Fusion protein and base editing tool and method and application thereof

A fusion protein and base technology, applied in the field of gene editing, can solve problems such as limiting the scope of genome targeting, and achieve the effect of broadening the scope of targeting

Active Publication Date: 2019-11-19
GUANGZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Cas9 (SpCas9) from Streptococcus pyogenes only recognizes the PAM of the NGG sequence, greatly limiting the extent of the genome that can be targeted

Method used

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  • Fusion protein and base editing tool and method and application thereof
  • Fusion protein and base editing tool and method and application thereof
  • Fusion protein and base editing tool and method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] First construct ancBE4max-NG and ABEmax-NG plasmids, and introduce 7 amino acid mutations (R1335V / L1111R / D1135V / G1218R / E1219F / A1322R / T1337R) into ancBE4max and ABEmax plasmids through Mut Express II FastMutagenesis Kit V2 (Vazyme, C214-02) , ancBE4max was synthesized from the whole gene by a commercial company, and the ABEmax plasmid was purchased from Addgene (#112095). The DNA sequence contained in the generated pCMV-ancBE4max-NG is shown in SEQ ID No.17; the DNA sequence contained in pCMV-ABEmax-NG is shown in SEQ ID No.18.

Embodiment 2

[0111] On the basis of embodiment 1 gained ancBE4max-NG and ABEmax-NG, construct as figure 1 and 2 Indicated pAAV-TRE-ancBE4max-NG 2-573-intein-N, pAAV-TRE-intein C-ancBE4max-NG 574-1368, pAAV-TRE-ABEmax-NG 2-573-intein-N, pAAV-TRE -intein C-ABEmax-NG 574-1368.

[0112] 2.1 pAAV-TRE-ancBE4max-NG 2-573-intein-N, pAAV-TRE-intein C-ancBE4max-NG 574-1368, pAAV-TRE-ABEmax-NG 2-573-intein-N, pAAV-TRE-intein Construction of C-ABEmax-NG574-1368 plasmid

[0113] The PCR primers whose sequences are shown in Table 1 were synthesized by Jinweizhi Biotechnology Co., Ltd., diluted to 10 μM as PCR primers, and the original pAAV-TRE was used as a template.

[0114] Table 1

[0115]

[0116]

[0117] The Novizym high-fidelity enzyme kit (Vazyme, p501-d2) was used to amplify the vector sequence fragment and the N-terminal or C-terminal fragment of ABEmax or ancBEmax, respectively. Amplification system (see Table 2) and PCR reaction conditions are as shown:

[0118] Table 2

[0119]...

Embodiment 3

[0128] Using the N-ancBE4max-NG+C-anc-BE4max-NG and N-ABEmax-NG+C-ABEmax-NG systems constructed in the above examples to transfect HEK293T cells, the process is as follows:

[0129] 3.1 HEK293T cells (from ATCC) were revived and cultured in a 10 cm culture dish (Corning, 430167). The medium was DMEM (HyClone, SH30243.01) mixed with 10% fetal bovine serum (HyClone, SV30087). The culture temperature was 37°C, and the carbon dioxide concentration was 5%. After multiple passages, when the cell density was 80%, the cells were divided into 12-well plates. The 12-well plate was coated with a 1:10 diluted polylysine solution (Sigma, P4707-50ML) before use.

[0130] 3.2 When the cell concentration is 80%, replace the medium with DMEM medium with 10% serum, and culture for 2 hours to restore the best cell state. The amount of plasmid transfected in each hole is N-ancBE4max-NG 0.5ug, C-anc-BE4max-NG 0.5ug, sgRNA0.5ug or N-ABEmax-NG 0.5ug, C-ABEmax-NG 0.5ug, sgRNA0. 5ug was co-transfec...

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Abstract

The invention discloses a fusion protein which comprises a segment N/C of intein and an N/C-terminal segment of a base editor. The base editor is polypeptides ancBE4max-NG or ABEmax-NG. The polypeptides ancBE4max-NG include the polypeptides APOBEC1 and the polypeptides SpCas9-NG D10A nickase. The polypeptides ABEmax-NG include the dimer polypeptides ecTad-ecTadA* and the polypeptides SpCas9-NG D10A nickase. The invention further discloses a gene editing tool or cell expression system with the fusion protein and application of the gene editing tool or cell expression system. The fusion proteinis a novel combined cytosine/adenine base editing tool, can recognize NG as the PAM and widens the base editing target range; moreover, the base editing tool can meet the packaging requirement of adenovirus, and the high-titer adenovirus can be obtained.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to an adenovirus-based base editing tool and method and its application. Background technique [0002] Gene editing is a technical means to achieve gene sequence changes or insertions by introducing sequence changes at specific sites on DNA. CRISPR / Cas9 is currently the most widely used gene editing technology 1 . The system is simple to operate, and only needs to pass the sgRNA targeting sequence to perform gene editing at the target site. This technology is widely used in gene function research, disease simulation, and gene therapy. The principle of CRISPR / Cas9 is that under the guidance of sgRNA, Cas9 reaches the designated DNA region to exert enzyme cleavage activity. The targeted recognition of the CRISPR / Cas9 system requires a protospacer adjacent motif (PAM) next to the target site. Then cut between 3bp and 4bp upstream of the PAM, resulting in a DNA double-strand bre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/861C12N5/10
CPCC07K14/775C07K2319/09C12N9/22C12N15/86C12N2710/10043
Inventor 乔云波
Owner GUANGZHOU UNIVERSITY
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