Application of VQRn-Cas9&PmCDA1&UGI base editing system in plant gene editing

A technology of DNA molecules and transgenic cell lines, applied in the direction of plant genetic improvement, application, plant products, etc., can solve problems such as limited scope

Inactive Publication Date: 2019-04-19
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

Method used

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  • Application of VQRn-Cas9&PmCDA1&UGI base editing system in plant gene editing
  • Application of VQRn-Cas9&PmCDA1&UGI base editing system in plant gene editing
  • Application of VQRn-Cas9&PmCDA1&UGI base editing system in plant gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Example 1, VQRn-Cas9&PmCDA1&UGI base editing system can mutate C into T in the rice genome

[0129] 1. Construction of recombinant expression vector

[0130]Artificially synthesize the following recombinant vectors, each of which is a circular plasmid:

[0131] VQRn-Cas9&PmCDA1&UGI-1, VQRn-Cas9&PmCDA1&UGI-2, VQRn-Cas9&PmCDA1&UGI-3, VQRn-Cas9&PmCDA1&UGI-4, and VRERn-Cas9&PmCDA1&UGI-1 and VRERn-Cas9&PmCDA1&UGI-2.

[0132] The vector sequence of VQRn-Cas9&PmCDA1&UGI-3 is sequence 1 in the sequence listing. The 131-467th position of sequence 1 is the OsU3 promoter sequence, the 474-550th position, the 647-723rd position, the 820-896th position, the 993-1069th position, the 1166-1242th position and the 1339-1415th position are all It is the DNA sequence for transcribing pre-tRNA, and positions 551-646, 724-819, 897-992, 1070-1165, 1243-1338, and 1416-1511 are transcriptional targeting OsWaxy The DNA sequence of the six sgRNAs of the gene, the common sgRNA backbone nucleot...

Embodiment 2

[0199] Embodiment 2, the synergy of the modified sgRNA (sgRNA (modified)) to the VQRn-Cas9&PmCDA1&UGI base editing system

[0200] This embodiment provides a sgRNA backbone named sgRNA backbone 2 (sgRNA (modified)), which can improve the efficiency of the VQRn-Cas9&PmCDA1&UGI base editing system. The sequence of the transcribed sgRNA backbone 2 is sequence 7 in the sequence listing.

[0201] 1. Construction of recombinant expression vector

[0202] Artificially synthesize the following recombinant vectors, each of which is a circular plasmid: VQRn-Cas9&PmCDA1&UGI-5, VQRn-Cas9&PmCDA1&UGI-6.

[0203] The sequence of the VQRn-Cas9&PmCDA1&UGI-5 vector is the sequence obtained by replacing the six sgRNA backbone 1 sequences in the VQRn-Cas9&PmCDA1&UGI-3 vector sequence with sequence 7 in the sequence listing.

[0204] The sequence of the VQRn-Cas9&PmCDA1&UGI-6 vector is the sequence obtained by replacing the six sgRNA backbone 1 sequences in the VQRn-Cas9&PmCDA1&UGI-4 vector sequ...

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Abstract

The invention discloses an application of a VQRn-Cas9&PmCDA1&UGI base editing system in plant gene editing. The VQRn-Cas9&PmCDA1&UGI base editing system consists of VQRn-Cas9, PmCDA1, UGI and sgRNA shown in a first formula; the target sequence transcribed RNA-sgRNA skeleton (the first formula), the sgRNA skeleton is RNA obtained by replacing T in sites 571-646 of sequence 1 or sequence 7 with U. The VQRn-Cas9&PmCDA1&UGI base editing system achieves the editing of target site sequences in plants, particularly achieves the replacement from C to T in the target site sequences, and has wide application prospect.

Description

technical field [0001] The invention relates to the application of the VQRn-Cas9&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 / Cas9protein-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 b...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/10C12N9/22C12N9/78C12N15/55A01H5/00A01H6/46
CPCC12N9/22C12N9/78C12N15/102C12N15/8213C12Y305/04014
Inventor 杨进孝张成伟徐雯王飞鹏赵思
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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