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Gene base editor

An editor, base technology, applied in chemical instruments and methods, antibody mimics/scaffolds, hybrid peptides, etc., can solve the problem of limiting the effective editing sites of base editors, unable to effectively correct, unable to effectively edit bases Base C, etc.

Active Publication Date: 2018-12-18
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, rA1-based base editors cannot efficiently edit base C in the GpC site, thus limiting the effective editing sites of existing base editors
For example, the mutation from GpT to GpC can lead to the deletion of RNA splicing sites, which can cause a variety of human diseases; while the existing rA1-based base editor cannot effectively correct the mutation from GpT to GpC

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0084] Example 1: Base editor

[0085] The expression plasmid of pCMV-hA3A-BE was constructed. Human apolipoprotein B messenger RNA deaminase catalytic subunit 3A (APOBEC3A, hA3A; SEQ ID NO: 1) with Cas9 nickase and a uracil DNA glycosidase inhibitor [Bacillus phage] (SEQ ID NO: 12 ) fused to an expression vector. The 10th aspartic acid of Cas9 nickase is mutated to alanine, thereby losing the activity of cutting double strands and ensuring a nick on one strand.

[0086] The fusion expression vector hA3A-nCas9-UGI (hA3A-BE, SEQ ID NO: 21) and the expression vector of single-stranded guide RNA were co-transformed into eukaryotic cells ( figure 1 , legend A), C-T base editing occurs at the site targeted by the guide RNA of the genome. The sequence of the genomic DNA target position was amplified by PCR, and the base editing efficiency of the target site C-T was detected by Sanger DNA sequencing. Compared with the co-expression of sgRNA and BE3, the method of co-expression of...

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Abstract

The invention provides a fusion expression protein (base editor) of human apolipoprotein B messenger RNA deaminase catalytic subunit 3A (APOBEC3A), CRISPR-related Cas protein and a uracil glycosidaseinhibitor (UGI) [addition depending on condition]. The base editor is capable of performing base editing in DNA, cytosine is deaminated to uracil, and the base editor still has high editing efficiencyeven if the cytosine is located at a GpC site or in a methylation state.

Description

technical field [0001] The invention relates to a gene base editor. Background technique [0002] Genome editing technology refers to a genetic engineering technology that uses designable nucleases (molecular scissors) to modify specific segments of the genome DNA of an organism through base insertion, deletion or substitution to achieve editing of the target gene. Using genome editing technology to genetically manipulate cells can be widely used in basic life science research, biotechnology development, agricultural technology development, and pharmaceutical research and development. For example: directly correcting the genetic mutations that cause genetic diseases in the body will be able to treat genetic diseases fundamentally; carry out precise genetic engineering on crops to increase their yield or resist environmental pollution or pathogen infection; precise microbial genome transformation, thereby promoting the development of renewable bioenergy, etc. [0003] Since...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12P19/30
CPCC07K2319/00C12N9/22C12N9/78C12N15/102C12P19/30
Inventor 陈佳杨力黄行许杨贝王潇李佳楠
Owner SHANGHAI TECH UNIV
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