Method for converting monocot plant genome sequence in which nucleic acid base in targeted DNA sequence is specifically converted, and molecular complex used therein

A monocotyledonous plant and nucleic acid base technology, applied in the field of genome sequence modification, can solve the problem of low frequency of insertion/deletion mutations, and achieve the effect of excellent safety and high mutation introduction efficiency

Active Publication Date: 2018-09-04
KOBE UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

In addition, in yeast and prokaryotes, as predicted from the use of deaminase, the mutation mode is mainly base substitution, and the frequency of insertion / deletion mutation is low. Therefore, it is necessary to develop mutant technology

Method used

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  • Method for converting monocot plant genome sequence in which nucleic acid base in targeted DNA sequence is specifically converted, and molecular complex used therein
  • Method for converting monocot plant genome sequence in which nucleic acid base in targeted DNA sequence is specifically converted, and molecular complex used therein
  • Method for converting monocot plant genome sequence in which nucleic acid base in targeted DNA sequence is specifically converted, and molecular complex used therein

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Experimental program
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Embodiment

[0137] 1. Vector construction

[0138] (1) Construction of Target-AID evaluation carrier

[0139] Produced by conventional methods with figure 1 The structure shown in A is pRIT3-EGFP (with EGFP ORF; SEQ ID NO: 9) and pRIT3-mEGFP (the stop codon is present directly after the EGFP start codon; SEQ ID NO: 10).

[0140] (2) Construction of Target-AID carrier

[0141] Generated by the following with figure 1Target-AID vectors 2408 (encoding dCas9; SEQ ID NO:11) and 2409 (encoding D10A mutant; SEQ ID NO:12) of the structure shown in B: pZH_OsU6gRNA_MMCas9 (PlantMol Biol (2015) 88:561-572 ) OS Opt.Cas9 was substituted with DNA encoding (with double mutations of H840A and D10A, or only D10A mutation) mutant Cas9, and a nuclear localization signal (NLS) encoding derived from SV40 was added at both ends downstream of it sequence and fused it to DNA encoding PmCDA1 optimized for codon usage in Arabidopsis.

[0142] 2. Introduction of Target-AID and vector for evaluation into Ag...

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Abstract

The present invention provides a method for modifying a targeted site in double-stranded DNA possessed by monocot plant cells, which includes a step for, by bringing a complex, in which a nucleic acidsequence-recognizing module that specifically bonds with a target nucleotide sequence in a selected double-stranded DNA is bonded with a nucleic acid base converting enzyme, into contact with the double-stranded DNA, converting one or more nucleotides at the targeted site to one or more different nucleotides, deleting one or more nucleotides at the targeted site, or inserting one or more nucleotides to the targeted site, without cleaving at least one of the strands of the double-stranded DNA at the targeted site, wherein the contact between the double-stranded DNA and the complex is achievedby introducing a nucleic acid coding for the complex to the monocot plant cells. In addition, also provided is the complex to be used in this method, in which the nucleic acid sequence-recognizing module that specifically bonds with a target nucleotide sequence in a double-stranded DNA possessed by monocot plant cells is bonded with a nucleic acid base converting enzyme.

Description

technical field [0001] The present invention relates to a method for modifying a genome sequence, and a complex of a nucleic acid base conversion enzyme and a nucleic acid sequence recognition module used therefor, which is not accompanied by DNA double-strand cutting (non-cutting or single-strand cutting), but can perform monocotyledonous plant Modification of nucleic acid bases within a specific region of the genome. Background technique [0002] The monocot refers to a group of plants having one cotyledon among angiosperms, and the three major grains of rice, wheat, and corn are classified in this group. Therefore, although molecular breeding of monocots has been intensively studied in the past, since monocots are not hosts of Agrobacterium, the most general Agrobacterium method cannot be used for a long time as a plant transformation method, and the direct introduction method is used . Since the mid-1990s, when it was reported that rice can be efficiently transformed b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N9/10C12N9/16C12N9/78
CPCC12N9/10C12N9/16C12N9/78C12N15/09C12N9/22C12Y305/04005C12N15/102C12N15/113C12N15/8205C12N2310/20C12N2523/00C12N15/82
Inventor 西田敬二岛古善平近藤昭彦
Owner KOBE UNIV
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