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Characterizing the collateral activity of CRISPR/Cas13 in mammalian cells: Implications for RNA editing and

Yanbin Wan1, Caimei Huang1, Dongyan Feng2

  • 1School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China.

International Journal of Biological Macromolecules
|November 21, 2024
PubMed

Insights

CRISPR/Cas13 RNA editing tools show varied collateral activity. While LwaCas13a demonstrated notable activity, its impact on cell viability and porcine embryo development was modest, offering insights for clinical applications.

Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • The CRISPR/Cas13 system is a promising RNA editing tool.
  • Concerns exist regarding collateral activity and cytotoxicity of Cas13 orthologs in mammalian cells, potentially limiting applications.

Purpose of the Study:

  • To investigate and compare the collateral activities of LwaCas13a and RfxCas13d in mammalian cells.
  • To assess the impact of Cas13 collateral activity on cell viability and early embryonic development.

Main Methods:

  • Utilized dual-fluorescence (mRuby/GFP) and dual-luciferase (Fluc/Rluc) reporter systems.
  • Tested LwaCas13a and RfxCas13d in 293T and U87 cell lines.
  • Evaluated effects on porcine embryo development.

Main Results:

  • Observed differential collateral RNA cleavage activities between LwaCas13a and RfxCas13d.
  • LwaCas13a exhibited significant collateral activity, contrary to some previous reports.
  • Collateral RNA cleavage had a modest impact on cell viability and only mildly impeded porcine embryo development.

Conclusions:

  • Distinct collateral RNA cleavage by Cas13 systems can mildly suppress mammalian cell proliferation and embryo development.
  • These findings may explain the lack of reported collateral effects in prior studies.
  • Provides new insights into the implications of Cas13 collateral activity for clinical applications.
Keywords:
CRISPR/Cas13Collateral activityMammalian cells

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