[go: up one dir, main page]

WO2015165274A1 - Protéine taler ayant un effet inhibiteur sur la transcription par le biais d'un empêchement stérique, et son application - Google Patents

Protéine taler ayant un effet inhibiteur sur la transcription par le biais d'un empêchement stérique, et son application Download PDF

Info

Publication number
WO2015165274A1
WO2015165274A1 PCT/CN2015/000138 CN2015000138W WO2015165274A1 WO 2015165274 A1 WO2015165274 A1 WO 2015165274A1 CN 2015000138 W CN2015000138 W CN 2015000138W WO 2015165274 A1 WO2015165274 A1 WO 2015165274A1
Authority
WO
WIPO (PCT)
Prior art keywords
plasmid
nucleotides
protein
promoter
target
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2015/000138
Other languages
English (en)
Chinese (zh)
Inventor
谢震
威斯
李寅青
蒋云
廖微曦
陈赫
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tsinghua University
Massachusetts Institute of Technology
Original Assignee
Tsinghua University
Massachusetts Institute of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tsinghua University, Massachusetts Institute of Technology filed Critical Tsinghua University
Publication of WO2015165274A1 publication Critical patent/WO2015165274A1/fr
Priority to TW105114331A priority Critical patent/TW201723168A/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to the TASTAT protein exerting transcriptional inhibition through steric hindrance and its use.
  • 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.
  • RNA-directed CRISPR cluster-like periodic interval short palindromic repeat
  • 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.
  • KRAB Krüppel-associated cassette
  • SID4 mSin interacting domain
  • 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).
  • CMV cytomegalovirus promoter
  • CAG promoter cytomegalovirus promoter
  • the expression system is lower than the prokaryotic expression system.
  • 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
  • TALEN Transcription Activator-Like Effector Nuclease
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • a target sequence of a TALER protein such as a T1 sequence, a T2 sequence, a T4 sequence, etc.
  • 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.
  • 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.
  • 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 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.
  • FIG. 2 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.
  • 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.
  • EYFP and TALER1 proteins 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.
  • the 1-549th nucleotide from the 5' end is the CMV promoter
  • the nucleotides 6013-1319 are the coding gene of EYFP (enhanced yellow fluorescent protein)
  • 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.
  • the nucleotides 4275-4367 from the 5' end are 5 ⁇ UAS sequences
  • 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
  • nucleotides 4532-5237 are genes encoding mKate2 (far red fluorescent protein).
  • the pEF1a-TagBFP-2A plasmid is shown in SEQ ID NO:53.
  • 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.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-1319 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A connecting peptide encoding genes
  • 1389- The 4220 nucleotide is the gene encoding the TALER2 protein
  • nucleotides 4227-4259 are the genes encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER4 plasmid is shown in SEQ ID NO:3.
  • nucleotides 1713 to 2301 are CMV promoters
  • nucleotides 2315 to 3031 are EYFP encoding genes
  • nucleotides 3038 to 3091 are encoding genes of 2A linked peptides
  • the 5932 nucleotide is the coding gene of the TALER4 protein
  • nucleotide 5939-5971 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER5 plasmid is shown in SEQ ID NO: 4.
  • nucleotides 5842-6430 are CMV promoters
  • nucleotides 6444-7160 are encoding genes of EYFP
  • nucleotides 7167-7220 are encoding genes of 2A linking peptides
  • 7230- The 2387 nucleotide is the coding gene of the TALER5 protein
  • nucleotide 2394-2426 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER9 plasmid is shown in SEQ ID NO: 5.
  • nucleotides 6148-6736 are the CMV promoter
  • nucleotides 6750-7466 are the coding gene of EYFP
  • nucleotides 7473-7526 are the coding genes of 2A junction peptide
  • 7536- The 2693 nucleotide is the coding gene of the TALER9 protein
  • 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.
  • the 1792-2380 nucleotide is the CMV promoter
  • the 2394-3110 nucleotide is the EYFP encoding gene
  • 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
  • the nucleotides 6630-6662 are the genes encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER11 plasmid is shown 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
  • the 3154- 4 The 6597 nucleotide is the coding gene of the TALER11 protein
  • 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.
  • the nucleotides 1705-2293 are the CMV promoter
  • the nucleotides 2307-3023 are the coding genes of EYFP
  • the nucleotides 3030-3083 are the coding genes of the 2A junction peptide
  • the 6332 nucleotide is the coding gene of the TALER12 protein
  • 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.
  • the nucleotides 1687-2275 are the CMV promoter
  • the nucleotides 2289-3005 are the coding genes of EYFP
  • 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
  • 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.
  • 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
  • the 3152- The 6289 nucleotide is the coding gene of the TALER14 protein
  • 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.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-1319 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A linking peptide encoding genes
  • 1394- The 4597 nucleotide is the coding gene of the TALER15 protein
  • nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER16 plasmid is shown in SEQ ID NO: 12.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-1319 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A linking peptide encoding genes
  • 1394- The 4597 nucleotide is the coding gene of the TALER16 protein
  • nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER17 plasmid is shown in SEQ ID NO: 13.
  • the nucleotides 1-549 are the CMV promoter
  • the nucleotides 6013-1319 are the coding genes of EYFP
  • 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
  • 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.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-13-119 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A linking peptide encoding genes
  • 1394- The 4597 nucleotide is the coding gene of the TALER18 protein
  • nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER19 plasmid is shown in SEQ ID NO: 15.
  • the nucleotides 1711-2299 are CMV promoters
  • the nucleotides 2313-3029 are EYFP encoding genes
  • the nucleotides 3036-3089 are 2A.
  • the gene encoding the linker peptide, the nucleotides 3099-6440 are the genes encoding the TALER19 protein
  • the nucleotides 6447-6479 are the genes encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER20 plasmid is shown in SEQ ID NO: 16.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-1319 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A linking peptide encoding genes
  • 1394- The 4597 nucleotide is the coding gene of the TALER20 protein
  • nucleotide 4605-4637 is the coding gene of the nuclear localization signal SV40NLS.
  • the pCMV-TALER21 plasmid is shown 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
  • 3004- The 6345 nucleotide is the coding gene of the TALER21 protein
  • 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.
  • the nucleotides 1-549 are the CMV promoter
  • the nucleotides 6013-1319 are the coding genes of EYFP
  • 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
  • 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.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-13-1 are EVFP encoding genes
  • nucleotides 1326-1379 are 2A linking peptide encoding genes
  • 1394- The 3985 nucleotide is the coding gene of the TALER23 protein
  • nucleotides 3993-4025 are the genes encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER24 plasmid is shown in SEQ ID NO: 20.
  • nucleotides 1-549 are CMV promoters
  • nucleotides 603-13-119 are EYFP encoding genes
  • nucleotides 1326-1379 are 2A connecting peptide encoding genes
  • 1394- The 3985 nucleotide is the coding gene of the TALER24 protein
  • nucleotides 3993-4025 are the genes encoding the nuclear localization signal SV40NLS.
  • the pCMV-TALER26 plasmid is shown in SEQ ID NO:21.
  • nucleotides 1679-2267 are CMV promoters
  • nucleotides 2281-2997 are EYFP encoding genes
  • nucleotides 3004-3057 are 2A connecting peptide encoding genes
  • 3064- The nucleotide at position 6009 is the gene encoding the TALER26 protein
  • 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.
  • 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
  • the 3303- The 5560 nucleotide is the coding gene of the TALER29 protein
  • 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.
  • the nucleotides 1838-2426 are the CMV promoter
  • the nucleotides 2440-3156 are the coding genes of EYFP
  • the nucleotides 3163-3216 are the coding genes of the 2A junction peptide
  • the 5760 nucleotide is the coding gene of the TALER30 protein
  • 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.
  • the nucleotides 3403-3991 are the CMV promoter
  • the nucleotides 4005-4721 are the coding genes of EYFP
  • 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
  • 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.
  • the nucleotides 1691-2279 are the CMV promoter
  • the nucleotides 2293-3009 are the coding genes of EYFP
  • 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
  • the nucleotide at position 5628-5649 is the gene encoding the nuclear localization signal SV40NLS.
  • pCMV-TALER35 is shown 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
  • 2992- The 5529 nucleotide is the coding gene of the TALER35 protein
  • 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.
  • 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
  • nucleotides 263-276 are the T2 sequence
  • nucleotides 355-1073 are the coding genes for mKate2.
  • the pT4+T4+ plasmid is shown in SEQ ID NO: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
  • nucleotides 355-1073 are genes encoding mKate2.
  • the pT5+T5+ plasmid is shown in SEQ ID NO: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
  • nucleotides 363-1081 are genes encoding mKate2.
  • the pT9+T9+ plasmid is shown in SEQ ID NO: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
  • nucleotides 369-1087 are genes encoding mKate2.
  • sequence 32 The pT10+T10+ plasmid is shown in sequence 32.
  • 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.
  • 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
  • nucleotides 367-1085 are the coding genes of mKate2.
  • sequence 34 The pT12+T12+ plasmid is shown as sequence 34.
  • 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.
  • the nucleotides from the 5' end of the 7069-7161 nucleotide are the 5 ⁇ UAS sequence
  • the nucleotides at the 7177-7193 are the T13 sequence (the target sequence of the TALER13 protein)
  • nucleotides 7266-7282 are T13 sequences
  • nucleotides 78-796 are genes encoding mKate2.
  • the pT14+T14+ plasmid is shown in SEQ ID NO:36.
  • 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
  • nucleotides 7266-7282 are T14 sequences
  • nucleotides 78-796 are genes encoding mKate2.
  • the pT15+T15+ plasmid is shown in SEQ ID NO: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
  • nucleotides 274-298 are the T15 sequence
  • nucleotides 377-1095 are the coding genes of mKate2.
  • sequence 38 The pT16+T16+ plasmid is shown in SEQ ID NO: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 274-298 are T16 sequences
  • nucleotides 377-1095 are genes encoding mKate2.
  • the pT17+T17+ plasmid is shown in SEQ ID NO: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
  • the nucleotides 377-1095 are the coding genes of mKate2.
  • sequence 40 The pT18+T18+ plasmid is shown 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
  • the nucleotides 377-1095 are the coding genes of mKate2.
  • the pT19+T19+ plasmid is shown in 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)
  • the nucleotides of the 7th to 7261th nucleotides are the T19 sequence (the target sequence of the TALER19 protein)
  • nucleotides 7268-7286 are T19 sequences
  • nucleotides 78-796 are genes encoding mKate2.
  • sequence 42 The pT20+T20+ plasmid is shown as 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 274-298 are T20 sequences
  • nucleotides 377-1095 are genes encoding mKate2.
  • sequence 43 The pT21+T21+ plasmid is shown in sequence 43.
  • nucleotides 7177-7195 are T21 sequences (target sequences for TALER21 protein)
  • nucleotides 7268-7286 are T21 sequences
  • nucleotides 78-796 are genes encoding mKate2.
  • sequence 44 The pT22+T22+ plasmid is shown as 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
  • nucleotides 365-1083 are genes encoding mKate2.
  • sequence 45 The pT23+T23+ plasmid is shown 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
  • nucleotides 365-1083 are genes encoding mKate2.
  • sequence 46 The pT24+T24+ plasmid is shown in SEQ ID NO: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
  • nucleotides 365-1083 are genes encoding mKate2.
  • 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.
  • 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
  • nucleotides 355-1073 are the coding genes of mKate2.
  • the pT30+T30+ plasmid is shown in SEQ ID NO:49.
  • 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.
  • sequence 50 The pT31+T31+ plasmid is shown as 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
  • nucleotides 353-1071 are genes encoding mKate2.
  • sequence 51 The pT32+T32+ plasmid is shown 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
  • nucleotides 355-1073 are the coding genes for mKate2.
  • sequence 52 The pT35+T35+ plasmid is shown 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
  • nucleotides 355-1073 are genes encoding mKate2.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • sequence 54 The pT1+T1+72-DsRed plasmid is shown 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
  • the nucleotides at positions 2668-3345 are genes encoding DsRed (red fluorescent protein).
  • sequence 55 The pT1+T2+72-DsRed plasmid is shown 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
  • 2635- The 2648 nucleotide is the T2 sequence (the target sequence of the TALER2 protein)
  • the 2668-3345 nucleotide is the coding gene of DsRed.
  • sequence 56 The pT2+T1+72-DsRed plasmid is shown as sequence 56.
  • nucleotides 2441-2533 from the 5' end are 5 x UAS sequences
  • nucleotides 2549-2562 are T2 sequences
  • positions 2569-2628 The nucleotide is the CMVmini promoter
  • the nucleotides at positions 2635-2648 are the T1 sequence
  • the nucleotides at positions 2668-3345 are the genes encoding DsRed.
  • sequence 57 The pT2+T2+72-DsRed plasmid is shown 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
  • 2635- The 2648 nucleotide is the T2 sequence
  • the 2668-3345 nucleotide is the DsRed coding gene.
  • the pT1+T1+72-mKate2 plasmid is shown in SEQ ID NO:58.
  • 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.
  • nucleotides 7161-7253 from the 5' end are 5 ⁇ UAS sequences
  • nucleotides 7269-7282 are T1 sequences
  • nucleotides 6-65 are CMVmini promoters
  • 78- The nucleotide at position 91 is the T1 sequence
  • 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.
  • nucleotides 7166-7258 from the 5' end are 5 x UAS sequences
  • nucleotides 7274-7287 are T1 sequences
  • nucleotides 6-65 are CMVmini promoters
  • 83- The 96th nucleotide is the T1 sequence
  • nucleotides 175-893 are the coding genes of mKate2.
  • sequence 61 The pT1+T1+89-mKate2 plasmid is shown in sequence 61.
  • nucleotides 7172-7264 from the 5' end are 5 ⁇ UAS sequences
  • nucleotides 7280-7293 are T1 sequences
  • nucleotides 6-65 are CMVmini promoters
  • 89- The 102 nucleotide is the T1 sequence
  • nucleotides 181-899 are the coding genes of mKate2.
  • sequence 62 The pT1+T1+94-mKate2 plasmid is shown as sequence 62.
  • nucleotides 7177-7269 from the 5' end are 5 x UAS sequences
  • nucleotides 7285-7298 are T1 sequences
  • nucleotides 6-65 are CMVmini promoters
  • 94- The 107th nucleotide is the T1 sequence
  • nucleotides 186-904 are the coding genes of mKate2.
  • sequence 63 The pT1+T1+100-mKate2 plasmid is shown as sequence 63.
  • nucleotides 7203-7295 from the 5' end are 5 ⁇ UAS sequences
  • nucleotides 6-19 are T1 sequences
  • nucleotides 26-85 are CMVmini promoters
  • 120- The nucleotide at position 133 is the T1 sequence
  • the nucleotides at positions 212-930 are the coding genes for mKate2.
  • sequence 64 The pT2+T2+72-mKate2 plasmid is shown as sequence 64.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-190 are T2 sequences
  • nucleotides 197-256 are CMVmini promoters
  • 263- The nucleotide at position 276 is the T2 sequence
  • the nucleotide at positions 355-1073 is the gene encoding mKate2.
  • sequence 65 The pT2+T2x3+72-mKate2 plasmid is shown as sequence 65.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-190 are T2 sequences
  • nucleotides 197-256 are CMVmini promoters
  • 263- 276 nucleotides are T2 sequences
  • nucleotides 279-292 are In the T2 sequence
  • nucleotides 295-308 are T2 sequences
  • nucleotides 384-1106 are genes encoding mKate2.
  • sequence 66 The pT4+T4+72-mKate2 plasmid is shown as 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
  • nucleotides 355-1073 are genes encoding mKate2.
  • the pT4+T4x3+72-mKate2 plasmid is shown in SEQ ID NO:67.
  • 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.
  • sequence 68 The pT5+T5+72-mKate2 plasmid is shown in SEQ ID NO: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
  • nucleotides 363-1081 are genes encoding mKate2.
  • sequence 69 The pT5+T5x3+72-mKate2 plasmid is shown in sequence 69.
  • nucleotides 69-161 from the 5' end are 5 x UAS sequences
  • nucleotides 177-194 are T5 sequences
  • 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
  • the nucleotides 399-1117 are the coding genes of mKate2.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • sequence 72 The plasmid shown in SEQ ID NO: 72 of the Sequence Listing was synthesized.
  • 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
  • the nucleotide is cHS4core
  • 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
  • the plasmid shown in SEQ ID NO: 72 was introduced into HEK293 cells to obtain recombinant cells.
  • DOX doxycycline
  • 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.
  • doxycycline 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.
  • a TALER protein ie, a TALEN fusion protein having no inhibitory domain
  • a TALER protein target ie, a TALEN fusion protein having no inhibitory domain
  • the invention has great value for the regulation of expression and function of proteins.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne une protéine TALER ayant un effet inhibiteur sur la transcription par le biais d'un empêchement stérique, et son application, comprenant l'utilisation d'une protéine TALER dans l'inactivation d'un promoteur cible ; les séquences amont et aval du promoteur d'intérêt comportent au moins une cible de protéine TALER, ou la séquence amont du promoteur d'intérêt ne comporte pas de cible de protéine TALER mais sa séquence aval comporte au moins une cible de protéine TALER. L'invention concerne également l'utilisation d'une protéine TALER dans l'inactivation d'un gène codant pour une protéine d'intérêt ; le gène codant pour la protéine d'intérêt est exprimé par le promoteur ; les séquences amont et aval du promoteur d'intérêt comportent au moins une cible de protéine TALER, ou la séquence amont du promoteur d'intérêt ne comporte pas de cible de protéine TALER mais sa séquence aval comporte au moins une cible de protéine TALER.
PCT/CN2015/000138 2014-04-30 2015-03-06 Protéine taler ayant un effet inhibiteur sur la transcription par le biais d'un empêchement stérique, et son application Ceased WO2015165274A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
TW105114331A TW201723168A (zh) 2014-04-30 2016-05-09 用於進行化學反應的設備和方法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201410183417.6 2014-04-30
CN201410183417 2014-04-30
CN201410341963.8A CN104178506B (zh) 2014-04-30 2014-07-17 Taler蛋白通过空间位阻发挥转录抑制作用及其应用
CN201410341963.8 2014-07-17

Publications (1)

Publication Number Publication Date
WO2015165274A1 true WO2015165274A1 (fr) 2015-11-05

Family

ID=51959853

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/000138 Ceased WO2015165274A1 (fr) 2014-04-30 2015-03-06 Protéine taler ayant un effet inhibiteur sur la transcription par le biais d'un empêchement stérique, et son application

Country Status (3)

Country Link
CN (1) CN104178506B (fr)
TW (1) TW201723168A (fr)
WO (1) WO2015165274A1 (fr)

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US12157760B2 (en) 2018-05-23 2024-12-03 The Broad Institute, Inc. Base editors and uses thereof
US12281338B2 (en) 2018-10-29 2025-04-22 The Broad Institute, Inc. Nucleobase editors comprising GeoCas9 and uses thereof
US12351837B2 (en) 2019-01-23 2025-07-08 The Broad Institute, Inc. Supernegatively charged proteins and uses thereof
US12390514B2 (en) 2017-03-09 2025-08-19 President And Fellows Of Harvard College Cancer vaccine
US12406749B2 (en) 2017-12-15 2025-09-02 The Broad Institute, Inc. Systems and methods for predicting repair outcomes in genetic engineering
US12435330B2 (en) 2019-10-10 2025-10-07 The Broad Institute, Inc. Methods and compositions for prime editing RNA
US12473543B2 (en) 2019-04-17 2025-11-18 The Broad Institute, Inc. Adenine base editors with reduced off-target effects

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104178506B (zh) * 2014-04-30 2017-03-01 清华大学 Taler蛋白通过空间位阻发挥转录抑制作用及其应用
JP6961788B2 (ja) * 2017-03-24 2021-11-05 ツィンファ ユニバーシティ プログラム可能な腫瘍溶解性ウイルスワクチン系及びその適用
CN108424934A (zh) * 2018-04-16 2018-08-21 和元生物技术（上海）股份有限公司 一种慢病毒cag-cmv双启动子改造载体构建及应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102325791A (zh) * 2009-01-12 2012-01-18 乌拉·博纳斯 模块式dna结合结构域及使用方法
CN104178506A (zh) * 2014-04-30 2014-12-03 清华大学 Taler蛋白通过空间位阻发挥转录抑制作用及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103184202B (zh) * 2011-12-28 2015-08-26 浙江大学 一对短肽、蛋白质和多核苷酸、其宿主细胞及其应用
CN102558309B (zh) * 2012-02-10 2014-09-17 浙江大学 一对转录激活子样效应因子核酸酶及其编码基因与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102325791A (zh) * 2009-01-12 2012-01-18 乌拉·博纳斯 模块式dna结合结构域及使用方法
CN104178506A (zh) * 2014-04-30 2014-12-03 清华大学 Taler蛋白通过空间位阻发挥转录抑制作用及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI, JI ET AL.: "Technology Development of Gene Engineering - Advances in TALE Research", PLANT PHYSIOLOGY JOURNAL, vol. 12, no. 48, 31 December 2012 (2012-12-31), pages 1141 - 1150 *
MAGDY, M.M. ET AL.: "Targeted Transcriptional Repression Using a Chimeric TALE-SRDX Repressor Protein", PLANT MOL BIOL, vol. 3, no. 78, 29 February 2012 (2012-02-29), pages 311 - 321 *

Cited By (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12006520B2 (en) 2011-07-22 2024-06-11 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US12473573B2 (en) 2013-09-06 2025-11-18 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US12215365B2 (en) 2013-12-12 2025-02-04 President And Fellows Of Harvard College Cas variants for gene editing
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US12398406B2 (en) 2014-07-30 2025-08-26 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US12043852B2 (en) 2015-10-23 2024-07-23 President And Fellows Of Harvard College Evolved Cas9 proteins for gene editing
US12344869B2 (en) 2015-10-23 2025-07-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11999947B2 (en) 2016-08-03 2024-06-04 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US12084663B2 (en) 2016-08-24 2024-09-10 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US12390514B2 (en) 2017-03-09 2025-08-19 President And Fellows Of Harvard College Cancer vaccine
US12435331B2 (en) 2017-03-10 2025-10-07 President And Fellows Of Harvard College Cytosine to guanine base editor
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US12359218B2 (en) 2017-07-28 2025-07-15 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US12406749B2 (en) 2017-12-15 2025-09-02 The Broad Institute, Inc. Systems and methods for predicting repair outcomes in genetic engineering
US12157760B2 (en) 2018-05-23 2024-12-03 The Broad Institute, Inc. Base editors and uses thereof
US12281338B2 (en) 2018-10-29 2025-04-22 The Broad Institute, Inc. Nucleobase editors comprising GeoCas9 and uses thereof
US12351837B2 (en) 2019-01-23 2025-07-08 The Broad Institute, Inc. Supernegatively charged proteins and uses thereof
US12281303B2 (en) 2019-03-19 2025-04-22 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US12473543B2 (en) 2019-04-17 2025-11-18 The Broad Institute, Inc. Adenine base editors with reduced off-target effects
US12435330B2 (en) 2019-10-10 2025-10-07 The Broad Institute, Inc. Methods and compositions for prime editing RNA
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US12031126B2 (en) 2020-05-08 2024-07-09 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Also Published As

Publication number Publication date
CN104178506B (zh) 2017-03-01
CN104178506A (zh) 2014-12-03
TW201723168A (zh) 2017-07-01

Similar Documents

Publication Publication Date Title
WO2015165274A1 (fr) Protéine taler ayant un effet inhibiteur sur la transcription par le biais d'un empêchement stérique, et son application
Orphanides et al. FACT, a factor that facilitates transcript elongation through nucleosomes
Llorca et al. bZIPs and WRKYs: two large transcription factor families executing two different functional strategies
US20160369268A1 (en) Transcription activator-like effector (tale) libraries and methods of synthesis and use
JP7705382B2 (ja) 新規ｃｒｉｓｐｒ ｄｎａターゲティング酵素及びシステム
WO2015165275A1 (fr) Utilisation de répresseur de transcription tale pour la construction modulaire d'une lignée de gènes synthétiques dans une cellule de mammifère
US8252535B2 (en) RNA interference tags
WO2015165276A1 (fr) Kit de réactifs utilisant le répresseur de transcription tale pour la construction modulaire d'une lignée de gènes synthétiques dans une cellule de mammifère
WO2017040786A1 (fr) Circuits de destruction de sécurité génétique multicouche basée sur une seule protéine cas9 et de multiples garn modifiés dans des cellules de mammifère
JP2018082709A5 (fr)
Liu et al. Altered replication in human cells promotes DMPK (CTG) n·(CAG) n repeat instability
CN106544351A (zh) CRISPR‑Cas9体外敲除耐药基因mcr‑1的方法及其专用细胞穿透肽
CA3042880A1 (fr) Inhibiteurs de crispr-cas9
JP2016521554A5 (fr)
US9127283B2 (en) Inducible expression system transcription modulators comprising a distributed protein transduction domain and methods for using the same
Piatek et al. Targeted genome regulation via synthetic programmable transcriptional regulators
Patel et al. Control of multigene expression stoichiometry in mammalian cells using synthetic promoters
Millau et al. CTCF, cohesin, and histone variants: connecting the genome
CA3189185A1 (fr) Represseurs de fusion krab et procedes et compositions pour la repression d'une expression genique
Urlinger et al. The p65 domain from NF-κB is an efficient human activator in the tetracycline-regulatable gene expression system
Tadauchi et al. Utilizing a regulated targeted integration cell line development approach to systematically investigate what makes an antibody difficult to express
CN109207518B (zh) 用于基因转录激活的药物诱导型CRISPR/Cas9系统
CN107245493A (zh) 一种表达受茶碱调控的适体核酶修饰型sgRNA的载体及应用
Gurova Can aggressive cancers be identified by the “aggressiveness” of their chromatin?
Anthony et al. Activation of silenced cytokine gene promoters by the synergistic effect of TBP-TALE and VP64-TALE activators

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15786356

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15786356

Country of ref document: EP

Kind code of ref document: A1