Cell line gene knock-out method for obtaining large fragment deletion through CRISPR/Cas9 system

A gene knockout and large fragment technology, applied in the field of genetic engineering and genetic modification, can solve problems such as unavailable bases, deletions, time-consuming and labor-intensive problems, and achieve the effect of improving work efficiency

Active Publication Date: 2017-12-05
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional cell line gene knockout requires multiple rounds of resistance screening and dilution after transfection to obtain positive monoclonal cells, which is very time-consuming and laborious, and the false positive rate is high
After the CRISPR/Cas9 system cuts genomic DNA, in most cases, a few bases will be deleted or inserted, and this small change cannot be detected by traditional gel electrophoresis; while other methods, such as: direct Sequencing method, PCR enzyme digestion detection method, T7E1 enzyme digest...

Method used

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  • Cell line gene knock-out method for obtaining large fragment deletion through CRISPR/Cas9 system
  • Cell line gene knock-out method for obtaining large fragment deletion through CRISPR/Cas9 system
  • Cell line gene knock-out method for obtaining large fragment deletion through CRISPR/Cas9 system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The all-in-one CRISPR / Cas9 system vector was transformed to obtain expression vectors with DsRed2 and ECFP respectively.

[0057] (1) The parent vector pX458 was purchased from addgene (ID: 48138). After expanding the culture, the vector DNA was linearized with the restriction enzyme EcoRI (NEB). The specific steps are: add 1 μg pX458 vector to a 1.5ml centrifuge tube in turn DNA; 3 μl of 10×NEB Buffer 2.1; 1 μl of EcoRI (NEB) and finally replenished with water to a total volume of 30 μl, and incubated at 37°C for 2 hours. After the digestion was completed, the digestion product was purified using QIAquick PCR Purification Kit and recovered into 30 μl ddH2O.

[0058] (2) The DNA sequences of DsRed2 (as shown in SEQ ID NO. 25) and ECFP (as shown in SEQ ID NO. 26) were submitted to the company for synthesis (Shanghai Bailiger Biotechnology Co., Ltd.), at the 5' end of the sequence Add the EcoRI restriction site and T2A sequence, and clone into the pUC57 vector; then dige...

Embodiment 2

[0061] A method to rapidly obtain the Gfi1b gene knockout in RAW264.7 cell line with large deletion by CRISPR / Cas9 system.

[0062] (1) Determine the specific target sites sgRNA1 and sgRNA2 of the gene to be knocked out Gfi1b (Gene ID: 1276578): find the mouse Gfi1b gene DNA sequence in the mouse genome database ensembl (http: / / asia.ensembl.org) (Transcript ID: ENSMUST00000028156.7), and then use the online design software CRISPOR (http: / / crispor.tefor.net / crispor.cgi) to determine the target sites of intron1-2 and exon2 in the mouse Gfi1b gene (exon ID: ENSMUSE00001307648) selected two specific sites as the target sequence of sgRNA, the two target sequences were: sgRNA1 (SEQ ID NO.1): 5'-AGTGACAAGCGCTAGTCCTTTGG-3', sgRNA2 (SEQ ID NO.2): 5'-TTACCACCAGCCCCGGGCACAGG-3'.

[0063] (2) Design primers: According to the sgRNA target sequence in step (1), design 2 pairs of 4 primers (Shanghai Bailiger Biotechnology Co., Ltd.), and add a BbsI restriction site at the 5' end of the prim...

Embodiment 3

[0080] A rapid knockout method of the Pparg gene in the RAW264.7 cell line with a large deletion by the CRISPR / Cas9 system.

[0081] (1) Determine the specific target sites sgRNA1 and sgRNA2 of the mouse gene Pparg (Gene ID: 97747) to be knocked out: find the mouse Pparg gene in the mouse genome database ensembl (http: / / asia.ensembl.org) DNA sequence (Transcript ID: ENSMUST00000171644.7), then use the online design software CRISPOR

[0082] (http: / / crispor.tefor.net / crispor.cgi), it is determined to select two specific sites in the target site intron2-3 of the mouse Pparg gene as the target sequence of the sgRNA, and the two target sequences are respectively :

[0083] sgRNA1 (SEQ ID NO.9): 5'-GTATACCTAACAAGATACTA TGG-3';

[0084] sgRNA2 (SEQ ID NO. 10): 5'-GTGAAGCTGTGCGTCATTTC AGG-3'.

[0085] (2) Design primers: According to step (1) sgRNA target sequence, design 2 pairs of 4 primers (Shanghai Bailig Biotechnology Co., Ltd., and add a BbsI restriction site at the 5' end o...

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Abstract

The invention relates to a cell line gene knock-out method for obtaining large fragment deletion through a CRISPR/Cas9 system, and belongs to the field of genetic engineering and genetic modification. A pX458 vector is modified, so that the pX458 vector is provided with DsRed2 and ECFP (Enhanced Cyan Fluorescence Protein); then, a plurality of specific sgRNA sites are designed by aiming at a target gene and are connected into the modified vector; after the cell line transfection, cell groups are sorted by a flow cytometry; single cells subjected to genome editing can be very fast obtained; then, a single-cell DNA (Deoxyribonucleic Acid) sequence is subjected to PCR (Polymerase Chain Reaction) amplification; and single cells with large fragment deletion can be picked out from the single cells through gel electrophoresis. The CRISPR/Cas9 system, the flow cytometry single-cell sorting and fluorescent protein screening on the expression vector are combined, so that the positive monoclonal cells with the large fragment deletion can be obtained in a short time; and the work efficiency of the cell line gene knock-out is greatly improved.

Description

technical field [0001] The invention relates to a cell line gene knockout method for rapidly obtaining large fragment deletion through a CRISPR / Cas9 system, and belongs to the fields of genetic engineering and genetic modification. Background technique [0002] The CRISPR-Cas9 system is the most widely studied and utilized genome editing technology in recent years. Compared with traditional genome editing methods: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), The CRISPR-Cas9 system has the following advantages: simple structure, very easy to transform and operate; high editing efficiency to the genome, easy to obtain gene knockout individuals; no species restrictions in actual use, so it is widely used in animals, plants and It has been widely used in the production of isogene knockout models in cell lines. [0003] Traditional cell line gene knockout requires multiple rounds of resistance screening and dilution after transfectio...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12N15/90
CPCC07K14/47C12N15/65C12N15/85C12N15/907C12N2810/10
Inventor 卢燎勋张黎琛梁银明黄蓉晁天柱郑前前罗静谷妍蓉袁鹏
Owner XINXIANG MEDICAL UNIV
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