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Base editing tools and their applications and methods for wide-window and sequence-bias-free base editing in eukaryotic cells

A technology of base editing and tools, applied in the field of base editing, which can solve problems such as limited applications

Active Publication Date: 2021-09-17
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This will greatly limit its application in regulatory regions

Method used

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  • Base editing tools and their applications and methods for wide-window and sequence-bias-free base editing in eukaryotic cells
  • Base editing tools and their applications and methods for wide-window and sequence-bias-free base editing in eukaryotic cells
  • Base editing tools and their applications and methods for wide-window and sequence-bias-free base editing in eukaryotic cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0040] According to the present invention, the sequence of the linker may be any amino acid sequence capable of connecting the amino acid sequences at both ends, and the present invention has no particular limitation on this. According to a preferred embodiment of the present invention, the linker The amino acid sequence of is shown in SEQ ID NO:1.

[0041]According to the present invention, the cytosine deaminase AID can be any amino acid sequence having cytosine deaminase AID activity known in the art, and according to a preferred embodiment of the present invention, the cytosine deaminase is The amino acid sequence of the full-length AIDfl is shown in SEQ ID NO:4.

[0042] However, the inventors of the present invention found in research that by performing point mutation and C-terminal truncation on the amino acid sequence shown in SEQ ID NO: 4, the base editing efficiency of the final base editing tool can be effectively improved, Wherein, the point mutation is selected f...

Embodiment 1

[0066] This example is used to illustrate the construction of a plasmid encoding scFv-AID-UGI-GB1 protein

[0067] 1. Amplification of nucleic acid sequence fragment (AID) encoding AID

[0068] The coding sequence hAID*Δ of the optimized version of cytosine deaminase AID hAID*Δ synthesized by Jinweizhi Biotechnology Co., Ltd. (the encoded amino acid sequence is shown in SEQ ID NO: 5, and the DNA sequence is shown in SEQ ID NO: 139) , cloned in the pUC57 vector to obtain the pUC57-hAID*Δ vector, diluted to 10 μM as a PCR template. Design forward primer (as shown in SEQ ID NO: 10), reverse primer (as shown in SEQ ID NO: 11), add water and dissolve to 10 μM. The hAID sequence fragment was amplified using Novizan High Fidelity Enzyme Kit (Vazyme, p501-d2). The amplification system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

[0069] Table 1

[0070]

[0071] Table 2

[0072]

[0073] 2. Amplification of the nucleic acid sequence fragment (...

Embodiment 2

[0087] This example is used to illustrate the construction of the plasmid encoding scFv-AIDfl-UGI-GB1 protein

[0088] The construction of the plasmid encoding the scFv-AIDfl-UGI-GB1 protein was carried out according to Example 1, except that the human codon-optimized hAID*Δ was replaced with the nucleotide sequence of the unoptimized full-length AIDfl, that is, the encoding For the nucleotide sequence (SEQ ID NO: 138) of the protein shown in SEQ ID NO: 4, the amplification forward primer and reverse primer are shown in SEQ ID NO: 143 and SEQ ID NO: 144, respectively, to obtain BE-PA2.

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Abstract

The invention relates to the field of base editing, and discloses a base editing tool and its application as well as a method for performing wide-window and sequence-free base editing in eukaryotic cells. The base editing tool includes: (1) GCN4-D10A protein formed by linking multiple copies of GCN4 and D10A-Cas9; (2) cytosine deaminase AID and uracil glycosylase inhibitor UGI linked to a single-chain antibody scFv-AID-UGI protein formed by scFv, or scFv-AID formed by cytosine deaminase AID, uracil glycosylase inhibitor UGI and thermostability domain GB1 to prevent multimerization linked to single-chain antibody scFv ‑UGI‑GB1 protein. The base editing tool provided by the present invention has the advantages of good sequence compatibility, wide editing window, high efficiency and high fidelity, and is more suitable for editing and functional research of regulatory elements in non-coding regions.

Description

technical field [0001] The present invention relates to the field of base editing, in particular to a base editing tool and its application and a method for performing wide-window and sequence-free base editing in eukaryotic cells. Background technique [0002] 98% of the DNA sequences in the human genome are non-coding sequences, 80% of which have regulatory functions. Changes in DNA sequences in non-coding regions are closely related to complex diseases (such as arthritis, ataxia, etc.), individual responses to drugs, and differences in susceptibility to diseases. It is currently believed that there are a large number of regulatory elements in non-coding DNA that regulate the activity of genes. These elements play an extremely important role in diseases such as cancer, heart disease, and autism, and may become potential targets for disease treatment . The study of regulatory sequences in non-coding regions is considered to be one of the next frontiers in genome research....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/22C12N15/113C12N15/90C12N15/11C07K19/00
CPCC07K16/00C07K2317/622C07K2319/00C12N9/22C12N9/78C12N15/113C12N15/902C12N2310/10C12Y305/04001C12N2310/20
Inventor 江雯冯松杰陈洁平黄行许
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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