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Wheat TaAGO4a gene CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats)/-CRISPR-associated protein 9) vector and application thereof

A wheat and gene technology, applied in the field of genetic engineering and genetic modification, can solve problems such as loss of function, gene frameshift mutation, etc.

Active Publication Date: 2016-02-10
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The CRISPR / Cas system generates a double strand DNA break (doublestrandbreak, DSB) at the target site, and the cell can be repaired by non-homologous end joining (NHEJ), resulting in a frameshift mutation of the gene and loss of function

Method used

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  • Wheat TaAGO4a gene CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats)/-CRISPR-associated protein 9) vector and application thereof
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  • Wheat TaAGO4a gene CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats)/-CRISPR-associated protein 9) vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 The gRNA design of wheat TaAGO4a gene CRISPR / Cas9

[0030] 1. Sequence alignment of three copies of wheat TaAGO4a gene

[0031] Using DNAMAN to compare the three copies of the AGO4a gene in Chinese spring wheat (Triticum aestivum L.), the results are as follows: Figure 1A-Figure 1E shown.

[0032] 2. Enzyme digestion analysis of wheat TaAGO4a gene

[0033] Primer5.0 was used to analyze the restriction site and quantity of the Chinese spring wheat AGO4a gene sequence, paying special attention to the sequence conserved in three copies and the single restriction position in the target interval.

[0034] 3. Find the PAM (protoadjacentmotif) motif

[0035] Look for the PAM motif, ie NGG, near the appropriate restriction site. Make sure that the enzyme cut position is exactly 4-6 bases from the 5' end of NGG.

[0036] 4. Determine the gRNA sequence

[0037] Determine the appropriate PAM position, and the 20bp sequence at the 5' end is the gRNA sequence. Generall...

Embodiment 2

[0040] Example 2 Wheat TaAGO4a gene gRNA is connected to the CRISPR / Cas9 carrier

[0041] 1. Synthesis system of double-stranded gRNA

[0042] The gRNA synthesized in Example 1 was added with deionized water as shown in SEQ ID NO.1 or SEQ ID NO.2 to make the concentration 10 μM, and then the double-stranded synthesis system was prepared as follows:

[0043]

[0044] 2. Double-stranded gRNA synthesis conditions

[0045]

[0046] 3. Digestion of CRISPR / Cas9-U6-sgRNA plasmid

[0047] The enzyme digestion system is as follows:

[0048]

[0049]

[0050] Mix well, and digest at 37°C for 3-16 hours.

[0051] 4. Recovery of enzyme-digested fragments

[0052] Use 1.2% agarose gel to detect the digested product, and the plasmid fragment with the correct size is about 2.9Kb. Then recover the digested fragments according to the operating instructions of the gel recovery kit.

[0053] 5. Link double-stranded sgRNA to U6-sgRNA

[0054]

[0055] overnight at 4°C.

[0...

Embodiment 3

[0066] Example 3 Wheat CRISPR / Cas9-AGO4a Vector Transformation of Wheat Protoplasts and Mutation Detection

[0067] 1. Extraction of high concentration CRISPR / Cas9-AGO4a plasmid

[0068] Shake 250 μl of bacteria (LB+Amp) to make its OD600 about 1.5, and then extract the U6-sgRNA plasmid connected with TaAGO4a-sgRNA prepared in Example 2 according to the instructions of the Promega plasmid extraction kit, so that the final plasmid concentration is greater than 1000ng / μl.

[0069] 2. Preparation and transformation of wheat protoplasts

[0070] Fresh leaves of wheat seedlings planted for 7-9 days were selected, and protoplasts were prepared according to the steps of wheat protoplast preparation (see literature Shanetal, naturebiotechnology, 2014(10): 2395-2410). Generally every 5×10 5 Wheat protoplast cells were transformed with plasmids. Then the Cas9 plasmid and the U6-sgRNA plasmid linked with TaAGO4a-sgRNA were transformed into wheat protoplasts under the induction of PE...

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Abstract

The invention provides a wheat TaAGO4a gene CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats) / -CRISPR-associated protein 9) vector and application thereof and belongs to the field of crop molecular biology. The wheat TaAGO4a gene CRISPR / Cas9 vector and the application thereof have the advantages that gRNA of a third exon of specificity-targeted TaAGO4a is provided firstly, a DNA sequence of the gRNA is shown as SEQ ID NO.1, and the gRNA contains an enzyme cutting site XmnI; subsequently, the CRISPR / Cas9 vector containing the gRNA is provided, and through co-transformation of Cas9 and the specific gRNA into a wheat protoplast as well as enzyme cutting and sequencing technologies, the condition that the gRNA can guide the Cas9 to cut three copies positioned on a chromosome 3A, a chromosome 3B and a chromosome 3D of the TaAGO4a respectively can be detected successfully so as to cause frameshift mutation of the gene and result in afunction or excalation of the gene; the wheat TaAGO4a gene CRISPR / Cas9 vector can be used for preparing TaAGO4a gene-deleted transgenic wheat.

Description

technical field [0001] The invention belongs to the field of genetic engineering and genetic modification, in particular, it relates to a CRISPR / Cas9 carrier of wheat TaAGO4a gene and its application. Background technique [0002] Wheat is an important food crop, and 35%-40% of the world uses wheat as the main food. At the same time, as one of the three major grains, almost all of its output is used for food, and it is the food crop with the world's total output second only to corn. With the continuous development of molecular biology technology and the gradual deepening of gene function research, epigenetics such as DNA methylation, histone modification, etc. can modify genes, regulate gene expression, and then control important agronomic traits such as thousand-grain weight and flowering. Play an important role. AGO4a is a member of the AGO family, which regulates the expression of related genes by mediating sRNAs to participate in the DNA methylation pathway. As the co...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/82A01H5/00C12Q1/68
Inventor 李爱丽宋高原耿帅锋贾美玲毛龙
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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