CRISPR-Cas9 targeted and knockout SLC30A1 gene and specific sgRNA of gene

A specific, MDA-MB-231 technology, applied in the field of sgRNA that specifically targets the SLC30A1 gene, can solve problems such as zinc toxicity

Inactive Publication Date: 2018-06-12
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Zinc supplementation can prevent and treat children with diarrhea, chronic hepatitis C, acute lower respiratory tract infection and colds to a certain extent, but too much zinc is toxic

Method used

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  • CRISPR-Cas9 targeted and knockout SLC30A1 gene and specific sgRNA of gene
  • CRISPR-Cas9 targeted and knockout SLC30A1 gene and specific sgRNA of gene
  • CRISPR-Cas9 targeted and knockout SLC30A1 gene and specific sgRNA of gene

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Experimental program
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Embodiment 1

[0043]1. Construction of a knockout SLC30A1 plasmid using CRISPR / Cas9 technology

[0044] 1 .1 sgRNA oligonucleotide chain synthesis.

[0045] Using the CRISPR online design tool (http: / / crispr.mit.edu / ) according to the scoring system, design a 20bp sgRNA on exon 2 of SLC30A1, and verify that there is no non-specific gene by BLAST. Add CACC to the 5' end of the coding strand template, and add AAAC to the 3' end of the non-coding strand template to complement the cohesive ends formed after digestion with BsmBI, and design a pair of CRISPR oligonucleotide chains, see Table 1 SLC30A1 targeting sites and sgRNA oligonucleotide sequence

[0046]

[0047] 1.2 Vector construction

[0048] 1.2.1 Use BsmBI to digest 2 μg lentiCRISPRv2 plasmid (purchased from Addgene), 2h, 37°C.

[0049]

[0050] 1.2.2 Use the GENRY Gel Recovery Kit to purify the digested plasmid product and operate according to the instructions

[0051] 1.2.3 Phosphorylation and annealing of sgRNA oligos:

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Abstract

The invention discloses a CRISPR-Cas9 targeted and knockout human breast cancer cell SLC30A1 gene and the specific sgRNA of the gene. Firstly, sgRNA for specific targeting of the SLC30A1 gene is obtained, and a base sequence of the sgRNA is shown in SEQ ID NO.1; secondly, the sgRNA of the SLC30A1 gene is constructed to a lentivirus vector system containing Cas9 proteins; finally, CRISPR/Cas9 lentivirus containing the sgRNA is infected with a human breast cancer cell MDA-MB-231 to obtain a cell strain of which the SLC30A1 protein expression level is obviously reduced. The CRISPR-Cas9 targeted and knockout human breast cancer cell SLC30A1 gene and the specific sgRNA of the gene disclosed by the invention have the advantages of simple operation steps, good sgRNA targetability and high cuttingefficiency on the SLC30A1 gene; in addition, the constructed CRISPR/Cas9 lentivirus system has the advantage of high knockout efficiency and can specifically knock out the SLC30A1 gene so as to obtain the human breast cancer cell having the SLC30A1 gene knocked out; therefore, a powerful tool is provided for further researching an action mechanism of the SLC30A1 in breast cancer cells.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a method for specifically knocking out the human SLC30A1 gene by CRISPR-Cas9 and an sgRNA for specifically targeting the SLC30A1 gene. Background technique [0002] SLC30A1 (zinc transporter) member 1 is a protein in humans encoded by the SLC30A1 gene. The essential trace element zinc participates in various enzyme and protein functions in the body through catalytic and structural effects, and is closely related to body development, brain function, bone growth, reproductive health, and immune function. Zinc supplementation can prevent and treat children with diarrhea, chronic hepatitis C, acute lower respiratory tract infection and common cold to a certain extent, but too much zinc is toxic. Therefore, there is a complex zinc ion homeostasis system in the body to maintain the balance process of zinc ion absorption, storage and loss. The SLC30A family, also kno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/90C12N5/10
CPCC07K14/47C12N5/0693C12N9/22C12N15/113C12N15/86C12N15/907C12N2310/10C12N2510/00C12N2740/15043C12N2800/107
Inventor 马佩敏孙子豪杨兴林潘讴东
Owner OBIO TECH SHANGHAI CORP LTD
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