RRC ID 77373
Author Hasan MN, Hyodo T, Biswas M, Rahman ML, Mihara Y, Karnan S, Ota A, Tsuzuki S, Hosokawa Y, Konishi H.
Title Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.
Journal PLoS One
Abstract 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.
Volume 18(11)
Pages e0294146
Published 2023-1-1
DOI 10.1371/journal.pone.0294146
PII PONE-D-23-14978
PMID 37943774
PMC PMC10635454
MeSH CRISPR-Cas Systems / genetics Cell Line Flow Cytometry Gene Editing* / methods Humans Neural Cell Adhesion Molecule L1* / genetics
Human and Animal Cells CHP-134(RCB0487) LA-N-5(RCB0485)