sgRNA specifically targeting CLCN7 and application of sgRNA

A specific and targeted technology, which is applied to sgRNA specifically targeting CLCN7 and its application field, can solve the problems of failure to apply benign osteolithiasis and limited effective treatment methods

Inactive Publication Date: 2020-06-05
SHENZHEN PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the effective treatment methods for osteolithiasis are relatively limited, among which allogeneic hematopoietic stem cell transplantation is one of the effective

Method used

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  • sgRNA specifically targeting CLCN7 and application of sgRNA
  • sgRNA specifically targeting CLCN7 and application of sgRNA
  • sgRNA specifically targeting CLCN7 and application of sgRNA

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0034] Example 1

[0035] This embodiment provides a sgRNA that specifically targets the CLCN7 gene and a recombinant plasmid including the sgRNA.

[0036] The sources of the materials in this embodiment are as follows:

[0037] CRISPR / Cas9 plasmid was purchased from Beijing Saibei Biotechnology Co., Ltd. See the plasmid map figure 1 .

[0038] The competent cell DH5α was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0039] BspQ I high-fidelity restriction endonuclease and T4 ligase were purchased from NEB.

[0040] DNA gel recovery kit, plasmid small extraction kit and TOP10 competent cells were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0041] The high-fidelity PCR enzyme was purchased from Baori Biotechnology (Beijing) Co., Ltd.

[0042] The DNA primers were provided by Suzhou Jinweizhi Biotechnology Co., Ltd., and the DNA sequencing was completed by Beijing Liuhe Huada Gene Technology Co., Ltd.

[0043] In this embodiment, the preparation m...

Example Embodiment

[0061] Example 2

[0062] The recombinant plasmids CLCN7-sg1RNA-CRISPR / Cas9 and CLCN7-sg2RNA-CRISPR / Cas9 in Example 1 were electrotransformed into mouse embryonic stem cells, and positive clones were obtained after G418 screening.

[0063] Take the mouse embryonic stem cells that have not been transferred into the recombinant plasmid as a control, put the positive cloned cells and the control cells into the cell lysate for lysis, and take the lysate supernatant to amplify the CLCN7 gene.

[0064] The results showed that the length of the amplified fragments of the positive cloned cells was significantly smaller than that of the control cells. The results showed that the recombinant plasmid provided in Example 1 can shear the CLCN7 gene sequence to achieve gene editing.

Example Embodiment

[0065] Example 3

[0066] A composition comprising the recombinant vector in Example 1. The composition can be used to edit the CLCN7 gene and be used for the knockout or modification of the CLCN7 gene.

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Abstract

The present invention provides a sgRNA specifically targeting a CLCN7 gene and an application of the sgRNA. A nucleotide sequence of the sgRNA specifically targeting the CLCN7 comprises any one of SEQID No.1-2. In order to realize editing of the CLCN7 gene, firstly the specific sgRNA is designed based on a recognition sequence of the gene. In order to avoid occurrence of off-target effects as much as possible, when the sgRNA is designed, the base pairing number of the sgRNA and off-target sites is reduced. Following the above-mentioned principle, two 20-bp sgRNAs are designed in an exon region of the CLCN7 gene, an interval of a target sequence is 3 bp, and a rear end is close to PAM (protospacer adjacent motif, NGG). The sgRNA constructs an expression of CRISPR/Cas9, can effectively realize gene editing of the CLCN7 and thus provides a reference for the next step to repair mutation of pathogenic sites of the CLCN7.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a sgRNA specifically targeting CLCN7 and its application. Background technique [0002] Osteopetrosis (OP), also known as marble bone disease, osteopetrosis and chalk-like bone, is a disease of abnormal bone metabolism with osteoclast differentiation or dysfunction as the main lesion, and it is also a typical human genetic disease , clinical manifestations of systemic osteosclerosis, bone plastic abnormalities, anemia, infection, hepatosplenomegaly, fractures, optic atrophy and deafness. There is no obvious gender difference in the disease, and the disease can occur in all age groups. Osteolithiasis can be divided into two types: autosomal recessive inheritance and autosomal dominant inheritance according to the genetic mode of osteoarthritis, and can be further subdivided into type 7 and type 2 according to the clinical manifestations. [0003] ClC-type chloride channel is...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/90
CPCC12N15/113C12N15/902C12N2310/20
Inventor 戴勇欧明林孙国平汤冬娥邱劲军朱鹏
Owner SHENZHEN PEOPLES HOSPITAL
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