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Cytosine base editing system and application thereof

A base editing and cytosine technology, applied in the field of cytosine base editing system, can solve the problems of limited operational range, complicated operation, low efficiency, etc., and achieve the effect of fast and efficient editing and great application potential

Pending Publication Date: 2022-08-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the above-mentioned technical problems, the present invention provides a CRISPR / Cpf1-based cytosine base editing system, which overcomes the limited operable range of the CRISPR / Cas9-based cytosine base editor on the genome, and in multiple The problem of low efficiency and complicated operation in the process of base editing (C→T) at a site

Method used

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  • Cytosine base editing system and application thereof
  • Cytosine base editing system and application thereof
  • Cytosine base editing system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Design and construction of a cytosine base editing system based on CRISPR / Cpf1

[0033] like figure 1 As shown in A, a CRISPR / Cpf1-based cytosine editing system was constructed, which consists of the following two basic elements:

[0034] (1) A fusion protein composed of cytosine deaminase, a DNase-inactive mutant of Cpf1 (D917A) dCpf1 and a uracil glycosidase inhibitor (UGI), namely the cytosine base editor dCpf1-CBE, this fusion Proteins can bind to specific targets in the genome with the help of dCpf1, and convert cytosine (C) to thymine (T) under the action of cytosine deaminase;

[0035] Specifically, the cytosine deaminase hAPOBEC3A (GenBank: KM266646.1), hAID (GenBank: AAM95402.1) and LjCDA1L2_1 (GenBank: MG495262.1) were respectively fused to the N-terminus of dCpf1 through the XTEN short peptide linker (SGSETPGTSESATPES). , and UGI (GenBank: YP_009283008.1) was fused to the C-terminus of dCpf1 through a short peptide linker (GSPKKKRKVSGGS), and the ...

Embodiment 2

[0037] Example 2 Application of CRISPR / Cpf1-based cytosine base editing system in multi-site base editing

[0038] Validation and application of a CRISPR / Cpf1-based cytosine base editing system in Bacillus subtilis. like figure 2 shown, using IPTG-induced P grac100 The promoter expresses the cytosine base editor dCpf1-CBE in different configurations and places the crRNA array insert into the constitutive promoter P veg Afterwards, in order to realize the expression of the crRNA array, the above two expression cassettes were placed on the plasmid containing the thermosensitive replicon pE194 for base editing (C→T) in Bacillus subtilis.

[0039] Specifically, the following steps are included: the plasmid backbone used is from pJOE8999 (Altenbuchner, J., 2016. Editing of the Bacillus subtilis genome by the CRISPR-Cas9 system. Applied and Environmental Microbiology 82, 5421–5427), the plasmid was produced in E. coli and Bacillus subtilis is kanamycin resistant (KanR), and has ...

Embodiment 3

[0051]Example 3 Influence of induction time on multi-site base editing efficiency

[0052] Since the cytosine base editor containing the cytosine deaminase hAID is the most efficient, simultaneous editing of five sites can be achieved. Therefore, the effect of the induction time of IPTG on the base editing efficiency of the base editor was also investigated. As shown in Table 4, with the increase of induction time, the editing efficiency of each site increased to a certain extent, but the amplitude was limited, and the editing window did not change with the prolongation of induction time. The above results indicated that induction for 12 h was sufficient to achieve relatively complete editing. Among them, the editing and sequencing results after 36h induction are as follows image 3 shown.

[0053] Table 4 The effect of induction time on the efficiency of multi-site base editing

[0054]

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Abstract

The invention relates to a cytosine base editing system and application thereof. The cytosine base editing system comprises a fusion protein with one of amino acid sequences as shown in SEQ ID NO.1-3, and a bacillus subtilis cytosine base editing system is designed and constructed based on the fusion protein. According to the constructed cytosine base editing system, the problems that the operable range of an existing cytosine base editor on a genome is limited, and the efficiency is low and the operation is complex in the multi-site base editing (C-T) process are solved, and a plurality of sites on the genome can be rapidly and efficiently edited.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cytosine base editing system and its application. Background technique [0002] Gene editing is a technical means to achieve gene knockout, insertion of exogenous DNA fragments or DNA base mutation by introducing sequence changes at specific sites on DNA. In recent years, the CRISPR / Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) system has been widely used in gene editing, especially the CRISPR / Cas9 system. In this system, immature precursor CRISPR RNA (pre-crRNA) can combine with tracrRNA (trans-activating crRNA) and form crRNA:tracrRNA complex under the action of RNase III, which guides Cas9 protein to recognize and cut Double-strand breaks are created at specific sites in the genome; then, homologous recombination repair (HDR) allows sequence changes on the homologous template to be introduced into the genome at the target site of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/113C12N15/62
CPCC12N15/75C12N15/113C12N9/78C12N9/22C07K14/005C12Y305/04001C12N2830/002C12N2310/20C07K2319/00C12N2795/10122
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰武耀康
Owner JIANGNAN UNIV