An Electroporation-Based Protocol for Ex Vivo Base Editing: From Design to Quantitative Assessment
Year: 2026
Type: book-chapter
Abstract: Base editing is a CRISPR variant approach that enables single-nucleotide conversions without generating double-strand breaks. Cytosine and adenine base editors mediate C•G to T•A and A•T to G•C transitions, respectively, by coupling a deaminase to a catalytically impaired Cas9, a modified nuclease that lacks DNA cleavage activity but retains DNA binding capability. By avoiding double-strand breaks, base editing limits reliance on unpredictable end-joining pathways and facilitates more precise outcomes. This chapter provides a practical protocol for base editing by electroporation in adherent and suspension stem cells. The workflow spans guide design aligned with the editor activity window, in vitro synthesis of mRNA-based editor components, and electroporation for transient delivery. We detail essential steps for cell handling and recovery, followed by standard readouts. Analytical endpoints focus on targeted sequencing to quantify base conversion and assess editing specificity, with guidance for basic interpretation of results. Troubleshooting notes address frequent pitfalls and practical remedies to optimize performance across cell types.
Source: Methods in molecular biology
Institutions: Centre for Biomedical Network Research on Rare Diseases, Hospital Universitario Fundación Jiménez Díaz, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Universidad Carlos III de Madrid, University of Freiburg, University Medical Center Freiburg
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
OpenAlex ID: W7213980910