Electroporation for High-Efficiency Delivery of CRISPR to Hematopoietic Cells.

Recent advances in genome editing technologies have enabled transformative therapeutic strategies for hematological disorders. Efficient implementation of these approaches requires reliable delivery of genome editing components into primary hematopoietic stem and progenitor cells (HSPCs), which are particularly sensitive and resistant to conventional transfection methods. Electroporation has emerged as the most widely used strategy for ex vivo delivery of genome editing reagents into CD34⁺ hematopoietic cells. Genome editing tools can be delivered in multiple formats, including plasmid DNA, messenger RNA (mRNA), and ribonucleoprotein (RNP) complexes. In HSPCs, mRNA- and RNP-based approaches are generally better tolerated than plasmid DNA and allow transient expression with reduced cytotoxicity. In this chapter, we describe optimized protocols for electroporation-based delivery of CRISPR/Cas9 RNP complexes and mRNA-encoded base editors into HSPCs using the Amaxa™ 4D-Nucleofector™ system. Detailed procedures are provided for cell preparation, guide RNA design, assembly of Cas9 RNPs, in vitro transcription and purification of base editor mRNA, electroporation parameters, and assessment of editing efficiency. Emphasis is placed on maximizing editing performance while preserving stem cell viability and functionality.