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CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) target knockout human Lin28A gene and specific gRNA (guide Ribonucleic Acid) of CRUSPR/Cas9 target knockout human Lin28A gene

A specific, genetic technology, applied in the fields of molecular biology and biomedicine

Inactive Publication Date: 2017-06-20
重庆高圣生物医药有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the exact mechanism by which Lin28A promotes tumorigenesis is still unclear, there has been a large amount of evidence that the Lin28 / let-7 bidirectional negative feedback pathway can combine with some factors (such as RAS, MYC, NF-κB) to form a complex network involved carcinogenesis

Method used

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  • CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) target knockout human Lin28A gene and specific gRNA (guide Ribonucleic Acid) of CRUSPR/Cas9 target knockout human Lin28A gene
  • CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) target knockout human Lin28A gene and specific gRNA (guide Ribonucleic Acid) of CRUSPR/Cas9 target knockout human Lin28A gene
  • CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) target knockout human Lin28A gene and specific gRNA (guide Ribonucleic Acid) of CRUSPR/Cas9 target knockout human Lin28A gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 gRNA synthesis and vector construction targeting human Lin28A gene

[0015] 1. Selection and design of gRNA targeting human Lin28A gene

[0016] Find the sequence of human Lin28A gene in Genebank, and design potential target sites in the exon region of human Lin28A gene;

[0017] Through the online design tool (http: / / crispr.mit.edu / ) and the design principles of gRNA, the target site with high score on the human Lin28A gene sequence was evaluated to design gRNA.

[0018] 2. Synthesis of gRNA oligonucleotide sequence targeting human Lin28A gene and construction of eukaryotic expression vector

[0019] The pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene plasmid ID: 48138, hereinafter referred to as PX458) was digested with BbSI, and after 1 h in a 37°C water bath, 1% agarose electrophoresis was performed to recover the digested product (TAKARA gel). recovery kit).

[0020] The enzyme digestion system is as follows:

[0021]

[0022] The two oligonucleotides ...

Embodiment 2

[0033] Example 2 Preparation of human melanoma cells and human hepatoma cells knockout Lin28A gene cell lines

[0034] Resuscitate human melanoma cells (A375 cell line, Shanghai Cell Bank of the Chinese Academy of Sciences), put the cells into a 10% FBS+DMEM culture flask, and store them at 37°C, 5% CO. 2 The cells were subcultured 1 day before transfection.

[0035] Aspirate the medium in the T75 flask for culturing A375 cells, add 2 mL of 0.25% trypsin from a 4°C refrigerator to cover the bottom of the bottle evenly, place it in a 37°C incubator for 3-5 min, take it out, and shake to find out The cells are detached from the bottom, shake them all off, add 3 mL of pre-warmed 10% DMEM in a 37°C water bath, and pipette with a 10 mL pipette for 6 to 8 times without leaving dead corners. It is difficult to pipette at the mouth of the bottle. The pipette can be aligned with the bottle mouth, and the medium can be covered with a small force to cover the cells close to the bottle m...

Embodiment 3

[0059] Example 3 Western blot detection of protein expression in A375-Lin28A gene-deficient cell lines and HepG2-Lin28A gene-deficient cell lines

[0060] Total protein extraction---cell lysis

[0061] (1) A375-KO-Lin28A and HepG2-KO-Lin28A cells were cultured in 10 cm dishes, and A375 and HepG2 cells were simultaneously cultured as a control group.

[0062] (2) Discard the culture supernatant and add 0.1 mL of RIPA buffer per 106 cells.

[0063] (3) Place on ice for a few minutes, and gently pipette with a pipette tip to fully lyse the cells. Gently tilt the Petri dish again so that the lysate flows to one side or corner of the dish, then transfer it to a 1.5 mL centrifuge tube and shake vigorously for 30 s.

[0064] (4) Centrifuge at 12,000 rpm and 4ºC for 15 min, and aspirate the supernatant for subsequent Western blot detection.

[0065] Protein Concentration Determination (BCA Determination of Protein Concentration)

[0066] Preparation of working fluid

[0067] Befo...

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Abstract

The invention belongs to the technical field of molecular biology and biomedicine and in particular relates to application of a gRNA (guide Ribonucleic Acid) sequence based on a CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated protein 9) system on knockout of a human Lin28A gene and application to tumor treatment. According to a design principle of CRISPR / Cas9, 6 guide RNAs (gRNA) are designed and a sequence table is shown as SEQ ID NO.1 to SEQ ID NO.6; the guide RNAs are constructed on a PX458 carrier and two efficient target gRNAs are obtained through activity detection and screening. The CRISPR / Cas9 system guided by the two gRNAs is utilized in human hepatocellular carcinoma cell lines (HepG2) and human melanoma cell lines (A375), and the human Lin28A gene can be effectively knocked out; the system is easy to operate and the human Lin28A gene knockout efficiency is high, so that the system is applicable to various tumor cell models. The gRNA provided by the invention is hopefully applied to a new drug for treating tumors.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and biomedicine, in particular to a method for CRISPR / Cas9 to specifically knock out the human genome Lin28A gene and a gRNA for targeting the human Lin28A gene. Background technique [0002] Gene knockout, that is, a technical means for a specific gene to lose its function by destroying or changing its gene sequence. Commonly used gene knockout methods include: zinc finger nucleases (ZFNs) (Miller et al., 2007; Porteus and Baltimore, 2003; Wood et al., 2011), transcription factor activator-like nucleases (TALENs) (Miller et al. ., 2011; Wood et al., 2011; Zhang et al., 2011), as well as the recently discovered prokaryotes type II adaptive immune system CRISPER / Cas9 system (Cong et al., 2013; Mali et al., 2013) Wait. [0003] The CRISPER / Cas9 system was originally used by the bacterial immune system to defend against foreign viruses or plasmids. In the second type of CRISPR system, Ca...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N5/10
Inventor 周勇申友锋
Owner 重庆高圣生物医药有限责任公司
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