{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown"],"volume":["31(1)"],"submitter":["Hermantara R"],"funding":["Indonesia Endowment Fund Scholarship"],"pubmed_abstract":["<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":["Journal of biomedical science"],"pagination":["32"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC10964699"],"repository":["biostudies-literature"],"pubmed_title":["Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration."],"pmcid":["PMC10964699"],"pubmed_authors":["West K","Jeantet V","Guerrini I","Richmond L","Hermantara R","Chilaka S","Taqi AF","West A"],"additional_accession":[]},"is_claimable":false,"name":"Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.","description":"<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.","dates":{"release":"2024-01-01T00:00:00Z","publication":"2024 Mar","modification":"2025-04-04T23:53:46.688Z","creation":"2025-04-04T23:53:46.688Z"},"accession":"S-EPMC10964699","cross_references":{"pubmed":["38532479"],"doi":["10.1186/s12929-024-01020-x"]}}