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The spcas9-nrch mutant for identifying specific sites in rice gene targeting and its application

A gene targeting, specific site technology, applied in the field of biotechnology and plant genetic engineering, can solve problems such as limited number

Active Publication Date: 2022-04-01
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 there are currently available mutants of individual SpCas9 proteins, the number is extremely limited and it is still unknown whether they can still maintain nuclease activity in rice

Method used

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  • The spcas9-nrch mutant for identifying specific sites in rice gene targeting and its application
  • The spcas9-nrch mutant for identifying specific sites in rice gene targeting and its application
  • The spcas9-nrch mutant for identifying specific sites in rice gene targeting and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1—The acquisition of SpCas9-NRCH mutant gene

[0053] The inventors of the present application tried to transform the SpCas9-NRCH gene from Escherichia coli in various ways, accidentally obtained a new DNA sequence, and added a rice-preferred stop codon TGA to the end of the DNA sequence to form A new gene, the gene is named as SpCas9-NRCH, the sequence is shown in SEQ ID NO:2.

[0054] The designed SpCas9-NRCH gene was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for synthesis, then connected to the PUC57-AMP vector to form the PUC57-AMP-SpCas9-NRCH vector, and loaded into E. coli XL-blue strain.

Embodiment 2

[0055] Embodiment 2—Containing the construction of SpCas9-NRCH gene plant targeting vector

[0056] From the Escherichia coli XL-blue containing the PUC57-AMP-SpCas9-NRCH vector above, use the Axygen plasmid extraction kit to extract the plasmid, digest it with NotI / SacI, and recover the SpCas9-NRCH fragment. At the same time, NotI / SacI enzyme was used to linearize pHUN600, and pHUN600 was recovered, and the above-mentioned SpCas9-NRCH fragment and pHUN600 fragment were connected with T4 ligase (purchased from TaKaRa Company), pHUN600-SpCas9-NRCH, and then SpCas9 matched The sgRNA expression cassette was connected to the pHUN600-SpCas9-NRCH vector to obtain a plant expression vector named pHUNCH ( figure 1 ).

[0057] The 20bp sequence at the 3' end of different PAMs of the rice PDS gene was selected as the targeting site, as shown in Table 2. The target site sequence was fused with pHUNCH to form a total of 12 different pHUNCH targeting vectors. The plant expression vector...

Embodiment 3

[0060] Example 3 - Genetic Transformation of Rice with pHUNCH Targeting Vector and Obtainment of Mutants

[0061] 1. Induction and pre-culture of mature embryo callus

[0062] The mature seeds of Nipponbare (the Paddy Rice Research Institute of Anhui Academy of Agricultural Sciences are preserved) are shelled, and the seeds with normal appearance and cleanness without mildew are selected, shaken for 90 sec with 70% alcohol, and pour off the alcohol; then use 50% sodium hypochlorite ( The concentration of available chlorine in the stock solution is greater than 4%. Add 1 drop of Tween20) solution per 100 milliliters to clean the seeds, and shake for 45 minutes (180 r / min) on a shaker. 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 ing...

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Abstract

The present invention provides a SpCas9‑NRCH mutant that recognizes a specific site in rice gene targeting and its application. In the course of the rice gene targeting experiment, the present invention accidentally obtained a new SpCas9‑NRCH mutant, and found that using the SpCas9‑NRCH gene to cut rice can identify specific sites. And the present invention provides an expression cassette and an expression vector constructed based on the gene, and the application of the expression cassette and the expression vector in rice gene editing. The present invention utilizes the obtained SpCas9‑NRCH to construct a plant expression vector, construct a rice targeting vector, and introduce into rice cells to cause DNA double-strand shearing of rice-specific gene loci, thereby realizing rice gene targeting, and obtaining transgenic rice with high mutation efficiency plants.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and plant genetic engineering. Specifically, the present invention relates to a SpCas9-NRCH mutant that recognizes a specific site in gene targeting and its application in rice gene targeting. Background technique [0002] The CRISPR / Cas gene editing system has become an important means for plant functional gene research and molecular breeding. In the process of gene editing, CRISPR / Cas not only needs single-stranded guide RNA (single guide RNA, sgRNA) to be complementary to the base pairing of the target sequence on the genome, but also needs to exist near the target sequence on the genome. Protospacer asjacent motif (PAM)-specific bases in the spacer sequence. Traditional CRISPR / Cas systems mostly use Cas9 protein as an endonuclease to cause site-specific cleavage at target sites in the plant genome, thereby introducing site-specific mutations. However, Cas9 (SpCas9) recognizes PAM mai...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/22C12N15/55C12N15/113C12N15/82A01H4/00A01H6/46
CPCC12N9/22C12N15/113C12N15/8218A01H4/005A01H4/008C12N2310/20
Inventor 李娟许蓉芳秦瑞英刘小双周桂林范家萌单调风魏鹏程
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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