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Related Experiment Video

Updated: Jun 16, 2026

Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System
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Stepwise Evaluation of Plasmid- and Adeno-Associated Virus-Based Knock-In Using A Triple-Reporter Platform.

Hiromasa Hara1,2,3,4, Hideki Uosaki1,5,6, Fumio Nakahara1

  • 1Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan.

Genes to Cells : Devoted to Molecular & Cellular Mechanisms
|June 15, 2026
PubMed
Summary

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Optimizing homology-directed repair (HDR) knock-in requires evaluating double-strand break (DSB) induction and repair pathway selection. A triple-reporter system revealed donor design impacts DSB levels and the balance between HDR and end-joining-mediated targeted insertion (EJ-TI).

Area of Science:

  • Molecular Biology
  • Gene Editing
  • Biotechnology

Background:

  • Homology-directed repair (HDR) mediated knock-in efficiency is crucial for gene editing but challenging to optimize.
  • Efficiency depends on double-strand break (DSB) induction and the selection of repair pathways.

Purpose of the Study:

  • To investigate how donor design and experimental parameters influence DSB induction and the balance between HDR and end-joining-mediated targeted insertion (EJ-TI).
  • To develop a comprehensive strategy for optimizing knock-in efficiency.

Main Methods:

  • Utilized a mouse embryonic stem cell-based triple-reporter platform.
  • Examined the impact of donor design, including Homology-Independent Targeted Integration (HITI) sequences and adeno-associated virus (AAV) donor formats.
Keywords:
CRISPR/Cas9adeno‐associated virus (AAV)break‐induced concurrent joining and recombination (bicJR)homology‐directed repair (HDR)homology‐independent targeted integration (HITI)knock‐intriple‐reporter platform

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  • Assessed DSB induction, total targeted insertion yield, and the ratio of HDR to EJ-TI.

    • Main Results:

    • Donor design significantly affects DSB induction; co-electroporation with Cas9/gRNA and HITI sequences reduced cleavage efficiency.
    • HITI sequences increased total targeted insertion yield with shorter homology arms and favored HDR with longer arms.
    • HITI's effect on knock-in and EJ-TI was donor-format dependent under AAV conditions, showing efficacy only with self-complementary AAV donors.

    Conclusions:

    • Evaluating DSB induction, total targeted insertion yield, and the HDR vs. EJ-TI balance separately is critical for optimizing knock-in strategies.
    • The triple-reporter platform provides a robust method for dissecting these factors.
    • Donor design and delivery method significantly influence gene editing outcomes.