Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.
CRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, ac...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2023-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0294146 |
| _version_ | 1797394514527125504 |
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| author | Muhammad Nazmul Hasan Toshinori Hyodo Mrityunjoy Biswas Md Lutfur Rahman Yuko Mihara Sivasundaram Karnan Akinobu Ota Shinobu Tsuzuki Yoshitaka Hosokawa Hiroyuki Konishi |
| author_facet | Muhammad Nazmul Hasan Toshinori Hyodo Mrityunjoy Biswas Md Lutfur Rahman Yuko Mihara Sivasundaram Karnan Akinobu Ota Shinobu Tsuzuki Yoshitaka Hosokawa Hiroyuki Konishi |
| author_sort | Muhammad Nazmul Hasan |
| collection | DOAJ |
| description | CRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, achieved with this system. To improve genome editing efficiency, researchers should first have a molecular assay that allows sensitive monitoring of genome editing events with simple procedures. In the current study, we demonstrate that genome editing events occurring in L1CAM, an X-chromosome gene encoding a cell surface protein, can be readily monitored using flow cytometry (FCM) in multiple human cell lines including neuroblastoma cell lines. The abrogation of L1CAM was efficiently achieved using Cas9 nucleases which disrupt exons encoding the L1CAM extracellular domain, and was easily detected by FCM using anti-L1CAM antibodies. Notably, L1CAM-abrogated cells could be quantified by FCM in four days after transfection with a Cas9 nuclease, which is much faster than an established assay based on the PIGA gene. In addition, the L1CAM-based assay allowed us to measure the efficiency of targeted knock-in (correction of L1CAM mutations) accomplished through different strategies, including a Cas9 nuclease-mediated method, tandem paired nicking, and prime editing. Our L1CAM-based assay using FCM enables rapid and sensitive quantification of genome editing efficiencies and will thereby help researchers improve genome editing technologies. |
| first_indexed | 2024-03-09T00:20:52Z |
| format | Article |
| id | doaj.art-ee84611c0553434eac87c559464f4cbf |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-03-09T00:20:52Z |
| publishDate | 2023-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-ee84611c0553434eac87c559464f4cbf2023-12-12T05:34:54ZengPublic Library of Science (PLoS)PLoS ONE1932-62032023-01-011811e029414610.1371/journal.pone.0294146Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.Muhammad Nazmul HasanToshinori HyodoMrityunjoy BiswasMd Lutfur RahmanYuko MiharaSivasundaram KarnanAkinobu OtaShinobu TsuzukiYoshitaka HosokawaHiroyuki KonishiCRISPR/Cas9 is a powerful genome editing system that has remarkably facilitated gene knockout and targeted knock-in. To accelerate the practical use of CRISPR/Cas9, however, it remains crucial to improve the efficiency, precision, and specificity of genome editing, particularly targeted knock-in, achieved with this system. To improve genome editing efficiency, researchers should first have a molecular assay that allows sensitive monitoring of genome editing events with simple procedures. In the current study, we demonstrate that genome editing events occurring in L1CAM, an X-chromosome gene encoding a cell surface protein, can be readily monitored using flow cytometry (FCM) in multiple human cell lines including neuroblastoma cell lines. The abrogation of L1CAM was efficiently achieved using Cas9 nucleases which disrupt exons encoding the L1CAM extracellular domain, and was easily detected by FCM using anti-L1CAM antibodies. Notably, L1CAM-abrogated cells could be quantified by FCM in four days after transfection with a Cas9 nuclease, which is much faster than an established assay based on the PIGA gene. In addition, the L1CAM-based assay allowed us to measure the efficiency of targeted knock-in (correction of L1CAM mutations) accomplished through different strategies, including a Cas9 nuclease-mediated method, tandem paired nicking, and prime editing. Our L1CAM-based assay using FCM enables rapid and sensitive quantification of genome editing efficiencies and will thereby help researchers improve genome editing technologies.https://doi.org/10.1371/journal.pone.0294146 |
| spellingShingle | Muhammad Nazmul Hasan Toshinori Hyodo Mrityunjoy Biswas Md Lutfur Rahman Yuko Mihara Sivasundaram Karnan Akinobu Ota Shinobu Tsuzuki Yoshitaka Hosokawa Hiroyuki Konishi Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene. PLoS ONE |
| title | Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene. |
| title_full | Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene. |
| title_fullStr | Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene. |
| title_full_unstemmed | Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene. |
| title_short | Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene. |
| title_sort | flow cytometry based quantification of genome editing efficiency in human cell lines using the l1cam gene |
| url | https://doi.org/10.1371/journal.pone.0294146 |
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