Plant high-efficiency homologous recombination method based on CRISPR/Cas9

A homologous recombination and plant technology, applied in the field of gene editing, can solve the problem of low efficiency of homologous recombination

Inactive Publication Date: 2018-09-04
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Homologous recombination uses exogenous nucleic acid fragments to accurately repair DNA, but compa

Method used

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  • Plant high-efficiency homologous recombination method based on CRISPR/Cas9
  • Plant high-efficiency homologous recombination method based on CRISPR/Cas9
  • Plant high-efficiency homologous recombination method based on CRISPR/Cas9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 PCR amplification of target sequence and vector digestion

[0061] The PCR amplification program used in the construction of the vector in the present invention is as follows: pre-denaturation at 98°C for 2 minutes, 35 PCR cycles (98°C for 10s, 55-60°C for 15s, 72°C for 1-2min). The PCR reaction system is: DNA polymerase ( FastPfu DNA Polymerase (Beijing Quanshijin Biotechnology Co., Ltd.) 1 μL, 10× buffer 5 μL, target template (100ng / μL) 1 μL, upstream and downstream primers (10pM) 1 μL each, and ultrapure water to 50 μL. After the PCR was completed, the PCR product was purified with a PCR purification kit (Beijing Quanshijin Biotechnology Co., Ltd.) for use.

[0062] Vector digestion system: 10U of each required endonuclease (New England Biolabs (NEB) company), 5μL of plasmid (200ng / μL), 1μL of 10×buffer, make up to 10μL with ultrapure water, 37℃ for about 2h, 80℃ Extinguish the fire for 10 minutes and set aside.

[0063] In-fusion cloning (method refer ...

Embodiment 2

[0064] The construction of embodiment 2 pHR04a vector

[0065] According to the reference sequence of BeYDV (NCBI NO.DQ458791), its replicon element LIR-RepA-SIR and multiple cloning site (MCS) were synthesized into pGH cloning vector (Shanghai Jierui Biotechnology Co., Ltd.). Firstly, a primer pair was designed (forward: 5-tatatcctgtcaaggcctgagggtcgtacgaataattcgtatccaacggaaatacc-3, reverse: 5-aacgttatcag cttgcatgcgatatcaggtacttttgttctgcga-3). According to the PCR conditions in Example 1, the target fragment LIR-RepA-SIR was amplified, and the pGreen0029 vector was digested with SphI and StuI to construct an intermediate vector. After the sequencing is correct, design primers (forward: 5-aacgttatcagcttgcatgcgagggtcgtacgaataattcgtatccaac-3, reverse: 5-aagtacctgatatcgcatggttgttgtga ctccgagggg-3). Amplify the target fragment LIR according to the PCR conditions in Example 1, cut the intermediate vector with SphI, and clone by In-fusion to obtain the carrier pHR04a( figure 1 ). ...

Embodiment 3

[0066] Example 3 Construction of pHR04a-AsRed Vector

[0067] The expression cassette CasMV35S-AsRed-Nos was recombined between the AscI and BstXI sites of pHR04a. Primer pairs (forward: 5-acgaccctcggcgcgcctgagacttttcaacaaagggtaatatccgga-3, reverse: 5-acgtgacgtacccaaagctctgggatctagtaacatagatgacaccgcgc-3) were designed. Amplify the target fragment CasMV35S-AsRed-TNos according to the PCR conditions in Example 1, cut the pHR04a vector with AscI and BstXI, and obtain the vector pHR04a-AsRed ( figure 2 ).

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Abstract

The invention provides a plant high-efficiency homologous recombination method based on CRISPR/Cas9. According to the plant high-efficiency homologous recombination method based on CRISPR/Cas9, a geneediting carrier and a gene homologous recombination carrier are constructed based on plant target genes for editing, the two carriers are adopted for conversion of plants at the same time so as to obtain transgenic plants; wherein the gene editing carrier at least comprises a Cas9 expression cassette and a gRNA expression cassette; the gene homologous recombination carrier at least comprises a copying sub-element, a homologous left arm, a gene for knocking in, and a homologous right arm which come from bean yellow dwarf virus. According to the plant high-efficiency homologous recombination method, a target donor fragment and a gene editing element are constructed onto the two carriers respectively, so that the load of replicors is reduced, the copy number of the target homologous segmentis increased, the copy number of a target sequence is increased on the base of effective editing on chromosome, and homologous recombination efficiency is increased greatly.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a CRISPR / Cas9-based high-efficiency homologous recombination method for plants. Background technique [0002] Bean yellow dwarf virus (BeYDV) was first isolated from bean and belongs to the genus Zeavirus of the family Geminiviridae (Liu et al., 1997, Journal / J Gen Virol, 78 (Pt 8): 2113-2117). Infect plants such as kidney bean, chickpea, tobacco, tomato, potato, Arabidopsis (Halley-Stott et al., 2007, Journal / Arch Virol, 152:1237-1240; Liu et al., 1999, Journal / Virology , 256:270-279; Liu et al., 1997, Journal / J Gen Virol, 78(Pt 8):2113-2117). Using its replicon elements, Agrobacterium-mediated infection of plants such as tobacco, tomato and lettuce can produce high-copy target DNA fragments in plant cells (Collens et al., 2007, Journal / Biotechnol Prog, 23:570-576 ; Hefferon and Dugdale, 2003, Journal / J GenVirol, 84:3465-3472; Hefferon and Fan, 2004, Journal / Vaccine, 23:4...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66A01H5/00A01H6/46A01H6/20
CPCC12N15/8213C12N2800/80C12N2810/10
Inventor 胡赞民范成明李冬冬王晓波陈宇红袁静韩方普张彦峰
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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