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sgRNA for target knockout of human NKG2A/KLRC1 genes, expression vector, reagent kit and application of sgRNA

A gene and targeting technology, applied in the field of genetic engineering, can solve the problems of short-acting antibody drugs, low gene editing efficiency, long drug development time, etc., achieve low cost, improve gene editing efficiency and the feasibility of clinical use, The effect of reducing off-target efficiency

Pending Publication Date: 2020-07-24
CHENGDU MEDGENCELL CO LTD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0009] One of the objectives of the present invention is to provide a sgRNA for targeted knockout of human NKG2A / KLRC1 gene, so as to overcome the short-acting effect of antibody drugs in the prior art, the diversity of immune checkpoints, the long time of drug development, the Expensive price and low gene editing efficiency of CRISPR / Cas9 technology

Method used

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  • sgRNA for target knockout of human NKG2A/KLRC1 genes, expression vector, reagent kit and application of sgRNA
  • sgRNA for target knockout of human NKG2A/KLRC1 genes, expression vector, reagent kit and application of sgRNA
  • sgRNA for target knockout of human NKG2A/KLRC1 genes, expression vector, reagent kit and application of sgRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Design and synthesis of sgRNA specifically targeting human NKG2A / KLRC1 gene in CRISPR / Cas9 specific knockout of human NKG2A / KLRC1 gene.

[0070] 1. Design of sgRNA targeting human NKG2A / KLRC1 gene:

[0071] (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;

[0072] (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;

[0073] (c) The targeting site of the sgRNA on the human NKG2A / KLRC1 gene is located on the common exon of different splicing forms;

[0074] (d) Use BLAST of online database NCBI or BLAT of UCSC to determine whether the target sequence of sgRNA is unique;

[0075] 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;

[0076] 2. Selection of sgRNA targeting hu...

Embodiment 2

[0098] 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, 23, 26 respectively sequence shown).

[0099] Include the following steps:

[0100] 1. A linearized sequence such as the pGL3-2U6-sgRNA vector of sequence SEQ ID NO.77

[0101] Enzyme digestion system and conditions are as follows:

[0102] 2 μg pGL3-2U6-sgRNA

[0103] 5μL CutSmart Buffer

[0104] 1 μL BsaI (NEB)

[0105] Replenish water to 50 μL, and react in PCR at 37°C for 1 hour;

[0106] After digestion, perform 1% agarose gel electrophoresis, and use EndoFree Plasmid Kits (QIAGEN) to recover the pGL3-2U6-sgRNA vector;

[0107] 2. The double-stranded sgRNA (2), sgRNA (8), sgRNA (21), and sgRNA (23) that can be connected to the U6 eukaryotic expression vector obtained after denaturation and annealing (respectively as sequence SEQ ID NO.2,8 , 21, 23) oligonucleot...

Embodiment 3

[0144] For experimental verification, 4 sequences (shown in sequence SEQ ID NO.20, 22, 26, and 27 respectively) were selected from the 23 sequences selected in step 2 of Example 1 again, and the sequences of Example 1 were taken successively. Step 3 and steps 1 to 8 of Example 2 were the same method to prepare pGL3-2U6-NKG2A / KLRC1-sg(22)-sg(20), pGL3-2U6-NKG2A / KLRC1-sg(26)-sg( 20), pGL3-2U6-NKG2A / KLRC1-sg(26)-sg(27); take the same method as step 9 of Example 2 to obtain cultured cells; finally take the same method as step 10 of Example 2 method to detect it.

[0145] Experimental results such as image 3 As shown, the expression of NKG2A on the cell surface decreased after treatment with pGL3-2U6-NKG2A / KLRC1-sg(2)-sg(6) and pGL3-2U6-NKG2A / KLRC1-sg(8)-sg(6) plasmids, respectively The rate (ie knockout rate) was 25.4% and 14.3% respectively; (20), pGL3-2U6-NKG2A / KLRC1-sg(23)-sg(20), pGL3-2U6-NKG2A / KLRC1-sg(26)-sg(20), and pGL3-2U6-NKG2A / KLRC1-sg( After 26)-sg(27) plasmid tre...

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Abstract

The invention relates to the field of genetic engineering, and discloses sgRNA for target knockout of human NKG2A / KLRC1 genes. The sgRNA has any nucleotide sequence as shown in SEQID NO.1-56. The invention also discloses an sgRNA composition for the sgRNA for target knockout of human NKG2A / KLRC1 genes, an expression vector, a CRISPR / Cas9 system, a reagent kit and the application of the sgRNA for preparation of immunocyte medicines for treating tumor diseases. Through utilizing specific target human NKG2A / KLRC1 genes prepared through the sgRNA disclosed by the invention, the human NKG2A / KLRC1 genes can be precisely targeted, effective knockout is realized, and besides, the obtained human NKG2A / KLRC1 gene knockout immunocyte is suitable for various tumor cell models. A preparation method hasthe advantages of being simple in steps, good in sgRNA target specificity and high in CRISPR / Cas9 system knockout efficiency.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an sgRNA for targeted knockout of human NKG2A / KLRC1 gene, expression vector, kit and application thereof. 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-Homologou...

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
Owner CHENGDU MEDGENCELL CO LTD
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