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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
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Related Experiment Video

Updated: Jul 3, 2025

Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates
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Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates

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Single-Stranded DNA with Internal Base Modifications Mediates Highly Efficient Gene Insertion in Primary Cells.

Karen L Kanke1, Rachael E Rayner2, Eli Abel1

  • 1Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, Columbus, OH.

Biorxiv : the Preprint Server for Biology
|February 14, 2024
PubMed
Summary
This summary is machine-generated.

Chemically modified single-stranded DNA (ssDNA) enhances gene insertion efficiency significantly compared to unmodified ssDNA. This improved gene editing holds promise for therapeutic applications and biological modeling.

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biochemistry

Background:

  • Single-stranded DNA (ssDNA) and Cas9 are used for gene insertion but exhibit low efficiency.
  • Existing methods require optimization for enhanced gene editing outcomes.

Approach:

  • Developed chemically modified ssDNA (eDNA) with 10-17% internal base modifications.
  • Tested eDNA compatibility with homologous recombination machinery.
  • Evaluated eDNA performance in various cell types including airway basal stem cells (ABCs) and hematopoietic stem and progenitor cells (HSPCs).

Key Points:

  • eDNA templates improve gene insertion 2-3 fold over unmodified ssDNA in multiple cell types.
  • Achieved over 50% allele gene insertion in clinically relevant loci (CFTR, HBB, CCR5) in ABCs.
  • Demonstrated up to 70% allele gene insertion in the HBB locus in HSPCs, comparable to adeno-associated virus (AAV) templates.
  • Identified that chemical modifications in eDNA inhibit TREX1 nuclease activity, unlike unmodified ssDNA.

Conclusions:

  • Chemically modified ssDNA (eDNA) represents a significant advancement in gene insertion efficiency.
  • This technology shows therapeutic relevance and potential for biological modeling applications.
  • The findings suggest eDNA is a promising alternative to viral vectors for gene therapy.