Plant site-directed recombination method mediated by repeated fragments

A technology for plant cells and transgenic plants, applied in the field of site-directed recombination of plants mediated by repeat fragments, can solve the problems of low efficiency

Active Publication Date: 2019-11-01
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Very inefficient to implement unless the knock-in or replacement sequence is a screening tag
So far, there has been a lack of efficient genomic precision knock-in / replacement methods in the plant field

Method used

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  • Plant site-directed recombination method mediated by repeated fragments
  • Plant site-directed recombination method mediated by repeated fragments
  • Plant site-directed recombination method mediated by repeated fragments

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0184] 1) For the preparation of shorter donor DNA (usually within 120bp), the modified oligonucleotide single strand can be directly synthesized and annealed directly to generate double-stranded donor DNA;

[0185] 2) For the preparation of longer donor DNA, primers modified by thiolation can be used to obtain by PCR amplification ( figure 1 );

[0186] 3) Or directly prepare by cutting exogenous DNA such as plasmids.

[0187] Preparation of site-directed cutting nuclease DNA construct

[0188] ZFN, Talen, and CRISPR / Cas9 technologies can all make site-directed cuts in plant genomes to generate double-strand DNA breaks (DSBs). Therefore, DNA elements expressing these three site-directed cutting nucleases can all be used in the present invention. The DNA element can be a plasmid or a linear fragment. Since the CRISPR / Cas9 technology is relatively simple and efficient, the present invention prefers CRISPR / Cas9 to make site-specific cuts on the plant genome.

[0189] Reagen...

Embodiment 1

[0228] Example 1 Rice SLR1 Gene Base Replacement

[0229] Using the modified DNA fragment synthesized in vitro as the donor DNA fragment, combined with CRISPR / Cas9 technology, the multiple bases in the rice SLR1 gene were precisely replaced and deleted. The specific operation process is as follows.

[0230] CRISPR / Cas9 vector preparation

[0231] The target gRNA-1 (SEQ ID NO.:) was designed for the region to be replaced of the rice SLR1 gene, and the gRNA-1 guide sequence was constructed into the rice CRISPR / Cas9 vector, wherein the OSU6-gRNA-1 sequence is shown in the sequence listing (SEQ ID NO. NO.: 2).

[0232] Donor fragment design and in vitro preparation

[0233] Such as image 3 As shown in A and 3B, the end of the donor DNA can be phosphorylated and thio-modified (5'P stands for phosphorylation modification at the 5' end, and * stands for inter-base thio-modification) to promote NHEJ; 82bp in the fragment and The sequence of the SLR1 target site is homologous; af...

Embodiment 2

[0246] Example 2 The fixed-point knock-in experiment of GFP

[0247] The DNA fragment amplified by PCR was used as the donor DNA fragment, and combined with CRISPR / Cas9 technology, the GFP gene was knocked into the 3' end of the rice highly expressed genes ACT1 and GST1 to form a fusion protein. The specific operation process is as follows.

[0248] CRISPR / Cas9 vector preparation

[0249] Target gRNA-2 and gRNA-3 (SEQ ID NO.: 9, 10) were designed for the 3' ends of rice ACT1 and GST1 genes respectively, and these two guide sequences were constructed into rice CRISPR / Cas9 vectors, wherein OSU6-gRNA- 2 and OSU6-gRNA-3 sequences are shown in the sequence listing (SEQ ID NO.: 11, 12).

[0250] Design and preparation of donor DNA fragments

[0251] Such as Figure 5 As shown in A, the donor DNA is amplified by PCR, and the primers used for the amplification are shown in the table below. Wherein, the donor DNA fragment (sequence 9, 1528bp) knocked in by primer ACT1-F1 and NOS-R1...

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Abstract

The invention provides a plant site-directed recombination method mediated by repeated fragments. Specifically, the present invention provides a donor DNA with a specific repeat sequence, and a reagent combination for gene editing. According to the invention, the donor DNA with a specific structure is adopted, and a specific site of a target gene is cut by means of a site-directed cutting nuclease, so that the donor DNA fragment is efficiently integrated into a cutting site.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a plant-directed recombination method mediated by repeated segments. Background technique [0002] Genome editing technologies include Zinc finger nuclease (ZFN), transcription activator-like (TAL) effector nucleases (Talen) and CRISPR / Cas technologies. These three technologies can specifically cut DNA at a specified site in the genome of an organism to generate a double-strand break (DSB), thereby utilizing the characteristics of non-homologous end joining or homologous recombination of the cell itself to perform site-directed editing. ZFN and Talen technologies use specific proteins to guide genome cutting, and their construction is relatively complex, and the editing efficiency is very low. [0003] It is generally believed that the DNA break repair mechanism in plant cells is dominated by NHEJ, and the probability of HDR is relatively very low. Therefore, when performing genome...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/22A01H5/00A01H6/46C12N5/10
CPCC12N15/8213C12N9/22C12N15/8261C12N5/10C12N15/63C12N15/85C12N15/8207C12N15/11
Inventor 朱健康陆钰明田益夫沈润东王木桂
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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