Co-transcription unit gene ABE-CBE system for bidirectional single base editing of rice and application thereof

An ABE-CBE, single-base technology, applied in the field of biotechnology and plant genetic engineering, can solve the problems of fixed-point modification dependence, low efficiency, and the ability to edit genes accurately, so as to achieve wide application value and reduce unexpected bases The effect of replacing

Active Publication Date: 2021-03-19
RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although CRISPR / Cas9 can efficiently target the target gene, its site-directed modification relies on the inefficient homologous recombination mechanism, so the ability to precisely edit genes needs to be improved

Method used

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  • Co-transcription unit gene ABE-CBE system for bidirectional single base editing of rice and application thereof
  • Co-transcription unit gene ABE-CBE system for bidirectional single base editing of rice and application thereof
  • Co-transcription unit gene ABE-CBE system for bidirectional single base editing of rice and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Example 1 - Splicing of the mutant TadA-nSpCas9-LjCDAL1 gene

[0120] The gene of the present application is named mutant TadA-nSpCas9-LjCDAL1, and its sequence is shown in SEQ ID NO:1.

[0121] First, through a large number of experiments, the applicant designed and screened a lamprey-like cytosine deaminase (cytidine deaminase, LjCDAL1) gene with a wide window and high editing efficiency, named Os-LjCDAL1 (sequence such as SEQ ID NO: 2 ), its editing efficiency can reach more than 80%. After the Os-LjCDAL1 gene was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis, it was connected to the PUC57-AMP vector to form the PUC57-AMP-LjCDAL1 vector, and loaded into E. coli XL-blue strain.

[0122] Secondly, the mutant adenine deaminase TadA suitable for rice was artificially synthesized, named mutant TadA, connected to the PUC57-AMP vector to form the PUC57-AMP-mutant TadA vector, and loaded into E. coli XL-blue strain .

[0123] According to the Gibson splic...

Embodiment 2

[0133] Example 2—Construction of plant targeting vector containing mutant TadA-nSpCas9-LjCDAL1 gene

[0134] From Escherichia coli XL-blue containing the mutant TadA-nSpCas9-LjCDAL1 vector, use the Axygen plasmid extraction kit to extract the plasmid, digest it with NotI / SacI, and recover the OsSpCas9-LjCDAL1 fragment. At the same time, pHUC411 was linearized using NotI / SacI enzymes, pHUC411 was recovered, and the above-mentioned mutant TadA-nSpCas9-LjCDAL1 fragment and pHUC411 fragment were connected with T4 ligase (purchased from TaKaRa Company) to obtain the plant expression vector pHUC411mutant TadA-nSpCas9 -LjCDAL1( figure 1 ), named pHUC411-TadA-LjCDAL1.

[0135] Select the nucleotide sequence CGGCGACGGCGAGCAAGTGG of position 3105-3124 in rice NRT1.1 gene (Os10g0554200) AGG , (the underlined part is the PAM sequence of the 5'NGG-3' structure), as the targeting site. The target site sequence was fused to pHUC411-TadA-LjCDAL1 to form pHUC411-TadA-LjCDAL1-NRT1.1. The p...

Embodiment 3

[0136] Example 3—Genetic transformation of rice using pHUC411-TadA-LjCDAL1-NRT1.1 as a targeting vector and acquisition of mutants.

[0137] 1. Induction and pre-culture of mature embryo callus

[0138] The mature seeds of Nipponbare are shelled, and the seeds with normal appearance and cleanness without mildew are selected, shaken for 90 sec with 70% alcohol, and poured off the alcohol; Add 1 drop of Tween20) solution to 1 ml to wash the seeds, and shake on the shaker for 45min (180r / min). Pour off the sodium hypochlorite, wash with sterile water 5-10 times until there is no smell of sodium hypochlorite, finally add sterile water, soak overnight at 30°C. Use a scalpel to separate the embryos along the aleurone layer, put the scutellum up on the induction medium (see Table 1 for ingredients), 12 embryos / dish, and culture in the dark at 30°C to induce callus.

[0139] Two weeks later, spherical, rough, light yellow secondary callus appeared, and pre-cultivation operation coul...

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Abstract

The invention provides a co-transcription unit gene ABE-CBE system for the bidirectional single base editing of rice, a vector and application thereof. A co-transcription unit gene ABE-CBE comprises an adenine deaminase coding gene TadA and a cytosine deaminase coding gene CDA, and the coding sequences of co-transcription unit genes mutant TadA and LjCDAL1 are constructed in the same expression vector. An expression vector containing a cytosine base editor and an adenine base editor is fused, so that the nucleotide replacement of specific sites of a genome is caused. Experiments prove that a designed fusion editing tool is used for constructing a plant expression vector, and then a rice targeting editing vector is constructed, so that after the gene is introduced into rice cells, the efficiency of the mutual replacement of A:T to G:C on DNA of a rice specific gene locus is improved, the simultaneous replacement of C to T and A to G can be realized at the specific locus of the gene, theapplication range is expanded, and meanwhile, off-target generation is reduced. Therefore, a fused A-C base editing system is enabled to have a wider application prospect.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and plant genetic engineering. Specifically, the present invention relates to the fusion and application of two base editors. Background technique [0002] Gene editing technology is one of the disruptive technologies that have made breakthroughs in recent years. The CRISPR / Cas9 gene editing system is simple and efficient, and is widely used in gene function research and utilization. Although CRISPR / Cas9 can efficiently target target genes, its site-directed modification relies on the inefficient homologous recombination mechanism, so the ability to precisely edit genes needs to be improved. Single base editing technology (base editor, BE) forms a fusion protein with Cas9 nickase (Cas9 nikase, Cas9n) or nuclease-free Cas9 (nuclease dead Cas9, dCas9) and cytosine deaminase, and through sgRNA ( Single-stranded guide RNA) targets the fusion protein to the target site, and performs precise a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/84C12N15/66C12N15/55C12N15/29C12N15/10A01H5/00A01H6/46
CPCC12N15/8218C12N15/8243C12N15/66C12N9/78C12N9/22C07K14/415C12N15/102C12Y305/04002C12Y305/04001
Inventor 许蓉芳李娟秦瑞英孔凡娜徐丽媛冯睿彤魏鹏程
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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