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Genome editing method

A genome editing and base sequence technology, applied in the field of genome editing, can solve the problem of low efficiency of flox mice, and achieve the effect of shortening the production time and reducing the production cost

Pending Publication Date: 2019-09-06
OSAKA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It is theoretically possible to insert the loxP sequence at two places simultaneously using the existing donor vector, but the efficiency of obtaining flox mice is not high in practice

Method used

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  • Genome editing method
  • Genome editing method
  • Genome editing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Example 1. Genome editing experiment 1 (wild type mouse, Serpina3n gene, microinjection)

[0140] Exon 4 and its surrounding region of the mouse Serpina3a (serine peptide inhibitor clade A member 3A) gene were used as the genome editing target region, and this region was replaced with a sequence added with loxP sequences at both ends of the region. Specifically according to figure 1 The protocol shown was carried out as follows.

Embodiment 1-1

[0141]

[0142] The design of the guide RNA was performed using a software tool (crispr.genome-engineering.org). Instructions designed to cleave one end of the region to be edited in the genome (SEQ ID NO: 1) synthesized using double-stranded DNA (synthesized by Integrated DNA Technologies) as a template using an in vitro transcription kit (MEGAshortscript T7Transcription Kit, manufactured by Life Technologies) RNA (Serpina3a gRNA-1, the sequence of the target site (target strand) is SEQ ID NO: 2), and a guide RNA designed to cut the other end (Serpina3a gRNA-2, the sequence of the target site (target strand) is SEQ ID NO: 3).

Embodiment 1-2

[0143]

[0144] A plasmid (pCas9-polyA, Addgene ID#72602) containing a sequence to which a polyA tail was added downstream of the Cas9 coding sequence was linearized, and used as a template DNA, using an in vitro transcription kit (MEGAshortscriptT7Transcription Kit, manufactured by Life Technologies, Inc. ) was synthesized and purified using a purification kit (MEGAClearkit, manufactured by Life Technologies).

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Abstract

This method for producing a genome-edited cell or non-human organism, comprises a step for introducing, into a cell or a non-human organism, (a) an artificial nuclease system which cleaves both ends of a region to be genome-edited, and (b) a single-stranded DNA including a base sequence in which a 5'-homology arm sequence, a donor DNA sequence, and a 3'-homology arm sequence are arranged in this order from the 5' end thereof, wherein the 5'-homology arm sequence is a homologous sequence to one base sequence at the outside of the region to be genome-edited, and the 3'-homology arm sequence is ahomologous sequence to the other base sequence at the outside of the region to be genome-edited.

Description

technical field [0001] The present invention relates to genome editing methods and the like. Background technique [0002] Genetically altered mice are used by researchers worldwide to analyze gene function at the individual level. In particular, conditional knockout mice are capable of tissue-specific and stage-specific control of gene expression and are therefore one of the most widely utilized genetically modified mice. [0003] A conditional knockout mouse is produced by mating a "Cre mouse" in which a Cre expression cassette is integrated and a "flox mouse" in which a loxP sequence sandwiches the target gene or a part thereof. In the production of Cre mice and flox mice, mouse ES cells have been mainly used so far, or a part of the Cre expression cassette has been knocked in by homologous recombination of ES cells, and the resulting cells are injected into mice. Blastocysts were transplanted into pseudopregnant female mice, and Cre mice and flox mice were produced thr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/82A01K67/027C12M1/00C12N1/15C12N1/19C12N1/21C12N5/10
CPCC12N15/90C12N15/102C12N15/907C12N2310/20A01K67/027
Inventor 真下知士宫坂佳树
Owner OSAKA UNIV