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A gene base editor

By fusing human cytosine deaminase 3A with the CRISPR/Cas system and combining it with a uracil glycosylase inhibitor, efficient base editing at the GpC site is achieved, solving the problem of HDR-mediated gene correction in the existing technology. The problems of low efficiency and inability to edit GpC sites have improved the accuracy and application scope of gene editing.

Active Publication Date: 2021-12-07
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
Comparison scheme
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

This invention provides a fusion-expressed protein (base editor) consisting of human apolipoprotein B messenger RNA deaminase catalytic subunit 3A (APOBEC3A), a CRISPR-associated Cas protein, and a uracil glycosidase inhibitor (UGI) [added or omitted depending on the situation]. This base editor is capable of base editing in DNA, deaminating cytosine to uracil, and maintains high editing efficiency even when cytosine is located at a GpC site or in a hypermethylated state.

Description

Technical Field [0001] This invention relates to a gene base editor. Background Technology [0002] Genome editing technology refers to a genetic engineering technique that uses programmable nucleases (molecular scissors) to modify specific segments of an organism's genomic DNA through base insertion, deletion, or substitution, thereby editing target genes. Genome editing technology, used for genetic manipulation of cells, has wide applications in basic life science research, biotechnology development, agricultural technology development, and pharmaceutical research. For example, directly correcting gene mutations that cause genetic diseases in vivo could fundamentally treat these diseases; precisely genetically modifying crops could increase yields or enhance resistance to environmental pollution or pathogen infection; and accurately modifying microbial genomes could promote the development of renewable bioenergy. [0003] The CRISPR / Cas (Clustered Regularly Interspaced Shor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12P19/30
CPCC07K2319/00C12N9/22C12N9/78C12N15/102C12P19/30
Owner SHANGHAI TECH UNIV