Journal of Biomedical Science (Mar 2024)

Improving CRISPR–Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration

  • Rio Hermantara,
  • Laura Richmond,
  • Aqeel Faisal Taqi,
  • Sabari Chilaka,
  • Valentine Jeantet,
  • Ileana Guerrini,
  • Katherine West,
  • Adam West

DOI
https://doi.org/10.1186/s12929-024-01020-x
Journal volume & issue
Vol. 31, no. 1
pp. 1 – 21

Abstract

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Abstract Background The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR–Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency. Method Here we report the development of a multicolour fluorescence assay for studying CRISPR–Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR–Cas9 strategies. Result We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration. Conclusion Our results highlight the need for a more stringent assessment of CRISPR–Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.

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