Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding
Michael Boettcher,
Sergio Covarrubias,
Anne Biton,
James Blau,
Haopeng Wang,
Noah Zaitlen,
Michael T. McManus
Affiliations
Michael Boettcher
Department of Microbiology and Immunology, UCSF Diabetes Center, University of California, San Francisco
Sergio Covarrubias
Department of Microbiology and Immunology, UCSF Diabetes Center, University of California, San Francisco
Anne Biton
Department of Medicine, Lung Biology Center, University of California, San Francisco
James Blau
Department of Microbiology and Immunology, UCSF Diabetes Center, University of California, San Francisco
Haopeng Wang
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
Noah Zaitlen
Department of Medicine, Lung Biology Center, University of California, San Francisco
Michael T. McManus
Department of Microbiology and Immunology, UCSF Diabetes Center, University of California, San Francisco
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.
