biostudies-literatureUnknown31(1)Hermantara RIndonesia Endowment Fund Scholarship<h4>Background</h4>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.<h4>Method</h4>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.<h4>Result</h4>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.<h4>Conclusion</h4>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.Journal of biomedical science32https://www.ebi.ac.uk/biostudies/studies/S-EPMC10964699biostudies-literatureImproving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.PMC10964699West KJeantet VGuerrini IRichmond LHermantara RChilaka STaqi AFWest AfalseImproving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.<h4>Background</h4>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.<h4>Method</h4>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.<h4>Result</h4>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.<h4>Conclusion</h4>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.2024-01-01T00:00:00Z2024 Mar2025-04-04T23:53:46.688Z2025-04-04T23:53:46.688ZS-EPMC109646993853247910.1186/s12929-024-01020-x