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Transcription activator-like effector nuclease (TALEN) target gene editing and optimizing method taking IDLV as template

A technology that targets genes and optimizes methods. It is applied in biochemical equipment and methods, genetic engineering, and plant gene improvement. It can solve the problems of difficult clone acquisition and screening, unsatisfactory results, and low efficiency of homologous recombination. Safety, the effect of not easy to insert mutagenesis

Active Publication Date: 2014-06-25
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the existing introduction methods can be applied to most cell types, the problems of cytotoxicity and low efficiency of homologous recombination during the operation make it difficult to obtain and screen clones, especially in cells with low transfection efficiency such as The effect of human pluripotent stem cells is very unsatisfactory (Giudice, A. and A. Trounson, Genetic modification of human embryonic stem cells for derivation of target cells. Cell Stem Cell, 2008. 2(5): p. 422-33.)

Method used

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  • Transcription activator-like effector nuclease (TALEN) target gene editing and optimizing method taking IDLV as template
  • Transcription activator-like effector nuclease (TALEN) target gene editing and optimizing method taking IDLV as template
  • Transcription activator-like effector nuclease (TALEN) target gene editing and optimizing method taking IDLV as template

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 An optimization method for TALEN targeted gene editing using IDLV as a template

[0036] (1) TALEN design and construction: According to the target sequence for gene editing, the upstream and downstream specific TALENs were respectively designed through the online TALEN design website (https: / / tale-nt.cac.cornell.edu / ). The TALEN assembly plasmid kit was purchased from Addgene (CAT. #1000000016). The TALEN mammalian expression plasmid can be obtained by transforming the TALEN yeast expression plasmid pTAL3 or pTAL4 in the kit. The TALEN construction method was carried out according to the kit instructions and literature reports (Cermak, T., et al., Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res, 2011. 39 (12): p. e82.);

[0037] (2) Construction of lentiviral plasmid template: Obtain homologous sequences of about 500 bp on both sides of the TALEN cut site of the target sequenc...

Embodiment 2

[0056] Example 2 Targeted gene editing optimization method of TALEN using IDLV as template for site-directed mutation of SMN2 gene

[0057] Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease, clinically manifested as progressive degeneration of motor neurons in the anterior horn of the spinal cord, muscle weakness, atrophy, etc., and the incidence rate of newborns is about 1 / 10000. SMN1 gene defect is an important cause of SMA. The SMN2 gene is highly homologous to the SMN1 gene. Mutation of the sixth nucleotide T in exon 7 of the SMN2 gene to C can change the splicing site of the SMN2 gene transcription product, allowing the SMN2 gene to express the full-length SMN protein. Compensate for the SMN1 gene defect and play a role in disease treatment.

[0058] First, based on the SMN2 gene sequence, design the upstream and downstream TALEN target sequences (SEQ ID No: 1-2) that can specifically cause DNA double-strand breaks near the 6th nucleotide ...

Embodiment 3

[0059] Example 3 The TALEN targeted gene editing optimization method using IDLV as a template was used to carry out targeted correction of WAS gene mutation sites.

[0060] IVS6+5 G>A is a common gene mutation site in patients with WAS syndrome, located at the fifth nucleotide of the splicing donor site in intron 6 of the WAS gene. The point mutation at this position causes abnormal splicing of WAS transcripts, resulting in functional defects of WAS protein. Using targeted gene editing technology to correct this gene mutation will hopefully cure patients with WAS syndrome.

[0061] First, based on the WAS gene sequence, design TALENs (SEQ ID No: 11-12) that can specifically cause DNA double-strand breaks near IVS6+5, and use the Survey test to verify and evaluate the shearing efficiency of TALENs. Then design a homologous sequence (SEQ ID No: 13-14) based on the wild-type gene sequence near the IVS6+5 site of the WAS gene, construct a lentiviral plasmid template, an...

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Abstract

The invention provides a transcription activator-like effector Nuclease (TALEN) target gene editing and optimizing method taking IDLV as a template. A homologous recombination template is inserted into a lentiviral vector, the intracellular delivery efficiency of the homologous recombination template is improved by using efficient transduction efficiency of lentivirus, so as to improve the efficiency of a TALEN target gene editing technique. The lentivirus is packaged by using packaging plasmids with integrase defect, the lentiviral vector containing the template is not integrated into a chromosome, insertion mutation is avoided and the safety of the target gene editing technique is ensured. A complete set of TALEN target gene editing and optimizing method in which the lentivirus with integrase defect is used as an intracellular import tool of the homologous recombination template is provided. Different TALENs and homologous recombination template sequences are designed according to the research targets by the method, and efficient target editing of any gene sequence is expected to be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a novel optimization method for efficient TALEN targeted gene editing using an integrase-deficient lentiviral vector (IDLV) as a template. technical background [0002] Targeted gene editing technology can cause site-specific gene knockout or gene correction. It is a powerful tool for modifying the genome of cells and has important significance in both basic research and clinical applications. Transcription activator-like effector (Transcription Activator-like Effector, TALE) is derived from the plant pathogen Xanthomonas, which can use its central domain to specifically bind to DNA targets in the nucleus, and activate transcription through the transcription activation domain . In 2009, Professor Bogdanove of Iowa State University and Professor Bonas of Halle-Wittenberg University in Germany published articles almost simultaneously, announcing that they had cracked the code of TALE b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867
Inventor 黄河王颖佳倪景宽
Owner ZHEJIANG UNIV
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