CRISPR–Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells—an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR–Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4⁺ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34⁺ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.

CRISPR–Cas9筛选允许对基因功能进行全基因组查询。当前，为了实现筛选所期望的高且均匀的Cas9表达，需要工程化稳定且克隆的表达Cas9的细胞-一种不适用于人类原代细胞的方法。通过利用出乎意料的发现，即当将Cas9与非靶向gRNA结合引入复合物中时，有效地进行了慢病毒递送的靶向性指导RNA（gRNA）的编辑，Guide Swap可以在人类原代细胞中进行基因组规模的CRISPR-Cas9筛选。我们在CD4 ⁺ T细胞的耗竭和富集筛选中验证了Guide Swap 。接下来，我们在离体造血模型中实施了Guide Swap，并确定了已知和以前未知的CD34 ⁺调节剂造血干细胞和祖细胞（HSPC）的扩增。我们预计该平台将广泛适用于其他具有挑战性的细胞类型，因此将使人们能够在以前无法接近但与生物学相关的人类原代细胞系统中进行发现。