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A base editor and its preparation method and application

An editor and gene editing technology, applied in the field of gene editing, can solve problems such as narrow application range and rare targets

Active Publication Date: 2021-07-16
湖北万德瑞生命科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this base editor only recognizes the PAM sequence of 5'-TTTV, which is relatively rare in the genome, resulting in a narrow range of applications

Method used

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  • A base editor and its preparation method and application
  • A base editor and its preparation method and application

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preparation example Construction

[0029]The invention provides the preparation method of the base editor, comprising the following steps: inserting the DNA fragment of the dCpf1-RR-eBE expression frame into the vector backbone pCMV-dCpf1-eBE to construct and obtain the pCMV-dCpf1-RR-eBE recombinant plasmid ; The sgRNA general expression frame DNA fragment was inserted into the vector backbone pUC57 to obtain the pLbCpf1-sgRNA recombinant plasmid.

[0030] In the present invention, the insertion site of the dCpf1-RR-eBE expression cassette DNA fragment is between the Pst I restriction site and the Apa I restriction site of the vector backbone pCMV-dCpf1-eBE, that is, the vector backbone The 2365bp-5178bp interval of pCMV-dCpf1-eBE; in the present invention, the insertion is preferably performed by performing double enzyme digestion on the dCpf1-RR-eBE expression cassette DNA fragment and pCMV-dCpf1-eBE respectively; The enzymes used for double enzyme cutting are Pst I enzyme and Apa I enzyme. In the present in...

Embodiment 1

[0040] Construction of base editors

[0041] 1. Construction of pCMV-dCpf1-RR-eBE recombinant plasmid

[0042] The DNA fragment of the dCpf1-RR-eBE expression cassette of 2814bp was synthesized (the nucleotide sequence is shown in SEQ ID NO: 1), and inserted into the pCMV-dCpf1-eBE vector by double digestion with Pst I enzyme and Apa I enzyme to obtain pCMV - dCpf1-RR-eBE vector.

[0043] The pCMV-dCpf1-eBE vector was purchased from addgene, Cat. No. 107688.

[0044] Pst I enzyme was purchased from Treasure Bioengineering (Dalian) Co., Ltd., product number 1624; ApaI enzyme was purchased from Treasure Bioengineering (Dalian) Co., Ltd., product number 1604.

[0045] Enzyme digestion system: 50 μL, reagents purchased from Treasure Bioengineering (Dalian) Co., Ltd.): Pst I enzyme 1 μL, Apa I enzyme 1 μL, dCpf1-RR-eBE expression cassette DNA fragment or pCMV-dCpf1-eBE backbone vector 1 μg, BufferH 5 μL , add double distilled water to 50 μL. The digestion temperature is 37°C, a...

Embodiment 2

[0070] Application of base editors in gene editing of mammalian cell lines

[0071] Diqing sheep skin epithelial cell line DQSHS1 was purchased from the Kunming Cell Bank of the Chinese Academy of Sciences, number: KCB 94026.

[0072] 1. sgRNA target design

[0073] The sequence of the sheep DKK2 gene including the first exon (DKK2-440, shown below) was extracted from the sequence of sheep chromosome 6 (NCBI GI:417531944), and the Cpf1 sgRNA target was designed.

[0074] agactgagttcacacggtgctgggcccccaaagc caagtg gggttgggggaacagagtctgcgagtcccggcgccccgagt gcagggccccgtgttggggtcctccttcccatttgt atccgt atccttgcgggctttgcgcctccccgggggaccccctcgccgggagatggccgcactgatgcggggcaaggactcctcccgctgcctgctcctactggccgcggtgctgatggtggagagctcacagttcggcagct c gcgggc caaactcaactccatcaagtcctctctgggcggggagacgcctgcccaggccgccaatcgatctgcgggcacttaccaag gactggctttcggcggcagtaagaagggcaaaaacctggggcaggtaggaaaatacccccaatacactcttcaaccagaagaggtag ggacccg (SEQ ID NO: 12)

[0075] Target site TF3: a tccgt (SEQ...

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Abstract

The invention provides a base editor and its preparation method and application, belonging to the field of gene editing technology, the base editor includes pCMV-dCpf1-RR-eBE recombinant plasmid and pLbCpf1-sgRNA recombinant plasmid; the application includes The following steps: determine the target sequence, design single-stranded oligonucleotide pairs; anneal to obtain double-stranded DNA fragments; connect to the pLbCpf1-sgRNA recombinant plasmid to obtain the target site sgRNA expression vector; target site sgRNA expression vector and pCMV-dCpf1- RR‑eBE recombinant plasmids are co-transfected into cells and cultured; the base editor of the present invention can specifically mutate cytosine C at the target site to thymine T without any effect on bases at non-target sites , The gene editing efficiency is between 20% and 30%.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a base editor and its preparation method and application. Background technique [0002] Although the traditional CRISPR / Cas9 gene editing technology has high gene knockout efficiency, its efficiency is usually low when performing base replacements (such as correcting point mutations that cause genetic diseases), which also limits CRISPR / Cas9 Applications of gene editing. In recent years, the new base editing system (Base Editor, BE) developed by integrating CRISPR / Cas9 and APOBEC (cytosine deaminase) can achieve high efficiency at the single base level (such as cytosine to thymine) genome targeted editing. This new base editing system can theoretically correct hundreds of genome point mutations that cause human diseases, so it has great potential for clinical application. The currently reported base editing systems all use Cas9 proteins (mainly Streptococcus p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/85C12N15/10
CPCC12N15/102C12N15/85C12N15/907C12N2800/107C12N2810/10
Inventor 李和刚张宁赵金山秦怀远辛京京郝小静
Owner 湖北万德瑞生命科学技术有限公司
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