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Target sequence, recognized by streptococcus thermophilus CRISPR-Cas9 system, of human CCR5 gene, sgRNA and application of CRISPR-Cas9 system

A technology for sequence recognition and system recognition, applied in the field of genetic engineering, can solve problems such as frameshift mutation, DNA insertion or deletion, and low efficiency, and achieve the effect of preventing and/or treating AIDS

Inactive Publication Date: 2016-03-16
张竞方
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2008, Sangamo successfully used ZFNs in CD4 + Knockout of CCR5 is achieved on T cells, but the efficiency of ZFN targeted knockout of CCR5 is low, people expect to find a more efficient knockout strategy of CCR5
In the absence of a template, non-homologous end joining (NHEJ) occurs, and DNA insertion or deletion (indel) often occurs during the NHEJ repair process, resulting in frameshift mutations and gene knockouts

Method used

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  • Target sequence, recognized by streptococcus thermophilus CRISPR-Cas9 system, of human CCR5 gene, sgRNA and application of CRISPR-Cas9 system
  • Target sequence, recognized by streptococcus thermophilus CRISPR-Cas9 system, of human CCR5 gene, sgRNA and application of CRISPR-Cas9 system
  • Target sequence, recognized by streptococcus thermophilus CRISPR-Cas9 system, of human CCR5 gene, sgRNA and application of CRISPR-Cas9 system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] This example is used to illustrate the design of the sgRNA of the present invention.

[0042] 120 sgRNAs were constructed according to the 5'-recognition sequence-recruiting Cas9 protein sequence-3' and the base sequence shown in Table 1 below.

[0043] Table 1

[0044]

Embodiment 2

[0046] This example is used to illustrate the construction of the CRISPR-Cas9 system of the present invention.

[0047] (1) Add CACC to the 5' end of the DNA sequence corresponding to the sgRNA (as shown in Example 1) to obtain a forward oligonucleotide (Forwardoligo). According to this sgRNA, its complementary strand is obtained, and at its 5' end Addition of AAAC results in a reverse oligonucleotide (Reverseoligo). synthesized separately. If the first base at the 5' end of the DNA sequence corresponding to the sgRNA recognition sequence is not G, you need to add a G after CACC, and correspondingly add a matching C at the 3' end of the reverse oligonucleotide. The above-mentioned forward oligonucleotide and reverse oligonucleotide are denatured and annealed in pairs, and after annealing, a double-stranded sgRNA oligonucleotide that can be connected to a U6 eukaryotic expression vector is formed. The denaturation and annealing system is:

[0048]

[0049] The conditions f...

Embodiment 3

[0065] This example is used to illustrate the method of using the CRISPR-Cas9 system of the present invention to edit the CCR5 gene.

[0066] (1) Cell culture and transfection

[0067] (1) HEK293T cells (CBR-130005, purchased from Shanghai Saiqi Bioengineering Co., Ltd.) were cultured in DMEM high-glucose medium (containing 10% FBS, penicillin (penicillin, 100U / ml) and streptomycin (streptomycin, 100μg / ml)) for cultivation;

[0068] (2) Divide into six-well plates before transfection, and perform transfection when the cell density reaches 70%;

[0069] (3) According to the dosage ratio of Lipofectamine3000 (Invitrogen, 11668-019), use 3 μg of U6-hCCR5sgRNA-EF1a-NLS-StCas9-NLS-2A-Puro-WPRE, or 1.5 μg of U6-hCCR5sgRNA-EF1a-Neo-WPRE and 1.5 μg of U6-EF1a-NLS-StCas9-NLS-2A-Puro-WPRE was combined to transfect each well of cells, and the medium was changed after 8 hours. Correspondingly, Puromycin (Puromycin) and G418 (Geneticin) were added for drug screening, 48 hours Cells wer...

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Abstract

The invention belongs to the field of genetic engineering, and discloses a target sequence, recognized by a streptococcus thermophilus CRISPR-Cas9 system, of a human CCR5 gene, sgRNA and an application of the CRISPR-Cas9 system. The target sequence is shown as any one of the nth bit to the 30th bit of the SEQ ID NO:1-30, and n is equal to 1-12. The invention further relates to the sgRNA of the sequence -3' with the sequence of 5'-recognition sequence-recruiting Cas9 protein and a coded DNA molecule thereof. The DNA sequence corresponding to the recognition sequence is identical to the target sequence. The invention further relates to the CRISPR-Cas9 system. The system comprises Cas9 protein, the sgRNA and / or the coded sequence carrying the Cas9 protein and a carrier of the coded sequence of the sgRNA. The invention further relates to the application of the CRISPR-Cas9 system in editing the CCR5 gene and preparing medicine for HIV infection. Editing of the human CCR5 gene can be achieved, and therefore Aids is prevented and treated.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a target sequence and sgRNA recognized by a CRISPR-Cas9 system and their related applications. Background technique [0002] Acquired immunodeficiency syndrome (acquiredimmune deficiency syndrome, AIDS), also known as AIDS, is a very harmful infectious disease. Since its discovery more than 30 years ago, AIDS has caused more than 20 million deaths, and has always been a huge public health problem, affecting the lives of 35.3 million people around the world. AIDS is caused by human immunodeficiency virus (Human Immunodeficiency Virus, HIV) infection, HIV is a lentivirus that infects human immune system cells, is a retrovirus, also known as HIV. HIV can be divided into two subtypes, HIV-1 and HIV-2. HIV-1 is highly pathogenic and is the main pathogen that causes AIDS. [0003] CD4 + T cells are the primary target of HIV-1 virus infection process, and subsequent studies have foun...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/113C12N9/22A61K31/7088A61P31/18
Inventor 张竞方秦小平
Owner 张竞方
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