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Method for building TALE (transcription activator-like effector) repeated sequences

A repeat sequence and tandem repeat sequence technology, applied in the field of molecular biology, can solve the problems of limited fragment length, limited rapid and routine construction of TALE, and complex condition control.

Active Publication Date: 2013-12-04
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the Golden Gate method is a one-step method for enzyme digestion and ligation, and the condition control is strict and complicated, which requires a long period of exploration and adjustment, and the efficiency and success rate need more practice and time testing
In addition, the length of fragments that can be connected based on one-step ligation is limited, and the efficiency of obtaining more than 10 repeat units is very low
Although more repetitions can be obtained through step-by-step connection, it also increases the difficulty of experimental operation
The existence of these problems limits known methods to construct TALE rapidly and routinely

Method used

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  • Method for building TALE (transcription activator-like effector) repeated sequences
  • Method for building TALE (transcription activator-like effector) repeated sequences
  • Method for building TALE (transcription activator-like effector) repeated sequences

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1 constructs a single side unit carrier

[0046] First, artificially synthesized DNA fragments (sequences listed in Table 1) encoding five kinds (classified according to the type of RVD) of the side unit sequences selected by the present invention based on the isotailase site and the typical TALE repeat unit coding sequence. The codons used by each side unit need to be carefully selected in advance to reduce the DNA sequence similarity between each side unit as much as possible. Simultaneously, since there are at least 3 possibilities, such as A, D, and E, for the amino acid residue at the +4 position in the natural repeating unit, the 15 sequence variants listed in this example are derived from five side units. Next, the five DNA fragments (15 variants) were amplified by PCR. The upstream primers are: Afwd:5'-ACTAGTAATATTGGTGGCAAACAGGCTCTTG-3'(SEQ ID No.39), Tfwd:5'-ACTAGTAATGGGGGTGGCAAACAGGCTCTTG-3'(SEQ ID No.40), Cfwd:5'-ACTAGTCATGACGGTGGCAAACAGGC TCTTG-3...

Embodiment 2

[0051] Example 2 Construction of a double side unit vector and a side unit tandem repeat vector containing n repeats

[0052] In order to construct a double side unit carrier, according to the two specified nucleotides (which can be the same or different) to be recognized, select two corresponding single side unit vectors, and use NheI+HindIII to carry out double enzymes on the carrier that recognizes the 5' end base The carrier that recognizes the base at the 3' end is digested with SpeI+HindIII, and then the two DNA fragments containing the sequence of the side unit are connected to obtain a double side unit vector with the side unit arranged in series ( image 3 c in and image 3 in d). Select five basic single side unit carriers that recognize the four nucleotides of A, T, C, and G, and connect them in pairs. A total of 25 double side unit combinations can be obtained, which can identify all 16 possible dual cores. combination of nucleotide target sites. Using a similar...

Embodiment 3

[0053] Example 3 Using the "unit assembly" method to construct a paraunit tandem repeat vector that recognizes the target sequence of the zebrafish endogenous gene tnikb

[0054] Through the analysis, a target site located in the zebrafish gene tnikb was found, which was located at the junction of the first intron and the second exon of tnikb. Since the FokI cleavage domain needs to cut DNA in the form of a dimer, when using TALEN for gene targeting, it is necessary to design two TALE binding sites on the left and right, and the length of the spacer in the middle is generally 12-21bp. In this embodiment, the length of the left TALE binding site is 15bp, the right side is 16bp, and the interval between the two binding sites is 15bp ( Image 6in a). The sequence of the left binding site is 5'-GTTATTTTCTCCCCT-3' (SEQ ID No.37). The steps of constructing the TALE repeat sequence combined with the above-mentioned sites are as follows: first step, use the double side unit carrier...

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Abstract

The invention discloses a side unit used for building TALE (transcription activator-like effector) repeated sequences. The side unit is a repeated unit DNA (deoxyribonucleic acid) segment containing isocaudarner or different flat terminase identification sites at two ends, and the preated unit DNA segment codes repeated units of RVD (repeated variable di-residue) containing NI, NG, HD, NK or NN or variants of the repeated units, wherein in the 5'end isocaudarner or flat terminase identification sites, the 3' end of the identification sites at least has one nucleotide participating in the amino acid coding the N end of the side unit; and in the 3' end isocaudarner or flat terminase identification sites, the 5' end of the identification sites at least has one nucleotide participating in the amino acid coding the C end of the side unit. The side unit has the advantages that the TALE repeated sequences containing any repeated unit number and any ranging sequences, plasmid vectors containing TALE repeated sequences, coding TALE protein DNA combined structural domains and various derived fusion protein plasmid vectors can be conveniently built.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a method for constructing TALE repeat sequences. Background technique [0002] The site-directed modification of endogenous genes is very attractive for both basic biological research and clinical treatment. Although the emergence of artificial zinc finger nuclease (zinc finger nuclease) has greatly promoted the genome-targeted modification technology, it is still a considerable technical problem to screen zinc finger proteins that can efficiently and specifically bind to specific DNA sequences. The transcription activator-like effector (TALE) from the plant pathogen Xanthomonas can infect plant hosts, regulate the expression of host plant endogenous genes by identifying specific DNA sequences, reduce the host's resistance, and increase its susceptibility ( figure 1 ). It is currently known that the TALE family has more than 100 gene members (Boch, J. & Bonas, U., 2010, Annu Rev P...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12R1/64
Inventor 黄鹏张博林硕肖安
Owner PEKING UNIV
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