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In-vitro cell platform for pig gene editing sgRNA screening

A technology for gene editing and porcine kidney epithelial cells, applied in the field of in vitro cell platforms, can solve the problem of no efficient gene editing sgRNA in vitro cell platform, etc., and achieve the effects of high efficiency, high throughput, and avoiding ineffective labor.

Pending Publication Date: 2022-01-28
WEST CHINA HOSPITAL SICHUAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently, there is no high-efficiency in vitro cell platform for gene editing sgRNA screening for gene editing in pigs

Method used

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  • In-vitro cell platform for pig gene editing sgRNA screening
  • In-vitro cell platform for pig gene editing sgRNA screening
  • In-vitro cell platform for pig gene editing sgRNA screening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Screening of cells stably expressing Cas9

[0042] Usually in CRISPR-Cas9 gene editing operation, sgRNA and Cas9 gene need to be transferred into cells at the same time. In order to improve the screening efficiency of gene editing sgRNA, the present invention constructs a cell stably expressing Cas9 protein.

[0043] To judge whether the Cas9 gene is successfully transferred into cells and can be stably expressed, it is first necessary to screen the marker gene on the lentiviral vector carrying the Cas9 gene.

[0044]The pHBLV-gRNA-Cas9-Puro vector used for Cas9 transformation carries a puromycin resistance gene. In order to find pig cells suitable for puromycin screening in this way, the inventors packaged the pHBLV-gRNA-Cas9-Puro vector as a lentivirus and transferred it into 9 target cells [Small Xiang pig skin cells (SSC-S2), Pig skin cells (SSC-S1), minipig kidney cells (SEPfk), minipig lung cells (SEP-L1), porcine kidney epithelial cells (PK15), porcin...

Embodiment 2

[0049] Example 2 RNA-seq (RNA sequencing) analysis of Cas9 stably transfected pig cells

[0050] Based on the Illumina sequencing platform, the inventors performed RNA-Seq on the four cell lines of PIEC / PK15 / IBRS-2 / ST and the corresponding Cas9 stable cells, namely PIEC-Cas9 / PK15-Cas9 / IBRS-2-Cas9 / ST-Cas9 Sequencing and comparative analysis. In order to reflect the correlation of gene expression between samples, this study calculated the Pearson correlation coefficients of all gene expressions between every two samples, and reflected these coefficients in the form of a heat map, as shown in image 3 shown. From image 3 A It can be seen that PIEC and PIEC-Cas9, PK15 and PK15-Cas9, IBRS-2 and IBRS-2-Cas9, ST and ST-Cas9 have the highest correlation, and the Pearson correlation coefficients are all above 0.9, while PIEC / PK15 / The gene expression correlation among the four kinds of IBRS-2 / ST cells was relatively low.

[0051] In order to display the number of genes in differen...

Embodiment 3

[0054] Example 3 Quantitative analysis of proteome of porcine Cas9 stable expression cell line

[0055] Using LC-MS / MS mass spectrometry and TMT labeling quantitative technology combined with bioinformatics analysis, the proteome quantitative analysis of porcine PIEC-Cas9 / PK15-Cas9 / IBRS-2-Cas9 / ST-Cas9 stably transfected cells was carried out, and the stable transfected cells were obtained. Cellular proteome expression. The total number of proteins identified by the four cell lines is 3824, and the protein expression differences of the four Cas9 stably transfected cells can be seen from the heat map of the protein map ( Figure 4 ). In order to facilitate the process-based retrieval, the inventor built a common porcine cell line gene and protein expression retrieval platform PiGenEditer. The link to the web page is: https: / / www.omicsolution.org / wukong / PiGenEditer / , the part of the URL page is as follows Figure 5 .

[0056] Using this platform, you can quickly query the gen...

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Abstract

The invention discloses an in-vitro cell platform for pig gene editing sgRNA screening, and belongs to the field of gene editing. The platform is suitable for sgRNA screening in a CRISPR-Cas9 gene editing technology, and mainly comprises a specific pig cell, a transcriptome of the specific pig cell, a proteome data platform and a gene editing sgRNA screening method. The cell is a pig hip artery endothelial cell PIEC, a pig kidney epithelial cell PK15, a pig kidney passage cell IBRS-2 or a pig testis cell ST capable of stably expressing a Cas9 gene. A user can select specific cells according to the expression level of a gene to be edited in the data platform, and screening of gene editing sgRNA is carried out. According to the in-vitro cell platform, the sgRNA screening efficiency is improved, and the in-vitro cell platform has a good application prospect.

Description

technical field [0001] The invention belongs to the field of gene editing. Specifically, it relates to an in vitro cell platform for sgRNA screening for pig CRISPR-Cas9 gene editing. Background technique [0002] Pigs are highly similar to humans in terms of anatomy, physiopathology, nutrient metabolism, and disease characteristics. Gene-edited pigs are now important animals needed in many fields such as disease pathogenesis, pathotoxicology research, and therapeutic drug evaluation. Model. [0003] CRISPR-Cas9 gene editing technology (sometimes also written as "CRISPR / Cas9 gene editing technology") is currently the most mainstream gene editing technology. The basic principle is: through sgRNA (single guide RNA), the Cas9 protein with nucleic acid cutting function is brought to the genomic DNA to be edited, so that the Cas9 protein can precisely cut the DNA. The sgRNA mainly has two subregions, one subregion is responsible for binding to the Cas9 protein, and its sequence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/55C12N15/867C12N15/90G16B20/30G16B25/20G16B30/10
CPCC12N5/069C12N5/0686C12N5/0683C12N9/22C12N15/86C12N15/907G16B20/30G16B25/20G16B30/10C12N2510/00C12N2740/15043
Inventor 包骥步宏高孟雨
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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