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Method for mutating base C into base T in plant genome

A plant genome and coding gene technology, which is applied in the field of mutating the base C in the plant genome to the base T, which can solve the problems of low editing ability and limited range

Pending Publication Date: 2022-04-12
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the SpCas9n(D10A)&rAPOBEC1 / PmCDA1&UGI base editing system has been widely used in rice to realize the conversion of C to T, but the editing target is mainly limited to the sequence of PAM (Protospacer Adjacent Motif) as NGG, which greatly limits the Editable range of C
The variant of SpCas9, SpCas9-NG, can recognize NGN (N=A, T, C or G) PAM targets, and was successfully developed into CBE (SpCas9-NG-CBE), which greatly expands the editable range in animal and plant genomes. C range, but SpCas9-NG-CBE has low editing capacity for NGC PAM targets relative to NGA, NGT and NGG PAM targets

Method used

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  • Method for mutating base C into base T in plant genome
  • Method for mutating base C into base T in plant genome
  • Method for mutating base C into base T in plant genome

Examples

Experimental program
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Effect test

Embodiment 1

[0125] Example 1. The SpRYn-CBE base editing system can realize base editing for targets whose PAM sequence is NGC in the rice genome

[0126] 1. Construction of recombinant expression vector

[0127] Artificially synthesize the following recombinant expression vectors, SpRYn-CBE-1 recombinant expression vector, SpRYn-CBE-2 recombinant expression vector, SpCas9n-NG-CBE-1 recombinant expression vector and SpCas9n-NG-CBE-2 recombinant expression vector. Schematic diagram of each element structure of SpRYn-CBE-1 recombinant expression vector and SpRYn-CBE-2 recombinant expression vector figure 1 shown. Schematic diagram of the structure of each element of the SpCas9n-NG-CBE-1 recombinant expression vector and SpCas9n-NG-CBE-2 recombinant expression vector figure 2 shown. Each vector is a circular plasmid, and the specific structure description is as follows:

[0128] The sequence of the SpRYn-CBE-1 recombinant expression vector is sequence 1 in the sequence list. The 131-5...

Embodiment 2

[0151] Example 2. The SpRYn-CBE base editing system can realize base editing of targets whose PAM sequence is NGA, NGT or NGG in the rice genome

[0152] 1. Construction of recombinant expression vector

[0153] Artificially synthesize the following recombinant expression vectors: SpRYn-CBE-3 recombinant expression vector, SpRYn-CBE-4 recombinant expression vector, SpRYn-CBE-5 recombinant expression vector, SpRYn-CBE-6 recombinant expression vector and SpRYn-CBE-7 recombinant expression vector carrier. Each vector is a circular plasmid.

[0154] The sequence of the SpRYn-CBE-3 recombinant expression vector is to replace the NGC-C1 target sequence in the SpRYn-CBE-1 recombinant expression vector sequence with the NGA-C1 target sequence, and replace the NGC-C4 target sequence with the NGA-C2 target sequence sequence, and the sequence obtained after keeping other sequences unchanged. NGA-C1 target sequence and NGA-C2 target sequence are shown in Table 3.

[0155] The sequence...

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Abstract

The invention discloses a method for mutating a basic group C into a basic group T in a plant genome. The method disclosed by the invention comprises the following steps: introducing SpRYn, cytosine deaminase, sgRNA and UGI into a plant body to realize mutation of C in a plant genome target sequence into T. Experiments prove that the method disclosed by the invention can be used for editing the base C in the target spot sequence of which the PAM sequence is NGN on the plant genome, so that the replacement from the base C to the base T is realized, and the base replacement efficiency is also improved while the range of editable C is expanded.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for mutating a base C in a plant genome into a base T. Background technique [0002] CRISPR-Cas9 technology has become a powerful genome editing method and has been widely used in many tissues and cells. The CRISPR / Cas9 protein-RNA complex is positioned on the target by the guide RNA (guide RNA), cuts and generates a DNA double-strand break (dsDNA break, DSB), and then the organism will instinctively initiate a DNA repair mechanism to repair the DSB. There are generally two repair mechanisms, one is non-homologous end joining (NHEJ), and the other is homologous recombination (homology-directed repair, HDR). Usually NHEJ accounts for the majority, so the random indels (insertions or deletions) generated by the repair are much higher than the precise repair. For precise base substitution, the application of HDR to achieve precise base substitution is greatly limi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/46
Inventor 王飞鹏赵思刘亚宋金岭贺晓庆
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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