FokI and dCpf1 fusion protein expression vector and fixed-point gene editing method mediated by fusion protein

A technology of fusion protein and expression vector, applied in the field of gene editing, can solve problems such as high off-target rate, achieve great application potential, improve specificity, and low off-target rate.

Pending Publication Date: 2021-03-02
YANGZHOU UNIV
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  • Application Information

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Problems solved by technology

Later, in the article of John et al., it was found that after comparing the fCas9 system, the Cas9nickases system and the CRISPR / Cas9 system, although the CRISPR / Cas9 system showed the highest editing activity, it also showed a high off-target rate

Method used

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  • FokI and dCpf1 fusion protein expression vector and fixed-point gene editing method mediated by fusion protein
  • FokI and dCpf1 fusion protein expression vector and fixed-point gene editing method mediated by fusion protein
  • FokI and dCpf1 fusion protein expression vector and fixed-point gene editing method mediated by fusion protein

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Embodiment 1

[0058] The experimental methods mentioned in the following examples are conventional methods unless otherwise specified; the implementation of the present invention is not limited thereto.

Embodiment I

[0059] Construction and application of embodiment 1, pFok1-dCpf1 fusion protein expression vector

[0060] 1. Construction of pLB-dCpf1 vector

[0061] dCpf1 was obtained by PCR amplification from the commercial vector WN10151 (Addgene plasmid #53369) (such as figure 1 ), cloned onto the LB vector, and constructed the pLB-dCpf1 vector, and the plasmid DNA was detected by 1% agarose gel electrophoresis (such as figure 2 ).

[0062] 2. Construction of pFokI-dCpf1 fusion protein expression vector

[0063] The pLB-dCpf1 was obtained by cutting pLB-dCpf1 with BsmBI, and the fragment dCpf1 was cut back; the commercial vector pSQT1601 (4849bp, 5474bp) was double cut with Acc65I and Not1 enzymes, and the CAG framework 4849bp was recovered. T2A-NLS and FokI fragments amplified by PCR from laboratory-preserved vectors (primer sequences are shown in Table 1); T2A-NLS, FokI, dCpf1 fragments and CAG frameworks were ligated together using the Golden Gate cloning method, T7 ligase 25 Re...

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Abstract

The invention belongs to the field of gene editing, and particularly relates to a FokI and dCpf1 fusion protein expression vector and a fixed-point gene editing method mediated by the vector. The vector fuses mutated and inactivated dCpf1 protein in a CRISPR / Cpf1 editing system with FokI nuclease to construct an efficient gRNA-mediated gene fixed-point editing system. According to the method, thesite-specific action of dCpf1 and the shearing action of nuclease FokI are utilized, and shearing is performed in a dimer form, so that the specificity of the gene editing system can be improved, anda relatively low off-target rate is achieved. Compared with Cas9 protein, the Cpf1 protein has greater advantages for gene editing and other operations. The invention provides the fusion-protein-mediated gene fixed-point editing method which is more convenient and efficient.

Description

technical field [0001] The invention belongs to the field of gene editing, in particular to a FokI and dCpf1 fusion protein expression vector and a site-directed gene editing method mediated by the same. Background technique [0002] Theoretically, site-specific endonucleases can perform directional operations on a single site in the genome, which is very useful for gene targeting in therapeutic and research applications. In many organisms, including mammals, site-specific endonucleases perform gene editing by stimulating NHEJ (non-homologous end joining) and HDR (homologous recombination), such as CRISPR / Cas9, Cpf1, etc. [0003] CRISPR / Cas is a defensive immune mechanism formed during the evolution of bacteria and archaea, which can be used to resist the invasion of viruses and foreign DNA. In the CRISPR / Cas system, CRISPR is the abbreviation of clustered regularly interspaced short palindromic repeats, and Cas is the abbreviation of CRISPR-associated proteins (Cas). The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/62C12N15/10
CPCC12N15/85C12N15/907C12N9/22C12N15/102C12N2800/107C07K2319/00
Inventor 高波留汉文王赛赛王亚丽宋成义陈才王宵燕
Owner YANGZHOU UNIV
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