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Method for efficiently knocking out CD96 gene in NK cell

A high-efficiency technology for NK cells, applied in the field of gene editing, to achieve high knockout efficiency, improved recognition and killing activity, obvious application prospects and clinical application value

Active Publication Date: 2021-01-05
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CRISPR / Cas9 is a new type of nuclease system derived from prokaryotes. It consists of two elements, the guide sequence sgRNA and the nuclease Cas9. At present, the CRISPR / Cas9 system has been successfully used in humans, mice, zebrafish, and Arabidopsis. , rice, Drosophila, silkworm and other species have achieved gene knockout, but so far there is no related report on the knockout of CD96 gene in NK cells

Method used

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  • Method for efficiently knocking out CD96 gene in NK cell
  • Method for efficiently knocking out CD96 gene in NK cell
  • Method for efficiently knocking out CD96 gene in NK cell

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Experimental program
Comparison scheme
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Embodiment 1

[0034] A method for efficiently knocking out the CD96 gene in NK cells, comprising the following specific steps:

[0035] 1. Expansion and culture of NK cells

[0036] 1) Take out the frozen human peripheral blood mononuclear cells (PBMC) from liquid nitrogen, and thaw them rapidly in a water bath at 37°C;

[0037] 2) Add 4 mL of RPMI-1640 complete culture solution containing 10% FBS and 1% penicillin / streptomycin double antibody to a new 15 mL centrifuge tube, and transfer 1 mL of PBMC suspension to the 15 mL centrifuge tube;

[0038] 3) Centrifuge at 250×g for 5 minutes at room temperature;

[0039] 4) Discard the supernatant and resuspend the cells in 1 mL RPMI-1640 culture medium;

[0040] 5) Add 19 mL of RPMI-1640 culture medium into a new 75 mL cell culture flask, and transfer the above cell suspension to the culture flask;

[0041] 6) Add human recombinant IL-2 protein to the culture flask, the final concentration is 200U / mL;

[0042] 7) Place the culture flask at 3...

Embodiment 2

[0073] Detection of gene knockout efficiency by flow cytometry:

[0074] 1) Collect wild-type or NK cells after knocking out the CD96 gene and wash twice with 1×PBS containing 1% fetal bovine serum (FBS);

[0075] 2) Resuspend the cells with PBS containing 1% FBS and count, and adjust the cell concentration to 3×10 6 a / mL;

[0076] 3) Add 50 μL of the above cell suspension into a new 1.5 mL centrifuge tube, add 1 μL of PE-anti TIGTI antibody, and incubate at 4°C in the dark for 30 min;

[0077] 4) Wash twice with 1% FBS 1×PBS;

[0078] 5) Resuspend the cells with 300 μL 1% FBS 1×PBS and perform flow cytometry detection.

[0079] Test results such as Figure 2A and 2B shown by Figure 2A and 2B The results shown can be seen: sgRNA-5, 9 and 10 have higher knockout efficiency, as shown in Table 1 for details:

[0080] Table 1 CD96 knockout efficiency detected by flow cytometry

[0081]

[0082]

[0083] It can be seen from the results in Table 1 that: under the sam...

Embodiment 3

[0085] The phosphorylation-modified gRNA targeting CD96 was purchased from GenScript Biotechnology Co., Ltd. The specific phosphorylation sites are: 3 thiol and methoxy modifications at the 3' end and 5' end respectively.

[0086] CD96-10:GCTGTCTATCATCCCAATA

[0087] Depend on image 3 The results shown can be seen: the percentage of CD56+ / CD96+NK cells in wild-type NK cells is 96.5%, and the CD96-10 gRNA modified by phosphorylation can reduce the percentage of CD56+ / CD96+NK cells to 67.4%, while the percentage without phosphorylation modification The CD96-10 gRNA can only be reduced to 90.4%, indicating that the sgRNA modified by phosphorylation has better stability and stronger activity. The specific results are shown in Table 2:

[0088] Table 2 The knockout efficiency of CD96 detected by flow cytometry

[0089] name CD56+ / CD96+NK cell percentage (%) Knockout rate (%) WT 96.5 0 Phosphorylated CD96-10 67.4 29.1 unmodified CD96-10 90.4 6.1...

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Abstract

The invention discloses a method for efficiently knocking out a CD96 gene in an NK cell. The method comprises the step of introducing a compound of sgRNA and Cas9 protein into an amplified NK cell inan electroporation transfection manner by using a CRISPR / Cas9 gene editing system, wherein the sequence of sgRNA is selected from any one of sequences as shown in SEQ ID NO. 1-10. By adopting the method disclosed by the invention, the CD96 gene in the NK cell can be knocked out, the knocking-out efficiency is relatively high, and the obtained NK cell which does not express CD96 can relieve the immunosuppression effect of CD155+ tumor cells on the NK cell. Compared with a wild type NK cell, the NK cell shows relatively strong anti-tumor activity in in-vivo and in-vitro experiments, the recognition and killing activity of the NK cell on tumor cells is obviously improved, and the NK cell is expected to be developed into a safe and effective anti-tumor biological preparation.

Description

technical field [0001] The invention relates to a method for efficiently knocking out the CD96 gene in NK cells, belonging to the technical field of gene editing. Background technique [0002] Natural killer cells (natural killer cells, NK cells) are important immune cells of the body, derived from bone marrow lymphoid stem cells, their differentiation and development depend on the microenvironment of bone marrow and thymus, and are mainly distributed in bone marrow, peripheral blood, liver, spleen, and lung and lymph nodes. NK cells are different from T and B cells. They are a type of lymphocytes that can non-specifically kill tumor cells and virus-infected cells without prior sensitization. They are not only related to anti-tumor, anti-viral infection and immune regulation, but also in some cases Participate in the occurrence of hypersensitivity and autoimmune diseases, and can identify target cells and killing mediators. [0003] Studies have shown that although NK cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N15/85C12N15/113C12N15/10
CPCC12N5/0646C07K14/47C12N15/85C12N15/113C12N15/10C12N2310/20
Inventor 朱诗国姚超于文霞
Owner SHANGHAI UNIV OF T C M
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