CRISPR/Cas9 targeted-knockout human TCAB1 gene and specificity gRNA thereof

A specific, -CCN-N technology, applied in the field of molecular biology and biomedicine, can solve the problems of cancer cell death, telomere formation and maintenance limitation, telomere shortening, etc.

Active Publication Date: 2016-05-11
重庆威斯腾生物医药科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the loss of TCAB1 expression does not affect the catalysis of the telomerase RNA component (TERC) by the active agent of telomerase reverse transcriptase (TERT), it can prevent the transport of the telomerase complex to the terminal

Method used

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  • CRISPR/Cas9 targeted-knockout human TCAB1 gene and specificity gRNA thereof
  • CRISPR/Cas9 targeted-knockout human TCAB1 gene and specificity gRNA thereof
  • CRISPR/Cas9 targeted-knockout human TCAB1 gene and specificity gRNA thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Synthesis of gRNA targeting human TCAB1 gene and vector construction

[0018] 1. Selection and design of gRNA targeting human TCAB1 gene

[0019] Find the sequence of the human TCAB1 gene in Genebank, and design potential target sites in the exon region of the human TCAB1 gene. Through the online design tool (http: / / crispr.mit.edu / ) and gRNA design principles, evaluate the target sites with higher scores on the human TCAB1 gene sequence to design gRNAs. The target site sequences are SEQIDNO.1-SEQIDNO.6 , and design the corresponding oligonucleotides.

[0020] 2. Synthesis of gRNA oligonucleotide sequence targeting human TCAB1 gene and construction of eukaryotic expression vector

[0021] The pSpCas9(BB)-2A-GFP(PX458) plasmid (AddgeneplasmidID: 48138, hereinafter referred to as pSpCas9(BB)) was digested with BbSI, and after 1 hour in 37°C water bath, 1% agarose electrophoresis was performed to recover the digested product ( TAKARA gel recovery kit).

[0022...

Embodiment 2

[0034] Example 2 Lipofection of HepG2 cells

[0035] Three days before transfection, resuscitate human liver cancer cells (HepG2 cells, Shanghai Cell Bank, Chinese Academy of Sciences), put the cells into a culture flask with 10% FBS+DMEM, and incubate at 37°C, 5% CO 2 Cultured in an incubator, the day before transfection, subculture the recovered cells.

[0036]Aspirate the culture medium in the T75 bottle of HepG2 cells, add 2ml of 0.25% trypsin taken out of the refrigerator at 4°C, make it evenly cover the bottom of the bottle, put it in the incubator at 37°C for 3-5min, take it out, shake it to find the cells at the bottom Remove it, shake it all down, add 3ml of 10% DMEM preheated in a 37°C water bath, and blow with a 10ml pipette for 6-8 times without leaving any dead ends. To prepare the mouth, push out the culture medium with a small force to cover the cells close to the mouth of the bottle. Afterwards, all the cells were aspirated and placed in a 15ml centrifuge tub...

Embodiment 3

[0047] Example 3 PCR product cloning sample sending and sequencing detection

[0048] Carry out the PCR reaction according to the method of Example 2, and the PCR product is connected to the PMD18-T carrier after being purified by the TAKARA kit, and the connection system is:

[0049]

[0050] Ligation was carried out at 16°C for 2 hours. Take competent cells DH5α, place in ice to melt for 5 minutes, add 10ul of ligation product and blow evenly, place in ice for 20 minutes. Heat shock at 42°C for 90s, quickly transfer to an ice bath and let stand for 3min, add 500ul LB liquid medium, place in a shaker, 37°C, 180rpm for 1h. Take 100 ul of the bacterial solution and evenly spread it on LB solid medium (containing 1 / 1000 AMP), and cultivate overnight at 37°C.

[0051] Pick 3 single colonies and put them into 3ml LB liquid medium (containing 3ulAMP) respectively, at 37°C and 200rpm for 12h. PCR identification was carried out using 1 ul bacterial liquid as a template, all of ...

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Abstract

The invention belongs to the technical field of the molecular biology and the biomedicine, and particularly relates to application of a gRNA sequence based on a CRISPR/Cas9 system to cause tumor cell apoptosis. According to the design principle of CRISPR/Cas9, two target points are designed on a human genome, and corresponding oligos is synthesized and established on a px458 carrier. The CRISPR/Cas9 system guided by the gRNA is established in a human hepatoma cell line (HepG2) according to the design of the two target points, a human TCAB1 gene can be effectively knocked out, the system is easy to operate, and the knockout efficiency of the human TCAB1 gene is high. The CRISPR/Cas9 system guided by the gRNA can be expected to be applied in novel tumor treating medicine.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and biomedicine, and specifically relates to the application of gRNA sequences based on CRISPR / Cas9 system in promoting tumor cell apoptosis. Background technique [0002] The CRISPR / Cas system is developed from the adaptive immune system of bacteria and archaea against foreign viruses or plasmids, including three different types, of which the Type II CRISPR / Cas system has only one subunit of the DNA endonuclease Cas9 , the structure is the simplest, so it is the most widely used. In addition to the Cas9 protein, the system also includes two short CRISPR RNAs (crRNAs) and trans-activatingcrRNAs (tracrRNA). The mature crRNA-tracrRNA complex can guide the Cas9 protein to the target sequence through complementary base pairing, and specifically cut the DNA double strand near the PAM (protospace radjacent motif) to form a DSB (double strand break). DSB can be repaired in two ways, one is no...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61P35/00
CPCC12N9/1276C12N15/1137C12N2310/10C12Y207/07049
Inventor 周勇申友锋
Owner 重庆威斯腾生物医药科技有限责任公司
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