Streptococcus thermophilus derived human CXCR3 gene target sequence recognizable by CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated 9) system and sgRNA (single guide ribonucleic acid) and application thereof

A technology for identifying sequences and systems, applied in the field of genetic engineering, can solve the problems of frameshift mutation, DNA insertion or deletion, low efficiency, etc., and achieve the effect of preventing and/or treating AIDS

Inactive Publication Date: 2016-02-10
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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  • Streptococcus thermophilus derived human CXCR3 gene target sequence recognizable by CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated 9) system and sgRNA (single guide ribonucleic acid) and application thereof
  • Streptococcus thermophilus derived human CXCR3 gene target sequence recognizable by CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated 9) system and sgRNA (single guide ribonucleic acid) and application thereof
  • Streptococcus thermophilus derived human CXCR3 gene target sequence recognizable by CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated 9) system and sgRNA (single guide ribonucleic acid) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] This example is used to illustrate the design of the sgRNA of the present invention.

[0043] According to the 5'-recognition sequence-recruiting Cas9 protein sequence-3' and the base sequence shown in Table 1 below, 992 sgRNAs were constructed.

[0044] Table 1

[0045]

[0046]

[0047]

Embodiment 2

[0049] This example is used to illustrate the construction of the CRISPR-Cas9 system of the present invention.

[0050] (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, obtain its complementary strand and place it at its 5'end Add AAAC to get Reverseoligo. Synthesize separately. If the first base at the 5'end of the DNA sequence corresponding to the sgRNA recognition sequence is not a G, a G must be added after CACC, and a matching C must be added to the 3'end of the reverse oligonucleotide. The above-mentioned forward oligonucleotide and reverse oligonucleotide are denatured and annealed in pairs. After annealing, a double-stranded sgRNA oligonucleotide that can be connected to the U6 eukaryotic expression vector is formed. The denaturation and annealing system is:

[0051]

[0052]

[0053] The PCR conditions are: 37°C for 30 minutes; 95°C for 5 minutes; ...

Embodiment 3

[0069] This example is used to illustrate the method of editing CXCR4 gene using the CRISPR-Cas9 system of the present invention.

[0070] (1) Cell culture and transfection

[0071] (1) HEK293T cells (CBR-130005, purchased from Shanghai Saiqi Biological Engineering Co., Ltd.) in DMEM high glucose medium (containing 10% FBS, penicillin (penicillin, 100U / ml) and streptomycin (100μg) / ml)) culture;

[0072] (2) Divide into a six-well plate before transfection, and perform transfection when the cell density reaches 70%;

[0073] (3) Use 3μg of U6-hCXCR4sgRNA-EF1a-NLS-StCas9-NLS-2A-Puro-WPRE or 1.5μg of U6-hCXCR4sgRNA-EF1a-Neo-WPRE according to the dosage ratio of Lipofectamine3000 (Invitrogen, 11668-019) 1.5μg of U6-EF1a-NLS-StCas9-NLS-2A-Puro-WPRE combination transfected cells per well, 8h later, the medium was changed, correspondingly, puromycin (Puromycin) and G418 (Geneticin) were added for drug screening, 48h After collecting the cells.

[0074] (2) TA clone sequencing detection:

[...

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Abstract

The invention belongs to the field of genetic engineering, discloses a streptococcus thermophilus derived target sequence recognizable by a CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated 9) system and further discloses a sgRNA (single guide ribonucleic acid) and a coding DNA (deoxyribonucleic acid) molecule thereof. The target sequence is shown as n-30th of any one of SEQ ID NO:1-248, wherein n=1-12. A sequence of the sgRNA is 5'-recognition sequence- Cas9 protein recruitment sequence-3', and a DNA sequence corresponding to the recognition sequence is identical to the target sequence. The invention also discloses the CRISPR-Cas9 system, and the CRISPR-Cas9 system comprises Cas9 proteins and sgRNA and / or comprises carriers carrying a Cas9 protein coding sequence and a sgRNA coding sequence. The invention further discloses application of the CRISPR-Cas9 system to CXCR4 gene editing and preparation of medicines for treating HIV (human immunodeficiency virus) infection. CXCR4 gene editing can be realized to protect cells from HIV infection.

Description

Technical field [0001] The invention belongs to the field of genetic engineering and relates to target sequences and sgRNA recognized by the CRISPR-Cas9 system and their related applications. Background technique [0002] Acquired immune deficiency syndrome (acquired immunodeficiency syndrome, AIDS), also known as AIDS, is a very harmful infectious disease. Over the past 30 years since its discovery, AIDS has caused more than 20 million deaths. It has always been a huge public health problem, affecting the lives of 35.3 million people worldwide. AIDS is caused by human immunodeficiency virus (HumanImmunodeficiencyVirus, HIV) infection. HIV is a kind of lentivirus that infects cells of the human immune system. It is a kind of retrovirus, also known as HIV. HIV can be divided into two subtypes: HIV-1 and HIV-2. HIV-1 has strong pathogenicity and is the main pathogen causing AIDS. [0003] CD4 + T cells are the primary target of the HIV-1 virus infection process. Later studies have ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/85C12N5/10A61K48/00A61P31/18
Inventor 张竞方秦小平
Owner 张竞方
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