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A spcas9-nrrh mutant that recognizes specific sites in rice gene targeting and its application

A gene targeting and specific site technology, applied in the fields of biotechnology and plant genetic engineering, can solve the problem of not being able to fully meet the editing situation.

Active Publication Date: 2022-04-01
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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  • A spcas9-nrrh mutant that recognizes specific sites in rice gene targeting and its application
  • A spcas9-nrrh mutant that recognizes specific sites in rice gene targeting and its application
  • A spcas9-nrrh mutant that recognizes specific sites in rice gene targeting and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1—The acquisition of SpCas9-NRRH mutant gene

[0050] The encoded protein sequence of 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. Amino acid changes from G to S, amino acid at position 1219 changes from E to V, amino acid at position 1221 changes from Q to H, amino acid at position 1249 changes from P to S, and amino acid at position 1253 changes from E to K , the amino acid at position 1...

Embodiment 2

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

[0055] From the Escherichia coli XL-blue containing the PUC57-AMP-SpCas9-NRRH vector above, use the Axygen plasmid extraction kit to extract the plasmid, digest it with NotI / SacI, and recover the SpCas9-NRRH fragment. At the same time, pHUN600 was linearized using NotI / SacI enzymes, pHUN600 was recovered, and the above-mentioned SpCas9-NRRH fragment and pHUN600 fragment were connected with T4 ligase (purchased from TaKaRa Company) to obtain pHUN600-SpCas9-NRRH, and then SpCas9 was matched to The sgRNA expression cassette was connected to the pHUN600-SpCas9-NRRH vector to obtain a plant expression vector named pHUN411NRRH ( figure 1 ).

[0056] The inventors of the present invention took 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 pHU...

Embodiment 3

[0059] Example 3 - Rice Genetic Transformation of pHUN411NRRH Targeting Vector and Obtainment of Mutants

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

[0061] 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 invention provides a SpCas9-NRRH gene for identifying specific sites in rice gene targeting and its application. In the process of rice gene targeting experiment, the present invention unexpectedly obtains a new SpCas9-NRRH mutant, and finds that using the SpCas9-NRRH mutant for rice shearing 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 invention utilizes the obtained SpCas9-NRRH to construct a plant expression vector, constructs a rice targeting vector, and after being introduced into a rice cell, causes DNA double-strand cleavage at a rice-specific gene site, realizes rice gene targeting, and obtains transgenic rice with high mutation efficiency plant.

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-NRRH mutant that recognizes a specific site in gene targeting and its application in rice gene targeting. Background technique [0002] CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins) system, as an important gene editing tool, has been widely used in crop trait improvement since its birth. The CRISPR / Cas9 gene editing technology mainly includes two core contents: 1) Construct the Cas9 / sgRNA expression vector, and introduce the vector into the recipient cell for expression; 2) Introduce the expressed and purified Cas9 protein and the synthesized sgRNA into the recipient cell for expression. Editing role. The Cas9 protein SpCas9 derived from Streptococcus pyogenes was first applied to gene editing. The protein contains a RuvC-like domain and an HNH nucleas...

Claims

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Application Information

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Patent Type & Authority Patents(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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