Hermantara, Rio and Richmond, Laura and Taqi, Aqeel Faisal and Chilaka, Sabari and Jeantet, Valentine and Guerrini, Ileana and West, Katherine and West, Adam (2024) Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration. Journal of Biomedical Science, 31 (1): 32. ISSN 1021-7770
AI Summary:
The study reports on the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. The results highlight the need for careful strategy design to maximise efficient and faithful transgene integration.AI Topics:
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.
Hermantara, Rio
Author
Donnelly, Hannah and Ross, Ewan and Xiao, Yinbo and Hermantara, Rio and Taqi, Aqeel and Doherty-Boyd, W. Sebastian and Cassels, Jennifer and Tsimbouri, Penelope M. and Dunne, Karen M. and Hay, Jodie and Cheng, Annie and Meek, R.M. Dominic and Jain, Nikhil and West, Christopher and Wheadon, Helen and Michie, Alison M. and Peault, Bruno and West, Adam G. and Salmeron-Sanchez, Manuel and Dalby, Matthew J. (2024) Bioengineered niches that recreate physiological extracellular matrix organisation to support long-term haematopoietic stem cells. Nature Communications, 15: 5791. ISSN 2041-1723
Hermantara, Rio and Richmond, Laura and Taqi, Aqeel Faisal and Chilaka, Sabari and Jeantet, Valentine and Guerrini, Ileana and West, Katherine and West, Adam (2024) Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration. Journal of Biomedical Science, 31 (1): 32. ISSN 1021-7770
See full publications listRichmond, Laura
Author
Hermantara, Rio and Richmond, Laura and Taqi, Aqeel Faisal and Chilaka, Sabari and Jeantet, Valentine and Guerrini, Ileana and West, Katherine and West, Adam (2024) Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration. Journal of Biomedical Science, 31 (1): 32. ISSN 1021-7770
See full publications listTaqi, Aqeel Faisal
Author
Donnelly, Hannah and Ross, Ewan and Xiao, Yinbo and Hermantara, Rio and Taqi, Aqeel and Doherty-Boyd, W. Sebastian and Cassels, Jennifer and Tsimbouri, Penelope M. and Dunne, Karen M. and Hay, Jodie and Cheng, Annie and Meek, R.M. Dominic and Jain, Nikhil and West, Christopher and Wheadon, Helen and Michie, Alison M. and Peault, Bruno and West, Adam G. and Salmeron-Sanchez, Manuel and Dalby, Matthew J. (2024) Bioengineered niches that recreate physiological extracellular matrix organisation to support long-term haematopoietic stem cells. Nature Communications, 15: 5791. ISSN 2041-1723
Hermantara, Rio and Richmond, Laura and Taqi, Aqeel Faisal and Chilaka, Sabari and Jeantet, Valentine and Guerrini, Ileana and West, Katherine and West, Adam (2024) Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration. Journal of Biomedical Science, 31 (1): 32. ISSN 1021-7770
See full publications listChilaka, Sabari
Author
Hermantara, Rio and Richmond, Laura and Taqi, Aqeel Faisal and Chilaka, Sabari and Jeantet, Valentine and Guerrini, Ileana and West, Katherine and West, Adam (2024) Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration. Journal of Biomedical Science, 31 (1): 32. ISSN 1021-7770
See full publications listJeantet, Valentine
Author
Hermantara, Rio and Richmond, Laura and Taqi, Aqeel Faisal and Chilaka, Sabari and Jeantet, Valentine and Guerrini, Ileana and West, Katherine and West, Adam (2024) Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration. Journal of Biomedical Science, 31 (1): 32. ISSN 1021-7770
See full publications listGuerrini, Ileana
Author
Hermantara, Rio and Richmond, Laura and Taqi, Aqeel Faisal and Chilaka, Sabari and Jeantet, Valentine and Guerrini, Ileana and West, Katherine and West, Adam (2024) Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration. Journal of Biomedical Science, 31 (1): 32. ISSN 1021-7770
See full publications listWest, Katherine
Author
Hermantara, Rio and Richmond, Laura and Taqi, Aqeel Faisal and Chilaka, Sabari and Jeantet, Valentine and Guerrini, Ileana and West, Katherine and West, Adam (2024) Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration. Journal of Biomedical Science, 31 (1): 32. ISSN 1021-7770
See full publications listWest, Adam
Author
Donnelly, Hannah and Ross, Ewan and Xiao, Yinbo and Hermantara, Rio and Taqi, Aqeel and Doherty-Boyd, W. Sebastian and Cassels, Jennifer and Tsimbouri, Penelope M. and Dunne, Karen M. and Hay, Jodie and Cheng, Annie and Meek, R.M. Dominic and Jain, Nikhil and West, Christopher and Wheadon, Helen and Michie, Alison M. and Peault, Bruno and West, Adam G. and Salmeron-Sanchez, Manuel and Dalby, Matthew J. (2024) Bioengineered niches that recreate physiological extracellular matrix organisation to support long-term haematopoietic stem cells. Nature Communications, 15: 5791. ISSN 2041-1723
Hermantara, Rio and Richmond, Laura and Taqi, Aqeel Faisal and Chilaka, Sabari and Jeantet, Valentine and Guerrini, Ileana and West, Katherine and West, Adam (2024) Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration. Journal of Biomedical Science, 31 (1): 32. ISSN 1021-7770
See full publications listAvailable under License Creative Commons Attribution.
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