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A method for targeted knockout of rice miRNA

A rice and carrier technology, applied in the field of plant genetic engineering, can solve the problems of large blindness, difficulty in obtaining mutants, time-consuming and labor-intensive problems, and achieve the effect of simple operation and high knockout rate

Active Publication Date: 2019-11-08
成都极谷基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are time-consuming, labor-intensive, blind, and difficult to implement in the study of miRNA functions, mainly because miRNAs are very small (about 21bp), and mutants are difficult to obtain

Method used

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  • A method for targeted knockout of rice miRNA
  • A method for targeted knockout of rice miRNA
  • A method for targeted knockout of rice miRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Using the method of the present invention to create a single miRNA mutant (taking rice OsmiR528 as an example)

[0040] 1. Vector construction of CRISPR-Cas9-miRNA mutant

[0041] (1) Primer design

[0042] The primers are designed according to the CRISPR / Cas9 recognition and cutting rules of the target site. For the precursor genome sequence of rice OsamiR528 (GenBank number: GQ419957.2), the primers were designed as OsmiR528-sgRNA1-F: 5'-gtgtGAAGGGGCATGCAGAGGAGC-3'; OsmiR528-sgRNA1-R: 5'-aaacGCTCCTCTGCA TGCCCCTTC-3'.

[0043] (2) Primer annealing

[0044] Dilute the upstream and downstream primers of each target site by 10 times, take 10 μL of each, at 98° C., denature for 5 minutes, cool naturally, and dilute the annealed product by 20 times for use.

[0045] (3) Enzyme digestion, glue recovery, and connection

[0046] The backbone vector used in the experiment is pZHY988 ( figure 1 ), constructed by our laboratory, the construction process is: first synthesis (by Sha...

Embodiment 2

[0090] Example 2 Using the method of the present invention to create multiple miRNA mutants (taking rice OsmiR397a and OsmiR97b as examples)

[0091] 1. Vector construction of CRISPR-Cas9-miRNAs mutant

[0092] (1) OsmiR397a and OsmiR397b double-site knockout vector construction method

[0093] The OsmiR397 family has two members, OsmiR397a (GenBank number: AP014962:28489785...28489898) and OsmiR97b (GenBank number: XM_015769221). In order to obtain the OsmiR397 knockout mutant, a two-site knockout mutation vector was constructed in which both OsmiR397a and OsmiR97b were knocked out simultaneously. According to the precursor sequences of OsmiR397a and OsmiR97b, design and synthesize OsmiR397a-sgRNA1, gRNA scaffold, U6 terminator, U6 promoter, OsmiR397b-sgRNA1 sequence OsmiR397a-gRNA1-OsmiR397b-gRNA1-F and OsmiR397a-gRNA1-OsmiR397a-gRNA1-Osmi R (synthesized by Shanghai Yingjun Biotechnology Co., Ltd.). OsmiR397a-gRNA1-OsmiR397b-gRNA1-F:

[0094] gtgtGAGTGCAGCGTTGATGAACAAGTTTTAGAGCTA...

Embodiment 3

[0105] Example 3 Using the method of the present invention to create a large-fragment deletion mutant of miRNA (taking rice OsmiR408 as an example)

[0106] 1. Vector construction of CRISPR-Cas9-miRNAs mutant

[0107] (1) OsmiR408-sgRNA1 and OsmiR408-sgRNA2 double-site knockout vector construction method

[0108] When considering knocking out all or most of the precursor miRNA or it is difficult to find a suitable PAM site near certain mature miRNA precursors, it is necessary to consider the strategy of using double-site knockout to create large fragment deletions.

[0109] According to the precursor sequence of OsmiR408, design and synthesize the sequence OsmiR408-gRNA1-gRNA2-F and OsmiR408-gRNA1-gRNA2-R containing sgRNA1, gRNA scaffold, U6 terminator, U6 promoter, and sgRNA2 (by Shanghai Yingjun Biotechnology) Co., Ltd. synthesis).

[0110] OsmiR408-gRNA1-gRNA2-F:

[0111] gtgtGATGAGGCAGAGCATGGGATGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGA...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for directional knockout of rice miRNA. The technical problem to be solved by the present invention is to provide methods and detection means for directional modification of plant miRNA sites. The invention discloses a method for obtaining rice micoRNA mutants, and the specific operation is as follows: using the CRISPR-Cas9 method to knock out a single miRNA, knock out multiple miRNAs or knock out large fragments of the miRNA. The method of the invention is suitable for creating and screening rice miRNA-directed knockout mutants.

Description

Technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a method for directed knockout of rice miRNA. Background technique [0002] miRNA (microRNA) is a type of non-coding small RNA that is ubiquitous in eukaryotes and is about 19-24 nucleotides in length. It is encoded by endogenous miRNA gene and is produced by RNA polymerase II transcription. It mainly regulates the expression level of its target gene mRNA by shearing degradation, inhibiting translation, and chromosome remodeling (methylation). miRNA plays an important role in plant growth and development, metabolism, epigenetics, and response to abiotic stress. [0003] The traditional methods of studying miRNA functions mainly include: (1) The indirect method of inhibiting the function of target genes by expressing miRNA by transgene is used to study the function of miRNA. However, because miRNAs often regulate multiple target genes with the same or diffe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/46
CPCC12N15/113C12N15/8216C12N2310/141C12N2800/80C12N2810/10
Inventor 周建平张勇郑雪莲唐旭程燕邓科君
Owner 成都极谷基因科技有限公司
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