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Gene modifying method for reserved locus of cell genome

A technology of genetic modification and predetermined sites, applied in the direction of cells modified by the introduction of foreign genetic material, recombinant DNA technology, introduction of foreign genetic material using a vector, etc., can solve problems to be improved, etc.

Inactive Publication Date: 2014-11-19
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the current methods of genome editing and gene expression regulation using the Cas9 system still need to be improved.

Method used

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  • Gene modifying method for reserved locus of cell genome
  • Gene modifying method for reserved locus of cell genome
  • Gene modifying method for reserved locus of cell genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Gene knockout in HELA cells

[0058] 1. Preparation of cells carrying exogenous genes

[0059] The H3F3A gene internal target destruction plasmid pDSB-H3.3 and the homologous recombination tool plasmid Donor-KI-1 / Donor-KI-2 with the inserted gene fragment were co-transfected into Hela cells.

[0060]Wherein, pDSB-H3.3 comprises the following sequences in turn: U6 promoter, H3F3A gRNA-1, U6 promoter, H3F3A gRNA-2, CBh promoter, coding gene of Flag tag protein, coding gene of Cas9 (D10A) protein. Wherein the Cas9 (D10A) protein is a derivative of the wild-type Cas9 protein, which has a D10A mutation relative to the wild-type Cas9 protein (the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1), and the coding gene of the Cas9 (D10A) protein , relative to the 74th base in the nucleotide sequence shown in SEQ ID NO: 1 is mutated from A to C.

[0061] Donor-KI-1 contains the following sequences in sequence: the left arm of the homologous recombinat...

Embodiment 2

[0095] Example 2 Gene expression regulation in Hela cells

[0096]According to a method similar to Example 1, Hela cells carrying Cas9 mutants (D10A, H840A) and transcriptional regulatory elements (SID4X or VP64) were constructed. Wherein, SID4X or VP64 is connected in series with the Cas9 mutant (D10A, H840A) on the carrier, and the carrier construction method also uses the commonly used methods of PCR and molecular cloning; the method and strategy of cell recombination are the same as in Example 1, only The Cas9 (D10A, H840A) DNA sequence in the Donor has more SID4X in front or more VP64 in the back; others, including transfection conditions, are exactly the same as in Example 1. Wherein the Cas9 (D10A, H840A) protein is a derivative of the wild-type Cas9 protein, which has both D10A and H840A mutations relative to the wild-type Cas9 protein (the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1).

[0097] Then, taking SID4X-Cas9(D10A, H840A), Cas9(D10A, H840...

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Abstract

The invention discloses a recombinant cell, a gene modifying reagent for a cell and a gene modifying method for a reserved locus of a cell genome. The recombinant cell is used for over-expressing a first nucleic acid molecule; the first nucleic acid molecule is used for encoding a Cas9 protein or a derivative of the Cas9 protein; and compared with the Cas9 protein, the derivative of the Cas9 protein has no DNA cleavage activity, but remains target recognition activity. By using the recombinant cell, Che-chiRNA or Che-gRNA and tracrRNA can be transferred into the recombinant cell, so that genome editing or gene expression control can be easily carried out, and furthermore the gene modification on the reserved locus of the cell genome can be effectively realized.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically, the present invention relates to recombinant cells, reagents for genetically modifying cells, and methods for genetically modifying predetermined sites in cell genomes. Background technique [0002] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 is a technology that has been widely recognized in the past two years for the specific DNA modification of targeted genes by RNA-guided Cas nucleases. In this technology, crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing to form a double-stranded RNA, resulting in a tracrRNA / crRNA binary complex. The resulting tracrRNA / crRNA binary complex can guide the Cas9 protein to cut double-stranded DNA at the target site matching the crRNA guide sequence, thereby achieving the purpose of modifying genomic DNA. The CRISPR / Cas9 system can precisely edit specific gene loci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/09C12N15/63
Inventor 李卓裴端卿
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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