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Method for targeted knockout of specific gene of Suqin yellow chicken embryonic stem cell

A technology for embryonic stem cells and specific genes, which can be applied to cells modified by the introduction of foreign genetic material, DNA/RNA fragments, and the introduction of foreign genetic material using vectors. It can solve problems such as off-target effects and no successful precedent, and achieve repeatability. strong effect

Inactive Publication Date: 2015-07-29
YANGZHOU UNIV
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Problems solved by technology

[0007] There are advantages, but there are also limitations. For example, CRISPR / Cas technology is still immature, and it is necessary to design gRNA plasmids with high specificity, serious off-target effects, and no successful precedents in stem cells that have not been established. With the development of CRISPR / Cas, these problems will eventually be solved

Method used

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Embodiment Construction

[0023] A method based on CRISPR / Cas9 technology for targeted knockout of specific genes in Suqin yellow chicken embryonic stem cells, comprising the following steps:

[0024] (1) Acquisition of the target gene

[0025] According to the sequence number of the gene, the CDS sequence of the target gene in the jungle chicken was queried in the NCBI database, specific primers were designed, and the single-stranded cDNA formed by the reverse transcription of the PGC RNA of the Suqin yellow chicken was used as a template to clone and obtain the target gene in the Suqin yellow chicken. The complete CDS sequence of the gene, and the complete exon sequence of the target gene is obtained by comparison;

[0026] (4) gRNA design and synthesis

[0027] Find the PAM sequence in the obtained exon sequence, and use the first 20 bp or so of the PAM sequence as the target sequence, all target sequences are compared in the whole genome, and the target sites without homology are used as gRNA and ...

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Abstract

The invention discloses a method for targeted knockout of a specific gene of a Suqin yellow chicken embryonic stem cell. The method comprises the following steps: firstly, checking out an exon sequence of a gene, cloning the gene, sequencing to obtain a complete exon sequence, designing a CRISPR / Cas9 knockout target site on the sequence as gRNA, and constructing a CRISPR / Cas9 dual-promoter knockout vector; performing SSA activity detection on the constructed Cas9 vector, transfecting a control group with an empty vector, detecting a luciferase signal, and obtaining a result that the more the luciferase activity is increased relative to the control group, the higher the gRNA shearing activity is shown; transfecting the CRISPR / Cas9 vector which is high in SSA activity with ESCs, using flow cytometry to screen a GFP-positive cell, extracting genome DNA, and designing a primer.

Description

Technical field: [0001] The present invention relates to the field of stem cell engineering, in particular to a method for targeted knockout of specific genes in Suqin yellow chicken embryonic stem cells based on CRISPR / Cas9 technology. technical background: [0002] At present, there are three main methods of genome editing technology, zinc finger nuclease (zinc finger endonuclease, ZFN), transcription activator-like effector nuclease (Transcription Activator-Like Effector Nuclease, TALEN) and clustered regularly spaced short palindromic repeats Sequence and related genes (clustered regularly interspaced short palindromic repeats / CRISPR-associated, CRISPR / Cas). All three approaches achieve genome editing by generating double-stranded nicks at exogenous DNA target sites, thereby inducing homologous recombination repair and non-homologous end joining. [0003] The ZFN structure includes a zinc finger protein (Zinc finger protein, ZFP) domain and a Fok I cleavage domain, wher...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/11C12N5/10
Inventor 李碧春张亚妮左其生李东王颖洁张蕾连超肖天荣汤贝贝
Owner YANGZHOU UNIV
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