Optimized Guide RNA Selection Improves Streptococcus pyogenes Cas9 Gene Editing of Human Hematopoietic Stem and Progenitor Cells.

Ribonucleoproteins (RNPs) are frequently applied for therapeutic gene editing as well as fundamental research because the method is fast, viral free, and shows fewest off target effects. We evaluated various parameters to genetically engineer human hematopoietic stem and progenitor cells (HSPCs) using Cas9 (Cas9) RNPs, and achieve gene editing efficiencies up to 80%. We find that guide RNA (gRNA) design is critical to achieve high gene editing efficiencies. However, finding effective gRNAs for HSPCs can be challenging, while the contribution of numerous models is unclear. By screening more than 120 gRNAs, our data demonstrate that gRNA prediction models are ineffective. In this study, we established a time- and cost-efficient transcribed gRNA screening model in K562 cells that predicts effective gRNAs for HSPCs. RNP based screening thus outperforms modeling and we report that gene editing is equally efficient in distinct CD34 HSPC subpopulations. Furthermore, no effects on cell proliferation, differentiation, or hematopoietic lineage commitment were observed. Finally, no upregulation of p21 expression was found, suggesting unperturbed HSPC homeostasis.