- 作者: Rio Hermantara, Laura Richmond, Aqeel Faisal Taqi, Sabari Chilaka, Valentine Jeantet, Ileana Guerrini, Katherine West & Adam West
- 作者服務機構: 1.Department of Biomedicine, School of Life Sciences, Indonesia International Institute for Life Sciences, Jakarta, Indonesia 2.School of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK 3.School of Molecular Biosciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
- 中文摘要:
- 英文摘要:
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. - 中文關鍵字:
- 英文關鍵字: CRISPR–Cas9, Knock-in, Of-target, Faithful genome editing, Self-cleaving, Homology arms