Double resistance CRISPR/Cas9 carrier and application

A dual-resistance, nls-cas9-nls technology, applied in the field of genetic engineering, can solve problems such as inability to remove, adverse effects on experimental results, and increasing the uncertainty of experimental results and the complexity of the experimental process.

Inactive Publication Date: 2016-05-04
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

Although the transgenic technology of rice is mature at present, the rice materials obtained through transgenic can not remove the transgenic T-DNA, which oft

Method used

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  • Double resistance CRISPR/Cas9 carrier and application
  • Double resistance CRISPR/Cas9 carrier and application
  • Double resistance CRISPR/Cas9 carrier and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 has the construction of hygroymcin and Basta double resistance CRISPR / Cas9 vector

[0028] The construction method of the double resistance CRISPR / Cas9 vector is as follows:

[0029] From the vector containing the 35S promoter and NOS terminator sequences, the 35S promoter and NOS terminator sequences were amplified by PCR, and the 35S promoter and NOS terminator sequences were concatenated with the 3×flag sequence synthesized in vitro by overlapping PCR, The tandem sequence was inserted between the EcoRI and HindIII sites of the vector pFGC5941 to obtain vector I; from the vector containing the gypsy insulator sequence, the gypsy insulator sequence was amplified by PCR, and the amplified sequence was inserted into the EcoRI and HindIII sites of the vector I. Between the HindIII sites, the vector II is obtained; from the vector containing the NLS-cas9-NLS sequence, the NLS-cas9-NLS sequence is amplified by PCR, and the amplified sequence is inserted into th...

Embodiment 2

[0033] Example 2 Construction of Rice CRY1a Gene Knockout Vector Oscry1a-sgRNA

[0034] According to the rice CRY1a gene sequence, through the online CRISP-P (http: / / cbi.hzau.edu.cn / crispr / ) design software, design the most efficient sgRNA sequence, the nucleotide sequence of the sgRNA action site is 5′- ACAGGCACCTGTCCCAGAACGG-3'.

[0035] From the vector containing the OsU3 promoter sequence, the OsU3 promoter sequence was amplified by PCR, and the primers for amplifying the OsU3 promoter sequence were shown in SEQ ID NO: 19-20.

[0036] Then, from the carrier containing the DNA fragment encoding the sgRNA sequence, the DNA fragment encoding the sgRNA sequence is amplified by PCR, and the primer sequences used are shown in SEQ ID NO: 21-22.

[0037] Insert the DNA fragment encoding the sgRNA sequence and the OsU3 promoter together between the XbaI and BglII sites of the double-resistant CRISPR / Cas9 vector of Example 1 (the DNA fragment encoding the sgRNA sequence is located ...

Embodiment 3

[0038] Example 3 Construction of Rice CRY1a Gene Homozygous Mutant Plants Not Containing Exogenous T-DNA Sequences

[0039] 1. Obtaining and phenotypic verification of transgenic rice plants

[0040] The mature seeds of rice 'kitaake' were taken, shelled manually or mechanically, and the seeds that were plump, smooth and free of plaque were selected, sterilized, and inoculated on the induction medium for induction culture. The rice callus with good appearance and good growth was selected as the recipient material, and the carrier Oscry1a-sgRNA of Example 2 was transferred into the rice callus by the Agrobacterium-mediated method, and the Oscry1a-sgRNA containing 100 μM acetosyringone and O.D. The AAM transformation solution of Agrobacterium 0.7 was transformed, and the calli soaked in the transformation solution were placed on the co-culture medium for co-cultivation. After 3 days of dark culture at 25°C, they were cultured on the screening medium for about 30 days, and contin...

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Abstract

The invention relates to a double resistance CRISPR/Cas9 carrier and application. The invention provides the double resistance carrier having both hygromycin B resistance and Basta resistance at the same time; the double resistance carrier is obtained by inserting sequences and fragments derived from multiple different carriers into a sequence of a carrier pFGC5941 by enzyme-cutting and linking up, and constructing. Hygromycin B is used for screening in a conversion process, and Basta is used for screening offspring transgenic plants not containing an external source T-DNA sequence in offspring plants. Two resistances, i.e., the hygromycin B and the Basta are used to respectively serve as screening marks of the transgenic plants for the first time, a feasible manner is provided for screening transgenic offspring not containing the external source T-DNA sequence, determining the existence of external source T-DNA by a complex molecular biology detection experiment is avoided, and the offspring transgenic plants not containing the external source T-DNA sequence can be obtained by directly screening the Basta resistance and performing phenotype combination.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a novel double-resistance CRISPR / Cas9 carrier and its application. Background technique [0002] As a model plant for the study of crop gene functions, rice has always been valued by researchers in its related genetics and molecular biology. Obtaining rice with improved target genes through transgenics has become the main method for studying the function of rice genes. Although the transgenic technology of rice is mature at present, the rice materials obtained through transgenic can not remove the transgenic T-DNA, which often has adverse effects on the experimental results, and also increases the uncertainty of the experimental results and the experimental process. complexity. [0003] At present, the technology of gene editing is developing rapidly. In the past two years, the gene editing technology mediated by the CRISPR / Cas9 system has become the mainstream technology fo...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66C12N1/21A01H5/00
CPCC12N15/66C12N15/8205C12N2800/80
Inventor 刘军于春生姬荣桓刘斌赵涛李宏宇林辰涛
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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