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Linearized CRISPR-CAS9 lentiviral vector-based gene knockout kit and application thereof

A technology of lentiviral vectors and kits, applied in applications, genetic engineering, plant genetic improvement, etc., can solve problems such as lack of kits, achieve the effects of eliminating inconsistencies, avoiding differences in connection efficiency, and reducing costs

Inactive Publication Date: 2018-01-19
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] CRISPR-Cas9-mediated genome editing technology has become a research hotspot due to its simple operation and high specificity, but there is still a lack of commercialized kits for the operation of gene editing technology. Therefore, the research and development is accurate, reliable, simple and practical, suitable for various The CRISPR-CAS9 lentiviral vector gene knockout kit operated by researchers is an urgent need at present. The present invention combines the high-efficiency transfection characteristics of lentiviral vectors and the efficient gene editing characteristics of CRISPR-CAS9 technology to solve important problems in this field. problem

Method used

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  • Linearized CRISPR-CAS9 lentiviral vector-based gene knockout kit and application thereof
  • Linearized CRISPR-CAS9 lentiviral vector-based gene knockout kit and application thereof
  • Linearized CRISPR-CAS9 lentiviral vector-based gene knockout kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 (CRISPR-CAS9 lentiviral vector gene knockout with puromycin resistance constructed by common cloning and recombination method, taking SNX10 gene as an example)

[0054] 1. Mouse SNX10 gene sgRNA design:

[0055] (1) Design the positive and antisense strand primer sequences of the SNX10 gene:

[0056] sgRNA1F: AAACGCAACGCATTACTTTGGAGTC SEQ ID No.1

[0057] sgRNA1R: CACCGCACGTGGATCAGCGTCGCCA SEQ ID No. 2

[0058] sgRNA2F: CACCGCACGTGGATCAGCGTCGCCA SEQ ID No.3

[0059] sgRNA2R: AAACTGGCGACGCTGATCCACGTGC SEQ ID No.4

[0060] (2) The linearized CRISPR-CAS9 lentiviral vector with puromycin resistance described in the kit;

[0061] (3) T4 ligase and T4 ligase buffer;

[0062] (4) High transformation activity Stbl3 competent;

[0063] 2. Recombination of CRISPR-CAS9 lentiviral cloning vector with puromycin resistance

[0064] (1) Anneal the sense strand and the antisense strand to form double strands: take equal amounts of the sense strand and the antisense str...

Embodiment 2

[0080] Example 2 (CRISPR-CAS9 lentiviral vector gene knockout with puromycin resistance constructed by common cloning and recombination method, taking HSF1 gene as an example)

[0081] 1. Human HSF1 gene sgRNA design:

[0082] (1) Design the positive and antisense strand primer sequences of the HSF1 gene:

[0083] sgRNA1F: TCGTGAGCGACCCGGACACCGTTTT SEQ ID No.5

[0084] sgRNA1R: GGTGTCCGGGTCGCTCACGACGGTG SEQ ID No. 6

[0085] sgRNA2F: CAGCTTCCACGTGTTCGACCGTTTT SEQ ID No. 7

[0086] sgRNA2R: GGTCGAACACGTGGAAGCTGCGGTG SEQ ID No.8

[0087] sgRNA3F: GGAGTCAATGAGGGCGGTCGGTTTT SEQ ID No. 9

[0088] sgRNA3R: CGACCGCCCTCATTGACTCCCGGTG SEQ ID No.10

[0089] (2) The linearized CRISPR-CAS9 lentiviral vector with puromycin resistance described in the kit;

[0090] (3) T4 ligase and T4 ligase buffer;

[0091] (4) High transformation activity Stbl3 competent;

[0092] 2. Recombination of CRISPR-CAS9 lentiviral cloning vector with puromycin resistance

[0093] (1) Anneal the sense st...

Embodiment 3

[0109] Example 3 (CRISPR-CAS9 lentiviral vector gene knockout with puromycin resistance constructed by common cloning and recombination method, taking PKA gene as an example)

[0110] 1. Rat PKA gene sgRNA design:

[0111] (1) Design and obtain the positive and antisense strand primer sequences of the PKA gene:

[0112] sgRNA1F: AACGCCGCCGCCGCCAAGAAGTTTT SEQ ID No.11

[0113] sgRNA1R: TTCTTGGCGGCGGCGGCGTTCGGTG SEQ ID No.12

[0114] sgRNA2F: CCTCCCAATCCGCCGTAAGTGTTTT SEQ ID No. 13

[0115] sgRNA2R: ACTTACGGCGGATTGGGAGGCGGTG SEQ ID No.14

[0116] sgRNA3F: CGATCTGCGCCGCGTAGAAAGTTTT SEQ ID No. 15

[0117] sgRNA3R: TTTCTACGCGGCGCAGATCGCGGTG SEQ ID No. 16

[0118] (2) The linearized CRISPR-CAS9 lentiviral vector with puromycin resistance described in the kit;

[0119] (3) T4 ligase and T4 ligase buffer;

[0120] (4) High transformation activity Stbl3 competent;

[0121] 2. Recombination of CRISPR-CAS9 lentiviral cloning vector with puromycin resistance

[0122] (1) Anneal the...

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Abstract

The invention discloses a linearized CRISPR-CAS9 lentiviral vector-based gene knockout kit. The kit comprises a linearized CRISPR-CAS9 lentiviral vector and a corresponding T4 ligase as well as competent cells; the kit is characterized in that the linearized CRISPR-CAS9 lentiviral vector is formed by a circular vector treated by using endonuclease; the T4 ligase is optimized T4 ligase; the competent cells are Stbl3 competent cells having the characteristic of inhibiting recombination. The kit can be directly used for constructing the CRISPR-CAS9 lentiviral vector; compared with commercial kitsat home and abroad, the linearized CRISPR-CAS9 lentiviral vector-based gene knockout kit is convenient to operate and rapid in construction and recombination, can effectively increase the current construction efficiency based on the CRISPR-CAS9 lentiviral vector, and reduces the cost of gene knockout.

Description

technical field [0001] The invention relates to the field of biology, in particular to a preparation for research, in particular to a gene knockout kit based on a CRISPR-CAS9 lentiviral vector. Background technique [0002] Gene modification is an important issue in the current biological field. In recent years, the rapid development of genome editing technology has brought a new era to biological research. Different from traditional gene cloning technology, genome editing technology can directly perform DNA sequence knockout, insertion, site-directed mutation, and combined editing on the genome to realize systematic research on gene function and regulatory elements, and has great potential in industrial bioengineering. Broad application prospects. In the early days, genome editing technology mainly used targeting technology mediated by homologous recombination, but its application was greatly limited due to its low efficiency. To solve this problem, a series of artificial...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/867C12N15/861
Inventor 姚新刚郭颂欣何世俊刘叔文
Owner SOUTHERN MEDICAL UNIVERSITY
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