Conditional gene knockout method based on CRISPR/Cas9 technology

A gene knockout and conditional technology, applied in the field of gene modification, can solve the problems that tissue, cell, and space-time specific knockout cannot be achieved, it is difficult to obtain double transgenic offspring mice, and the identification and screening process is complicated, etc. Short cycle, reduced cytotoxicity, short cycle effect

Active Publication Date: 2017-12-01
CYAGEN BIOSCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although the Cre-loxp recombinase system can mediate the conditional knockout of genes and avoid the disadvantages of traditional gene targeting techniques such as premature death of embryos caused by missing functional genes, there are still many uncertainties:
[0010] ①There is a hidden / false LoxP sequence in the mammalian genome, and its sequence may not be exactly the same as the conserved sequence of LoxP, but it can be recognized by Cre recombinase and recombined, causing unnecessary DNA damage
[0011] ② Poor specificity, it is difficult to obtain offspring mice with double transgenes, and the success rate is <50%
[0012] ③ The identification and screening process is complex and time-consuming
[0013] Although the existing CRISPR / Cas9 system is simple to operate and has high targeting accuracy, it cannot achieve specific knockout of tissues, cells, and time and space

Method used

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  • Conditional gene knockout method based on CRISPR/Cas9 technology
  • Conditional gene knockout method based on CRISPR/Cas9 technology
  • Conditional gene knockout method based on CRISPR/Cas9 technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] 1. Experimental materials

[0075] ① HD Clinging Kit (639648): purchased from Clontech;

[0076] ②Premix Taq (D331A), Primerstar (DRO44A), dNTP (4030Q): purchased from Yubao Bioengineering (Dalian) Co., Ltd. (Takara);

[0077] ③ Restriction endonucleases BbsI (R0539L), AsisI (R0630L), HpaI (R0105S): purchased from Beijing Bomeisi Biotechnology Co., Ltd. (NEB); Church Cloning Vector is a commercial plasmid, purchased from Church through Addgene laboratory; pCR-Blunt II-TOPO is also a commercial plasmid, purchased from Invitrogen through Addgene; plasmid with Cre recombinase: purchased from Albee Messing Laboratories through Addgene; source of Rosa26 expression vector, purchased from Addgene at Liqun Luo's lab.

[0078] 2. Gene structure and sequence analysis

[0079] Target gene: mouse eukaryotic translation initiation factor 3 (Eif3h gene);

[0080] Ensembl gene code number: ENSMUSG00000022312;

[0081] Eif3h gene structure: Eif3h gene contains 8 exons, including...

Embodiment 2

[0113] Example 2 Construction of Cas9 expression vector and production of Cas9 tool mouse

[0114] (1) Amplification of Cas9 protein

[0115] ① Design primers for amplifying Cas9 protein, as follows:

[0116] Cas9-F: CGCGGTCTTTCCAGTGATCGATTAGTTATTAATAGTAATCAA

[0117] Cas9-R: CTCTAGTCCGCGGGTGCGATAGCTCACACCTTCCTCTTCTTTCTTG

[0118] ②PCR amplified the full sequence of Cas9 protein, the system is as follows:

[0119]

[0120] The PCR program is as follows:

[0121]

[0122]

[0123] ③ PCR products were subjected to agarose gel electrophoresis, such as Figure 9 shown.

[0124] (2) Cas9 protein connected to Rosa26 carrier

[0125] ① Use Clontech's The HD Clinging Kit will detect the correct Cas9 sequence and connect it to the vector linearized by AsisI and HpaI enzymes. The vector is transformed by the company, and it has a broad-spectrum expression promoter CMV, the 5'arm of Rosa26, and the Rosa26 vector backbone of the 3'arm. The map is as follows Figure 10 s...

Embodiment 3

[0137] Example 3 Obtaining conditional knockout mice

[0138] The Cas9 tool mouse was crossed with the sgRNA mouse, and finally 15 mice were obtained. Different tissues of the mice were taken, and the genome was extracted. After PCR amplification with Eif3h-F / Eif3h-R primers, they were sent to Suzhou Jinweizhi Co., Ltd. Sequencing results showed that the gene knockout rate in the liver tissue reached over 80%, while the genes in other tissues remained intact.

[0139] The above primer pair: Eif3h-F:ATCATATATTTAATTTTCAACAAGT

[0140] Eif3h-R: CTTTCCTACAGAGCTTCACCT

[0141] Analysis of gene knockout results:

[0142] liver tissue:

[0143]

[0144] Wild type refers to the liver tissue of mice without any modification;

[0145] Samples 1 to 15 refer to experiments on different tissues of 15 mice. The genomes of different tissues were extracted, amplified by PCR, and then sent for sequencing. The results showed that only the Eif3h gene of the liver tissue was modified while...

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Abstract

The invention discloses a primer for gene knockout, including five sets of primers, such as SEQ NO:1 and SEQ NO:2, SEQ NO:3 and SEQ NO:4, SEQ NO:5 and SEQ NO:6 , SEQ NO:7 and SEQ NO:8, SEQ NO:9 and SEQ NO:10. The invention also discloses the application of the primer in gene knockout. The invention also discloses a conditional gene knockout method based on CRISPR / Cas9 technology. The invention can carry out condition specificity, space-time specificity, and drug-induced gene modification; reduce the harm to other cells, and study the function of constitutively expressed genes in specific tissues; only the Cas9 tool mouse can realize tissue and space specificity Sexual gene knockout or inducible gene knockout; the test cycle is short, saving time and cost.

Description

technical field [0001] The invention belongs to the technical field of gene modification, in particular to a conditional gene knockout method based on CRISPR / Cas9 technology. Background technique [0002] With the advancement of science and technology and the continuous exploration of the field of life sciences, it is more urgent to study the expression of a gene in a specific tissue, cell and time in vivo. The site-specific recombination technology developed rapidly in recent years is a key gene manipulation tool to meet this need. It can induce target gene inactivation at a certain developmental stage or in a specific tissue, avoiding embryonic damage caused by target gene deletion. In case of early death or complex phenotype, delete the screening marker gene, so as to make a complete functional analysis of the target gene. Currently widely used recombinase systems include Cre-LoxP system, FLP-FRT system, R-RS system and I-SceI system, but the most widely used system is C...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/85
Inventor 郑敦武营婷姜莎莎俞晓峰欧阳应斌徐文静黎妃凤朱道云王正龙
Owner CYAGEN BIOSCI INC
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