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Nat Methods|一种单细胞谱系追踪方法,用于CRISPR-Cas9 池筛选...

已有 3529 次阅读 2017-12-17 08:51 |个人分类:CRISPR-UMI: single-cell lineage tracing|系统分类:科研笔记| CRISPR, 编辑方法

2017 Dec;14(12):1191-1197. doi: 10.1038/nmeth.4466. Epub  2017 Oct 16.

CRISPR-UMI: single-cell lineage tracing of pooled CRISPR-Cas9 screens.

Abstract

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.

PMID:
29039415
DOI:
10.1038/nmeth.4466




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