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A Gene Knockout Method for Rapidly Acquiring Large Deletions in Cell Lines Using the CRISPR/Cas9 System

A gene knockout and large fragment technology, applied in the fields of genetic engineering and genetic modification, can solve the problems of inability to obtain bases, complicated operation of PAGE electrophoresis detection method, and deletion, and achieve the effect of improving work efficiency.

Active Publication Date: 2020-06-02
XINXIANG MEDICAL UNIV
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  • Abstract
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  • Claims
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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 digestion detection method, high resolution melting curve detection method can detect whether base changes have occurred at the target site, but cannot obtain the specific number of base deletions or insertions; PAGE Electrophoretic detection method and fluorescent PCR method can obtain detailed information of base changes more accurately, but PAGE electrophoretic detection method is complicated to operate, and fluorescent PCR method requires expensive equipment, which is not suitable for high-throughput screening and large-scale promotion

Method used

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  • A Gene Knockout Method for Rapidly Acquiring Large Deletions in Cell Lines Using the CRISPR/Cas9 System
  • A Gene Knockout Method for Rapidly Acquiring Large Deletions in Cell Lines Using the CRISPR/Cas9 System
  • A Gene Knockout Method for Rapidly Acquiring Large Deletions in Cell Lines Using the CRISPR/Cas9 System

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0057] (1) The mother vector pX458 was purchased from addgene (ID: 48138). After expanding the culture, use the restriction endonuclease EcoRI (NEB) to linearize the vector DNA; the specific steps are: add 1 μg pX458 vector to a 1.5ml centrifuge tube in sequence DNA; 3 μl 10×NEB Buffer 2.1; 1 μl EcoRI (NEB) and finally rehydrated to a total volume of 30 μl, and incubated at 37°C for 2 hours. After digestion, the digested 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 Bailig Biotechnology Co., Ltd.), at the 5' end of the sequence Add EcoRI restriction site and T2A sequence, and clone into the pUC57 vector; then digest the two DNA sequences separately, the spe...

Embodiment 2

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

[0062] (1) Determine the specific target sites sgRNA1 and sgRNA2 of the gene Gfi1b (Gene ID: 1276578) to be knocked out: 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 intron1-2 and exon2 (exon ID: ENSMUSE00001307648) select two specific sites as the target sequence of sgRNA, the two target sequences are: sgRNA1 (SEQ ID NO.1): 5'-AGTGACAAGCGCTAGTCCTTTGG-3', sgRNA2 (SEQ ID NO.2): 5'-TTACCACCAGCCCCGGGCACAGG-3'.

[0063] (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 of the primer sequence:

[0064]...

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 knockout method for rapidly obtaining large fragment deletion through a CRISPR / Cas9 system, and belongs to the fields of genetic engineering and genetic modification. In the present invention, the pX458 vector is modified to carry DsRed2 and ECFP, and then multiple specific sgRNA sites are designed for the target gene, which are connected to the modified vector. Sorting, single cells with edited genomes can be obtained very quickly, and then single-cell DNA sequences are amplified by PCR, and single cells with large deletions can be selected by gel electrophoresis. By combining the CRISPR / Cas9 system, flow cytometry single cell sorting and fluorescent protein screening on the expression vector, the present invention can obtain a positive single clone with large fragment deletion in a short time, and greatly improve the cell line Gene knockout work efficiency.

Description

technical field [0001] The invention relates to a cell line gene knockout method for rapidly obtaining a large fragment deletion through a CRISPR / Cas9 system, and belongs to the field 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 transfecti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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