CD34+ cells are a prime target for therapeutic strategies for gene editing because modified progenitor cells have the capacity to differentiate through an erythropoietic lineage. While experimental advances have been reported, by and large, the associated experimental protocols have been less than clear or robust. As such, we evaluated the relationship among cellular delivery, nuclear uptake, often viewed as the benchmark metric of successful gene editing, and single base repair. We took a combinatorial approach using single stranded oligonucleotide and a CRISPR/Cas9 ribonucleoprotein to convert wild type HBB into the Sickle Cell genotype by evaluating conditions for two common delivery strategies of gene editing tools into CD34+ cells. Confocal microscopy data show that the CRISPR/Cas9 ribonucleoprotein tends to accumulate at the outer membrane of the CD34+ cell nucleus when NEON Transfection System is employed, while the ribonucleoproteins do pass into the cell nucleus when Nucleofection is used. Yet, despite the high efficiency of cellular transformation, and the traditional view of success in efficient nuclear uptake, neither delivery methodology enabled gene editing activity. Our results indicate that more stringent criteria must be established to facilitate the clinical translation and scientific robustness of gene editing for Sickle Cell Disease.

CD34 +细胞是基因编辑治疗策略的主要靶标，因为修饰的祖细胞具有通过促红细胞系分化的能力。虽然总体上已经报道了实验进展，但是相关的实验方案还不够清楚或可靠。因此，我们评估了细胞递送，核摄取（通常被视为成功进行基因编辑和单碱基修复的基准指标）之间的关系。我们采用单链寡核苷酸和CRISPR / Cas9核糖核酸蛋白的组合方法，通过评估将基因编辑工具导入CD34 +细胞的两种常见递送策略的条件，将野生型HBB转化为镰状细胞基因型。共聚焦显微镜数据显示，当使用NEON转染系统时，CRISPR / Cas9核糖核蛋白倾向于积聚在CD34 +细胞核的外膜上，而当使用核转染时，核糖核蛋白确实会进入细胞核中。然而，尽管细胞转化效率很高，并且传统观点认为有效的核吸收成功，但两种递送方法均未启用基因编辑活性。我们的结果表明必须建立更严格的标准，以促进镰状细胞病的基因翻译的临床翻译和科学鲁棒性。和传统的成功吸收核能的观点一样，两种递送方法都无法实现基因编辑活动。我们的结果表明必须建立更严格的标准，以促进镰状细胞病的基因翻译的临床翻译和科学鲁棒性。和传统的成功吸收核能的观点一样，两种递送方法都无法实现基因编辑活动。我们的结果表明必须建立更严格的标准，以促进镰状细胞病的基因翻译的临床翻译和科学鲁棒性。