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Method for specifically removing pig SALL1 gene by CRISPR-Cas9 and sgRNA used for specific targeting SALL1 gene

A specific and genetic technology, applied in the field of genetic engineering and gene knockout, to achieve the effect of high solution cost and long solution cycle

Active Publication Date: 2016-04-20
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the key technical problem of this approach is to design and prepare precisely targeted sgRNA, because the targeting accuracy of genes is highly dependent on the sgRNA target sequence, and whether the precise targeted sgRNA can be successfully designed becomes a key technical issue for knocking out the target gene , the present invention is intended to solve this technical problem so as to provide a solid foundation for knocking out the SALL1 gene

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  • Method for specifically removing pig SALL1 gene by CRISPR-Cas9 and sgRNA used for specific targeting SALL1 gene
  • Method for specifically removing pig SALL1 gene by CRISPR-Cas9 and sgRNA used for specific targeting SALL1 gene
  • Method for specifically removing pig SALL1 gene by CRISPR-Cas9 and sgRNA used for specific targeting SALL1 gene

Examples

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Effect test

Embodiment 1

[0066] Example 1, Selection and design of Susscrofa (pig) SALL1 gene sgRNA target sequence

[0067] The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors.

[0068] 1. Selection of sgRNA target sequence for SALL1 gene

[0069] For the SALL1 gene, the following principles should be followed in the selection of target sequences:

[0070] (1) Find the target sequence in the exon coding region of the SALL1 gene that conforms to the 5'-N(20)NGG-3' rule, where N(20) represents 20 consecutive bases, and each N represents A or T Or C or G, the target sequence that meets the above rules is located on the sense strand or the antisense strand;

[0071] (2) Select the 3 exon coding region sequences near the N-terminal, the target sequence can be located in the 3 exon coding re...

Embodiment 2

[0087] Example 2: Construction of the sgRNA expression vector of the SALL1 gene

[0088] 1. Synthesis of DNA Inserts

[0089] (1) Synthesize the forward and reverse oligonucleotide sequences designed above

[0090] The oligonucleotide sequence can be specifically synthesized by a commercial company (such as Invitrogen) according to the provided sequence. In this example and the following examples, the effect of the target sequences listed in the No. 3 and No. 4 sequences listed in Table 1 on the knockout effect of the SALL1 gene was studied.

[0091] The forward oligonucleotide sequence and reverse oligonucleotide sequence corresponding to No. 3 target sequence are as follows:

[0092] CACCGTTTCCAATCCGACCCCGAAG (SEQ ID NO: 51);

[0093] AAACCTTCGGGGTCGGATTGGAAAC (SEQ ID NO: 52).

[0094] The forward oligonucleotide sequence and reverse oligonucleotide sequence corresponding to No. 4 target sequence are as follows:

[0095] CACCGGAGCGAGGCCACTTCGGGGT (SEQ ID NO: 53);

[0096...

Embodiment 3

[0132] Example 3. Obtaining a pseudotyped lentivirus expressing SALL1 sgRNA

[0133] 1. Material preparation

[0134] Amplify and extract packaging plasmids pLP1, pLP2, and pLP / VSVG (purchased from Invitrogen, whose maps are shown in figure 2 , image 3 and Figure 4 shown); amplify and extract vector plasmid lentiCRISPRv2-SALL1; culture packaging cell line HEK293T cells (purchased from ATCC); DMEM medium, Opti-MEM medium and fetal bovine serum FBS (purchased from Gibco); Lipofectamine2000 (purchased from from Invitrogen); HEK293T cells were cultured in 5% CO 2 In the culture environment of 37°C, the culture medium is DMEM medium containing 10% FBS.

[0135] 2. Transfection and Viral Packaging

[0136] Day 1: Passage the packaging cell line HEK293T to 10cmdish, about 30% confluence;

[0137] The next day: when HEK293T reaches 80% confluence, transfect according to the following recipe:

[0138] Prepare Mixture 1, containing:

[0139] lentiCRISPRv2-SALL1: 6 μg

[0140...

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Abstract

The invention discloses a method for specifically removing a pig SALL1 gene by CRISPR-Cas9 and sgRNA used for the specific targeting SALL1 gene. The sgRNA of the specific targeting SALL1 gene has a target sequence on the SALL1 gene which accords with a sequence arrangement rule of 5'-N(20)NGG-3', wherein N(20) represents 20 continuous basic groups, each N represents A or T or C or G. The target sequence on the SALL1 gene is positioned in the three exon code areas at the N end of the SALL1 gene or at the junction of the exons and the adjacent intrones. The target sequence on the SALL1 gene is unique. The sgRNA is used in the method for specifically removing the pig SALL1 gene by the CRISPR-Cas9, the pig SALL1 gene can be removed, and the problems if long period and high cost for SALL1 gene removal are effectively solved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the technical field of gene knockout, in particular to a method for specifically knocking out pig SALL1 gene by CRISPR-Cas9 and an sgRNA for specifically targeting the SALL1 gene. Background technique [0002] Organ transplantation is the most effective treatment for diseases of organ failure. So far, nearly one million patients around the world have extended their lives through organ transplantation. With the aging of the population and the advancement of medical technology, more and more patients need organ transplantation, but the shortage of donor organs seriously restricts the development of organ transplantation. Taking kidney transplantation as an example, as many as 300,000 patients need kidney transplantation in my country every year, but no more than 10,000 donated kidneys can be used for transplantation, and most patients die of kidney failure. Relying on...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867
CPCC12N5/10C12N15/113C12N15/86
Inventor 蔡志明牟丽莎刘璐谢崇伟陈鹏飞张军方高汉超陆赢
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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