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Adenine base editor fusion protein without PAM limitation and application

A fusion protein and gene editing technology, applied in the field of biomedicine, can solve the problems of unresolved off-target problems, low editing efficiency, and restrictions on the application of base editors

Active Publication Date: 2021-11-26
国家卫生健康委科学技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ABEmax-SpRY can target all sequences of the genome, but the editing efficiency of ABEmax-SpRY is low, and it does not solve the off-target problem of ABE at the transcriptome level, which limits the application of this base editor and needs to be improved and optimization

Method used

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  • Adenine base editor fusion protein without PAM limitation and application
  • Adenine base editor fusion protein without PAM limitation and application
  • Adenine base editor fusion protein without PAM limitation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1 Construction of base editor plasmid

[0112]First construct 8e-SpRY and corresponding mutants. Refer to the instructions of ClonExpress MultiS One StepCloning Kit (Vazyme, C113-01) to design primers to amplify the TadA8e fragment in ABE8e (Addgene #138489), and replace the TadA dimer in ABEmax-SpRY (Addgene #140003) with TadA8e to construct 8e-SpRY plasmid.

[0113] First delete TadA8e in 8e-SpRY from the original position, and then replace the 1048th to 1063rd amino acids in SpRY D10A with TadA8e to construct the CE-8e-SpRY plasmid, and the sequence from the 5' end to the 3' end is SpRY (D10A) N-terminal, TadA8e and SpRY (D10A) C-terminal, wherein the nucleotide sequence of SpRY (D10A) N-terminal is as shown in SEQ ID NO: 2 (amino acid sequence is shown in SEQ ID NO: 1), the nucleotide of TadA8e The sequence is shown in SEQ ID NO: 4 (the amino acid sequence is shown in SEQ ID NO: 3), and the nucleotide sequence at the C-terminal of SpRY (D10A) is shown in S...

Embodiment 2

[0131] In this example, ABEmax-SpRY, 8e-SpRY and their mutants were used to edit endogenous sites in 293T cells.

[0132] 2.1 Construction of sgRNA plasmid

[0133] Referring to the human genome sequence, 48 sgRNAs were designed according to the PAM characteristics of SpRY nuclease, covering 16 different PAM sequences. The sgRNA sequence is shown in SEQ ID NO: 18-65. ACCG is added to the 5' end of the sgRNA sequence as the upstream sequence. The sgRNA AAAC is added to the 5' end of the reverse complementary sequence as the downstream sequence, and the upstream and downstream sequences are annealed after the oligo is synthesized (program: 95°C, 5min; 95°C-85°C at-2°C / s; 85°C-25°C at-0.1 ℃ / s; hold at 16℃) and then ligated with the pGL3-U6-sgRNA (Addgene #51133) vector digested with BsaI (NEB: R3733L). The enzyme digestion system is: pGL3-U6-sgRNA 2μg; CutSmart buffer (NEB: B7204S) 6μL; BsaI 1μL; ddH 2 Make up to 60 μL with O, digest overnight at 37°C. The ligation system is: ...

Embodiment 3

[0152] In this example, the RNA off-target situation of ABEmax-SpRY, 8e-SpRY and their mutants in 293T cells was compared.

[0153] 3.1 Construction of sgRNA

[0154] The sgRNA sequence used for RNA off-target detection is 5'-CTGGAACACAAAGCATAGAC-'3 (SEQ ID NO:66), which was constructed according to the plasmid construction method described in 2.1.

[0155] 3.2 Cell culture and transfection

[0156] Cell culture was carried out as described in 2.2. The day before transfection, 293T cells were used to spread a 6cm dish, so that the cell density at the time of transfection reached about 80%. The amount of plasmid transfected per dish is 4 μg of base editor plasmid and 2 μg of sgRNA plasmid. Dilute the plasmid in 250 μL of DMEM, and dilute 18 μL of EZ Trans cell transfection reagent (Shanghai Lee Kee Bio: AC04L092) in 250 μL of DMEM Finally, add the diluted EZ transfection reagent to the diluted plasmid, mix well and let stand at room temperature for 15 minutes. Add the DMEM m...

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Abstract

The invention belongs to the field of biological medicines, and relates to an adenine base editor fusion protein without PAM limitation and an application. The invention provides a mutant polypeptide. The polypeptide sequentially comprises an N-terminal fragment of SpRY (D10A), a TadA8e fragment and a C-terminal fragment of SpRY (D10A) polypeptide from an N terminal to a C terminal. The fusion protein containing the mutant polypeptide can target a whole genome, so that the editable range of the genome is widened. Basic group conversion from A: T to G: C can be more efficiently introduced, and great application potential including but not limited to simulation or repair of pathogenic sites of gene diseases is achieved. The miss target on the transcriptome level is lower, and the mutant form with high efficiency and low miss target is considered.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a PAM-free adenine base editor fusion protein and its application. Background technique [0002] The CRISPR / Cas9 system was originally discovered in bacteria and archaea, and has been optimized and modified to form a powerful gene editing tool, which is widely used in the research of DNA knockout, knockin, and modification. The CRISPR / Cas9 system is composed of Cas9 nuclease and sgRNA that recognizes the target sequence. The sgRNA and the target sequence are complementary and paired to mediate the directional cutting of the genome by the Cas9 nuclease, resulting in double strand DNA breaks (double strand break, DSB). Repair mechanisms in cells Homologous recombination (with a template) and non-homologous end joining (without a template) enable editing at targeted sites [1,2] . Subsequently, David Liu et al. constructed nickase Cas9 (nCas9) with inactivated RuvC domain, and on this basi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N9/78C12N15/62C07K19/00C12N15/85C12N5/10A61K48/00A61K38/50A61K38/46A61P3/00
CPCC12N9/22C12N9/78C12N15/85C12N5/0686A61K48/005A61P3/00C12Y305/04004C07K2319/00C07K2319/09C12N2800/107C12N2510/00A61K38/00C07K14/4702C12N15/102C12N15/63
Inventor 马旭曹小芳金孝华
Owner 国家卫生健康委科学技术研究所
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