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

Michlits, Georg and Hubmann, Maria and Wu, Szu-Hsien and Vainorius, Gintautas and Budusan, Elena and Zhuk, Sergei and Burkhard, Thomas and Novatchkova, Maria and Aichinger, Martin and Lu, Yiqing and Reece-Hoyes, John and Nitsch, Roberto and Schramek, Daniel and Hoepfner, Dominic and Elling, Ulrich (2017) CRISPR-UMI : single-cell lineage tracing of pooled CRISPR–Cas9 screens. Nature methods, 14 (12). pp. 1191-1197.

Abstract

Pooled CRISPR screens are a powerful tool to assign gene function. However, conventional analysis is based on the relative abundance of integrated sgRNAs and thus falls short in accounting for heterogeneity within screened cells due to background mutations and variable editing outcomes. Based on the conceptual notion that that genetics at single cell level can be considered binary -loss-of-function or neutral/other- we present CRISPR DigDeep (Differentiating genotypes deduced from enrichment of phenotypes), a single cell lineage tracing methodology for pooled screening, that accounts for both cell heterogeneity as well as various editing outcomes. We achieve single cell-level resolution by generating complex sgRNA retroviral libraries combined with unique molecular identifiers (UMIs) and clonal expansion of individually infected and tagged cells, thereby tracking single editing outcomes. This method overcomes the limit of maximal achievable sgRNA depletion levels for negative selection screens, that are due to incomplete generation of loss of function alleles. We performed a pooled CRISPR screen to identify genes that rendered cells vulnerable to treatment with the chemotherapeutic drug etoposide. CRISPR DigDeep displayed increased sensitivity and robustness of screening outcomes compared to conventional analysis due to presence of outlier clones, that typically remain undetected and interfere with conventional analysis. Furthermore, we used CRISPR DigDeep to analyze a stochastic positive selection scenario. We identified new potent roadblocks for reprogramming mouse embryonic fibroblasts to induced pluripotent stem cells and gained biological insight by comparing reprogramming frequency to speed of reprogramming revealed by CRISPR DigDeep. Taken together, CRISPR DigDeep boosts the predictive power and sensitivity of pooled CRISPR screens.

Item Type: Article
Date Deposited: 19 Jan 2018 00:45
Last Modified: 19 Jan 2018 00:45
URI: https://oak.novartis.com/id/eprint/32647

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