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Related Concept Videos

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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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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.
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Related Experiment Video

Updated: Sep 9, 2025

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors
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An integrated enzymatic and computational pipeline for quantifying off-target base-editing.

Alexander G McFarland1, Soon-Keat Ooi2, Davin Tafuri2

  • 1Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.

Biorxiv : the Preprint Server for Biology
|September 5, 2025
PubMed
Summary
This summary is machine-generated.

A new method identifies unintended DNA changes from base editing by marking double-stranded DNA breaks. This approach helps monitor off-target edits in patient cells, improving the safety of gene editing therapies.

Keywords:
ABE8eCRISPR/Cas9DNA base editingcell-type specific off-target sitesgene modificationiGUIDEoff-target analysis

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • DNA base editing offers precise genetic modification but lacks robust methods for detecting off-target effects.
  • Ensuring the safety of base editing technologies requires reliable tools to identify unintended genomic alterations.

Purpose of the Study:

  • To develop a generalizable and model-independent workflow for genome-wide identification of off-target base editing sites.
  • To establish a method for monitoring unintended DNA modifications in patient-derived cells for clinical applications.

Main Methods:

  • Utilized an ABE8e base editor derivative with restored double-stranded DNA (dsDNA) cleavage activity to mark off-target cleavage sites.
  • Employed dsDNA oligonucleotide incorporation at enzyme-generated dsDNA breaks, followed by DNA sequencing to pinpoint off-target locations.
  • Integrated a cellular assay (BEiGUIDE-Seq) with a computational workflow (CRISPRito) to create optimized amplicon panels for monitoring.

Main Results:

  • Successfully identified genomic locations of off-target cleavage by the ABE8e editor.
  • Quantified the extent of base editing in proximity to off-target cleavage sites.
  • Demonstrated the ability to detect off-target edits in primary target cells, including patient-derived cells.

Conclusions:

  • Introduced BEiGUIDE-Seq, a novel platform for detecting off-target base editing with high accuracy.
  • Addressed a critical safety gap in clinical base editing by providing a method to evaluate unintended edits.
  • Established a generalizable strategy for assessing the safety and specificity of base editing tools in relevant cellular contexts.