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Human CCR5 gene target sequence recognized by streptococcus thermophilus CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated protein 9) system, sgRNA (single guide ribonucleic acid) and application

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: 2016-02-17
张竞方
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This technology allows for editing or alteration of genes involved in immune response (CRC) pathways such as CR1A, CD4, T lymphocyte function-associated proteins like CCR5. By changing these sequences, cellular communication between them becomes blocked, making it difficult for viruses from entering their body's bloodstream without causing harm.

Problems solved by technology

This patents discuss how researchers aim to develop effective methods to silence certain parts of proteins involved in diseases like cancer, autoimmunity, neurologisms, chronic illnesses, and other conditions related to aggravability issues associated with traditional medicaments. However, these technical solutions require expensive chemical agents and may result in side effects due to their high risk profile.

Method used

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  • Human CCR5 gene target sequence recognized by streptococcus thermophilus CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated protein 9) system, sgRNA (single guide ribonucleic acid) and application
  • Human CCR5 gene target sequence recognized by streptococcus thermophilus CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated protein 9) system, sgRNA (single guide ribonucleic acid) and application
  • Human CCR5 gene target sequence recognized by streptococcus thermophilus CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated protein 9) system, sgRNA (single guide ribonucleic acid) and application

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

[0043]

[0044]

Embodiment 2

[0046] This example is used to illustrate the construction of the CRISPR-Cas9 (CRISPR-St1Cas9) 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]...

Embodiment 3

[0065] This example is used to illustrate the method of using the CRISPR-Cas9 (CRISPR-St1Cas9) 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-St1Cas9-NLS-2A-Puro-WPRE, or 1.5 μg of U6-hCCR5sgRNA-EF1a-Neo-WPRE and 1.5 μg of U6-EF1a-NLS-St1Cas9-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, 4...

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Abstract

The invention belongs to the field of gene engineering and discloses a target sequence recognized by a CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated protein 9) system stemming from streptococcus thermophilus. The target sequence is shown as the n-20th position of any one of SEQ ID NO: 1-88, wherein n ranges from 1 to 5. The invention further discloses sgRNA (single guide ribonucleic acid) with a sequence being 5'-recognition sequence-recruitment Cas9 sequence-3' and a coding DNA (deoxyribonucleic acid) molecule thereof, and a DNA sequence corresponding to the recognition sequence is as same as the target sequence. The invention further discloses the CRISPR-Cas9 system comprising Cas9 and the sgRNA and/or Cas9-carried coding sequence and an sgRNA coding sequence vector. The invention further discloses application of the CRISPR-Cas9 system to CCR5 gene editing and HIV (human immunodeficiency virus) infection drug preparation. CCR5 gene editing can be achieved to protect cells from HIV infection.

Description

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Claims

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Application Information

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Owner 张竞方
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