Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Vienna, Austria.
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC),Vienna, Austria.
Nat Methods. 2017 Dec;14(12):1191-1197. doi: 10.1038/nmeth.4466. Epub 2017 Oct 16.
Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.
基于 CRISPR 的文库筛选是评估基因功能的有力工具。然而，传统的分析方法完全依赖于群体间整合的单指导 RNA（sgRNA）的相对丰度，无法区分由相同 sgRNA 产生的不同表型和编辑结果。在这里，我们提出了 CRISPR-UMI，这是一种用于基于池的筛选的单细胞谱系追踪方法，可以解决细胞异质性问题。我们生成了具有独特分子标识符（UMI）的复杂 sgRNA 文库，允许对克隆扩增的、单独标记的细胞进行筛选。通过考虑潜在的细胞和编辑结果异质性以及检测异常克隆，与传统分析相比，基于原理验证的 CRISPR-UMI 负选择筛选提供了更高的灵敏度和稳健性。此外，CRISPR-UMI 正选择筛选揭示了重新编程小鼠胚胎成纤维细胞为多能干细胞过程中的新障碍，区分了重编程的频率和速度（即效应大小和概率）。CRISPR-UMI 提高了基于池的 CRISPR 筛选的预测能力、灵敏度和信息量。
