As CRISPR-based therapies enter the clinic, evaluation of safety remains a critical and active area of study. Here, we employ a clinical next generation sequencing (NGS) workflow to achieve high sequencing depth and detect ultra-low frequency variants across exons of genes associated with cancer, all exons, and genome wide. In three separate primary human hematopoietic stem and progenitor cell (HSPC) donors assessed in technical triplicates, we electroporated high-fidelity Cas9 protein targeted to three loci (AAVS1, HBB, and ZFPM2) and harvested genomic DNA at days 4 and 10. Our results demonstrate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP in a gRNA spacer sequence is sufficient to eliminate Cas9 off-target activity in primary, repair-competent human HSPCs.

随着基于 CRISPR 的疗法进入临床，安全性评估仍然是一个关键且活跃的研究领域。在这里，我们采用临床下一代测序 (NGS) 工作流程来实现高测序深度并检测与癌症相关的基因外显子、所有外显子和全基因组的超低频变异。在以技术三次重复评估的三个独立的原代人类造血干细胞和祖细胞 (HSPC) 供体中，我们电穿孔针对三个基因座（AAVS1、 HBB和ZFPM2 ）的高保真 Cas9 蛋白，并在第 4 天和第 10 天收获基因组 DNA。证明临床相关的高保真 Cas9 递送至原代 HSPC 和离体培养长达 10 天不会引入或富集致瘤变异，并且即使 gRNA 间隔序列中的单个 SNP 也足以消除 Cas9 脱靶活性具有修复能力的初级人类 HSPC。