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DNA containing mutational endonuclease identification section and application of DNA in genome editing

A technology of genome editing and endonuclease, which is applied in recombinant DNA technology, DNA/RNA fragments, gene therapy, etc., can solve the problems that affect wide application, low efficiency of mutation or repair, and low efficiency of genome editing, so as to prevent Base mutation, improve editing efficiency, improve the effect of low efficiency and easy introduction of mutations

Inactive Publication Date: 2015-12-16
TSINGHUA UNIV
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Problems solved by technology

[0012] Although there are successful examples, the efficiency of genome editing is currently low, especially the targeted editing (mutation or repair) efficiency of specific single bases or a small number of bases using HR is even worse, and it is easy to introduce additional or wrong bases mutation
The existing problems of the current technology have seriously affected its wide application in the gene therapy of human diseases and the construction of genetically modified cells and animal models. It is urgent to overcome its potential problems and release its maximum efficacy

Method used

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  • DNA containing mutational endonuclease identification section and application of DNA in genome editing
  • DNA containing mutational endonuclease identification section and application of DNA in genome editing
  • DNA containing mutational endonuclease identification section and application of DNA in genome editing

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Embodiment 1

[0070] The following example introduces the operating principle of the present invention by taking the introduction of PTEN-C124S and PTEN-G129E point mutations in 293FT cells as an example.

[0071] The codon of the 124th amino acid-cysteine ​​corresponding to the human PTEN gene coding region is TGT, and by changing one base to TCT, the conversion from cysteine ​​to serine can be realized. Similarly, by converting GGA to GAA, the glycine at position 129 of PTEN can be changed to glutamic acid. through the website http: / / crispr.mit.edu , identified two target RNAs whose recognition regions cover the PTEN-C124 site and the PTEN-G129 site respectively. The innovation of the present invention is that, when preparing the homologous recombination template, in addition to the mutation of the target base, the 20 bases of the target RNA recognition region and the subsequent NGG motif will be carried out according to the rules of codon degeneracy Mutations to protect the genome seq...

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Abstract

The invention provides DNA containing a mutational genome editing endonuclease targeted identification section and application of the DNA in genome editing. The DNA comprises a target input sequence (1) which is edited artificially, a mutational endonuclease identification section sequence (2) and a two-side homologous arm sequence (3). The invention further provides carriers, systems and cells containing the DNA and application of the above products in improving genome editing efficiency, preparing genetically modified cells and animal models and preparing drug for diseases needing gene treatment. By adopting the DNA and the subsequent products thereof, double-strand break and NHEJ repair induced by endonuclease can be avoided reoccurring or occurring for many times on specific genome sequences where DNA editing is completed successfully already, so that gene editing efficiency is improved greatly, and wrong mutation is avoided.

Description

technical field [0001] The invention relates to the field of genetic engineering and gene therapy, in particular to a method for improving gene knock-in efficiency and effectively reducing mutation frequency by mutating the DNA sequence of the target recognition site of genome editing endonuclease, thereby improving genetically modified cells, Construction of animal models and gene therapy of human diseases. Background technique [0002] Genome editing is the use of genetic engineering methods to modify specific sequences on the genome to achieve specific point mutations (base repair), sequence knockout or knockin. In recent years, genome editing technology has been greatly developed and shines in many fields. Currently, the most important applications of genome editing include the gene therapy of human diseases and the construction of genetically modified cells and animal models in basic scientific research. [0003] Gene therapy is a treatment method that introduces exog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/85C12N5/10A61K48/00A61P35/00A61P31/18A61P7/04A61P7/06
Inventor 郭伟何华斌
Owner TSINGHUA UNIV
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