In this publication Wu et al. are using Nucleofection to obtain highly efficient and selection-free method for genome editing in human hematopoietic stem cells. CD34+ HSPCs isolated from mobilized peripheral blood were thawed into X-VIVO 15 (Lonza, 04–418Q) supplemented with 100ng/ml human stem cell factor (SCF), 100ng/ml human thrombopoietin (TPO), and 100ng/ml recombinant human Flt3-ligand (Flt3-L).
HSPCs were electroporated with Cas9 RNP 24h afer thawing and maintained in X-VIVO media with cytokines. Electroporation was performed using Lonza P3 Primary Cell 4D Nucleofector Kit (V4XP-3032 for 20µl Nucleocuvette Strips or V4XP-3024 for 100µl Nucleocuvettes) using the program EO-100.
For 20µl Nucleocuvette Strips, the RNP complex was prepared by mixing 200 pmol Cas9 and 200 pmol sgRNA and incubating for 15min at room temperature immediately before electroporation. For indicated experiments in which glycerol was supplemented, 30% glycerol solution was added to Cas9 protein before addition of sgRNA. HSPCs (50000 ) resuspended in 20µl P3 solution were mixed with RNP and transferred to a cuvette for electroporation.