An Electroporation-Based Protocol for Ex Vivo Base Editing: From Design to Quantitative Assessment.
TL;DR
This chapter provides a practical protocol for base editing by electroporation by electroporation in adherent and suspension stem cells and details essential steps for cell handling and recovery, followed by standard readouts.
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.