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A base editing tool and its application

A base editing and tool technology, applied in the field of gene editing, can solve the problems of insufficient editing efficiency, small editing window, and low recognition efficiency, and achieve increased window and accuracy, high base editing accuracy, and targeted highly specific effect

Active Publication Date: 2022-06-24
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing base editing tools generally cannot edit more sites of interest due to the small editing window and the limitation of PAM sequences
Although the coverage can be improved by modifying SpCas9 to recognize different PAMs, compared with the specific recognition of the PAM sequence NGG by wild-type SpCas9, the recognition efficiency of the modified version of SpCas9-BE3 to other PAMs is still low, resulting in the loss of Editing efficiency is not efficient enough

Method used

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  • A base editing tool and its application
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  • A base editing tool and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] 1. Construction of BE-PLUS System Plasmid

[0057] 10 copies of the GCN4 sequence (10XGCN4) were synthesized by Jinweizhi Biotechnology Co., Ltd. and cloned into the pUC57 vector to obtain the pUC57-10XGCN4 vector, which was diluted to 10 μM as a PCR template. Design forward primer with NheI restriction site GCTGGCTAGCACCATGGGACCCAAGAAAAAACGCAAGGTGGAAG, reverse primer with XbaI restriction site GTCTCTAGACACCTTGCGCTTCTTCTTGGGTCC, add water to dissolve to 10 μM. The 10XGCN4 sequence fragment was amplified using the Novozyme High-Fidelity Enzyme Kit (Vazyme, p501-d2). The amplification system and PCR reaction conditions are as follows:

[0058]

[0059]

[0060] The PCR amplification product was purified and recovered by AxyPrep PCR Clean-up Kit (Axygen, AP-PCR-500G), and then 1 μg was taken, digested with NheI (NEB, R0131S) and XbaI (NEB, R0145S), and incubated at 37°C for 2h . Another 1 μg of pST1374-N-NLS-flag-linker-Cas9-D10A (Addgene#51130) vector was taken, ...

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Abstract

The invention provides a base editing tool and its application. The base editing tool is characterized in that it includes: GCN4-D10A formed by connecting multiple copies of GCN4 and D10A-Cas9; cytosine deaminase APOBEC and uracil glycosylase inhibitor UGI are connected to the single-chain antibody ScFv-APOBEC-UGI vector formed by scFv or cytosine deaminase APOBEC and uracil glycosylase inhibitor UGI and the thermostability domain GB1 to prevent multimerization linked to scFv-APOBEC-formed single-chain antibody scFv UGI‑GB1 vector; and sgRNA targeting specific sites. Compared with commonly used base editing tools, the present invention significantly increases the window of base editing, thereby expanding the application range of base editing in genomes.

Description

technical field [0001] The invention relates to a novel base editing tool, which belongs to the field of gene editing, and more specifically relates to a tool for specific base replacement based on the combination of a CRISPR system and a base deaminase. Background technique [0002] Gene editing is a technical means to achieve gene knockout or insertion of foreign DNA fragments by introducing sequence changes at specific sites on DNA. At present, there are mainly three kinds of programmable nucleases, the early zinc finger ribonuclease (ZFN) system, the transcription activator-like effector nuclease (TALEN) system and the now commonly used CRISPR-Cas9 system. 1 . The CRISPR-Cas9 gene editing system is easy to operate, and only needs to change the target sequence of sgRNA to perform gene editing at the new target site, so it is rapidly and widely used in gene function research, disease modeling at the cell or animal level, and Gene therapy 2-7 . [0003] In the process o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85
CPCC12N15/85C12N2810/10
Inventor 冯松杰江雯黄行许
Owner SHANGHAI TECH UNIV