Split SpCas9 lentiviral vector and application thereof in stem cell gene editing

A lentiviral vector, lentivirus technology, applied in the field of gene editing

Pending Publication Date: 2020-02-07
保定诺未科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as mentioned above, the huge Cas9 protein is an obstacle to the application of the CRISPR-Cas9 system in gene therapy

Method used

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  • Split SpCas9 lentiviral vector and application thereof in stem cell gene editing
  • Split SpCas9 lentiviral vector and application thereof in stem cell gene editing
  • Split SpCas9 lentiviral vector and application thereof in stem cell gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0069] 1. Prepare the split SpCas9 vector;

[0070] (1) The Cas9-C vector can be used directly without additional molecular cloning process;

[0071] (2) Design sgRNA targeting GYPA and AAVS1 genes and annealing oligonucleotide chains (Table 1), and perform annealing reaction to obtain sgRNA double-stranded DNA;

[0072] Table 1 sgRNA, primers and annealed oligonucleotide chains

[0073] GYPA sgRNA targeting sequence atctttgtattactattgtc GYPA sgRNA forward annealed oligonucleotide strand caccgatctttgtattactattgtc GYPA sgRNA reverse annealed oligonucleotide strands caccgagcattaagtaccactgagg GYPA upstream detection primers cattagaaatgagaaggtccatggc GYPA downstream detection primers aaactggatttggccataaatactg AAVS1 sgRNA targeting sequence gcaaactctccaagtgacc AAVS1 sgRNA forward annealed oligonucleotide strand caccgagcaaactctccaagtgacc AAVS1 sgRNA reverse annealed oligonucleotide strands aaacggtcacttggagagtttgct AAV...

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Abstract

The invention relates to the technical field of gene editing, in particular to a split SpCas9 lentiviral vector and an application thereof in stem cell gene editing. The preparation method comprises the following steps: respectively expressing SpCas9-N-Intein-N and Intein-C-SpCas9-C on two lentiviral vectors, after an organism is infected, Intein-N and Intein-C recognize each other to form complete intein, self-shearing is carried out, and adjacent Cas9-N and Cas9-C are connected to form complete Cas9, so that gene editing can be realized in vivo. The experiments show that the lentiviral vector can effectively infect human mobilized peripheral blood derived hematopoietic stem cells and perform gene editing on the hematopoietic stem cells, and the maximum editing efficiency reaches 50%.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a split-type SpCas9 lentiviral vector and its application in stem cell gene editing. Background technique [0002] CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRIPSR associated protein 9) system has been widely used in various scientific research fields, such as gene modification, gene expression regulation and epigenetic modification. The greatest advantage of the CRISPR-Cas9 system lies in its simplicity of application: under the guidance of a specific single guide RNA (sgRNA), Cas9 can target almost any sequence in the human genome. However, there are still many challenges in the application of the CRISPR-Cas9 system in the treatment of human diseases, one of which is that the huge Cas9 protein is not convenient for the delivery of viral vectors. The most widely used Cas9 protein is S.pyogenes Cas9 (SpCas9). The gene encoding the SpCas9 prot...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/65C12N15/113C12N15/90C12N5/10C12N7/01A61K35/14A61P37/04A61P7/06
CPCA61K35/14A61P7/06A61P37/04C12N5/0647C12N7/00C12N15/113C12N15/65C12N15/86C12N15/907C12N2310/20C12N2510/00C12N2740/15021C12N2740/15043
Inventor 孙志娟刘德芳罗天明齐海龙郭潇年聚会杨欢孙忠杰
Owner 保定诺未科技有限公司
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