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Chimeric protein pAgoE, construction method and applications thereof, chimeric protein pAgoE using guide, and construction method and applications thereof

A chimeric protein and construction method technology, applied in chemical instruments and methods, biochemical equipment and methods, hybrid peptides, etc., can solve problems such as unclear methods and reproducibility crises

Active Publication Date: 2017-04-26
仪宏
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On May 2, 2016, Feng Gao et al. reported NgAgo from the halophilic microorganism Natronobacterium gregoryi. The paper claimed that the NgAgo guided by the 24nt 5' phosphorylated single-stranded DNA can produce mammalian cell target at room temperature. Double-strand breaks (DSBs) to genomic loci trigger efficient genome editing, yet the paper suffers from a serious reproducibility crisis
[0013] Existing research results show that Argonaute, which retains the PAZ domain and maintains PIWI nuclease activity in the original microbial host, plays the role of defending against foreign invading DNA such as viruses in the way of DNAi; while retaining the PAZ domain but losing PIWI in the original microbial host The Argonaute with nuclease activity is likely to participate in the defense against foreign invading DNA in a way and mechanism that is not yet clear, and even monitors overactive genetic elements by continuously sampling the entire DNA of the host

Method used

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  • Chimeric protein pAgoE, construction method and applications thereof, chimeric protein pAgoE using guide, and construction method and applications thereof
  • Chimeric protein pAgoE, construction method and applications thereof, chimeric protein pAgoE using guide, and construction method and applications thereof
  • Chimeric protein pAgoE, construction method and applications thereof, chimeric protein pAgoE using guide, and construction method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0173] Example 1: Construction and genome editing of the chimeric protein Fok-RsAgo.

[0174] From the 319 candidate pAgos in Table 3, RsAgo was selected as the pAgo domain; FokI in the endonuclease was selected as the E domain to construct the N-pAgo type pAgoE (hereinafter referred to as Fok-RsAgo), and the Fok-RsAgo was used to carry the corresponding Combine the plasmids of gNA and HR fragments, transform yeast and subculture, and detect gene editing.

[0175] 1. Construction and expression of Fok-RsAgo expression plasmid:

[0176]According to the amino acid sequence of the RsAgo protein (NCBI accession number: ABP72561.1) of Rhodobacter sphaeroides ATCC 17025, such as SEQ ID NO: 1, according to the base preference of yeast, the rsago gene sequence encoding the RsAgo protein was designed, and the overlapping extension PCR was used technology, artificially synthesized and T-cloned the gene sequence, and obtained rsago‐pEASY. According to the amino acid sequence of the end...

Embodiment 2

[0185] Example 2: Construction and genome editing of the chimeric protein Fok-MpAgo.

[0186] From the 319 candidate pAgos in Table 3, MpAgo was selected as the pAgo domain; FokI in the endonuclease was selected as the E domain to construct the N‐pAgo type pAgoE (hereinafter referred to as Fok‐MpAgo), and the Fok‐MpAgo was used to carry the corresponding The plasmids of gNA and HR fragments are combined to transform yeast and edit its genome.

[0187] 1. Construction and expression of Fok-MpAgo expression plasmid:

[0188] According to the amino acid sequence of the MpAgo protein of Marinitoga piezophila (NCBI accession number: WP_014295921.1), such as SEQ ID NO: 2, according to the base preference of yeast, the mpago gene sequence encoding the MpAgo protein was designed, and the overlapping extension PCR technique was used to The gene sequence was artificially synthesized and T-cloned to obtain mpago-pEASY. Using the foki-pEASY obtained in step 1 of Example 1, select GGGGS ...

Embodiment 3

[0195] Example 3: Construction and genome editing of the chimeric protein Fok-dMpAgo.

[0196] From the 319 candidate pAgos in Table 3, the PIWI inactivated mutant dMpAgo of MpAgo was selected as the pAgo domain, and the FokI in the endonuclease was selected as the E domain to construct the N‐pAgo type pAgoE (hereinafter referred to as Fok‐dMpAgo). Fok‐dMpAgo cooperates with plasmids carrying corresponding gNA and HR fragments to transform yeast and edit its genome.

[0197] 1. Construction and expression of Fok‐dMpAgo expression plasmid:

[0198] According to the amino acid sequence of the MpAgo protein of Marinitoga piezophila (NCBI accession number: WP_014295921.1), such as SEQ ID NO: 2, according to the base preference of yeast, the mpago gene sequence encoding the MpAgo protein was designed, and the overlapping extension PCR technique was used to The gene sequence was artificially synthesized and T-cloned to obtain mpago-pEASY. Using the obtained mpago-pEASY plasmid as ...

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Abstract

The invention relates to biological technology, specifically relates to a chimeric protein pAgoE, a construction method and applications thereof, and at the same time, relates to a chimeric protein pAgoE using a guide, a construction method and applications thereof, and a chimeric protein, which is based on the chimeric protein pAgoE and has a genome targeting editing function. The chimeric protein pAgoE contains a pAgo structural domain with a genome targeting positioning function and an effect structural domain E, which is connected to the pAgo structural domain and has a genome cutting or modifying activity. The pAgo structural domain with a genome targeting positioning function and the effect structural domain E with a genome cutting or modifying activity are fused to establish the chimeric protein pAgoE; the chimeric protein pAgoE maintains the original targeting binding performance of the genome (DNA) having a targeting positioning function in the pAgo structural domain and the genome catalytic or modifying performance of the effect structural domain E at the same time, and an sufficient space is provided for the structural domain E to fully exert the effect.

Description

technical field [0001] The present invention relates to biotechnology, specifically, the present invention relates to a kind of chimeric protein pAgoE and its construction method and application, meanwhile, the present invention also relates to a kind of chimeric protein pAgoE using guide and its construction method and application, and based on Chimeric protein pAgoE is a chimeric protein with genome-targeted editing function. [0002] technical background [0003] Targeted genome editing technology and targeted genome modification technology are a general term for a type of high-efficiency genome rewriting technology. Its main advantages over gene recombination technology in the general sense are: high genome targeting accuracy, high gene editing efficiency, and a wide range of applications , Mammalian cells can be manipulated. Targeted genome editing technology and targeted genome modification technology can carry out functional research and genetic modification of the ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00
CPCC07K14/195C07K2319/33C12N9/0071C12N9/1007C12N9/16C12N9/2497C12N9/78C12Y114/11C12Y201/01037C12Y301/21004C12Y302/02021C12Y305/04005
Inventor 仪宏冯惠勇李天明刘金雷
Owner 仪宏
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