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Screening report vector and screening method for enriching CRISPR/Cas9-mediated homologous recombination repairing cells

A homologous recombination and carrier technology, applied in the field of genetic engineering, can solve problems such as the impact of cell functions, and achieve the effect of improving screening efficiency

Active Publication Date: 2019-06-14
NORTHWEST A & F UNIV
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Problems solved by technology

However, this approach still leads to intrinsic integration of the selection cassette at an irrelevant site on the genome of the edited cell, with consequences for cellular function

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  • Screening report vector and screening method for enriching CRISPR/Cas9-mediated homologous recombination repairing cells
  • Screening report vector and screening method for enriching CRISPR/Cas9-mediated homologous recombination repairing cells
  • Screening report vector and screening method for enriching CRISPR/Cas9-mediated homologous recombination repairing cells

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Embodiment Construction

[0045] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The examples are only used to explain the present invention, not to limit the protection scope of the present invention.

[0046] (1) HDR-USR vector construction

[0047] The present invention proposes a screening reporter carrier HDR-USR (CRISPR / Cas9mediated HDR-Universal Surrogate Reporter) for enriching CRISPR / Cas9-mediated HDR editing cells.

[0048] HDR-USR is mainly composed of four parts, namely: universal sgRNA expression cassette, Cas9 expression cassette, Puro expression cassette interrupted by the target sequence corresponding to universal sgRNA, and Puro coding sequence (ie ΔPuro) with ATG removed. After HDR-USR is transfected into cells, it can express universal sgRNA and Cas9 protein; then, under the guidance of universal sgRNA, Cas9 protein cuts the target sequence region between the Puro left arm (PuroL) and Puro right arm (Puro...

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Abstract

The invention discloses a screening report vector and screening method for enriching CRISPR / Cas9-mediated homologous recombination repairing cells. The screening report vector includes a universal sgRNA expression cassette, a Cas9 expression cassette, a resistance gene expression cassette and a resistance gene homologous recombination repair template, and the screening reporter vector itself can be repaired by homologous recombination of the resistance gene sequence to make the cells have resistance. The screening report vector, a targeting vector for cell genome HDR editing and a donor vectorform a co-transfection system which can be applied to HDR positive cell cloning with efficient enrichment point editing, fragment insertion and fragment deletion, and can be widely applied to a variety of cells of mammals. The screening efficiency of HDR-edited cells is significantly improved, the reintroduction of double-strand breaking and DNA integration in the unrelated positions of the cellgenome is not required, and an effective way is provided to promote research and application of accurate gene editing.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to the construction and application of a screening reporter vector that can be used to enrich CRISPR / Cas9-mediated homologous recombination repair cells. Background technique [0002] CRISPR / Cas9 nuclease technology is the third-generation targeted editing technology after ZFNs and TALENs. Jinek et al. reported for the first time in June 2012 the use of a CRISPR / Cas9 system (clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein-9nuclease (Cas9)) derived from S.pyogenes (S.pyogenes) Proteins and guide RNAs (guide RNA, gRNA) can target and cleave plasmid DNA in vitro. The CRISPR / Cas9 system uses a Cas9 nuclease and a guide RNA (sgRNA) containing a 20nt target sequence to generate DNA double strand breaks (DSBs) at specific sites in the genome. DSBs can activate a variety of DNA break repair mechanisms in cells, mainly non-homologous e...

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Application Information

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IPC IPC(8): C12N15/85C12N15/113C12N15/65C12N15/90C12N5/10
Inventor 张智英闫娜娜孙永森徐坤房圆圆邓竞荣穆璐
Owner NORTHWEST A & F UNIV
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