CRISPR-Cas9-specific method for knocking out pig Fah and Rag2 genes

A specific, double-gene technology, applied in the field of gene editing, to achieve the effect of reasonable design and intuitive screening

Active Publication Date: 2022-04-19
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is no report on double knockout of Fah and Rag2 genes in pigs using this technology

Method used

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  • CRISPR-Cas9-specific method for knocking out pig Fah and Rag2 genes
  • CRISPR-Cas9-specific method for knocking out pig Fah and Rag2 genes
  • CRISPR-Cas9-specific method for knocking out pig Fah and Rag2 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Selection and design of Sus scrofa (pig) Fah and Rag2 gene sgRNA target sequences

[0051] (1) Method

[0052] 1. Selection of sgRNA target sequences for Fah and Rag2 genes

[0053] To find sgRNA target sequences in the exon coding regions of Fah and Rag2 genes, the principles are as follows:

[0054] (1) The general sequence formula is 5'-N(20)NGG-3', where N(20) represents 20 consecutive bases, and each N represents A or T or C or G, and targets that meet the above rules The sequence is on the sense or antisense strand;

[0055] (2) For the Fah gene, select the 5 exon coding region sequences near the N-terminal of the gene Fah, the target sequence can be located at the 5 exon coding regions at the N-terminal of the Fah gene, or a part of the target sequence is located at the N-terminal of the Fah gene 5 exons, the rest spans the junction with the adjacent intron and is located in the adjacent intron; the cutting of such coding region sequence will cause t...

Embodiment 2

[0075] Example 2 Construction of sgRNA expression vectors of Fah gene and Rag2 gene and adenovirus packaging

[0076] 1. Synthesis of DNA Inserts

[0077] (1) Synthesize the Fah-sgRNA and Rag2-sgRNA forward and reverse oligonucleotide sequences designed above

[0078] The oligonucleotide sequence can be specifically synthesized by a commercial company (such as Invitrogen) according to the provided sequence. The forward oligonucleotide sequence and reverse oligonucleotide sequence corresponding to No. 17 target sequence in Table 1 are as follows:

[0079] 5'-CACCGGCGATTGGTGACCAGATCC-3' (SEQ ID NO.91):

[0080] 5'-AAACGGATCTGGTCACCAATCGCC-3' (SEQ ID NO. 92).

[0081] The forward oligonucleotide sequence and reverse oligonucleotide sequence corresponding to No. 1 target sequence in Table 2 are as follows:

[0082] 5'-CACCGGACTGTGCAAGTCACAGCT-3' (SEQ ID NO.93);

[0083] 5'-AAACAGCTGTGACTTGCACAGTCC-3' (SEQ ID NO. 94).

[0084] The gene knockout pattern after the sgRNA corresp...

Embodiment 3

[0136] Example 3 Obtaining a pseudotyped lentivirus expressing Cas9

[0137] 1. Material preparation

[0138] Virus packaging system: three-plasmid system, pSPAX2, pMD2G, pHBLV-Cas9-Puro (purchased from Hanbio, Figure 5 ); culture packaging cell line HEK293T cells (purchased from ATCC); DMEM medium, Opti-MEM medium and fetal bovine serum FBS (purchased from Gibco); Lipofectamine2000 (purchased from Invitrogen); HEK293T cells were cultured in 5% CO2 containing In a 37°C culture environment, the culture medium is DMEM medium containing 10% FBS.

[0139] 2. Transfection and Viral Packaging

[0140] Day 1: Passage the packaging cell line HEK293T to T75, about 30% confluence;

[0141] The next day: When HEK293T reaches 50%-60% confluence, transfect according to the following recipe:

[0142] Prepare Mixture 1, containing:

[0143] pSPAX2 10 μg

[0144] pMD2G 10 μg

[0145] pHBLV-Cas9-Puro vector 10 μg

[0146] Opti-MEM: 500 μL

[0147] Prepare Mixture 2, containing:

[0...

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Abstract

In order to solve the problems of low efficiency and difficult screening of pig Fah and Rag2 double gene knockout, the present invention provides a set of target sequences of Fah and Rag2 double genes and their specific knockout of pig Fah and Rag2 double genes by CRISPR‑Cas9 use. The present invention also provides a method for knocking out pig Fah and Rag2 genes by CRISPR-Cas9. Through verification, the invention can effectively realize the knockout of pig Fah and Rag2 double genes, the double knockout efficiency is as high as 50%, and the screening is also very simple.

Description

technical field [0001] The invention relates to the field of gene editing, in particular to a method for specifically knocking out pig Fah and Rag2 genes by CRISPR-Cas9. Background technique [0002] my country is a country with a large number of liver diseases. It is the country with the highest incidence of viral hepatitis and liver cancer and the largest number of patients in the world. At present, there are 30 million patients with chronic liver disease in the country, and more than 500,000 patients die of liver cancer every year, and the annual treatment cost for liver disease reaches 100 billion yuan. Liver transplantation is still the most effective way to treat liver failure, but the shortage of donors and life-long immunosuppressant treatment after transplantation seriously limit the clinical application of liver transplantation. It is an urgent clinical need to find alternative treatment options for whole or partial liver transplantation. Therefore, hepatocyte tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/113C12N15/861C12N15/90C12N5/10
CPCC07K14/47C12N15/113C12N15/86C12N15/907C12N2510/00C12N2310/10C12N2310/20C12N2710/10043
Inventor 包骥高孟雨步宏
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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