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Chimeric protein pagoe and its construction method, application, and chimeric protein pagoe using a wizard, its construction method, and application

A chimeric protein and targeting technology, applied in chemical instruments and methods, biochemical equipment and methods, hybrid peptides, etc., can solve problems such as unclear methods and reproducibility crises, and achieve the goal of enriching technology platforms Effect

Active Publication Date: 2021-04-20
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 and its construction method, application, and chimeric protein pagoe using a wizard, its construction method, and application
  • Chimeric protein pagoe and its construction method, application, and chimeric protein pagoe using a wizard, its construction method, and application
  • Chimeric protein pagoe and its construction method, application, and chimeric protein pagoe using a wizard, its construction method, and application

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 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. The chimeric protein pAgoE contains a pAgo structural domain with genome targeting function, and an effector domain E connected with the pAgo structural domain and having genome cutting or modifying activity. The chimeric protein pAgoE of the present invention fuses the pAgo structural domain with genome targeting function and the effector domain with catalytic activity such as genome cutting or modification activity to construct a chimeric protein (pAgoE). pAgoE also maintains the targeting binding ability of the original genome (DNA) with targeting positioning function in the pAgo domain and the genome catalysis or modification ability of the effector domain E, so as to provide the basis for the spatial position guarantee of the effector domain E. .

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 Patents(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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