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SpCas9-NRRH mutant for identifying specific sites in rice gene targeting and application thereof

A technology of gene targeting and specific sites, which is applied in the field of biotechnology and plant genetic engineering, and can solve the problems that cannot fully meet the editing conditions

Active Publication Date: 2021-04-09
RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these PAM extended editing tools still cannot fully meet the various editing situations encountered in the actual application process

Method used

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  • SpCas9-NRRH mutant for identifying specific sites in rice gene targeting and application thereof
  • SpCas9-NRRH mutant for identifying specific sites in rice gene targeting and application thereof
  • SpCas9-NRRH mutant for identifying specific sites in rice gene targeting and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1—Obtaining of SpCas9-NRRH mutant gene

[0050] The protein sequence encoded by the SpCas9-NRRH mutant of the present application is shown in SEQ ID NO: 1. It differs from the protein SpCas9 in that the amino acid at position 322 is changed from I to V, the amino acid at position 409 is changed from S to I, the amino acid at position 427 is changed from E to G, and the amino acid at position 654 is changed from R to L, the amino acid at position 753 is changed from R to G, the amino acid at position 1114 is changed from R to G, the amino acid at position 1135 is changed from D to N, the amino acid at position 1180 is changed from D to G, and the amino acid at position 1218 is changed from D to G. The amino acid at position 1219 is changed from G to S, the amino acid at position 1219 is changed from E to V, the amino acid at position 1221 is changed from Q to H, the amino acid at position 1249 is changed from P to S, the amino acid at position 1253 is changed fro...

Embodiment 2

[0054] Embodiment 2——Contain the construction of SpCas9-NRRH gene plant targeting vector

[0055] From the E. coli XL-blue containing the PUC57-AMP-SpCas9-NRRH vector above, the plasmid was extracted with the Axygen plasmid extraction kit, digested with NotI / SacI, and the SpCas9-NRRH fragment was recovered. At the same time, pHUN600 was linearized with NotI / SacI enzyme, pHUN600 was recovered, and the above-mentioned SpCas9-NRRH fragment and pHUN600 fragment were ligated with T4 ligase (purchased from TaKaRa Company) to obtain pHUN600-SpCas9-NRRH, and then SpCas9 was matched The sgRNA expression box was connected on the pHUN600-SpCas9-NRRH carrier to obtain a plant expression vector, named pHUN411NRRH ( figure 1 ).

[0056] The inventors of the present invention selected rice PDS and BADH2 genes as target genes, and selected 24 targets for different combinations of PAM sequences of NRRH. The target sequences are shown in Table 2. The target sequence was fused with pHUN411NRRH...

Embodiment 3

[0059] Example 3 - Rice genetic transformation of pHUN411NRRH targeting vector and acquisition of mutants

[0060] 1. Induction and pre-culture of mature embryo callus

[0061] The mature seeds of Nippon Harvest (preserved by the Rice Research Institute of Anhui Academy of Agricultural Sciences) were hulled, and the seeds with normal appearance, clean and mildew-free were selected, and the alcohol was shaken for 90 sec with 70% alcohol; then 50% sodium hypochlorite containing Tween20 ( If the available chlorine concentration of the stock solution is greater than 4%, add 1 drop of Tween20) solution per 100 ml to wash the seeds, and shake them on a shaking table for 45 min (180 r / min). Pour out the sodium hypochlorite, wash with sterile water for 5-10 times until there is no smell of sodium hypochlorite, finally add sterile water and soak overnight at 30°C. The embryos were separated along the aleurone layer with a scalpel blade, and the scutellum was placed on the induction me...

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Abstract

The invention provides a SpCas9-NRRH gene for identifying specific sites in rice gene targeting and application thereof. In the rice gene targeting experiment process, a novel SpCas9-NRRH mutant is accidentally obtained, and the specific sites can be identified when the SpCas9-NRRH mutant is used for rice shearing. The invention further provides an expression cassette and an expression vector which are constructed based on the gene and application of the expression cassette and the expression vector in the aspect of rice gene editing. The obtained SpCas9-NRRH is used for constructing a plant expression vector, a rice targeting vector is constructed, DNA double-strand shearing of the rice specific gene sites is caused after the vector is introduced into rice cells, rice gene targeting is achieved, and transgenic rice plants are obtained with high mutation efficiency.

Description

technical field [0001] The present invention relates to the technical field of biotechnology and plant genetic engineering. Specifically, the present invention relates to a SpCas9-NRRH mutant that recognizes specific sites in gene targeting and its application in rice gene targeting. Background technique [0002] As an important gene editing tool, CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins) system has been widely used in crop trait improvement since its birth. CRISPR / Cas9 gene editing technology mainly includes two core contents: 1) constructing a Cas9 / sgRNA expression vector, and introducing the vector into recipient cells for expression and editing; 2) introducing the expressed and purified Cas9 protein and synthetic sgRNA into recipient cells to play a role editorial action. The Cas9 protein SpCas9 derived from Streptococcus pyogenes was first used for gene editing. The protein contains a RuvC-like domain and an HNH ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/55C12N15/82C12N15/66A01H5/00A01H6/46
CPCC12N9/22C12N15/8218C12N15/8221
Inventor 李娟许蓉芳秦瑞英刘小双单调风魏鹏程
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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