WO2015192379A1 - Procede de synthese de segments tale repetes en vue d'une modification genetique specifique de site - Google Patents

Procede de synthese de segments tale repetes en vue d'une modification genetique specifique de site Download PDF

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WO2015192379A1
WO2015192379A1 PCT/CN2014/080423 CN2014080423W WO2015192379A1 WO 2015192379 A1 WO2015192379 A1 WO 2015192379A1 CN 2014080423 W CN2014080423 W CN 2014080423W WO 2015192379 A1 WO2015192379 A1 WO 2015192379A1
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tale
template
pcr
bsmbi
primer
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PCT/CN2014/080423
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Chinese (zh)
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魏文胜
杨君娇
郭生杰
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北京大学
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates to biological genetic engineering techniques, and in particular to the application of fixed-point modification to genes (especially eukaryotic genes)
  • TALEs Transcription activator like effectors
  • Xanthomonas to activate host gene expression or promote self-community in nature
  • the TALEs protein contains a highly conserved tandem repeat sequence consisting of 33-35 amino acids.
  • the 12- and 13-position amino acids are responsible for recognizing and binding a specific DNA base (such as NI recognition A, HD recognition C, NG recognition ⁇ , NN recognition G and A, etc.).
  • TALEs can specifically bind to target DNA (Boch et al., 2009; Moscou and Bogdanove, 2009). TALEs successfully solved the problem that zinc finger proteins could not recognize any target gene sequence, and the recognition sequence was often affected by upstream and downstream sequences, and it is one of the most effective methods to bring effector proteins to specific nucleic acid sequences ( Bogdanove and Voytas, 2011). ).
  • TALE Tranuclease chimeras
  • Miller et al., 2011 TALE-nuclease chimeras
  • TALE-mediated transcriptional activation of specific genes Miller et al., 2011
  • inhibition Cong et al., 2012; Garg et al., 2012; Mahfouz et al., 2012
  • TALE is fused to a fluorescent protein to indicate the position of the target DNA, etc.
  • any custom TALE typically contains more than ten highly repeating fragments (34 amino acid residues per fragment), the synthesis of its expression vector is difficult.
  • a number of research institutions have used this amino acid to correspond to nucleic acid sequences, modularly assembling TALEs, and specifically binding to the nucleic acid sequence of interest (Brigs et al., 2012; Cermak et al., 2011; Garg Et al., 2012; Huang et al., 2011; Li et al., 2012; Li et al., 2011; Morbitzer et al., 2011; Reyon et al., 2012; Sanjana et al., 2012; Weber et Al., 2011; Zhang et al., 2011; Schmid-Burgk et al., 2013), but there are still various problems such as low efficiency, long cycle time, cumbersome operation, various types of precursor materials or high cost.
  • the Rene Geibler team established a synthetic method called "Golden TALE Technology", which reduced the number of vectors using subclones to produce cohesive ends at specific positions to six, and optimized the cloning vectors between different groups. To achieve a more efficient and accurate method without prolonging the time (Geissler et al., 2011).
  • the Bing Yang team proposed a synthesis method called "modular assembly", which uses dense Degeneracy of the code
  • the BsmBI digestion method allows eight different sticky ends to be obtained for each repeating unit, so the TALE synthesis is based on eight groups of repeating units that differ from each other in position, which improves efficiency to some extent. (Li et al., 2011). All of the above methods have their own advantages and disadvantages, but at the same time, one problem is that it is difficult to achieve mass production of TALE.
  • connection-independent TALE cloning method developed by Schmid-Burgk and colleagues was published in Nature-Biotechnology, which uses longer sticky ends and can be used without connection.
  • the fragments are integrated and more than 600 TALEN genes can be synthesized in one day, provided that 3072 precursors containing five repeat units are synthesized (Schmid-Burgk et al., 2013).
  • the object of the present invention is to provide a method for synthesizing a TALE repeat of a gene (especially a eukaryotic gene), and to provide 512 TALE four repeat unit templates based on the Chinese patent (ZL201210380206.2). And related PCR primers for high throughput production.
  • the 512 TALE four-repeat unit template of the present invention is a uracil-specific excision reagent (USER) manufactured by uracil-specific excision reagent (USER) using a uracil deoxyribonucleotide excision reagent (USER) to make the sticky ends of the DNA fragments, and is used for PCR.
  • the amplified DNA template and primer design were used to synthesize 512 template plasmids containing four TALE repeat units based on 16 monomers containing the TALE repeat.
  • the present invention utilizes 512 template plasmids containing four TALE repeat units to synthesize TALE coding sequences using two technical routes: 1. Amplification of tetrameric templates using a set of immobilized PCR primers containing uracil deoxyribonucleotides Using a uracil excision reagent to make a cohesive end of the DNA fragment, and ligating multiple DNA fragments containing four or fewer TALE repeat units into a TALE protein coding sequence; 2. using a fixed set of BsmBI-containing cleavage sites The PCR primers amplify the tetrameric template, and the multiple fragments are combined into a TALE coding sequence by a B smBI digestion-ligation cycle. After the complete TALE coding sequence of multiple vectors is introduced into the cell, the target gene locus can be specifically combined to complete target gene knockout, knock-in, expression regulation, fluorescent labeling and the like.
  • TALE repeat means that the amino acid sequence of each TALE repeat sequence is the same except for the region of variable di-nucleotides (RVDs).
  • TALE repeat A DNA fragment used to encode a TALE repeat.
  • the present invention utilizes the characteristics of codon degeneracy to design four DNA monomer templates, which are named W, X, Y and Z, respectively. Outside the RVDs coding region, these four sequences differ greatly, but the amino acid sequences they encode are identical.
  • linking monomer a monomer for synthesizing a TALE repeat sequence, which is designed to amplify the above four DNA monomer templates by using 16 pairs of primers designed to obtain a uracil deoxyribonucleotide or A nucleotide sequence containing a BsmBI recognition site.
  • quadruplet the above four linking monomers are sequentially joined to form a quadruplet, which can be used as a PCR template.
  • the present invention pre-assembles 512 quadruplexes based on 16 monomers used to synthesize TALE repeats.
  • the monomer and the quadruplet encode a TALE repeat sequence containing RVDs, and after PCR by the uracil-containing primer, the uracil deoxyribonucleotides are present at both ends; after cleavage by uracil cleavage reagent, adjacent two
  • the body/quadruplex has mutually matching cohesive ends and one-to-one correspondence.
  • the monomer and the quadruplex are subjected to primers containing the BsmBI recognition site without uracil deoxyribonucleotides, and the two segments have a BsmBI cleavage site; after BsmBI digestion, the phase
  • the adjacent two monomers/quadruplex have mutually matching sticky ends and one-to-one correspondence.
  • the monomer of the present invention is the same as the Chinese patent ZL201210380206.2. Except for the intermediate DNA base recognition region, the amino acid sequence of each TALE fragment is the same, so the DNA sequence encoding TALE can be serially and sequentially synthesized.
  • the present invention utilizes the characteristics of codon degeneracy to design four DNA sequences, which are named W, X, Y and Z, respectively. Outside the RVDs coding region, these four sequences differ greatly, but the amino acid sequences they encode are identical.
  • the specific sequences of the W, X, Y and Z types are shown in Table 1 of the Chinese patent ZL201210380206.2.
  • the four repeating units in the quadruplet are WXYZ or XYZW, each with 256 quadruplets, including all possible four RVD combinations.
  • the plasmid vector shown in Fig. 2 conforming to Chinese Patent ZL201210380206.2 can be applied to the method of the present invention.
  • dUTP uracil deoxyribonucleotides
  • dUTP uracil deoxyribonucleotides
  • the nucleotide sequence of the primer containing the BsmBI restriction endonuclease recognition site is shown in Table 1.
  • the underlined sequence is the BsmBI recognition site, and the italicized bold sequence is the sticky end of the BsmBI cut.
  • F-W5, R-W3, R-X3, R-Y3, and R-Z3 are used to link TALE repeats and vectors, and other primers in the table are used to connect the individual TALE repeats.
  • the invention is based on the Chinese patent ZL201210380206.2, using TALE repeat monomer as a template, using uracil-containing primers for PCR, USER enzyme cleavage, T4 DNA ligase ligation, pre-assembled 512 tetramer templates .
  • there are 256 tetramers of WXYZ structure and XYZW structure, covering TALE repeat sequences for all four-base DNA fragments see Table 2 and Table 3). Table 2 Tetramer of WXYZ structure
  • T, C, and G indicate the DNA bases bound by each TALE monomer in the tetramer, as shown in Table 3.
  • All tetramers in the table are XYZW structures, that is, the first monomer is the X structure, the second is the Y structure, and the rest is analogous.
  • two different techniques can be used to construct complete TALE repeats: (1) using uracil deoxyribonucleotides Primer PCR amplification of the tetrameric template, and continued construction of sticky ends between different tetramers using uracil deoxyribonucleotide-specific cleavage, constructing complete repeats by BsmBI digestion-ligation cycle; (2) The tetramer template was PCR-amplified using BsmBI-recognizing site primers, the cohesive ends were digested by BsmBI digestion, and the complete repeats were constructed by BsmBI digestion-ligation cycle.
  • the primers used in the construction of the TALE sequence with the same number of repeating units are fixed, and therefore are very suitable for large-scale, high-throughput TALE construction.
  • the time, cost and sequencing mutation rate of the TALE sequence of the present invention are also lower than the previous Chinese patent ZL201210380206.2.
  • Using the first technical route to construct a TALE sequence specifically includes the following steps (see Figure 3):
  • the primer pair shown by the shade is used to amplify the most downstream TALE repeat, in which the BsmBI cleavage site carried by the R primer matches the sticky end to the vector, and the repeat length may be 1/2/3/4 Repeat the unit, and the remaining primer pairs are amplified into fragments containing four repeating units.
  • PCR amplification reaction The reaction can only be carried out using PfuTurbo Cx (Agilent) or Taq polymerase, 30 cycles (95 ° C, 30 s denaturation; 60 ° C, 30 s annealing; 72 ° C, 30 s extension).
  • the fourth step of the ligation product can be purified by ordinary PCR products. If the vector also carries ampicillin resistance, the ligation product in the fourth step is subjected to agarose gel electrophoresis and recovered.
  • the use of the second technical route to construct the TALE sequence specifically includes the following steps (see Figure 4):
  • PCR amplification reaction Commonly used high-fidelity PCR enzymes can be used in the PCR amplification reaction of the second route. Take TaqHiFi enzyme (Transgen) as an example, the reaction is 30 cycles (95 C, 30 s denaturation; 60 C, 30 s annealing; 72 ° C, 30 s extension).
  • the ligation product of the third step only needs to be purified by ordinary PCR products; if the vector is only resistant to ampicillin, the ligation product in the third step is subjected to agar. Glycogel electrophoresis and recovery.
  • the general steps of the second technical route are similar to the first one, except that (1) the primers used in the PCR are the same as the first route except for the primers of the ligation vector, and the other primers are all containing the BsmBI site. GG version (see Table 1); (2) PCR enzyme has no special requirements, common high-fidelity enzymes can be; (3) no need to use USERTM enzyme treatment, PCR reaction directly after purification of mixed PCR products.
  • the present invention retains the advantages of the TALE repeating unit having a long adhesive end, high connection accuracy, easy expansion, simple steps, high efficiency and rapidity in the Chinese patent ZL201210380206.2.
  • the TALE sequence is mainly connected by a fragment containing four repeating units, requiring fewer fragments and a lower error rate.
  • each PCR template structure and primer are fixed, and it is not necessary to separately design a template and a primer pair for each TALE, and is more suitable for high-throughput, automated operation than the Chinese patent ZL201210380206.2.
  • Figure 1 is a diagram of the pIRES2-EGFP-TALEN vector
  • Figure 2 is a diagram of the pGL3 - TALE- Venus carrier
  • FIG. 3 is a flow chart of constructing a TALE sequence by using the first technical route of the present invention
  • FIG. 5 is a PCR result of a TALEN fragment according to Embodiment 1 of the present invention.
  • Figure 6 shows the results of treatment with diphtheria toxin by wild type and HBEGF knockout cells.
  • Example 1 Using TALENs to achieve knockout of HBEGF gene in human cells
  • the human HBEGF gene also known as DTR3 ⁇ 4, is a receptor for diphtheria toxin in host cells. After the gene is knocked out, the host cell should be completely resistant to diphtheria toxin.
  • the exon sequence information of the human HBEGF gene was searched in the NCBI (http://www.ncbi.nlm.nih.gov/gene/) database, and the TALENs used were designed for the sequence. This region is located at positions 81-126 of the second exon of the HBEGF gene.
  • the sequence is as follows:
  • the uppercase letters are TALENs combined with the lowercase letters and the lowercase letters are Spacer.
  • the required synthesis TALE is CCCACTGTATCCACG, CCCGGCCGCCTCCTA.
  • the Spacer region contains a PvuII restriction site (underlined).
  • the TALE repeat region to be synthesized can be split into four four-unit segments: CCCA CTGT ATCC ACG; CCCG GCCG CCTC CTA.
  • the DNA marker followed by the left to right, is the four fragments of TALEN-L and TALEN-R. Two different TALEN fragments were separately mixed, treated with USER enzyme, and the PCR product was purified using a PCR product purification kit (Transgen).
  • the ligation product and the pIRES2-EGFP-TALEN vector were treated with BsmBI endonuclease and T4 ligase, and then transformed into E. coli competent DH50 PCR to identify the recombinant, and the plasmid was extracted and verified by sequencing.
  • This experiment used HeLa cells cultured in DMEM (Invitrogen) medium. Transfect 0.9 upstream TALEN, 0.9 downstream TALEN, and 0.2 eGFP plasmid to 1 ⁇ using the AAD-1001S Nucleofector II ( Lonza ) electrocycler
  • the cells were cultured at 37 ° C for 24 hours (C 0 2 5% ), and then transferred to 30 ° C for further 72 hours. Subsequently, the cells were cultured in a medium containing 2 ⁇ ⁇ / ⁇ 1 puromycin for 48 hours at 37 ° C, and the cells not transfected into the plasmid were killed, and the next detection was carried out.
  • the genomic DNA of the HeLa cells obtained in the previous step was extracted, and primers were designed for genomic PCR amplification.
  • TALEN binding region The genomic DNA of the HeLa cells obtained in the previous step was extracted, and primers were designed for genomic PCR amplification.
  • Upstream primer GTGGCCGCCGCTTCGAAAGTGAC
  • Downstream primer GTCCAAGGATGGGGGGCCTCCA; annealing temperature 65 °C, product length 503 bp, and only contains a PvuII restriction site in Spacer, and digested to produce two fragments of 104 bp and 399 bp.
  • PvuII TAKARA
  • agarose gel electrophoresis determined that the probability of fragment deletion in the designed Spacer region was more than 50%.
  • HeLa cells were cultured using a medium containing 50 ng/ml of diphtheria toxin (this concentration was 5 times the lethal concentration of diphtheria toxin in HeLa cells), and the resistant phenotype was observed. The results are shown in Fig. 6.
  • A is the result of toxin treatment of wild-type HeLa cells, and all the cells are dead.
  • B is the result of treatment of HeLa cells transfected with HBEGF TALEN, and there are many healthy cells. Cells successfully knocked out for HBEGF.
  • telomere structure A highly repetitive sequence in human telomeres was found: TTAGGGTTAGGG...TTAGGG, TALE- Venus fusion fluorescent protein with target site TAGGGTTAGGGTTAGG was prepared to indicate telomere structure.
  • the TALE sequence to be synthesized contains 16 repeating units and can be separated into four tetramers of TAGG GTTA GGGT TAGG. According to Table 5, the structure of the four tetramers and the desired primers were found, and the primers with the GG type in Table 1 were used, and the specific templates were determined according to Table 2 and Table 3. The results are shown in Table 8. Table 8 TALE- Venus template, primer selection table
  • the PCR amplification was carried out by TaqHiFi, and the PCR products were mixed and subjected to agarose electrophoresis, and the 400 bp band obtained by PCR was removed and purified.
  • the purified fragment and the pGL3-TALE-Venus vector were co-transformed into a BsmBI digestion-ligation cycle, transformed into Trans-1-T1 E. coli competent (Transgen), coated with an ampicillin plate, and cultured overnight at 37 °C.
  • the method for synthesizing a TALE repeat sequence for gene (especially eukaryotic gene) site-directed modification disclosed in the present invention uses a uracil deoxyribonucleotide excision method to produce a sticky end of a DNA fragment at both ends of a DNA fragment by using a uracil excision reagent. Based on the DNA template amplified by P CR and the design of the primers, 512 template plasmids containing four TALE repeat units were synthesized on the basis of 16 monomers containing TALE repeats, which can be used for high-throughput production.

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Abstract

La présente invention concerne un procédé de synthèse à haut rendement de segments répétés TALE en vue d'une modification génétique spécifique de site, qui fournit 512 modèles d'unités TALE en quatre exemplaires et des amorces de PCR correspondantes, et peut être utilisé à des fins de production à haut rendement. Dans la présente invention, une séquence de codage TALE est synthétisée au moyen de 512 plasmides de matrice comprenant quatre unités TALE répétées, et à l'aide de deux moyens techniques : 1) l'expansion d'une matrice de tétramères au moyen d'un ensemble d'amorces de PCR fixes comprenant l'uracil désoxyribonucléotide, la fabrication d'extrémités de queue adhésives à deux extrémités de segments d'ADN à l'aide d'un réactif d'excision d'uracile, et la liaison de multiples segments d'ADN comprenant une quantité inférieure ou égale à quatre unités TALE répétées pour former une séquence de codage de protéine TALE; et 2) l'expansion de la matrice de tétramères à l'aide d'un ensemble d'amorces de PCR fixes comprenant des sites de coupe d'enzyme BsmBI, et la combinaison de multiples segments pour former une séquence de codage TALE au moyen d'un cycle de coupe et de liaison d'enzyme BsmBI. Après l'introduction dans des cellules des séquences de codage TALE entièrement liées à de multiples supports, des applications de knock-out, de knock-in, d'expression et de régulation, de génération de fluorescence et analogue de gènes cibles sont mises en oeuvre par une liaison spécifique à des loci de gènes cibles.
PCT/CN2014/080423 2014-06-20 2014-06-20 Procede de synthese de segments tale repetes en vue d'une modification genetique specifique de site WO2015192379A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102864158A (zh) * 2012-09-29 2013-01-09 北京大学 用于基因定点修饰的tale重复片段的高效合成方法
CN103146735A (zh) * 2012-12-28 2013-06-12 西北农林科技大学 Tale重复单元四聚体库的构建方法、talen表达载体的构建方法及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102864158A (zh) * 2012-09-29 2013-01-09 北京大学 用于基因定点修饰的tale重复片段的高效合成方法
CN103146735A (zh) * 2012-12-28 2013-06-12 西北农林科技大学 Tale重复单元四聚体库的构建方法、talen表达载体的构建方法及其应用

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