Protocol for clonal isolation of gene-edited hiPSCs using droplet and microfluidic sorting

Genetically engineered human induced pluripotent stem cells (hiPSCs) are vital for disease modeling and drug discovery, yet generating clonal lines efficiently post-editing remains challenging. Here, we present a protocol to generate clonal hiPSC lines after gene editing using either electrostatic droplet- or microfluidics-based sorting platforms. We describe steps for culturing hiPSCs, CRISPR-RNP electroporation, single-cell sorting, and expansion of gene-edited clones. Using this protocol, we generated over 100 clonal lines across seven knock-in/knock-out experiments, demonstrating broad utility and reproducibility. For additional details on the use and execution of this protocol, please refer to Patel et al. 1 • Clonal isolation and expansion of gene-edited hiPSCs using single-cell sorting • Single-cell sorting efficiencies in droplet- vs. microfluidics-based sorters • Post-sort recovery media supplements for efficient generation of clonal lines Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Genetically engineered human induced pluripotent stem cells (hiPSCs) are vital for disease modeling and drug discovery, yet generating clonal lines efficiently post-editing remains challenging. Here, we present a protocol to generate clonal hiPSC lines after gene editing using either electrostatic droplet- or microfluidics-based sorting platforms. We describe steps for culturing hiPSCs, CRISPR-RNP electroporation, single-cell sorting, and expansion of gene-edited clones. Using this protocol, we generated over 100 clonal lines across seven knockin/knockout experiments, demonstrating broad utility and reproducibility.