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 Streptococcus pyogenes Cas9 (spCas9) 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 in silico models is unclear. By screening more than 120 gRNAs, our data demonstrate that in silico gRNA prediction models are ineffective. In this study, we established a time- and cost-efficient in vitro transcribed gRNA screening model in K562 cells that predicts effective gRNAs for HSPCs. RNP based screening thus outperforms in silico modeling and we report that gene editing is equally efficient in distinct CD34⁺ HSPC subpopulations. Furthermore, no effects on cell proliferation, differentiation, or in vitro hematopoietic lineage commitment were observed. Finally, no upregulation of p21 expression was found, suggesting unperturbed HSPC homeostasis.

中文翻译：

---

优化的向导 RNA 选择改善化脓性链球菌 Cas9 基因编辑人类造血干细胞和祖细胞

核糖核蛋白 (RNP) 经常用于治疗性基因编辑和基础研究，因为该方法速度快、无病毒且脱靶效应最少。我们使用化脓性链球菌Cas9 ( sp Cas9) RNP评估了基因工程人类造血干细胞和祖细胞 (HSPC) 的各种参数，并实现了高达 80% 的基因编辑效率。我们发现向导 RNA (gRNA) 设计对于实现高基因编辑效率至关重要。然而，为 HSPC 寻找有效的 gRNA 可能具有挑战性，而许多计算机模型的贡献尚不清楚。通过筛选 120 多个 gRNA，我们的数据表明在计算机中gRNA 预测模型无效。在这项研究中，我们在 K562 细胞中建立了一种时间和成本效益高的体外转录 gRNA 筛选模型，可预测 HSPC 的有效 gRNA。因此，基于 RNP 的筛选优于计算机建模，我们报告说，基因编辑在不同的 CD34 ⁺ HSPC 亚群中同样有效。此外，未观察到对细胞增殖、分化或体外造血谱系定型的影响。最后，未发现 p21 表达上调，表明 HSPC 稳态不受干扰。