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sgRNA sequence for specifically targeting arabidopsis ILK2 gene and application of sgRNA sequence

A DNA sequence, Arabidopsis technology, applied in the field of genetic engineering, can solve problems such as hindering wide application

Inactive Publication Date: 2017-10-10
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] This technology has attracted widespread attention because it can quickly, easily and efficiently target any gene in the genome. However, subsequent studies have found that this system also has certain limitations, that is, DNA mutations will occur in non-target regions. That is, the off-target effect, which hinders the wide application of this technology to a certain extent (High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology. Fu et al, 2013; High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology. Pattanayak et al, 2013)

Method used

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  • sgRNA sequence for specifically targeting arabidopsis ILK2 gene and application of sgRNA sequence
  • sgRNA sequence for specifically targeting arabidopsis ILK2 gene and application of sgRNA sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Reality Example 1, Obtaining the Target Sequence of the Arabidopsis ILK2 Gene

[0028] Obtain the coding region sequence of the first exon of the ILK2 gene on the Phytozome website (https: / / phytozome.jgi.doe.gov / pz / portal.html), and determine the target sequence according to the principle of sgRNA designed for knockout , located in the first exon, see figure 1 , the sequence is as follows:

[0029] SEQ ID NO.3: 5'-CGGCGAGATTCAAGCTAGGTAGG-3'

[0030] The sgRNA sequence targeting to edit this DNA sequence is as follows:

[0031] SEQ ID NO.1: 5'-CGGCGAGAUUCAAGCUAGGU-3'

[0032]The design principles of sgRNA are as follows:

[0033] 1. The length of sgRNA should generally be around 20nt

[0034] 2. The best content of GC% is 40%~60%,

[0035] 3. The binding position of the sgRNA targeting gene should be as close as possible to the downstream of ATG in the coding region of the gene, generally located in the first or second exon.

[0036] 4. The number of matches bet...

Embodiment 2

[0039] Embodiment 2, construction of Arabidopsis Cas9-ILK2KO vector

[0040] (1) sgRNA sequence: The 3-terminal PAM sequence AGG is removed from the target target sequence to obtain the sgRNA coding sequence SEQ ID NO. 2: 5'-CGGCGAGATTCAAGCTAGGT-3'.

[0041] (2) The reverse complementary sequence of the sgRNA coding sequence: the reverse complementary sequence of the sgRNA sequence was obtained to obtain the reverse complementary sequence SEQID NO.4: 5'-ACCTAGCTTGAATCTCGCCG-3'.

[0042] (3) Synthesis of single-stranded DNA oligonucleotides: according to the Bbs I restriction site of the selected Cas9 vector, 4 additional bases GATT are introduced at the 3 ends of the sgRNA sequence to obtain the sequence of SEQ ID NO.5; Four additional bases AAAC were introduced into the 5-end of the sgRNA reverse complementary sequence to obtain the sequence of SEQ ID NO.6. The sequences of SEQ ID NO.5 and SEQ ID NO.6 were handed over to Sangon Bioengineering (Shanghai) Co., Ltd. for artif...

Embodiment 3

[0068] Example 3, Cas9-ILK2KO transfected Agrobacterium EHA105

[0069] (1) Preparation of Agrobacterium Competent Cells:

[0070] Pick a single colony of Agrobacterium EHA105 and inoculate it in 5ml of YEB medium, cultivate overnight at 28°C on a shaker at 200rpm, inoculate it into 50ml of YEB medium at a ratio of 1:100 for expansion, and continue to cultivate at 28°C for about 6-7h until OD600=0.4 -0.6. Put the bacterial solution on ice for 30min; centrifuge at 5000rpm at 4°C for 5min, discard the supernatant, and suspend the bacteria in 10ml of 0.15M CaCl2; Suspended, distributed in 50 μl per tube, added sterile glycerol with a final concentration of 20%, and stored at -70°C.

[0071] (2) Transformation and identification of Agrobacterium:

[0072] Add 2 μl carrier Cas9-ILK2KO DNA to 50 μl Agrobacterium competent, mix well, ice bath for 30 minutes, freeze in liquid nitrogen for 4 minutes, 37°C water bath for 6 minutes, add 1ml YEB medium, 28°C, 200rpm shaking table for...

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Abstract

The invention discloses a sgRNA sequence for specifically targeting an arabidopsis ILK2 gene and application of the sgRNA sequence. The nucleotide sequence of the sgRNA sequence is represented by SEQ ID NO.1. Two single-chain oligo DNA sequences are designed and synthesized according to a sgRNA guide sequence, a double chain is formed through annealing and is linked with a Cas9 carrier, a sgRNA coding sequence and a CRISPR system are introduced into Arabidopsis by using an agrobacterium-mediated genetic transformation technique, a target sequence is shorn by a Cas9 protein under the guidance of sgRNA, and therefore, the knockout of an ILK2 gene is realized. According to the sgRNA sequence provided by the invention, the ILK2 gene can be knocked out or edited by virtue of a CRISPR-Cas9 system, so as to analyze the functions of the arabidopsis ILK2 gene.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a sgRNA sequence specifically targeting Arabidopsis ILK2 gene and its application. Background technique [0002] ZFN, TALEN, and CRISPR gene targeting technologies are the core technologies of gene editing. Compared with the cumbersome construction procedures of the first two technologies, for example, each site needs to construct a pair of corresponding nucleases, which are highly dependent on the upstream and downstream sequences of the target. CRISPR The / Cas9 system's recognition of specific sites is guided by small sgRNAs. The sgRNA region can be composed of a series of multiple sgRNAs targeting different genes. Each sgRNA targeting a specific site is only 20 bases, and the entire carrier is small. , CRIEPR vectors are easier to construct, and their gene editing is more efficient. [0003] CRISPR / Cas9 was originally a defense system for bacteria and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N9/22C12Q1/68C12N15/11A01H5/00
CPCC12N9/22C12N15/113C12N15/8213C12Q1/6895
Inventor 左开井王俊吕萌荔
Owner SHANGHAI JIAO TONG UNIV
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