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Method for specifically knocking out human CCR5 (Chemokine Receptor 5) gene by CRISPR (clustered regularly interspaced short palindromic repeat-associated)-Cas 9 and SgRNA (single guide RNA) for specifically targeting CCR5 gene

A pgl3-u6-hccr5sg, specific technology, applied in the field of genetic engineering, can solve the problems of low efficiency, high cost, time-consuming ZFN, etc., and achieves high efficiency, improved efficiency, and good therapeutic effect.

Active Publication Date: 2014-07-16
AOMIAO BIOTECH GUANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] For the existing problems of using ZFNs to target CCR5 for AIDS treatment: (1) The efficiency is low, and only a small amount of CCR5 can be knocked out; (2) A single target site may only cause non-frameshift mutations, so CCR5 cannot be effectively knocked out (3) Designing and synthesizing a pair of specific ZFNs is time-consuming, labor-intensive, and expensive, making it expensive, etc.

Method used

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  • Method for specifically knocking out human CCR5 (Chemokine Receptor 5) gene by CRISPR (clustered regularly interspaced short palindromic repeat-associated)-Cas 9 and SgRNA (single guide RNA) for specifically targeting CCR5 gene
  • Method for specifically knocking out human CCR5 (Chemokine Receptor 5) gene by CRISPR (clustered regularly interspaced short palindromic repeat-associated)-Cas 9 and SgRNA (single guide RNA) for specifically targeting CCR5 gene
  • Method for specifically knocking out human CCR5 (Chemokine Receptor 5) gene by CRISPR (clustered regularly interspaced short palindromic repeat-associated)-Cas 9 and SgRNA (single guide RNA) for specifically targeting CCR5 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Design and synthesis of sgRNA for specific targeting of CCR5 gene in CRISPR-Cas9 specific knockout of human CCR5 gene

[0067] 1. Design of sgRNA targeting human CCR5 gene:

[0068] (1) Select the 5'-GGN(19)GG sequence on the CCR5 gene. If there is no 5'-GGN(19)GG sequence, 5'-GN(20)GG or 5'-N(21)GG is also Can.

[0069] (2) The target site of sgRNA on the CCR5 gene is located in the exon of the gene, which is more likely to cause the deletion of the fragment or the frame shift mutation, so as to achieve the purpose of complete gene inactivation.

[0070] (3) The targeting sites of sgRNA on the CCR5 gene are located on the common exons of different splicing forms.

[0071] (4) Use BLAT in the UCSC database or BLAST in the NCBI database to determine whether the target sequence of the sgRNA is unique and reduce potential off-target sites.

[0072] According to the above method, we designed a total of 100 sgRNAs targeting the human CCR5 gene, and the ...

Embodiment 2

[0089] Example 2 Using CRISPR-Cas9 to specifically knock out the human CCR5 gene (the sgRNA used to target the CCR5 gene is shown in Sequence Table 53)

[0090] 1. Linearization sequence such as sequence table SEQ ID NO. 6 The indicated pGL3-U6-sgRNA plasmids.

[0091] Enzyme digestion system and conditions are as follows:

[0092] 2 μg pGL3-U6-sgRNA (400 ng / μl);

[0093] 1 μl CutSmart Buffer;

[0094] 1 μl BsaI (NEB, R0535L);

[0095] Add water to 50 μl, incubate at 37°C for 3-4 hours, shake and centrifuge at intervals to prevent droplets from evaporating onto the tube cap.

[0096] After digestion, use AxyPrep PCR Clean up Kit (AP-PCR-250) to purify and recover to 20-40 μl sterilized water.

[0097] 2. The double-stranded sgRNA oligonucleotides obtained after denaturation and annealing that can be connected to the U6 eukaryotic expression vector (the sequences of its Forward oligo and Reverse oligo are respectively as shown in the sequence table S...

Embodiment 3

[0128] Example 3 Using CRISPR-Cas9 to specifically knock out the human CCR5 gene (the sgRNA used to target the CCR5 gene is shown in the sequence table as SEQ ID NO. 58)

[0129] 1. Linearization sequence such as sequence table SEQ ID NO. 6 The indicated pGL3-U6-sgRNA plasmids.

[0130] Enzyme digestion system and conditions are as follows:

[0131] 2 μg pGL3-U6-sgRNA (400 ng / μl);

[0132] 1 μl CutSmart Buffer;

[0133] 1 μl BsaI (NEB, R0535L);

[0134] Add water to 50 μl, incubate at 37°C for 3-4 hours, shake and centrifuge at intervals to prevent droplets from evaporating onto the tube cap.

[0135] After digestion, use AxyPrep PCR Clean up Kit (AP-PCR-250) to purify and recover to 20-40 μl sterilized water.

[0136] 2. The double-stranded sgRNA oligonucleotides obtained after denaturation and annealing that can be connected to the U6 eukaryotic expression vector (the sequences of its Forward oligo and Reverse oligo are respectively as shown in th...

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Abstract

The invention belongs to the field of gene engineering and particularly relates to a method for specifically knocking out a human CCR5 (Chemokine Receptor 5) gene by CRISPR (clustered regularly interspaced short palindromic repeat-associated)-Cas 9 and SgRNA (single guide RNA) for specifically targeting the CCR5 gene as well as an intermediate carrier and application thereof. The sgRNA for specifically targeting the human CCR5 gene, prepared by the method disclosed by the invention, can be used for accurately targeting the human CCR5 gene and realizing gene knockout. The preparation method is simple in steps and good in sgRNA targeting property; the knockout efficiency of a CRISPR-Cas 9 system is high.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a CRISPR-Cas9 method for specifically knocking out the human CCR5 gene and an sgRNA for specifically targeting the CCR5 gene. Background technique [0002] Human Immunodeficiency Virus (HIV) is a lentivirus that infects cells of the human immune system and is a type of retrovirus. It is generally believed that the infection of human immunodeficiency virus leads to AIDS, and AIDS is a fatal disease caused by defects in acquired cellular immune function that lead to severe random infection and secondary tumors. Since AIDS was identified in the United States in 1981 and developed into a global pandemic until the end of 2003, it has caused more than 20 million deaths. Human Immunodeficiency Virus is also commonly referred to colloquially as "AIDS". Twenty-five years since the first case of AIDS caused by HIV was discovered in 1981, although the clinical treatment ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85
Inventor 杜忆南黄行许
Owner AOMIAO BIOTECH GUANGZHOU CO LTD
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