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Multi-target sequence sgRNA expression vector based on endogenous tRNA processing system and application of sgRNA expression vector in plant gene editing

A technology for expressing vector and RNA polymerase, applied in the direction of DNA/RNA fragment, introduction of foreign genetic material and vector using vector, etc., which can solve the problems of dicotyledonous plant report and increase of cost, etc.

Inactive Publication Date: 2017-12-15
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, direct chemical synthesis of the target sequence of interest together with the tRNA-sgRNA sequence greatly increases the cost
Currently, multi-site gene editing using the tRNA-sgRNA / Cas9 system has not been reported in dicotyledonous plants

Method used

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  • Multi-target sequence sgRNA expression vector based on endogenous tRNA processing system and application of sgRNA expression vector in plant gene editing
  • Multi-target sequence sgRNA expression vector based on endogenous tRNA processing system and application of sgRNA expression vector in plant gene editing
  • Multi-target sequence sgRNA expression vector based on endogenous tRNA processing system and application of sgRNA expression vector in plant gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Using tRNA-sgRNA sequence expression to realize mutation of cabbage self-incompatibility related gene SRK

[0057] 1. Artificially synthesize the U6-26::tRNA-sgRNA expression cassette sequence shown in the present invention.

[0058] 2. According to the SRK3 haplotype gene sequence of the cabbage self-incompatibility line F416 and the target site sequence requirements of the CRISPR / Cas9 gene editing system, four target sites of the SRK3 gene A, B, C, and D were selected and designed. The following target site complementary primers anneal to double-stranded DNA fragments.

[0059] (1) SRK3 gene target site A complementary primer sequence:

[0060] sgSRK-1F:5-TGCAGTGGCAGAGCTTCTCGCCAA-3

[0061] sgSRK-1R:5-AAACTTGGCGAGAAGCTCTGCCAC-3

[0062] (2) SRK3 gene target site B complementary primer sequence:

[0063] sgSRK-2F:5-TGCAAACCCTATATCCAACTCCAC-3

[0064] sgSRK-2R:5-AAACGTGGAGTTGGATATAGGGTT-3

[0065] (3) SRK3 gene target site C complementary primer sequence...

Embodiment 2

[0076] Example 2 Using tRNA-sgRNA sequence expression to realize the mutation of tobacco PDS gene

[0077] 1. Artificially synthesize the U6-26::tRNA-sgRNA expression cassette sequence shown in the present invention.

[0078] 2. According to the PDS gene sequence of common tobacco and the target site sequence requirements of CRISPR / Cas9 gene editing, three target sites of PDS gene A, B, and C were selected, and the complementary primers for the target sites were designed as follows to anneal to double-stranded DNA fragments .

[0079] (1) NtPDS gene target site A complementary primer sequence:

[0080] sgNtPDS1F:5-TGCAGCCGTTAATTTGAGAGTCCA-3

[0081] sgNtPDS1R:5-AAACCGGACTCTCAAATTAACGGC-3

[0082] (2) NtPDS gene target site B complementary primer sequence:

[0083] sgNtPDS2F:5-TGCAGCTGCATGGAAAGATGATGA-3

[0084] sgNtPDS2R:5-AAACTCATCATCTTTCCATGCAGC-3

[0085] (3) NtPDS gene target site C complementary primer sequence:

[0086] sgNtPDS3F:5-TGCAGAGGCAAGAGATGTCCTAGG-3

[0...

Embodiment 3

[0095] Example 3 Using tRNA-sgRNA sequence expression to realize the double mutation of cabbage MS1 gene and SRK gene

[0096] 1. Artificially synthesize the U6-26::tRNA-sgRNA expression cassette sequence shown in the present invention.

[0097] 2. According to the MS1 gene (male sterility-related gene) sequence of the cabbage self-incompatibility line F416 and the sequence requirements of the target site of the CRISPR / Cas9 gene editing system, four of the MS1 genes, A, B, C, and D, were selected. For the target target site, design the following target site complementary primers to anneal to double-stranded DNA fragments.

[0098] (1) MS1 gene target site A complementary primer sequence:

[0099] sgMS1-1F: 5-TGCAGCCTTTCTAAAACTAGAAGG-3

[0100] sgMS1-1R: 5-AAACCCTTTCTAGTTTTAGAAAGGC-3

[0101] (2) MS1 gene target site B complementary primer sequence:

[0102] sgMS1-2F: 5-TGCAGAGTAGCAAAAGGAGAGCCG-3

[0103] sgMS1-2R: 5-AAACCGGCTCTCCTTTTGCTACTC-3

[0104] (3) MS1 gene target...

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Abstract

The invention provides a tRNA-sgRNA expression sequence. The expression sequence is characterized in that tRNA, type II endonuclease sites and sgRNA are sequentially connected to form a unit sequence, four unit sequences are arranged in series, and the type II endonuclease sites in the four unit sequences which are arranged in series are BbsI, BsaI, BsmBI and BfuAI sequentially. According to the tRNA-sgRNA expression sequence, multiple target sequences can be assembled in a tRNA-sgRNA expression vector step by step, and multi-site gene editing of multiple plants such as tobacco, cabbages and the like can be realized; editing of multiple genes or combined editing of different genes according to targets can be realized by repeatedly inserting tRNA-sgRNA expression sequence units on which different target sequences are assembled in a Cas9 gene expression vector.

Description

technical field [0001] The invention belongs to the field of design and application of sgRNA expression vectors of the CRISPR / Cas9 system, and in particular relates to a method for constructing sgRNA expression vectors with multiple target site sequences by using an enzyme-cut insertion method to realize multi-gene editing in plants. Background technique [0002] In 2013, clustered regularly interspaced short palindromic repeats and the related nuclease Cas9 were reported to be used for gene editing. After several years of development, this system has quickly become the hottest genome editing technology. For polyploid crops such as tetraploid rapeseed and hexaploid wheat, most genes have multiple copies, and it is very important to analyze their gene functions or improve their traits based on traditional forward or reverse genetics. difficulty. Therefore, the development of a CRISPR / Cas9 gene editing system capable of simultaneously modifying multiple sites is of great sig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N5/10
CPCC12N15/113C12N15/8261C12N2800/80C12N2810/10
Inventor 宋洪元马存发朱陈曾郑敏任雪松司军李勤菲
Owner SOUTHWEST UNIVERSITY
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