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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Updated: Dec 10, 2025

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Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions.

Zhiquan Liu1, Siyu Chen1, Huanhuan Shan1

  • 1Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin University, Changchun, 130062, China.

BMC Biology
|September 2, 2020
PubMed
Summary
This summary is machine-generated.

Researchers developed a new cytidine base editor (CBE) with enhanced precision by reducing unwanted bystander mutations. This engineered APOBEC3G-based editor shows high efficiency and context-specificity for precise gene modification.

Keywords:
Base editorCRISPR/Cas9PrecisioneA3G

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • Cytidine base editors (CBEs) facilitate C-to-T conversions but suffer from imprecise bystander edits.
  • Existing CBEs can cause unintended C-to-T conversions within the deaminase's activity window, limiting precision.
  • There is a need for base editors with improved specificity to reduce off-target mutations.

Purpose of the Study:

  • To develop a novel base editor with significantly reduced unwanted bystander activities.
  • To engineer a CBE with enhanced precision and context-specificity for gene modification.
  • To create a base editor capable of recognizing relaxed NG PAM sequences.

Main Methods:

  • Utilized an engineered human APOBEC3G (eA3G) C-terminal catalytic domain for preferential cytidine deaminase activity.
  • Developed an eA3G-based editor (eA3G-BE) with specific CC context preference.
  • Combined hA3G with an engineered SpCas9-NG variant to achieve relaxed NG PAM recognition.

Main Results:

  • Achieved targeted editing efficiencies of 18.3-58.0% in human cells and 54.5-92.2% in rabbit embryos with excellent CC context-specificity.
  • Demonstrated reduced generation of bystander mutations compared to existing CBEs.
  • Induced accurate single-base substitutions, leading to nonsense mutations with 83-100% efficiency and minimal bystander mutations in rabbit Founder (F0) at Tyr loci.

Conclusions:

  • The novel A3G-BEs exhibit improved precision and CC context-specificity.
  • These engineered base editors expand the toolkit for precise gene modification in various organisms.
  • The developed base editors offer a more accurate approach to gene editing applications.