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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 methods for the treatment of severe osteolithiasis, but this method is limited by many factors and cannot be obtained in the treatment of benign osteolithiasis. application

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
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Effect test

Embodiment 1

[0035] This embodiment provides an sgRNA specifically targeting the CLCN7 gene and a recombinant plasmid comprising the sgRNA.

[0036] The source of each material in this embodiment is as follows:

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

[0038] Competent DH5α cells were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

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

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

[0041] High-fidelity PCR enzymes were purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.

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

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

Embodiment 2

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

[0063] The mouse embryonic stem cells that were not transferred with the recombinant plasmid were taken as a control, the positive cloned cells and the control cells were respectively put into the cell lysate and lysed, and the supernatant of the lysate was taken to amplify the CLCN7 gene.

[0064] The results showed that the length of the amplified fragment of the positive clone cells was significantly smaller than that of the control cells, which indicated that the recombinant plasmid provided in Example 1 could cut the CLCN7 gene sequence, thereby realizing gene editing.

Embodiment 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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