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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 system capable of simultaneously knocking out KRAS genes and EGFR (Epidermal Growth Factor Receptor) genes and application thereof

A gene, the same technology, applied in the field of CRISPR-Cas9 system, to achieve the effect of simple operation, high knockout efficiency, and many choices

Active Publication Date: 2017-09-05
浙江卫未生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no report on the CRISPR-Cas9 system that simultaneously knocks out the KRAS gene and the EGFR gene

Method used

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  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 system capable of simultaneously knocking out KRAS genes and EGFR (Epidermal Growth Factor Receptor) genes and application thereof
  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 system capable of simultaneously knocking out KRAS genes and EGFR (Epidermal Growth Factor Receptor) genes and application thereof
  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 system capable of simultaneously knocking out KRAS genes and EGFR (Epidermal Growth Factor Receptor) genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. sgRNA design

[0035] According to the human KRAS gene sequence (Sequence ID: NM_033360.3) and EGFR gene sequence (Sequence ID: NM_201282.1) given in GeneBank, 10 sgRNAs (corresponding DNA sequences are shown in SEQ ID NO. 1-10) And SEQ ID NO. 11-20). The sgRNAs of both genes are targeted on their exon regions, and blast is used for alignment in UCSC or NCBI to ensure the uniqueness of their target sequences. Among the 10 sgRNAs designed separately, only 2 can effectively knock out KRAS and EGFR, and the first kras-sg1 (corresponding DNA sequence is shown in SEQ ID NO.1) and egfr-sg1 (corresponding DNA sequence) (As shown in SEQ ID NO.11) for detailed explanation, such as figure 1 with figure 2 Shown.

[0036] 2. Construction of sgRNA oligonucleotide duplex

[0037] Add CACC to the 5'end of the upstream primer of the determined KRAS target sequence, and add AAAC to the 5'end of the downstream primer, so that the double-stranded DNA sticky end formed after annealing and ...

Embodiment 2

[0063] T7EN1 restriction experiment:

[0064] The cells collected in Example 1 were lysed, genomic DNA was extracted with the kit, and finally dissolved in 50 μL of deionized water.

[0065] According to the KRAS gene sequence and EGFR gene sequence published by GenBank, specific primers were designed with Primer 5.0 software, as shown in Table 2:

[0066] Table 2

[0067]

[0068] Use the kit to extract part of the cell genomic DNA, use the extracted cell genomic DNA as a template, and use the specific primers designed in Table 2 to amplify the target fragment. Amplification system: 2×PCR Mix 10μL, genomic DNA 1μL, upstream and downstream primers 1μL each, add dd H 2 O to 20μL. Reaction procedure: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 35 seconds, total 30 cycles; extension at 72°C for 10 minutes; storage at 4°C.

[0069] After denaturing and renaturing 100 ng of the purified PCR product in ...

Embodiment 3

[0071] Western blot experiment:

[0072] Take 1x10 of the lung cancer cells KRAS and EGFR before and after knockout 6 After collecting the cells, add 20μL of lysis buffer (50mM HEPES, PH7.0, 1% NP-40, 5mM EDTA, 450mM Nacl, 10mM Na pyrophosphate and 50mM NaF), and add various fresh inhibitors (1mM Na orthovanadate, 1mM) to the lysis buffer. PMSF, 10μg / ml Aprotinin, Leupeptin, pepstatin). After sonication at room temperature, 1% mercaptoethanol was added, and the mixture was boiled and denatured at 100°C for 5 min. In SDS-PAGE gel, 10 μL was loaded per well. After electrophoresis, the protein sample is transferred to the nitrocellulose membrane by membrane transfer. After the transfer, the membrane was washed once with TBST, blocked with 5% skimmed milk powder for 1 hour, and washed with TBST once, and the diluted primary antibody was hybridized with the membrane at room temperature for 2 hours or overnight at 4°C. After washing three times with TTBS, hybridize the diluted second...

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Abstract

The invention discloses a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 system capable of simultaneously knocking out KRAS genes and EGFR genes. The system comprises sgRNA for specifically targeting KRAS genes and sgRNA for specifically targeting EGFR genes, wherein a corresponding DNA sequence of the sgRNA for specifically targeting KRAS genes is shown as SEQ ID NO.1 or / and SEQ ID NO.2; and a corresponding DNA sequence of the sgRNA for specifically targeting EGFR genes is shown as SEQ ID NO.11 or / and SEQ ID NO.12. The invention further discloses application of the system in preparation of medicines for treating cancers. The CRISPR-Cas9 system disclosed by the invention is capable of simultaneously and efficiently knocking out two cancer driving factors KRAS and EGFR which are highly-expressed in lung cancer. The system is simple in operation and high in knockout efficiency and is expected to be applied to treatment of the lung cancer. The system disclosed by the invention is applicable to multiple cancers with abnormal expressions of the EGFR and KRAS.

Description

Technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a CRISPR-Cas9 system for simultaneously knocking out KRAS gene and EGFR gene and its application. Background technique [0002] Lung cancer is currently the malignant tumor with the highest morbidity and mortality in the world, and the global morbidity and mortality of lung cancer are on the rise. In our country, with the acceleration of industrialization, serious environmental pollution and increasing population aging, the cancer burden of lung cancer is increasing. According to histopathology, lung cancer can be divided into two categories: non-small cell lung cancer (NSCLC, 85%) and small cell lung cancer (SCLC, 15%). Its treatment is still one of the most challenging tasks in the medical field. [0003] Tumor cells continue to grow under the action of driver genes and are highly sensitive to the inhibition of driver genes. Among them, KRAS and EGFR are common mu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22A61K48/00A61K31/713A61P35/00
CPCA61K31/713C07K14/71C07K14/82C12N9/22C12N15/102C12N15/907
Inventor 陈锦阳邓兆群
Owner 浙江卫未生物医药科技有限公司
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