Two-RVD (repeat variant diresidue) unite module library for efficient construction of TALEN (transcription activator-like effectors nuclease) and TALEN construction method

A unit module and module library technology, applied in the field of genetic engineering, can solve the problems of increasing experimental consumption, prolonging the construction period, and limiting the high-throughput application of TALEN technology, so as to shorten the construction time, simplify the construction steps, and reduce the experimental consumption. Effect

Active Publication Date: 2016-09-21
UNIV OF ELECTRONIC SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The complete TALEN assembly process had to be split into 2 independent stages, which took about 5 working days
This undoubtedly increases the experimental consumption, prolongs the construction cycle, and greatly limits the high-throughput application of TALEN technology

Method used

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  • Two-RVD (repeat variant diresidue) unite module library for efficient construction of TALEN (transcription activator-like effectors nuclease) and TALEN construction method
  • Two-RVD (repeat variant diresidue) unite module library for efficient construction of TALEN (transcription activator-like effectors nuclease) and TALEN construction method
  • Two-RVD (repeat variant diresidue) unite module library for efficient construction of TALEN (transcription activator-like effectors nuclease) and TALEN construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The basic vector construction and detection of the double RVD unit library of embodiment 1

[0036] By means of artificial synthesis (commissioned by GenScript Biotechnology Co., Ltd.), the modular sequences of 144 double RVD units (SEQ ID No.1D01-AA to SEQ ID No.144D09-TT) were fully synthesized and constructed into On the pUC57 vector (purchased from GenScript), a double RVD unit module plasmid vector ( figure 1 ). A series of plasmid vectors were introduced into Escherichia coli DH5a strain by heat shock method to obtain a double RVD unit module library, and the double RVD unit region of each plasmid was amplified by colony PCR to verify the accuracy of the library. Taking the 16 carriers from D04-AA to D04-TT in column D04 as an example, the verification method is introduced. Using the D04-AA bacterial liquid as a template, and the corresponding oligonucleotides M13F and M13R as upstream and downstream primers, the following PCR system was established: 10×Taqbuffe...

Embodiment 2

[0042] Embodiment 2 Method and rules for assembling TALEN based on dual RVD unit modules

[0043] When using double RVD unit modules to organize TALENs, first select the target sequence (generally 15-20 bp in length), then design the corresponding RVD, and then select from the double RVD unit module library, single RVD module library, and the last RVD backbone expression vector. Select an appropriate library vector and construct it according to the GoldenGate reaction.

[0044]The double RVD unit module is designed according to the rules of RVD recognition nucleotides and all possible positions. For example, the NI-NI double RVD unit can recognize AA nucleotides. When the AA base is located at the first and second positions of the target sequence, its corresponding The RVD module carrier is D01-AA:NI-NI(D01-01-AA); when the AA base is located at the third and fourth positions of the target sequence, the corresponding RVD module carrier is D02-AA:NI-NI(D02 -01-AA); and so on, ...

Embodiment 3

[0051] Example 3 Assembly and activity detection of plant endogenous gene-directed modification TALEN vector based on double RVD unit module

[0052] 1. Design and assembly of TALEN vectors for targeted modification of plant endogenous genes

[0053] In order to test the efficiency of directional splicing and mutation of plant endogenous genes using double RVD unit modules, rice varieties Nipponbare's OsDEP1 (GenBank NO.: FJ039904), OsBADH2 (GenBank NO.: KT993490), OsCKX2 (GenBank NO. : AB205193) gene and the wheat TaMLO (GenBank NO.: KF009556) gene were used as target genes, and 4 pairs of TALEN were designed to specifically cut their corresponding DNA target sequences. Target gene name, target site DNA sequence (SEQ ID No.177~184), corresponding TALEN name, RVD number, RVD sequence, and the corresponding dibody, monomer and last RVD that should be selected in the double RVD unit library Plasmid vector numbers are listed in Table 2.

[0054] Table 2 Construction of target g...

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Abstract

The invention belongs to the technical field of genetic engineering and relates to a two-RVD (repeat variant diresidue) unite module library for efficient construction of TALEN (transcription activator-like effectors nuclease). The TALEN assembly strategy of the two-RVD unite is redesigned aiming to overcome various shortcomings of the existing GG (golden gate)-Vector TALEN assembly method. The two-RVD unite module library comprises individually-packed 144 two-RVD unite modules, 8 one-RVD unite modules and 24 end RVD unites. The two-RVD unite module library based on Golden Gate cloning method can construct TALEN expression vectors targeted against 15-19bp arbitrary DNA sequences through primary reaction.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a double RVD (repeated variation double residue) unit module library for efficient construction of TALEN (transcription activator-like effector nuclease) and a TALEN construction method. Background technique [0002] Genome editing technology refers to a technology for genetic modification such as site-directed mutation, site-directed integration, and site-directed replacement for specific sequences in the genome. Using genome editing technology to introduce gene sequence changes in situ in the genome provides convenience for studying gene functions. In terms of application, the use of genome editing technology to create new varieties of animals and plants can avoid expression uncertainty and damage to the original genome caused by random insertion of genes in existing transgenic technologies. With the advent of targeted nuclease technology, the precise targeted knocko...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N15/82C40B40/06C40B40/02
CPCC12N15/113C12N15/63C12N15/8216C12N2310/10C12N2800/80C40B40/02C40B40/06
Inventor 郑雪莲张勇邓科君仲昭辉
Owner UNIV OF ELECTRONIC SCI & TECH OF CHINA
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