We varied the sgRNA concentrations from 4–8 µg, Cas9 concentrations from 7.5–15 µg, and ssODNs from 1.35–8.1 µg in 2.0 × 105 CD34+ cells and used program ER-100 on the Lonza 4D nucleofector. The Lonza P3 Primary cell 4DNucleofector X-Kit was used for these experiments. The optimized RNP condition and ssODN used in all the experiments in the manuscript was 8 µg sgRNA to 15 µg Cas9, which is a 2:1 pmol ratio of sgRNA to Cas9. In addition, a 5.4 µg concentration of the 72-mer ssODN was the optimal concentration. Preliminary work showed optimization of nucleofection kit on a variety of CD34+ cell sources (i.e., donors) based on the pmax GFP plasmid uptake analyzed by flow cytometry. From the optimized parameters of the nucleofector and Lonza kit, the optimal transfection efficiency was chosen, and the same protocol was used for subsequent CD34+ targeted samples.