear localization signal SV40NLS.

The pCMV-TALER26 plasmid is shown in SEQ ID NO:21. In SEQ ID NO: 21, nucleotides 1679-2267 are CMV promoters, nucleotides 2281-2997 are EYFP encoding genes, and nucleotides 3004-3057 are 2A connecting peptide encoding genes, 3064- The nucleotide at position 6009 is the gene encoding the TALER26 protein, and the nucleotide at position 6024-6045 is the gene encoding the nuclear localization signal SV40NLS.

The pCMV-TALER29 plasmid is shown in SEQ ID NO:22. In SEQ ID NO:22, the 1638-2226 nucleotide is the CMV promoter, the 2240-2956 nucleotide is the EYFP coding gene, the 2963-3016 nucleotide is the 2A junction peptide coding gene, and the 3303- The 5560 nucleotide is the coding gene of the TALER29 protein, and the nucleotides 5575-5596 are the coding genes of the nuclear localization signal SV40NLS.

The pCMV-TALER30 plasmid is shown in SEQ ID NO:23. In SEQ ID NO:23, the nucleotides 1838-2426 are the CMV promoter, the nucleotides 2440-3156 are the coding genes of EYFP, and the nucleotides 3163-3216 are the coding genes of the 2A junction peptide, pp. 3223- The 5760 nucleotide is the coding gene of the TALER30 protein, and the nucleotides 5775-5796 are the coding genes of the nuclear localization signal SV40NLS.

The pCMV-TALER31 plasmid is shown in SEQ ID NO:24. In SEQ ID NO: 24, the nucleotides 3403-3991 are the CMV promoter, the nucleotides 4005-4721 are the coding genes of EYFP, and the nucleotides 4728-4781 are 2A. The coding gene of the ligation peptide, the nucleotide of 4788-7325 is the coding gene of TALER31 protein, and the nucleotide of 7340-7361 is the coding gene of nuclear localization signal SV40NLS.

The pCMV-TALER32 plasmid is shown in SEQ ID NO:25. In SEQ ID NO: 25, the nucleotides 1691-2279 are the CMV promoter, the nucleotides 2293-3009 are the coding genes of EYFP, and the nucleotides 3016-3069 are the coding genes of the 2A junction peptide, 3076- The nucleotide at position 5613 is the gene encoding the TALER32 protein, and the nucleotide at position 5628-5649 is the gene encoding the nuclear localization signal SV40NLS.

pCMV-TALER35 is shown in SEQ ID NO:26. In SEQ ID NO: 26, nucleotides 1607-2195 are CMV promoters, nucleotides 2209-2925 are EYFP encoding genes, nucleotides 2932-2985 are 2A linking peptide encoding genes, and 2992- The 5529 nucleotide is the coding gene of the TALER35 protein, and the 5544-5565 nucleotide is the coding gene of the nuclear localization signal SV40NLS.

The pT2+T2+ plasmid is shown in SEQ ID NO: 28. In SEQ ID NO: 28, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T2 sequences (target sequence of TALER2 protein), nucleotides 197-256 For the CMVmini promoter, the nucleotides 263-276 are the T2 sequence, and the nucleotides 355-1073 are the coding genes for mKate2.

The pT4+T4+ plasmid is shown in SEQ ID NO:29. In sequence 29, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T4 sequences (target sequence of TALER4 protein), nucleotides 197-256 For the CMVmini promoter, nucleotides 263-276 are T4 sequences, and nucleotides 355-1073 are genes encoding mKate2.

The pT5+T5+ plasmid is shown in SEQ ID NO:30. In sequence 30, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-194 are T5 sequences (target sequence of TALER5 protein), nucleotides 201-260 For the CMVmini promoter, nucleotides 267-284 are T5 sequences, and nucleotides 363-1081 are genes encoding mKate2.

The pT9+T9+ plasmid is shown in SEQ ID NO:31. In sequence 31, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-197 are T9 sequences (target sequences of TALER9 protein), nucleotides 204-263 For the CMVmini promoter, nucleotides 270-290 are T9 sequences, and nucleotides 369-1087 are genes encoding mKate2.

The pT10+T10+ plasmid is shown in sequence 32. In sequence 32, the nucleotides from the 5' end of the 7069-7161 nucleotide are 5×UAS sequences, and the nucleotides from the 7177-7196 nucleotides are the T10 sequence (target sequence of the TALER10 protein), and nucleotides 7203-7262 For the CMVmini promoter, nucleotides 7269-7288 are T10 sequences, and nucleotides 78-796 are genes encoding mKate2.

The pT11+T11+ plasmid is shown in SEQ ID NO:33. In sequence 33, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-196 are T11 sequences (target sequences of TALER11 protein), nucleotides 203-262 For the CMVmini promoter, the nucleotides 269-288 are the T11 sequence, and the nucleotides 367-1085 are the coding genes of mKate2.

The pT12+T12+ plasmid is shown as sequence 34. In sequence 34, the nucleotides from the 5' end of the 7069-7161 nucleotide are 5×UAS sequences, and the nucleotides at positions 7177-7194 are the T12 sequence (target sequence of the TALER12 protein), and nucleotides 7201-7260 For the CMVmini promoter, nucleotides 7267-7284 are The T12 sequence, nucleotides 78-796 is the gene encoding mKate2.

The pT13+T13+ plasmid is shown in SEQ ID NO:35. In SEQ ID NO: 35, the nucleotides from the 5' end of the 7069-7161 nucleotide are the 5×UAS sequence, and the nucleotides at the 7177-7193 are the T13 sequence (the target sequence of the TALER13 protein), and the nucleotides 7200-7259 For the CMVmini promoter, nucleotides 7266-7282 are T13 sequences, and nucleotides 78-796 are genes encoding mKate2.

The pT14+T14+ plasmid is shown in SEQ ID NO:36. In sequence 36, the nucleotides from the 5' end of the 7069-7161 nucleotides are 5×UAS sequences, and the nucleotides at positions 7177-7193 are the T14 sequences (target sequences of the TALER14 protein), and nucleotides 7200-7259 For the CMVmini promoter, nucleotides 7266-7282 are T14 sequences, and nucleotides 78-796 are genes encoding mKate2.

The pT15+T15+ plasmid is shown in SEQ ID NO:37. In sequence 37, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-201 are T15 sequences (target sequences of TALER15 protein), nucleotides 208-267 For the CMVmini promoter, the nucleotides 274-298 are the T15 sequence, and the nucleotides 377-1095 are the coding genes of mKate2.

The pT16+T16+ plasmid is shown in SEQ ID NO:38. In sequence 38, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-201 are T16 sequences (target sequences of TALER16 protein), nucleotides 208-267 For the CMVmini promoter, nucleotides 274-298 are T16 sequences and nucleotides 377-1095 are genes encoding mKate2.

The pT17+T17+ plasmid is shown in SEQ ID NO:39. In sequence 39, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-201 are T17 sequences (target sequences of TALER17 protein), nucleotides 208-267 For the CMVmini promoter, the nucleotides 274-298 are the T17 sequence, and the nucleotides 377-1095 are the coding genes of mKate2.

The pT18+T18+ plasmid is shown in sequence 40. In sequence 40, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-201 are T18 sequences (target sequence of TALER18 protein), nucleotides 208-267 For the CMVmini promoter, the nucleotides 274-298 are the T18 sequence, and the nucleotides 377-1095 are the coding genes of mKate2.

The pT19+T19+ plasmid is shown in sequence 41. In the sequence 41, the nucleotides from the 5' end of the 7069-7161 nucleotide are 5×UAS sequences, the nucleotides at the 7177-7195 nucleotides are the T19 sequence (the target sequence of the TALER19 protein), and the nucleotides of the 7th to 7261th nucleotides. For the CMVmini promoter, nucleotides 7268-7286 are T19 sequences, and nucleotides 78-796 are genes encoding mKate2.

The pT20+T20+ plasmid is shown as sequence 42. In sequence 42, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-201 are T20 sequences (target sequences of TALER20 protein), nucleotides 208-267 For the CMVmini promoter, nucleotides 274-298 are T20 sequences, and nucleotides 377-1095 are genes encoding mKate2.

The pT21+T21+ plasmid is shown in sequence 43. In sequence 43, from nucleotides 7069-7161 at the 5' end, 5 x UAS sequences, nucleotides 7177-7195 are T21 sequences (target sequences for TALER21 protein), nucleotides 7202-7261 For the CMVmini promoter, nucleotides 7268-7286 are T21 sequences, and nucleotides 78-796 are genes encoding mKate2.

The pT22+T22+ plasmid is shown as sequence 44. In sequence 44, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-195 are T22 sequences (target sequences of TALER22 protein), nucleotides 202-261 For the CMVmini promoter, nucleotides 268-286 are T22 sequences, and nucleotides 365-1083 are genes encoding mKate2.

The pT23+T23+ plasmid is shown in sequence 45. In sequence 45, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-195 are T23 sequences (target sequences of TALER23 protein), nucleotides 202-261 For the CMVmini promoter, nucleotides 268-286 are T23 sequences, and nucleotides 365-1083 are genes encoding mKate2.

The pT24+T24+ plasmid is shown in SEQ ID NO:46. In sequence 46, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-195 are T24 sequences (target sequences of TALER24 protein), nucleotides 202-261 For the CMVmini promoter, nucleotides 268-286 are T24 sequences, and nucleotides 365-1083 are genes encoding mKate2.

The pT26+T26+ plasmid is shown in SEQ ID NO:47. In sequence 47, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-194 are T26 sequences (target sequences of TALER26 protein), nucleotides 201-260 For the CMVmini promoter, nucleotides 267-284 are T26 sequences, and nucleotides 363-1081 are genes encoding mKate2.

The pT29+T29+ plasmid is shown in SEQ ID NO:48. In sequence 48, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T29 sequences (target sequences of TALER29 protein), nucleotides 197-256 For the CMVmini promoter, the nucleotides 263-276 are the T29 sequence, and the nucleotides 355-1073 are the coding genes of mKate2.

The pT30+T30+ plasmid is shown in SEQ ID NO:49. In sequence 49, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T30 sequences (target sequence of TALER30 protein), nucleotides 197-256 For the CMVmini promoter, the nucleotides 263-276 are the T30 sequence, and the nucleotides 355-1073 are the coding genes of mKate2.

The pT31+T31+ plasmid is shown as sequence 50. In sequence 50, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-189 are T31 sequences (target sequences of TALER31 protein), nucleotides 196-255 For the CMVmini promoter, nucleotides 262-274 are T31 sequences, and nucleotides 353-1071 are genes encoding mKate2.

The pT32+T32+ plasmid is shown in sequence 51. In sequence 51, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T32 sequences (target sequence of TALER32 protein), nucleotides 197-256 For the CMVmini promoter, nucleotides 263-276 are T32 sequences, and nucleotides 355-1073 are the coding genes for mKate2.

The pT35+T35+ plasmid is shown in sequence 52. In sequence 52, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T35 sequences (target sequences of TALER35 protein), nucleotides 197-256 For the CMVmini promoter, nucleotides 263-276 are T35 sequences, and nucleotides 355-1073 are genes encoding mKate2.

First, experiment one

The pCMV-TALER1 plasmid, pT1+T1+ plasmid and pEF1a-TagBFP-2A plasmid were co-transfected into HEK293 cells (transfected with 200 ng pCMV-TALER1 plasmid, 50 ng pT1+T1+ plasmid and 30 ng pEF1a-TagBFP-2A plasmid per well), transfected. Flow cytometry analysis was performed after 48 hours to measure the fluorescence intensity of EYFP, the fluorescence intensity of mKate2, and the fluorescence intensity of TagBFP. A control treatment without the addition of the pCMV-TALER1 plasmid was set. Inhibition ratio = control group mKate2 fluorescence intensity correction value ÷ experimental group mKate2 fluorescence intensity correction value. Percent inhibition = (control group mKate2 fluorescence intensity correction value - experimental group mKate2 fluorescence intensity correction value) ÷ control group mKate2 fluorescence intensity correction value. mKate2 fluorescence intensity correction value = fluorescence intensity of mKate2 / fluorescence intensity of TagBFP.

The pCMV-TALER1 plasmid was replaced with the pCMV-TALER2 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with the pT2+T2+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER4 plasmid, and the above procedure was carried out by substituting the pT4+T4+ plasmid for the pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER5 plasmid, and the above procedure was carried out by substituting the pT5+T5+ plasmid for the pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER9 plasmid, and the above procedure was carried out by substituting the pT9+T9+ plasmid for the pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER10 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT10+T10+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER11 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT11+T11+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER12 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT12+T12+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER13 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT13+T13+ plasmid. The pCMV-TALER1 plasmid was replaced with pCMV-TALER14 plasmid, and the above procedure was carried out using pT14+T14+ plasmid instead of pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with pCMV-TALER15 plasmid, and the above procedure was carried out by substituting pT15+T15+ plasmid for pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with pCMV-TALER16 plasmid, and the above procedure was carried out using pT16+T16+ plasmid instead of pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER17 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT17+T17+ plasmid. The pCMV-TALER1 plasmid was replaced with pCMV-TALER18 plasmid, and the above procedure was carried out by substituting pT18+T18+ plasmid for pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER19 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT19+T19+ plasmid. The pCMV-TALER1 plasmid was replaced with pCMV-TALER20 plasmid, and the above procedure was carried out by substituting pT20+T20+ plasmid for pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER21 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT21+T21+ plasmid. The pCMV-TALER1 plasmid was replaced with pCMV-TALER22 plasmid, and the above procedure was carried out using pT22+T22+ plasmid instead of pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with pCMV-TALER23 plasmid, and the above procedure was carried out by substituting pT23+T23+ plasmid for pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with pCMV-TALER24 plasmid, and the above procedure was carried out by substituting pT24+T24+ plasmid for pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER26 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT26+T26+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER29 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT29+T29+ plasmid. The pCMV-TALER1 plasmid was replaced with pCMV-TALER30 plasmid, and the above procedure was carried out by substituting pT30+T30+ plasmid for pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with the pCMV-TALER31 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT31+T31+ plasmid. Replace pCMV-TALER1 plasmid with pCMV-TALER32 plasmid The above procedure was carried out by substituting the pT32+T32+ plasmid for the pT1+T1+ plasmid. The pCMV-TALER1 plasmid was replaced with pCMV-TALER35 plasmid, and the above procedure was carried out by replacing the pT1+T1+ plasmid with pT35+T35+ plasmid.

The inhibition doubling results and percent inhibition results are shown in Figure 3 (the bar graph represents the inhibition fold, the dot plot represents the percent inhibition) and Table 1. Twenty-three of the 26 TALER proteins showed greater than 90% transcriptional repression, 16 of which Each TALER protein has a transcriptional inhibition effect greater than 100-fold. The results indicate that in mammalian cells, the TALER protein (ie, the TALEN fusion protein without the inhibitory domain) can also exert efficient transcriptional inhibition through steric hindrance.

Table 1 inhibition fold results and percent inhibition results

Second, experiment two

On the basis of Experiment 1, orthogonality was detected by measuring the transcriptional inhibition of the verification module (Tx-CMVmini promoter-Tx-mKate2 gene) by the TARG protein of the top ten most potent inhibitory effects.

For example, the pCMV-TALER1 plasmid, pT35+T35+ plasmid and pEF1a-TagBFP-2A plasmid were co-transfected into HEK293 cells (200 ng pCMV-TALER1 plasmid, 50 ng pT1+T1+ plasmid and 30 ng pEF1a-TagBFP-2A plasmid per well). Flow cytometry analysis 48 hours after transfection, detection of EYFP Fluorescence intensity, fluorescence intensity of mKate2, and fluorescence intensity of TagBFP. A control treatment without the addition of the pCMV-TALER1 plasmid was set.

The results are shown in Fig. 4 (10 ¹ to 10 ^{-2.5 in} Fig. 4 represents the fluorescence intensity of mKate 2 / the fluorescence intensity of TagBFP) and Table 2. All of the TALER proteins tested showed a strong inhibitory effect on the promoter between their respective targets and had little effect on the promoter between the other targets. For example, the TALER1 protein, the TALER9 protein, the TALER10 protein, the TALER12 protein, the TALER14 protein, and the TALER21 protein are more than 100-fold stronger than the promoter between their respective targets and the promoter between the other targets.

Table 2 Results of Figure 4 (fluorescence intensity of mKate2 / fluorescence intensity of TagBFP)

Example 2, further ductility study

The pEF1a-TagBFP-2A plasmid was the pEF1a-TagBFP-2A plasmid of Example 1.

The pCMV-TALER1 plasmid was the pCMV-TALER1 plasmid of Example 1.

The pCMV-TALER2 plasmid was the pCMV-TALER2 plasmid of Example 1.

The pCMV-TALER4 plasmid was the pCMV-TALER4 plasmid of Example 1.

The pCMV-TALER5 plasmid was the pCMV-TALER5 plasmid of Example 1.

The pCMV-TALER32 plasmid is the pCMV-TALER32 plasmid of Example 1.

The pT1+T1+72-DsRed plasmid is shown in sequence 54. In sequence 54, the nucleotides 2441-2533 from the 5' end are 5 x UAS sequences, the nucleotides 2549-2562 are T1 sequences (target sequences of TALER1 protein), nucleotides 2569-2628 For the CMVmini promoter, the nucleotides at positions 2635-2648 are T1 sequences, and the nucleotides at positions 2668-3345 are genes encoding DsRed (red fluorescent protein).

The pT1+T2+72-DsRed plasmid is shown in sequence 55. In sequence 55, nucleotides 2441-2533 from the 5' end are 5 x UAS sequences, nucleotides 2549-2562 are T1 sequences, nucleotides 2569-2628 are CMVmini promoters, and 2635- The 2648 nucleotide is the T2 sequence (the target sequence of the TALER2 protein), and the 2668-3345 nucleotide is the coding gene of DsRed.

The pT2+T1+72-DsRed plasmid is shown as sequence 56. In sequence 56, nucleotides 2441-2533 from the 5' end are 5 x UAS sequences, nucleotides 2549-2562 are T2 sequences, and positions 2569-2628 The nucleotide is the CMVmini promoter, the nucleotides at positions 2635-2648 are the T1 sequence, and the nucleotides at positions 2668-3345 are the genes encoding DsRed.

The pT2+T2+72-DsRed plasmid is shown in sequence 57. In sequence 57, nucleotides 2441-2533 from the 5' end are 5 x UAS sequences, nucleotides 2549-2562 are T2 sequences, nucleotides 2569-2628 are CMVmini promoters, and 2635- The 2648 nucleotide is the T2 sequence, and the 2668-3345 nucleotide is the DsRed coding gene.

The pT1+T1+72-mKate2 plasmid is shown in SEQ ID NO:58. In sequence 58, the nucleotides 4275-4367 from the 5' end are 5×UAS sequences, the nucleotides 4383-4396 are T1 sequences, and the nucleotides 4403-4462 are CMVmini promoters, 4469- The 4482 nucleotide is the T1 sequence, and the nucleotides 4532-5237 are the coding genes of mKate2.

The pT1+T1+78-mKate2 plasmid is shown in SEQ ID NO:59. In sequence 59, nucleotides 7161-7253 from the 5' end are 5×UAS sequences, nucleotides 7269-7282 are T1 sequences, and nucleotides 6-65 are CMVmini promoters, 78- The nucleotide at position 91 is the T1 sequence, and the nucleotide at positions 170-888 is the coding gene for mKate2.

The pT1+T1+83-mKate2 plasmid is shown in SEQ ID NO:60. In sequence 60, nucleotides 7166-7258 from the 5' end are 5 x UAS sequences, nucleotides 7274-7287 are T1 sequences, and nucleotides 6-65 are CMVmini promoters, 83- The 96th nucleotide is the T1 sequence, and the nucleotides 175-893 are the coding genes of mKate2.

The pT1+T1+89-mKate2 plasmid is shown in sequence 61. In sequence 61, nucleotides 7172-7264 from the 5' end are 5×UAS sequences, nucleotides 7280-7293 are T1 sequences, and nucleotides 6-65 are CMVmini promoters, 89- The 102 nucleotide is the T1 sequence, and the nucleotides 181-899 are the coding genes of mKate2.

The pT1+T1+94-mKate2 plasmid is shown as sequence 62. In sequence 62, nucleotides 7177-7269 from the 5' end are 5 x UAS sequences, nucleotides 7285-7298 are T1 sequences, and nucleotides 6-65 are CMVmini promoters, 94- The 107th nucleotide is the T1 sequence, and the nucleotides 186-904 are the coding genes of mKate2.

The pT1+T1+100-mKate2 plasmid is shown as sequence 63. In sequence 63, nucleotides 7203-7295 from the 5' end are 5×UAS sequences, nucleotides 6-19 are T1 sequences, and nucleotides 26-85 are CMVmini promoters, 120- The nucleotide at position 133 is the T1 sequence, and the nucleotides at positions 212-930 are the coding genes for mKate2.

The pT2+T2+72-mKate2 plasmid is shown as sequence 64. In sequence 64, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T2 sequences, and nucleotides 197-256 are CMVmini promoters, 263- The nucleotide at position 276 is the T2 sequence, and the nucleotide at positions 355-1073 is the gene encoding mKate2.

The pT2+T2x3+72-mKate2 plasmid is shown as sequence 65. In sequence 65, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T2 sequences, and nucleotides 197-256 are CMVmini promoters, 263- 276 nucleotides are T2 sequences, and nucleotides 279-292 are In the T2 sequence, nucleotides 295-308 are T2 sequences, and nucleotides 384-1106 are genes encoding mKate2.

The pT4+T4+72-mKate2 plasmid is shown as sequence 66. In sequence 66, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T4 sequences (target sequence of TALER4 protein), nucleotides 197-256 For the CMVmini promoter, nucleotides 263-276 are T4 sequences, and nucleotides 355-1073 are genes encoding mKate2.

The pT4+T4x3+72-mKate2 plasmid is shown in SEQ ID NO:67. In sequence 67, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T4 sequences, and nucleotides 197-256 are CMVmini promoters, 263- 276 nucleotides are T4 sequences, nucleotides 277-290 are T4 sequences, nucleotides 291-304 are T4 sequences, and nucleotides 383-1101 are mKate2 encoding genes.

The pT5+T5+72-mKate2 plasmid is shown in SEQ ID NO:68. In sequence 68, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-194 are T5 sequences (target sequence of TALER5 protein), nucleotides 201-260 For the CMVmini promoter, nucleotides 267-284 are T5 sequences, and nucleotides 363-1081 are genes encoding mKate2.

The pT5+T5x3+72-mKate2 plasmid is shown in sequence 69. In sequence 69, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-194 are T5 sequences, and nucleotides 201-260 are CMVmini promoters, 267- The nucleotide number 284 is the T5 sequence, the nucleotides 285-302 are the T5 sequence, the nucleotides 303-320 are the T5 sequence, and the nucleotides 399-1117 are the coding genes of mKate2.

The pT32+T32+72-mKate2 plasmid is shown in SEQ ID NO:70. In sequence 70, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T32 sequences (target sequences of TALER32 protein), nucleotides 197-256 For the CMVmini promoter, nucleotides 263-276 are T32 sequences, and nucleotides 355-1073 are the coding genes for mKate2.

pT32+T32x3+72-mKate2 is shown as in sequence 71. In sequence 71, nucleotides 69-161 from the 5' end are 5 x UAS sequences, nucleotides 177-190 are T32 sequences, and nucleotides 197-256 are CMVmini promoters, 263- 276 nucleotides are T32 sequences, nucleotides 277-290 are T32 sequences, nucleotides 291-304 are T32 sequences, and nucleotides 383-1101 are mKate2 encoding genes.

First, experiment one

The pCMV-TALER1 plasmid, pT1+T1+72-DsRed plasmid and pEF1a-TagBFP-2A plasmid were co-transfected into HEK293 cells (200 ng pCMV-TALER1 plasmid, 50 ng pT1+T1+72-DsRed plasmid and 30 ng pEF1a-transfected per well). TagBFP-2A plasmid), flow cytometry analysis 48 hours after transfection, detection of EYFP fluorescence intensity, DsRed fluorescence intensity and TagBFP fluorescence intensity. A control treatment without the addition of the pCMV-TALER1 plasmid was set.

The above procedure was carried out by substituting pT1+T2+72-DsRed plasmid, pT2+T1+72-DsRed plasmid or pT2+T2+72-DsRed plasmid for pT1+T1+72-DsRed plasmid, respectively.

The inhibition multiples and percent inhibition results are shown in Figures 5 and 3. The results indicate that the 3' binding site of the TALER protein is essential for strong inhibitory ability, while the 5' binding site is much less potent and has a stronger inhibitory effect when both binding sites are present.

Table 3 inhibition fold results and percent inhibition results

Second, experiment two

The pCMV-TALER1 plasmid, pT1+T1+72-mKate2 plasmid and pEF1a-TagBFP-2A plasmid were co-transfected into HEK293 cells (200 ng pCMV-TALER1 plasmid, 50 ng pT1+T1+72-mKate2 plasmid and 30 ng pEF1a-transfected per well). TagBFP-2A plasmid), flow cytometry analysis 48 hours after transfection, detection of EYFP fluorescence intensity, mKate2 fluorescence intensity and TagBFP fluorescence intensity. A control treatment without the addition of the pCMV-TALER1 plasmid was set.

Replace pT1+T1 with pT1+T1+78-mKate2 plasmid, pT1+T1+83-mKate2 plasmid, pT1+T1+89-mKate2 plasmid, pT1+T1+94-mKate2 plasmid or pT1+T1+100-mKate2 plasmid The +72-mKate2 plasmid was subjected to the above procedure.

The inhibition multiples and percent inhibition results are shown in Figures 6 and 4. The TALER protein has no periodic inhibitory behavior, and the closer the TALER binding site is to the miniCMV promoter, the stronger the inhibitory effect.

Table 4 inhibition fold results and percent inhibition results

Third, experiment three

The pCMV-TALER2 plasmid, pT2+T2+72-mKate2 plasmid (or T2+T2x3+72-mKate2 plasmid) and pEF1a-TagBFP-2A plasmid were co-transfected into HEK293 cells (transfected with 200 ng pCMV-TALER2 plasmid per well, 50 ng pT2 per well). +T2+72-mKate2 plasmid or T2+T2x3+72-mKate2 plasmid, 30ng pEF1a-TagBFP-2A plasmid), flow cytometry analysis 48 hours after transfection, detection of EYFP fluorescence intensity, mKate2 fluorescence intensity and TagBFP Fluorescence intensity. A control treatment without the addition of the pCMV-TALER2 plasmid was set.

Replacing the pCMV-TALER2 plasmid with pCMV-TALER4 plasmid and the pT2+T2+72-mKate2 plasmid with pT4+T4+72-mKate2 plasmid (or replacing with pT4+T4x3+72-mKate2 plasmid) The T2+T2x3+72-mKate2 plasmid) was subjected to the above steps.

Replacing the pCMV-TALER2 plasmid with pCMV-TALER5 plasmid and the pT2+T2+72-mKate2 plasmid with pT5+T5+72-mKate2 plasmid (or replacing the T2+T2x3+72-mKate2 plasmid with pT5+T5x3+72-mKate2 plasmid ) Carry out the above steps.

Replacing the pCMV-TALER2 plasmid with pCMV-TALER32 plasmid and the pT2+T2+72-mKate2 plasmid with pT32+T32+72-mKate2 plasmid (or replacing the T2+T2x3+72-mKate2 plasmid with pT32+T32x3+72-mKate2 plasmid ) Carry out the above steps.

The inhibition fold results and percent inhibition results are shown in Figures 7 and 5. The TALER protein showed a stronger inhibitory effect when it had 3 targets downstream of the miniCMV promoter compared to having 1 target downstream of the miniCMV promoter. In some highly efficient TALERs, the extra binding sites unexpectedly resulted in a slight decrease in inhibition. The inventors noted that the inhibitory ability of the TALER protein with strong inhibition by the additional binding site is significantly lower, but the background expression of the promoter is also due to the insertion sequence between the miniCMV promoter and the reporter gene. And lower. These results indicate that transcriptional repression ability can be optimized by balancing the background expression level of the promoter and the ability of TALER to inhibit the miniCMV promoter.

Table 5 inhibition fold results and percent inhibition results

Fourth, experiment four

The plasmid shown in SEQ ID NO: 72 of the Sequence Listing was synthesized. In sequence 72, the nucleotides 4766-5033 from the 5' end are the doxycycline response element TRE (wherein nucleotides 4766-4961 are tetO and nucleotides 4976-5033 are CMVmini promoters), The 5113-8250 nucleotide is the coding gene of TALER14 protein, the nucleotides of 9306-9549 are cHS4core, the nucleotides of 9625-9868 are cHS4core, and the nucleotides of 9998-10079 are 5×UAS sequences. The nucleotides 10095-10111 are the T14 sequence (the target sequence of the TALER14 protein), the nucleotides 10118-10177 are the CMVmini promoter, and the nucleotides 10184-10200 are the T14 sequence, 10201-10217 The nucleotide is the T14 sequence, the nucleotides 10218-10234 are the T14 sequence, the nucleotides 10313-11031 are the coding genes of mKate2, the nucleotides 11979-12222 are cHS4core, and the 12298-12541 The nucleotide is cHS4core, and the nucleotides 12658-12925 are the doxycycline response element TRE (wherein nucleotides 112658-12853 are tetO, nucleotides 12868-12925 are CMVmini promoter), 12982 -13701 nucleotide is the coding gene of EYFP, nucleotides 14612-14855 are cHS4core, and nucleotides 14431-15174 are cHS4core Of nucleotides 15292-16465 of pEF1a (promoter), the nucleotides of nucleotides 16539-17219 are the genes encoding Gal4/vp16, the nucleotides of 17220-17285 are the genes encoding the 2A linker peptide, and the nucleotides of 17292-17996 are the genes of rtTA. . A schematic representation of the elements of the plasmid shown in SEQ ID NO: 72 is shown in Figure 8A.

The plasmid shown in SEQ ID NO: 72 was introduced into HEK293 cells to obtain recombinant cells. In the absence of doxycycline (DOX), Gal4/vp16 and rtTA were expressed under the action of pEF1a, and Gal4/vp16 was bound to the 5×UAS sequence, thereby activating the transcription initiation of the CMVmini promoter, and mKate2 was expressed. After the addition of doxycycline, doxycycline binds to rtTA, activates the doxycycline response element TRE, TALER14 protein and EYFP are expressed, TALER14 protein binds to the T14 sequence, and transcriptional inhibition is achieved by steric hindrance, CMVmini between T14 sequences The promoter is inactivated, whereby mKate2 is inhibited from expression. The expression level of TALFP14 was estimated to be under Dox-induced expression, and the change in the expression level of mKate2 reflects the inhibition of the CMVmini promoter between two T14 sequences by TALER14.

Recombinant cells were first cultured in a Dox environment until mKate2 expression was maximally inhibited and then replaced with Dox-free medium. After 3 days of Dox removal, the expression level of EYFP decreased to 16% of the maximum, while the expression level of mKate2 almost returned to the level of the control group without Dox induction. Then, Dox induction was added on the 8th day, so that the mKate2 expression level was inhibited again, and the Dox-free medium was again replaced on the 16th day, and the mKate2 expression level was still restored. The specific results are shown in Figures 8B and 8C. These results indicate that the TALER protein can achieve rapid and reversible transcriptional repression.

Industrial application

The present inventors have found that a TALER protein (ie, a TALEN fusion protein having no inhibitory domain) can utilize steric hindrance to inhibit the binding of a transcriptional initiation element to a promoter near a TALER protein target, thereby inhibiting transcription and inhibiting efficiency by a TALER protein target. The number and the effect of the distance between the TALER protein target and the CMVmini promoter. The invention has great value for the regulation of expression and function of proteins.

Claims (13)

1. Use of a TALER protein for inactivating a promoter of interest; each of the upstream and downstream of the promoter of interest has at least one target of the TALER protein, or the upstream of the promoter of interest does not have the TALER protein The target, but downstream thereof, has at least one target for the TALER protein.
2. The use according to claim 1, wherein the target promoter has a target of one of the TALER proteins upstream of the target promoter and has 1-3 targets of the TALER protein downstream thereof.
3. The use according to claim 2, wherein the distance between the target of the TALER protein upstream of the promoter of interest and the target of the TAREL protein nearest to the downstream of the promoter of interest is 72-100 bp.
4. Use of a TALER protein for inactivating a gene encoding a protein of interest; the gene encoding the gene of interest is primingly expressed by a promoter of interest; each of the upstream and downstream of the promoter of interest has at least one target of the TALER protein, Alternatively, the upstream of the promoter of interest does not have a target of the TALER protein but has at least one target of the TALER protein downstream thereof.
5. The use according to claim 4, wherein the target promoter has a target of one of the TALER proteins upstream of the target promoter and has 1-3 targets of the TALER protein downstream thereof.
6. The use according to claim 5, wherein the distance between the target of the TALER protein upstream of the promoter of interest and the target of the TAREL protein nearest to the downstream of the promoter of interest is 72-100 bp.
7. Use of a TALER protein for inactivating a functional fragment of interest; the functional fragment of interest is a DNA fragment; each of the upstream and downstream of the functional fragment of interest has at least one target of the TALER protein, or the functional fragment of interest The upstream does not have a target for the TALER protein but has at least one target for the TALER protein downstream thereof.
8. The use according to claim 7, wherein the target functional fragment has a target of one of the TALER proteins upstream of the target functional fragment and has 1-3 targets of the TALER protein downstream thereof.
9. The use according to claim 8, wherein the distance between the target of the TALER protein upstream of the target functional fragment and the target of the TAREL protein nearest to the downstream of the target functional fragment is 72-100 bp.
10. A DNA molecule combination comprising a DNA molecule A, a DNA molecule B and a DNA molecule C; the DNA molecule A comprises a promoter and a gene encoding Gal4/vp16 from upstream to downstream; the DNA molecule B is sequentially from upstream to downstream The following elements are included: a 5×UAS sequence, a target of a TALER protein, a CMVmini promoter, 1-3 targets of the TALER protein, and a gene encoding a protein of interest; the DNA molecule C includes a promoter from upstream to downstream. And a gene encoding the TALER protein.
11. A plasmid combination comprising a plasmid A, a plasmid B and a plasmid C; the plasmid A having the DNA molecule A according to claim 7; the plasmid B having the DNA molecule B according to claim 7; The DNA molecule C of claim 7.
12. A method of regulating expression of a protein of interest, comprising the steps of: The DNA molecule A and the DNA molecule of claim 10 are transfected into a host cell such that the protein of interest is expressed; by transfecting the DNA molecule of claim 10 and the DNA molecule of claim 10. The DNA molecule C as claimed in claim 10 is transfected into the cells of B to inhibit expression of the protein of interest.
13. A method for regulating expression of a protein of interest, comprising the steps of: expressing said protein of interest by transfecting a plasmid A according to claim 11 and a plasmid B according to claim 11 into a host cell; The plasmid A described in claim 11 and the plasmid B described in claim 11 were transfected with the plasmid C described in claim 11, thereby inhibiting the expression of the protein of interest.

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