Application of SpCas9n&PmCDA1&UGI base editing system to editing of plant genes

A technology for encoding genes and editing, applied in applications, plant peptides, plant products, etc., can solve problems such as limited scope, and achieve the effect of wide application prospects

Inactive Publication Date: 2019-03-12
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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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

Method used

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  • Application of SpCas9n&PmCDA1&UGI base editing system to editing of plant genes
  • Application of SpCas9n&PmCDA1&UGI base editing system to editing of plant genes
  • Application of SpCas9n&PmCDA1&UGI base editing system to editing of plant genes

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Embodiment 1

[0071] Example 1. The SpCas9n&PmCDA1&UGI base editing system can mutate C into T in the rice genome

[0072] 1. Construction of recombinant expression vector

[0073] Artificially synthesize the following recombinant vector SpCas9n&PmCDA1&UGI, which is a circular plasmid.

[0074] The sequence of SpCas9n&PmCDA1&UGI is sequence 1 in the sequence listing, the vector can express the protein SpCas9n shown in the sequence 2 in the sequence listing, the protein PmCDA1 shown in the sequence 3, the protein UGI shown in the sequence 4, and the six transcription targeting OsWaxy genes sgRNA (target points are marked as W-T1, W-T2, W-T3, W-T4, W-T5 and W-T6), the common sequence of these six sgRNAs is the sgRNA backbone, which is composed of the 571-646th sequence of sequence 1 bits are transcribed. Among them, the 1809-3522th position of sequence 1 is the OsUbq3 promoter sequence, the 3529-7797th position of sequence 1 is the coding sequence of SpCas9n, the 8089-8712th position is the...

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Abstract

The invention discloses application of a SpCas9n&PmCDA1&UGI base editing system to editing of plant genes. The SpCas9n&PmCDA1&UGI base editing system is introduced into organisms or biological cells,so that encoding genes of SpCas9n, sgRNA, PmCDA1 and UGI in the system are expressed, and editing of genome target site sequences is realized; the target site sequences are targeted through sgRNA; PAMsequences of the target site sequences are NAG; N is A, T, C or G. The SpCas9n&PmCDA1&UGI base editing system is used for achieving substituting C by T in more target site sequences in the plants, for example, the PAM sequences are target site sequences of NAG; the editing range of a PmCDA1-mediated base substitution system is expanded; the SpCas9n&PmCDA1&UGI base editing system has broad application prospects.

Description

technical field [0001] The invention relates to the application of SpCas9n&PmCDA1&UGI base editing system in plant gene editing in the field of biotechnology. 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 limited becaus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N5/10A01H5/00A01H6/46
CPCC12N15/113C12N15/8213C07K14/415C12N2310/20C12N2310/10C12N2510/00
Inventor 杨进孝张成伟武莹冯峰宋金岭
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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