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CRISPR/dCas9-mediated transposition with specificity and efficiency of site-directed genomic insertions

Lena Goshayeshi1,2, Sara Yousefi Taemeh1,2, Nima Dehdilani1,2

  • 1Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
|January 26, 2021
PubMed

Insights

Researchers developed a novel RNA-guided piggyBac transposase system for precise DNA integration. This method overcomes limitations of traditional gene editing, enabling targeted genomic insertions with potential applications in cancer therapy.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Current targeted nucleases for genome editing have limited efficiency for sequence replacements or insertions.
  • Limitations include reliance on DNA repair pathways, potential cellular toxicity, and proto-oncogene activation.
  • The piggyBac (PB) transposase offers efficient, albeit random, DNA integration into the genome.

Purpose of the Study:

  • To develop a method for site-directed DNA integration using a fusion of catalytically inactive Cas9 (dCas9) and PB transposase.
  • To enhance precision and efficiency in genomic insertions by guiding the PB transposase to specific DNA targets.
  • To establish a system for recovering cells with successful targeted integrations.

Main Methods:

  • Fused catalytically inactive Cas9 (dCas9) to the piggyBac (PB) transposase.
  • Utilized dual single-guide RNAs (sgRNAs) to direct the dCas9-PB fusion to specific genomic locations.
  • Employed a promoter/reporter complementation assay to identify and select cells with accurate genomic integrations.

Main Results:

  • Achieved site-directed integrations into the human ROSA26 safe harbor region using the RNA-guided PB transposase system.
  • Demonstrated successful targeting with an efficiency of 0.32% of cells.
  • Validated the functionality of the promoter/reporter complementation assay for detecting specific integrations.

Conclusions:

  • The developed RNA-guided piggyBac transposase methodology enables precise targeting of specific genomic regions.
  • This approach offers a potential solution to the limitations of existing gene editing technologies.
  • Potential applications include targeted sequence insertion in cancer cells for therapeutic purposes, such as activating tumor suppressor genes or inserting sequences for destruction.
Keywords:
CRISPR-associated protein 9Insertional mutagenesisgene insertionpiggyBac transposasetransposases

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