Genetic screens in isogenic mammalian cell lines without single cell cloning

Peter C. DeWeirdt, Annabel K. Sangree, Ruth E. Hanna, Kendall R. Sanson, Mudra Hegde, Christine Strand, Nicole S. Persky and John G. Doench ()
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Peter C. DeWeirdt: Broad Institute of MIT and Harvard
Annabel K. Sangree: Broad Institute of MIT and Harvard
Ruth E. Hanna: Broad Institute of MIT and Harvard
Kendall R. Sanson: Broad Institute of MIT and Harvard
Mudra Hegde: Broad Institute of MIT and Harvard
Christine Strand: Broad Institute of MIT and Harvard
Nicole S. Persky: Broad Institute of MIT and Harvard
John G. Doench: Broad Institute of MIT and Harvard

Nature Communications, 2020, vol. 11, issue 1, 1-15

Abstract: Abstract Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs of mammalian cells, however, is labor-intensive, time-consuming, and, in some cell types, essentially impossible. Here, we present an approach to create isogenic pairs of cells that avoids single cell cloning, and screen these pairs with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We query the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, identifying both expected and uncharacterized buffering and synthetic lethal interactions. Additionally, we compare acute CRISPR-based knockout, single cell clones, and small-molecule inhibition. We observe that, while the approaches provide largely overlapping information, differences emerge, highlighting an important consideration when employing genetic screens to identify and characterize potential drug targets. We anticipate that this methodology will be broadly useful to comprehensively study gene function across many contexts.

Date: 2020
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DOI: 10.1038/s41467-020-14620-6

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