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Adenine base editing tool and application thereof

A technology of adenine deaminase and purpose, applied in the direction of targeting specific cell fusion, introducing foreign genetic material using vectors, antibody mimics/scaffolds, etc., can solve problems such as limiting the application of base editors

Pending Publication Date: 2019-11-05
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recent studies have found that the ABEmax base editor will produce serious RNA off-target effects (off-Target) 5 , which greatly limits the application of this base editor

Method used

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  • Adenine base editing tool and application thereof
  • Adenine base editing tool and application thereof
  • Adenine base editing tool and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] In this example, the hokB overexpression reporter system was used to verify the editing ability of eABEmax and the RNA off-target effect on HEK293T. The results are as follows figure 2 Shown.

[0062] 1.1 HokB overexpression reporter system plasmid construction

[0063] The hokB overexpression vector was constructed by Mut Express II Fast Mutagenesis Kit V2 (Vazyme, C214-02). The constructed hokB overexpression reporter system plasmid sequence is shown in SEQ ID NO.2.

[0064] 1.2 Construction of sgRNA plasmid

[0065] Design sgRNA and synthesize oligos, the upstream sequence is: 5'-accgGGCCCAGACTGAGCACGTGA-3' (SEQ ID NO. 3), the downstream sequence is: 5'-aaacTCACGTGCTCAGTCTGGGCC-3' (SEQ ID NO. 4), the upstream and downstream sequences are through the program (95 ℃, 5min; 95℃-85℃at-2℃ / s; 85℃-25℃at-0.1℃ / s; hold at 4℃) Annealing, connect to pGL3-U6 linearized by BsaI (NEB: R0539L) -sgRNA (Addgene#51133) vector. The linearization system is as follows: pGL3-U6-sgRNA 2μg; buffe...

Embodiment 2

[0073] In this example, eABEmax was used to edit endogenous gene loci on HEK293T cells.

[0074] 2.1 Construction of sgRNA plasmid

[0075] Eight human endogenous base sites were selected and sgRNA was designed. The polynucleotide sequences of oligos used are shown in SEQ ID NOs. 9 to 26. The positions of the eight sgRNAs used in the genome are NC_000004.12:52670026- 52670045; NC_000022.11: 19401208-19401227; NC_000015.10: 47301029-47301048; NC_000014.9: 88099864-88099883; NC_000001.11: 154311091-154311110; NC_000001.11: 179826686-179826705; NC_000001.11: 184974900-184974919; NC_000001.11: 184974901-184974920. Follow 1.2 to construct sgRNA plasmid.

[0076] 2.2 Cell culture, transfection and identification

[0077] HEK293T cells were cultured and transfected according to 1.3. The amount of transfected plasmid was 0.6 g of eABEmax and 0.3 g of sgRNA expression vector plasmid. ABEmax was used as a control. After 12 hours of transfection, puromycin was added according to the above 1.3...

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Abstract

The invention relates to the field of biotechnology, in particular to an adenine base editing tool and application thereof. The invention provides a fusion protein. The fusion protein includes an ecTadA-ecTadA* dimer fragment and an SpCas9-D10A nickase fragment, and the ecTadA-ecTadA* dimer fragment includes an ecTad fragment and an ecTadA* fragment. According to the provided adenine base editingtool, in the ecTadA-ecTadA* dimer fragment, compared with wild-type existing R153P and N46A amino acid mutations, binding of ecTadA and RNA can be restricted, and during base editing of A at the 4-7 position of sgRNA 5' end mutating to G, the RNA off-target effect of the adenine base editing tool can be greatly lowered or even eliminated.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an adenine base editing tool and its application. Background technique [0002] CRISPR / Cas9 (Clustered regularly interspaced short palindromic repeats / CRISPR-associated protein) is currently the most effective and convenient genome editing technology. Under the guidance of guide RNA (guide RNA, sgRNA), Cas9 nuclease can reach the specific target of the genome and cut it, thereby generating DNA double strand breaks (double strand breaks, DSB), and then through the endogenous DNA repair mechanism to repair Implement editing. DNA repair mechanisms include non-homologous end joining (Non-Homologous End Join, NHEJ) and homologous recombination repair (Homologous Directly Repair, HDR). Among them, the result of NHEJ repair is the random introduction of insertions and deletions, leading to gene inactivation, which plays a major role in genome repair. However, HDR can use templates for ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/78C12N9/22C12N15/62C12N15/85
CPCC07K14/00C12N9/78C12N9/22C12N15/85C07K2319/00C07K2319/33
Inventor 黄行许李佳楠李广磊
Owner SHANGHAI TECH UNIV
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