Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA.
Novartis Institutes for BioMedical Research, Cambridge, MA, USA.
Nat Methods. 2018 Nov;15(11):941-946. doi: 10.1038/s41592-018-0149-1. Epub 2018 Oct 8.
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研究。目前，为了实现筛选所需的高表达和均一的 Cas9 表达，需要对稳定且克隆的 Cas9 表达细胞进行工程改造——这种方法不适用于人原代细胞。Guide Swap 通过利用意想不到的发现，即当 Cas9 与非靶向 gRNA 一起引入时，通过慢病毒递送的靶向向导 RNA (gRNA) 进行编辑会非常有效，从而在人原代细胞中实现了基于基因组规模的 pooled 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，我们在体外造血模型中实施了 Guide Swap，并鉴定了已知和以前未知的 CD34 造血干细胞和祖细胞 (HSPC) 扩增的调节因子。我们预计这个平台将广泛适用于其他具有挑战性的细胞类型，因此将能够在以前无法进入但具有生物学相关性的人原代细胞系统中进行发现。
