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Related Concept Videos

Long-patch Base Excision Repair01:02

Long-patch Base Excision Repair

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Since the discovery of the two BER pathways, there has been a debate about how a cell chooses one pathway over the other and the factors determining this selection. Numerous in vitro experiments have pointed out multiple determinants for the sub-pathway selection. These are:
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Base Excision Repair01:54

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One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
The first step of...
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Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
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RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
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Split complementation of base editors to minimize off-target edits.

Xiangyu Xiong1, Kehui Liu2, Zhenxiang Li1

  • 1State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.

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Summary
This summary is machine-generated.

A new Split Deaminase for Safe Editing (SAFE) system improves base editors (BEs) by preventing unintended DNA and RNA edits. This breakthrough enhances precision and purity in genome editing across various cell types.

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Area of Science:

  • Molecular Biology
  • Genome Editing
  • Biotechnology

Background:

  • Base editors (BEs) are crucial for introducing precise point mutations in genomes.
  • Conventional BEs exhibit off-target editing in both the genome and transcriptome.
  • Existing methods to mitigate off-target effects often require deaminase mutagenesis or external regulators.

Purpose of the Study:

  • To develop a novel system for enhancing the safety and specificity of base editors.
  • To minimize both guide RNA (gRNA)-independent and gRNA-dependent off-target edits.
  • To improve the overall on-target editing efficiency and product purity of BEs.

Main Methods:

  • Development of a split deaminase for safe editing (SAFE) system.
  • Splitting BEs within the deaminase domain of a Cas9 nickase to inactivate both components.
  • Demonstrating gRNA-conditioned reassembly for targeted editing in plant, human, and yeast cells.

Main Results:

  • The SAFE system effectively suppressed gRNA-independent and gRNA-dependent off-target DNA and RNA edits.
  • Robust on-target editing was achieved across diverse cell types (plant, human, yeast).
  • Significant increase in product purity due to the elimination of indels.

Conclusions:

  • The SAFE system offers a generalizable strategy to reduce off-target editing in base editors.
  • This approach enhances the precision and reliability of genome editing applications.
  • SAFE provides a robust solution for safer and more efficient base editing.