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CRISPR/Cas9 system-containing targeted knockout vector and adenovirus and applications thereof

A knockout carrier and targeting technology, applied in the field of expression system, can solve problems such as gene modification and achieve high knockout rate

Inactive Publication Date: 2014-08-27
重庆高圣生物医药有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

but not for accurate modification of certain genes

Method used

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  • CRISPR/Cas9 system-containing targeted knockout vector and adenovirus and applications thereof
  • CRISPR/Cas9 system-containing targeted knockout vector and adenovirus and applications thereof
  • CRISPR/Cas9 system-containing targeted knockout vector and adenovirus and applications thereof

Examples

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Embodiment 1

[0023] 1. Construction of Crispr / Cas9 adenoviral vector

[0024] (1) The pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Addgene plasmid ID: 42230) was digested with EcoRI and SacII, and the digested product was subjected to 1% agarose electrophoresis, and the fragment containing hSpCas9 was recovered and named U6 -Chimeric_BB-CBh-hSpCas9.

[0025] (2) The pAdTrack-CMV adenoviral vector was digested with BstXI to remove the CMV promoter and GFP fragment, and the digested product was subjected to 1% agarose electrophoresis to recover the vector backbone, which was named pAdTrack vector backbone.

[0026] (3) The recovered product obtained in step (1) and the vector skeleton obtained in step (2) were filled with klenow enzyme, and then ligated with T4 ligase overnight at 16°C to obtain a recombinant vector, which was named pAdTrack-U6- Chimeric_BB-CBh-hSpCas9.

[0027] The obtained recombinant vector pAdTrack-U6-Chimeric_BB-CBh-hSpCas9 was transformed into competent Escherichia coli...

Embodiment 2

[0069] Example 2. Adenovirus-mediated CRISPR / Cas9 targeted knockout of c-myc gene mice

[0070] 1. Construction of adenovirus expression vector

[0071] Using the online tool ZiFiT Targeter version4.0, design 3 CRISPR / Cas9 targets on the second exon of the c-myc gene (GenBank: AH005318.1), and design the corresponding oligonucleotides, specifically:

[0072] The first target is T4, the nucleotide sequence is 5'-tcgctacgtccttctcccca-3' (SEQ ID NO.12); the oligonucleotide pair designed by it is 5'-caccgtcgctacgtccttctcccca-3' (SEQ ID NO.13 ); 5'-aaactggggagaaggacgtagcgac-3' (SEQ ID NO. 14);

[0073] The second target is T5, the nucleotide sequence is 5'-gcagccgcccgcgcccagtg-3' (SEQ ID NO.15); the oligonucleotide pair designed for it is: 5'-caccgcagccgcccgcgcccagtgg-3' (SEQ ID NO. 16); 5'-aaaccactgggcgcgggcggctgcg-3' (SEQ ID NO.17);

[0074] The third target is T6, the nucleotide sequence is 5'-cagatgatgaccgagttact-3' (SEQ ID NO.18); the oligonucleotide pair designed for it is...

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Abstract

The invention discloses a CRISPR / Cas9 system-containing targeted knockout vector and adenovirus and applications thereof. The targeted knockout vector is prepared through the following steps: after a pX330 U6-Chimeric_BB-CBh-hSpCas9 plasmid is subjected to enzyme digestion and filling-in by using EcoRI and SacII, connecting the pX330 U6-Chimeric_BB-CBh-hSpCas9 plasmid with a pAdTrack-CMV plasmid subjected to enzyme digestion and filling-in by using BstXI; after the obtained product is linearized by using BbsI, connecting the obtained product to a specific target sequence of a desired gene; and after the obtained object is linearized by using PmeI and dephosphorylated by using CIAP alkaline phosphatase, recombining the obtained product with a pAdEasy-1 plasmid. The targeted knockout vector can mutate gene sequences in target sequence areas, and the mutation rate is high, up to 30.6-45.8%, therefore, the targeted knockout vector can be used for gene site-directed mutation, and lays a foundation for gene therapy.

Description

technical field [0001] The invention belongs to the field of expression systems, and in particular relates to a targeted knockout vector containing a CRISPR / Cas9 system, an adenovirus and applications thereof. Background technique [0002] The CRISPR / Cas9 system is an acquired immune system that exists in bacteria and archaea to eliminate foreign nucleic acids or phages, and leave a small gene that can express the genome sequence of the invading virus at the CRISPR site in its own genome. Molecular RNA that protects bacteria and archaea from viruses. When bacteria and archaea containing the CRISPR / Cas9 system are infected with viruses, CRISPR RNA can bind to the viral genome through complementary sequences and express CRISPR-related enzymes. Since CRISPR-related enzymes are nucleases, they can cut viral DNA molecules, thereby preventing virus replication. . Since 2013, researchers have published a number of articles introducing the CRISPR / Cas9 system in journals such as Sc...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N7/01A61K48/00A61P35/00
Inventor 周勇温平申友锋杨晓芳李建均何久香王蜀英牟浪陈小雨
Owner 重庆高圣生物医药有限责任公司
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