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Method for human CTLA4 gene specific knockout through CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat) and sgRNA(single guide RNA)for specially targeting CTLA4 gene

A pgl3-u6-hctla4sg, specific technology, applied in the field of genetic engineering, can solve the problems of expensive, time-consuming and expensive antibody drugs

Active Publication Date: 2014-05-28
AOMIAO BIOTECH GUANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] In view of the problems existing in the current use of CTLA4 antibody for immunodetection and blocking treatment of tumors: (1) The effect of the antibody is only a temporary blocking effect; (2) There are many kinds of inhibitory receptors, how to use multiple antibodies to block multiple There is no countermeasure for inhibitory receptors; (3) It is not easy to develop effective antibodies; (4) It only targets extracellular targets; (5) The development of antibody drugs is time-consuming, laborious, and expensive, making antibody drugs expensive, etc.

Method used

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  • Method for human CTLA4 gene specific knockout through CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat) and sgRNA(single guide RNA)for specially targeting CTLA4 gene
  • Method for human CTLA4 gene specific knockout through CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat) and sgRNA(single guide RNA)for specially targeting CTLA4 gene
  • Method for human CTLA4 gene specific knockout through CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat) and sgRNA(single guide RNA)for specially targeting CTLA4 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Design and synthesis of sgRNA for specific targeting of CTLA4 gene in CRISPR-Cas9 specific knockout of human CTLA4 gene

[0060] 1. Design of sgRNA targeting human CTLA4 gene:

[0061] (1) Select the 5'-GGN(19)GG sequence on the CTLA4 gene. If there is no 5'-GGN(19)GG sequence, 5'-GN(20)GG or 5'-N(21)GG is also Can.

[0062] (2) The targeting site of the sgRNA on the CTLA4 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.

[0063] (3) The targeting sites of sgRNA on the CTLA4 gene are located on the common exons of different splicing forms.

[0064] (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.

[0065] According to the above method, we designed a total of 67 sgRNAs targeting the human CTLA4 gene, the ...

Embodiment 2

[0085] Example 2 Using CRISPR-Cas9 to specifically knock out the human CTLA4 gene (the sgRNA used to target the CTLA4 gene is shown in the sequence table as SEQ ID NO.36)

[0086] 1. The linearized sequence is the pGL3-U6-sgRNA plasmid shown in the sequence table SEQ ID NO.10.

[0087] Enzyme digestion system and conditions are as follows:

[0088] 2 μg pGL3-U6-sgRNA (400ng / μl);

[0089] 1μl CutSmart Buffer;

[0090] 1 μl BsaI (NEB, R0535L);

[0091] Replenish water to 50 μl, incubate at 37°C for 3-4 hours, shake and centrifuge at regular intervals to prevent droplets from evaporating onto the tube cap.

[0092] After digestion, use AxyPrep PCR Clean up Kit (AP-PCR-250) to purify and recover to 20-40 μl sterilized water.

[0093] 2. Combine the double-stranded sgRNA oligonucleotides obtained after denaturation and annealing that can be connected to the U6 eukaryotic expression vector (the sequences of their Forward oligo and Reverse oligo are shown in the sequence table SE...

Embodiment 3

[0123] Example 3 Using CRISPR-Cas9 to specifically knock out the human CTLA4 gene (the sgRNA used to target the CTLA4 gene is shown in the sequence table SEQ ID NO.84)

[0124] 1. The linearized sequence is the pGL3-U6-sgRNA plasmid shown in the sequence table SEQ ID NO.10. Enzyme digestion system and conditions are as follows:

[0125] 2 μg pGL3-U6-sgRNA (400ng / μl);

[0126] 1μl CutSmart Buffer;

[0127] 1 μl BsaI (NEB, R0535L);

[0128] Replenish water to 50 μl, incubate at 37°C for 3-4 hours, shake and centrifuge at regular intervals to prevent droplets from evaporating onto the tube cap.

[0129] After digestion, use AxyPrep PCR Clean up Kit (AP-PCR-250) to purify and recover to 20-40 μl sterilized water.

[0130]2. The double-stranded sgRNA oligonucleotides obtained after denaturation and annealing that can be connected to the U6 eukaryotic expression vector (the sequences of their Forward oligo and Reverse oligo are shown in the sequence table SEQ ID NO.3 and 4, resp...

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Abstract

The invention belongs to the field of genetic engineering, particularly relates to a method for human CTLA4 gene specific knockout through CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat) and sgRNA(single guide RNA)for specially targeting a CTLA4 gene, and provides a method for human CTLA4 gene specific knockout through CRISPR-Cas9 and sgRNA(single guide RNA)for specially targeting a CTLA4 gene. The sgRNA(single guide RNA)for specially targeting a human CTLA4 gene, prepared through adopting the method provided by the invention, can be used for accurately targeting the human CTLA4 gene and realizes gene knockout; the preparing method has simple steps, the targeting performance of the sgRNA is good, and the knockout efficiency of a CRISPR-Cas9 system is high.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a method for specifically knocking out human CTLA4 gene by CRISPR-Cas9 and an sgRNA for specifically targeting the CTLA4 gene. Background technique [0002] The clustered regularly interspaced short palindromic repeat system (clustered regularly interspaced short palindromic repeat; CRISPR-associated, CRISPR-Cas9) is a complex with endonuclease activity that recognizes a specific DNA sequence and cuts it at a specific site. Double-strand breaks (Double-strand breaks, DSB), in the absence of template conditions, non-homologous end joining (Non-homologous end joining, NHEJ), resulting in frameshift mutation (frameshift mutation), resulting in gene knockout ( figure 1 ). [0003] This technology has attracted widespread attention because it can quickly, easily and efficiently target any gene in the genome, and it became popular like an explosion in 2012. Cas9 ha...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85
Inventor 胡边黄行许
Owner AOMIAO BIOTECH GUANGZHOU CO LTD
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