Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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
View PDF0 Cites 2 Cited by
  • 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 multiple positions in the editing window, and cannot efficiently edit a single cytosine base, which will greatly affect the cytosine base Application of base editing tools

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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
Effect test

Embodiment 1

[0035] Example 1. Construction of cytosine editor in vitro expression vector and purification of cytosine editor in vitro expression

[0036] First, insert the coding sequence of MSB tag protein (SEQ ID NO.: 7) between the NcoI and BamHI restriction sites of the prokaryotic expression vector pET28a, the purpose is to enhance the expression of the prokaryotic protein in Escherichia coli, and then between BamHI and SalI respectively The coding sequences for AID-CBE, A1-CBE, A3A-CBE, A3B-CBE and A3Bctd-CBE (SEQ ID Nos.: 8, 9, 10, 11 and 12, respectively) were inserted. Among them, AID-CBE represents the fusion protein of human AID-XTEN-nxCas9-UGI, A1-CBE represents the fusion protein of rat APOBEC1-XTEN-nxCas9-UGI, and A3A-CBE represents the fusion protein of human APOBEC3A-XTEN-nxCas9-UGI , A3B-CBE indicates the fusion protein of human APOBEC3B-XTEN-nxCas9-UGI, and A3Bctd-CBE indicates the fusion protein of the C-terminal domain of human APOBEC3B-XTEN-nxCas9-UGI, both of which...

Embodiment 2

[0038] Example 2. Preparation of guide RNA

[0039] Between the HindIII and EcoRI restriction sites of plasmid pUC19, the insertion sequence is SEQ ID NO.: 13, the 20 bases at the 5' end are the T7 promoter sequence, and the 76 bases at the 3' end are the scaffold structure of the guide RNA sequence.

[0040] Using the T7 promoter and target-specific forward primers in Table 1 and the general reverse primers specific to the guide RNA scaffold structure, the three sgRNA in vitro transcription templates were obtained by PCR amplification from the above-mentioned recombinant pUC19 vector, that is, each amplification An additional primer pair is used to amplify an sgRNA template for in vitro transcription. The specific PCR system is: 10×KOD-Plus-Neo buffer, 5 μl; 2mM dNTP, 5 μl; 25mM MgSO 4 , 3μl; 10pM / μl forward and reverse primers, 1.5μl each; template plasmid, 10ng; KOD-Plus-Neo (1.0U / μl), 1μl; water up to 50μl. The PCR program is: 94 degrees for 2 minutes; 98 degrees for ...

Embodiment 3

[0044] Example 3. In vitro cytosine editing assays

[0045] In this example, different guide RNAs were used to test the cytosine deamination reaction of genomic targets AT4G22970, AT3G13784 and AT1G30950 in vitro.

[0046] Specifically, genomic DNA was extracted from two-week-old Arabidopsis seedlings using DNeasy Plant Maxi Kit (QIAGEN). 500 ng of genomic DNA was reacted with 1 μg of each fusion protein obtained in Example 1 and 1 μg of each guide RNA obtained in Example 2 in 10×CutSmart buffer (NEB) at 37° C. for 3 hours, and the total reaction volume was 20 μl. At the same time, the reaction system without cytosine base editor fusion protein and guide RNA was used as a negative control. After the reaction, use ddH 2 O dilute the reaction system to 40 μl, and add 1 μg of restriction endonuclease to digest at 37°C for at least 8 hours (AT4G22970 target is digested with MspI, the recognition site is CCGG, AT3G13784 target is digested with KpnI, the recognition site The po...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com