Cytosine deaminase and cytosine editor comprising same

A technology of cytosine deaminase and cytosine, which is applied in the direction of enzymes, hydrolases, chemical instruments and methods, and can solve problems affecting the efficiency of cytosine base editing tools and the application of cytosine base editing tools

Pending Publication Date: 2021-07-23
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And studies have shown that methylation of cytosine affects the efficiency of cytosine base editing tools
Second, the cytosine base editing tool will edit cytosine at mul

Method used

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  • Cytosine deaminase and cytosine editor comprising same
  • Cytosine deaminase and cytosine editor comprising same
  • Cytosine deaminase and cytosine editor comprising same

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0035] Example 1. Construction of the intrapamine editing device and the purification of the outer destination of cytosine editing instruments

[0036] First, an encoding sequence of the MSB tag protein (SEQ IDNO.: 7) is inserted between the NCOI and the BamHikase sort of the prokaryotic expression vector PET28A, and the purpose is to enhance the rootenin in E. coli, which is next to BamHi and SALI respectively. The encoding sequence (SEQ ID NOS: 8, 9, 10, 11, respectively) is inserted into the AID-CBE, A1-CBE, A3A-CBE, A3B-CBE, and A3BCTD-CBE (SEQ ID NOS.: 8, 9, 10, 11, and 12). Among them, the AID-CBE represents the fusion protein of human AID-Xten-NXCas9-UGi, A1-CBE represents the fusion protein of rat apobec1-xten-NXCas9-UGi, A3a-CBE represents the fusion protein of human apobec3a-xten-NXCas9-UGi A3B-CBE represents the fusion protein of human apobec3b-xten-nxcas9-UGi, A3BCTD-CBE represents the fusion protein of human apobec3b C-terminal domain-tTEN-NXCAS9-UGi, all belong to c...

Example Embodiment

[0038] Example 2. Preparation of the guide RNA

[0039] Between the HindIII and ECORI-sized sites of the plasmid PUC19, the sequence SEQ ID No.:13, 5 'end 20 bases is a T7 promoter sequence, and the 3' end 76 base is a stent structure of the wizard RNA. sequence.

[0040] Three SGRNA vitro transcription templates were amplified from the recombinant PUC19 support using T7 promoter and target specific positive primer and a guide RNA bracket structure specific, and each PCR was amplified from the recombinant PUC19 support. A primer pair is used to amplify an SGRNA in vitro transcription template. The specific PCR system is: 10 × KOD-PLUS-NEO buffer, 5 μL; 2mm DNTP, 5 μL; 25mm MgSO 4 3 μL; 10 pm / μL positive reverse primer, 1.5 μl of each of the templates, 10 ng; KOD-PLUS-NEO (1.0 U / μl), 1 μL; hydrating to 50 μl. The PCR program is: 94 degrees for 2 minutes; 98 degrees 10 seconds, 58 degrees 30 seconds, 68 degrees 30 seconds, 35 cycles.

[0041] The PCR reaction product was separa...

Example Embodiment

[0044] Example 3. Sports osteosine editing test

[0045] In the present embodiment, cytosine deamination reaction test is carried out in vitro to genomic target AT4G22970, AT3G13784, and AT1G30950 in vitro using different guide RNA.

[0046] Specifically, genomic DNA was extracted from a Dneasy Plant Maxi kit (Qiagen) from two weeks large Arabidopsis seedlings. The 500 ng of genomic DNA was used as 1 μg Example 1, respectively, the respective director RNA obtained by 1 μg Example 2 at 10 × CUTSMART buffer (NEB) at 37 ° C for 3 hours, and the entire reaction system was 20 μl. At the same time, the reaction system without the fusion protein and the wizard RNA without the addition of cytosine base editor is used as a negative control. After the reaction is completed, use DDH 2 O Dilute the reaction system to 40 μL, respectively, 1 μg of restriction endonuclease 37 ° C, for at least 8 hours (AT4G22970 target digestion, the recognition site is CCGG, the AT3G13784 target with KPNi enzy...

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Abstract

The invention discloses a cytosine deaminase formed by a C-terminal domain of cytosine deaminase APOBEC3B, and a cytosine editor fusion protein containing the cytosine deaminase, a CRISPR/Cas system related protein with DNA (Deoxyribose Nucleic Acid) single-chain cleavage activity, and an optional uracil glycosylase inhibitor.

Description

technical field [0001] The present application relates to a truncated cytosine deaminase, which has higher cytosine deamination efficiency than the full-length cytosine deaminase. The present application also relates to a cytosine editor comprising the above-mentioned truncated cytosine deaminase, which can efficiently and accurately edit methylated or unmethylated cytosine (C) into uracil ( U) or thymine (T). Background technique [0002] Precise editing of bases at a specific site in the genome is of great significance to basic research, modern crop breeding and medical fields. Many adenine and cytosine (C) single base editing tools have been developed. Adenine base editors rely on the function of adenine deaminase to mutate adenine to hypoxanthine, while cytosine base editors use the function of cytosine deaminase to mutate cytosine to uracil. During the process of DNA replication of the cell genome, the hypoxanthine base and uracil base formed by the mutation match wit...

Claims

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Application Information

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IPC IPC(8): C12N9/78C07K19/00C12N15/113C12N15/82
CPCC12N9/22C12N9/78C12N15/113C12N15/8216C12Y305/04001
Inventor 曹晓风王东刘志红唐善杰宋显伟赵庆华
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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