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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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A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors
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CRISPR-associated transposon for programmable viral vector engineering and prime editing.

Quyen T Dang1, Chin-Wei Chang1, Pin-Yan Chen1

  • 1Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300044, Taiwan.

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|February 23, 2026
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Summary

SHOT 2.0 is a new CRISPR-associated transposon platform for flexible bacmid engineering in E. coli. This tool enables efficient gene delivery and prime editing in various cell types, expanding baculovirus applications.

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Area of Science:

  • Molecular Biology
  • Gene Editing
  • Virology

Background:

  • Baculovirus is widely used for recombinant protein expression and gene delivery.
  • Current bacmid engineering methods lack flexibility and programmability.
  • CRISPR-associated transposon systems offer potential for advanced genetic manipulation.

Purpose of the Study:

  • To develop an optimized CRISPR-associated transposon platform for flexible and programmable bacmid engineering.
  • To enable efficient integration of large DNA cargos into specific bacmid loci.
  • To demonstrate the utility of the platform for constructing advanced baculovirus vectors for gene editing.

Main Methods:

  • Development of SHOT 2.0, a CRISPR-associated transposon system for RNA-guided bacmid editing in Escherichia coli.
  • Site-specific integration of large DNA payloads (≥14 kb) into bacmid loci (v-cath, ODVe56).
  • Construction and testing of an all-in-one baculovirus encoding a prime editor (PE5max).
  • Evaluation of prime editing efficiency in HEK293T cells, induced pluripotent stem cells (iPSCs), and liver cancer cells.

Main Results:

  • SHOT 2.0 enables site-specific integration of large DNA cargos (≥14 kb) into defined bacmid loci.
  • Integration at the ODVe56 locus significantly enhances transgene stability during serial virus passaging.
  • The system is compatible with the Bac-to-Bac® workflow, allowing dual-gene insertion.
  • Vector-mediated prime editing achieved up to 85.6% efficiency in HEK293T cells and 37.1% in iPSCs and liver cancer cells.

Conclusions:

  • SHOT 2.0 significantly expands the baculovirus engineering toolbox, offering a flexible platform for genome editing.
  • The platform facilitates the creation of advanced baculovirus vectors for diverse applications, including gene delivery.
  • Demonstrated robust prime editing capabilities in various cell types, including challenging ones like iPSCs and cancer cells.