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Production application of mono-subunit RNA polymerase KP34RP in long-chain mRNA synthesis

An RNA polymerase and subunit technology, which is used in the production and application of single-subunit RNA polymerase KP34RP in the synthesis of long-chain mRNA, and can solve problems such as off-target effects

Active Publication Date: 2018-12-21
RNASYN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, transfection of mRNA is safer than plasmid DNA, and there is no risk that plasmid DNA may be integrated into the genome; secondly, direct transfection of mRNA does not need to consider promoters and terminators that affect gene transcription, and the long-term existence of plasmids in cells , the continuous expression of the target protein will cause some risks of unknown effects. For example, if the cas9 protein is continuously expressed in the cell, it will inevitably increase the risk of off-target effects

Method used

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  • Production application of mono-subunit RNA polymerase KP34RP in long-chain mRNA synthesis
  • Production application of mono-subunit RNA polymerase KP34RP in long-chain mRNA synthesis
  • Production application of mono-subunit RNA polymerase KP34RP in long-chain mRNA synthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction and linearization and purification of three template plasmids

[0040] (1) Construction of cap-cas9 template plasmid

[0041] On the basis of the number: 72247 cas9 plasmid purchased by addgene, the KP34RP promoter was designed into the PCR primers by seamless cloning and directly inserted into the 5' end of the Kozak sequence (GCCGCCACC) immediately before the cas9 gene. An additional nuclear localization signal sequence NLS derived from the nucleoplasmic protein of Xenopus laevis (Xenopus laevis) was inserted between the promoter sequence and the cas9 gene: see SEQ ID NO. In order to improve the genome editing efficiency of the cas9 protein; in addition, we inserted a continuous poly(A) sequence of 67 A after the 3xFlag tag at the 3' end of the cas9 gene followed by a reverse BapQ I for plasmid linearization Restriction site, the sequence of the 67 A and reverse BapQ I restriction site is shown in the sequence table SEQ ID NO.3, which maximize...

Embodiment 2

[0053] Example 2: Using KP34RP to transcribe and synthesize three long-chain mRNAs in vitro and purify

[0054] On the basis of the linearized template plasmid constructed in Example 1, a large amount of mRNA was synthesized by in vitro transcription with KP34RP. The schematic diagrams of the sequence structures of the three mRNAs are shown in Figure 4 , Figure 4 -A represents the schematic structure of the cap-cas9 mRNA sequence, and its corresponding nucleic acid sequence is shown in the sequence listing: SEQ ID NO.4; Figure 4 -B represents the schematic structure of the IRES-cas9 mRNA sequence, and its corresponding nucleic acid sequence is shown in the sequence listing: SEQ ID NO.5; Figure 4 -C represents the schematic structure of the IRES-sox7 mRNA sequence, and its corresponding nucleic acid sequence is shown in the sequence listing: SEQ ID NO.6.

[0055] Since the product of in vitro transcription is RNA, it is easily degraded by RNase in the environment, so in t...

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Abstract

The invention discloses a production application of mono-subunit RNA polymerase KP34RP in long-chain mRNA synthesis. The mono-subunit RNA polymerase is bacteriophago-like particle mono-subunit RNA polymerase (a formula is as shown in the description). The KP34RP is used for preparing long-chain cap-cas9mRNA which is as show in a sequence list SEQ ID NO.4, IRES-cas9mRNA which is as show in a sequence list SEQ ID NO.5, and IRES-sox7mRNA which is as show in a sequence list SEQ ID NO.6, wherein cas9 is extensively applied to gene editing, and sox7 plays an important role in embryonic development.While three types of mRNA transcription template plasmids are constructed, at the gene sequence 5'-terminal and 3'-terminal, sequence elements of a nucleoplasmin positioning signal sequence NLS derived from xenopus laevis which is as shown in a sequence list SEQ ID NO.1, an internal ribosome entry site sequence IRES from hepatitis C virus which is as shown in a sequence list SEQ ID NO.2 and poly(A)+BspQI which is as shown in a sequence list SEQ ID NO.3 are added. It is guaranteed that the in-vitro synthesized mRNA can be efficiently translated to a function protein with activity in a cell. Theproduction application is uniform in product, high in purity, reaches a milligram level in average, and is capable of completely meeting market requirements to mRNA research and application.

Description

technical field [0001] The invention belongs to the field of long-chain RNA synthesis research and production, and specifically relates to the production and application of single-subunit RNA polymerase KP34RP in long-chain mRNA synthesis, using KP34 bacteriophage single-subunit RNA polymerase to synthesize cap-cas9 mRNA that can be used for cell gene editing , IRES-cas9 mRNA, the production technology of transcription factor IRES-sox7 mRNA that affects embryonic development. Background technique [0002] In the past few decades, with the rapid development of RNA technologies such as siRNA, miRNA, mRNA, long non-coding RNA, RNA aptamers, and ribozymes, RNA has received more and more attention in biology (Burnett J C., 2012 ). Some of the previous experimental methods have gradually been unable to meet the needs of scientific research and clinical experiments on RNA, and more and more RNA biological research needs to use tens of milligrams or even hundreds of milligrams of h...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12P19/34
CPCC12N9/1247C12P19/34C12Y207/07006
Inventor 朱斌陆雪玲夏恒吴慧余兵兵
Owner RNASYN BIOTECH CO LTD
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