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SgRNA, expression vector and kit used for targeted knockout of human NKG2A/KLRC1 gene and application of sgRNA used for targeted knockout of human NKG2A/KLRC1 gene

A technology of expression vectors and kits, applied in the field of genetic engineering, can solve the problems of short-acting effects of antibody drugs, long drug development time, expensive antibodies, etc., achieve low cost, reduce off-target efficiency, and reduce safety risks.

Inactive Publication Date: 2019-08-02
CHENGDU MEDGENCELL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many unfavorable factors such as the short-term effect of antibody drugs, the diversity of immune checkpoints, the long time of drug development, and the high price of antibodies.

Method used

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  • SgRNA, expression vector and kit used for targeted knockout of human NKG2A/KLRC1 gene and application of sgRNA used for targeted knockout of human NKG2A/KLRC1 gene
  • SgRNA, expression vector and kit used for targeted knockout of human NKG2A/KLRC1 gene and application of sgRNA used for targeted knockout of human NKG2A/KLRC1 gene
  • SgRNA, expression vector and kit used for targeted knockout of human NKG2A/KLRC1 gene and application of sgRNA used for targeted knockout of human NKG2A/KLRC1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Design and synthesis of sgRNA specifically targeting human NKG2A / KLRC1 gene in CRISPR / Cas9 specific knockout of human NKG2A / KLRC1 gene:

[0057] 1. Design of sgRNA targeting human NKG2A / KLRC1 gene:

[0058] (a) select the sequence of 5'-GGN(19)GG-3', 5'-GN(20)GG-3' or 5'-N(21)GG-3' on the human NKG2A / KLRC1 gene;

[0059] (b) Select the sequence where the target site is located on the exon of the human NKG2A / KLRC1 gene to ensure that it is easier to cause complete gene inactivation;

[0060] (c) The targeting site of the sgRNA on the human NKG2A / KLRC1 gene is located on the common exon of different splicing forms;

[0061] (d) Use BLAST of online database NCBI or BLAT of UCSC to determine whether the target sequence of sgRNA is unique;

[0062] According to the above method, a total of 56 sgRNAs targeting human NKG2A / KLRC1 gene were designed, the sequences of which are shown in the sequence table SEQ ID NO.1-56 respectively;

[0063] 2. Selection of sgRNA targeting hu...

Embodiment 2

[0084] Use CRISPR / Cas9 to specifically knock out the human NKG2A / KLRC1 gene (the pair of sgRNAs used in this example to target the human NKG2A / KLRC1 gene are shown in the sequences SEQ ID NO.2, 6, 8, 20, 21, 22, 26 sequence shown)

[0085] Include the following steps:

[0086] 1. A linearized sequence such as the pGL3-2U6-sgRNA vector of sequence SEQ ID NO.77.

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

[0088] 2 μg pGL3-2U6-sgRNA

[0089] 5μl CutSmart Buffer

[0090] 1 μl BsaI (NEB)

[0091] Replenish water to 50 μl, and react for 1 hour at 37°C for PCR.

[0092] After digestion, perform 1% agarose gel electrophoresis, and use EndoFree Plasmid Kits (QIAGEN) to recover the pGL3-2U6-sgRNA vector;

[0093]2. The double-stranded sgRNA (2), sgRNA (8), sgRNA (21), and sgRNA (22) obtained after denaturation and annealing that can be connected to the U6 eukaryotic expression vector (respectively as the sequences of SEQ ID NO.2,8 , shown in 21, 22) oligonuc...

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Abstract

The invention discloses sgRNA used for targeted knockout of a human NKG2A / KLRC1 gene, and belongs to the technical field of genetic engineering. The sgRNA has an arbitrary nucleotide sequence as shownin SEQ ID NO.1-56. The invention further discloses a sgRNA compound, expression vector, CRISPR / Cas9 system and kit used for targeted knockout of the human NKG2A / KLRC1 gene, and application of the sgRNA compound in preparing an immune cell medicine for treating neoplastic diseases. By means of the prepared specific targeted human KNG2A / KLRC1 gene, the human NKG2A / KLRC1 gene can be precisely targeted, effective knockout can be achieved, and meanwhile, an obtained human NKG2A / KLRC1 gene knockout immune cell is suitable for multiple tumor cell models; and a preparation method has simple steps, the targeting specificity of the sgRNA is good, and the knockout efficiency of the CRISPR / Cas9 system is high.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to sgRNA, expression vector, kit and application thereof for targeted knockout of human NKG2A / KLRC1 gene. Background technique [0002] The CRISPR / Cas system is developed from the adaptive immune system of bacteria and archaea against foreign viruses or plasmids. The CRISPR sequence and its related gene (Cas gene) work together because of its unique RNA-mediated endonuclease Activity has received extensive attention from the biological community. Among them, the type II DNA endonuclease Cas9 represented by Streptococcus Pyogenes has only one subunit and the simplest structure, so it is the most widely used. The CRISPR / Cas9 system locates the target gene by identifying the small-guide RNA (sgRNA) sequence of a specific sequence, guides Cas9 to cut the target sequence, and causes double-strand breaks (Double-strand breaks, DSB). Under the condition of no template, Non-Ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N9/22A61K48/00A61P35/00
CPCA61K48/005A61P35/00C12N9/22C12N15/113C12N15/907C12N2310/10C12N2830/008
Inventor 邓涛王越喻堃李倩成俊杰
Owner CHENGDU MEDGENCELL CO LTD
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