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BMC Genomics. 2019 Feb 6;20(1):107. doi: 10.1186/s12864-019-5480-0.

Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding.

Author information

1
Department of Microbiology and Immunology, UCSF Diabetes Center, University of California, San Francisco, San Francisco, CA, 94143, USA.
2
Department of Medicine, Lung Biology Center, University of California, San Francisco, San Francisco, 94143, CA, USA.
3
Institut Pasteur, Hub Bioinformatique et Biostatistique, Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI, USR 3756 Institut Pasteur et CNRS), Paris, France.
4
Departments of Medicine and of Microbiology & Immunology, the Rosalind Russell-Ephraim P. Engleman Medical Research Center for Arthritis, and the Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, 94143, USA.
5
Department of Microbiology and Immunology, UCSF Diabetes Center, University of California, San Francisco, San Francisco, CA, 94143, USA. Michael.McManus@ucsf.edu.

Abstract

BACKGROUND:

While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have thus far not investigated the influence of cellular heterogeneity on screen results. Instead most screens are analyzed by averaging the abundance of perturbed cells from a bulk population of cells.

RESULTS:

Here we developed multi-level barcoded sgRNA libraries to trace multiple clonal Cas9 cell lines exposed to the same environment. The first level of barcoding allows monitoring growth kinetics and treatment responses of multiplexed clonal cell lines under identical conditions while the second level enables in-sample replication and tracing of sub-clonal lineages of cells expressing the same sgRNA.

CONCLUSION:

Using our approach, we illustrate how heterogeneity in growth kinetics and treatment response of clonal cell lines impairs the results of pooled genetic screens.

KEYWORDS:

CRISPR; Clonal heterogeneity; Genome editing; Screening

PMID:
30727954
PMCID:
PMC6364396
DOI:
10.1186/s12864-019-5480-0
[Indexed for MEDLINE]
Free PMC Article

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