A short small small nuclear RNA promoter and its construction method and application in genome editing

A genome editing and promoter technology, applied in DNA/RNA fragments, applications, genetic engineering, etc., can solve the problems of unfavorable sgRNA expression cassette limitations, long promoter length, scattered MSP positions, etc. short, efficiency-enhancing effects

Active Publication Date: 2020-12-11
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved in the present invention is that the MSP positions of the existing U3 and U6 promoters are scattered, resulting in the longer length of such promoters used, which is not conducive to the limitation of cloning multiple sgRNA expression cassettes in plant genome editing , designed to create a short and small modified U3 and U6 promoters and apply it to multigene target editing in plants

Method used

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  • A short small small nuclear RNA promoter and its construction method and application in genome editing
  • A short small small nuclear RNA promoter and its construction method and application in genome editing
  • A short small small nuclear RNA promoter and its construction method and application in genome editing

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1 Transformation of small nuclear RNA gene short small promoter

[0037] Using the plasmids pYLgRNA-OsU6a, pYLgRNA-OsU6b, pYLgRNA-OsU6c and pYLgRNA-OsU3 (Ma et al., 2015, Molecular Plant, 8:1274-1284) containing the wild-type promoter of rice small nuclear RNA gene as templates, respectively The primer pairs (Table 1) were amplified by reverse PCR, and the PCR products were purified, and the plasmids pYLgRNA-mOsU6a, pYLgRNA-mOsU6b, and pYLgRNA-mOsU6c containing short and small promoters mOsU6a, mOsU6b, mOsU6c, mOsU3, and sgRNA were respectively obtained by Gibson reaction and pYLgRNA-mOsU3, and finally sequenced to determine the short small promoter sequence ( figure 1 and figure 2 ).

[0038] Table 1 is used for small nuclear RNA gene promoter modification

[0039]

[0040] The specific procedure is as follows:

[0041] 1. Transformation of the short small promoter mOsU6a

[0042] Use mU6a F / mU6aR (SEQ ID NO.5 and SEQ ID NO 6) as primers, and pYLgRN...

Embodiment 2

[0052] Example 2, short small promoter-driven sgRNA has high editing efficiency

[0053] Refer to the literature previously published by the inventor team (Ma et al., 2015, Molecular Plant, 8: 1274-1284; Ma and Liu, 2016, Current Protocols in Molecular Biology, 115: 31.6.1-31.6.21; Zeng Dongchang et al. , 2018, Chinese Science: Life Sciences, 48:783-794), respectively constructed short small promoters (mOsU6a, mOsU6b, mOsU6c and mOsU3) and wild-type small nuclear RNA gene promoters (OsU6a, OsU6b, OsU6c and OsU3) drives the sgRNA expression cassettes of different targets, inserts the pYLCRISPR / Cas9Pubi-H binary vector (Ma et al., 2015, Molecular Plant, 8:1274-1284) in the "Golden Gate assembly, Golden Gate" method, and transforms rice And sequence the target of the transformant to analyze the editing efficiency of the sgRNA driven by the short small promoter.

[0054] 1. Primer design for different targets

[0055] Use the online program CRISPR-GE webpage (http: / / skl.scau.edu...

Embodiment 3

[0064] Example 3, short promoters improve the efficiency of multiple sgRNA expression cassettes for ligated cloning

[0065] The "Golden Gate Assembly" method can assemble multiple DNA fragments. The number of connected fragments and the connection efficiency are related to the size of the connected fragments. When the connected fragments are smaller, the number of connected DNA fragments is more and the connection efficiency is higher. In order to verify the use of short promoters to improve the efficiency of connecting and cloning multiple sgRNA expression cassettes, according to the literature published by the inventor's team (Ma and Liu, 2016, Current Protocols in Molecular Biology, 115:31.6.1-31.6.21; Zeng Dongchang et al. , 2018, Chinese Science: Life Science, 48:783-794), using "Golden Gate Assembly" to simultaneously clone 12 or 20 different promoter-driven sgRNA expression cassettes in the pYLCRISPR / Cas9Pubi-H vector ( Figure 4 A), the proportion of clones successful...

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Abstract

The invention provides a short small-nuclear RNA promoter as well as a construction method and application thereof. The length of the short small-nuclear RNA promoter is 150-300bp, and the short small-nuclear RNA promoter sequentially contains one TATA box conservative element, one USE conservative element and at least one MSP conservative element in the direction from an end 3' to an end 5'. Compared with an existing wild type small-nuclear RNA gene promoter, the short small-nuclear RNA promoter constructed in the invention has the advantages that the promoter has stronger transcriptional activity and obviously higher editing efficiency of driving the sgRNA and is shorter, the length of each sgRNA expression cassette can be reduced, the efficiency of constructing a vector by multi-targetsgRNA cloning is improved, the simultaneous editing of multiple targets and multiple genes is facilitated, and the promoter has a good application prospect in the aspect of genome editing.

Description

technical field [0001] The invention belongs to the field of plant biotechnology. More specifically, it relates to a short small nuclear RNA promoter, its construction method and its application in genome editing. Background technique [0002] In recent years, the genome editing technology represented by CRISPR / Cas9 and its various derivative gene editing systems (such as single base editors) have developed rapidly, and have been widely used in animals, plants and other organisms. The CRISPR / Cas9 system needs to express Cas9 protein and single guide RNA (single guider RNA, sgRNA, whose 5' contains a specific target sequence of about 19-21 bases), which combine to form a Cas9 / sgRNA complex to target the target gene. Cas9 cuts the target DNA to generate double-strand breaks, and induces base mutations during DNA repair to achieve gene-specific mutations. If multiple sgRNAs with different targets are simultaneously expressed or introduced into the recipient biological cells, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/46
CPCC12N15/113C12N15/8213C12N2310/10
Inventor 刘耀光郭晶心祝钦泷郝雨宗伍辈
Owner SOUTH CHINA AGRI UNIV
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