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.
Highlights
Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function
Genetic screens with CRISPR technology often start with the creation of a cell line stably expressing Cas[9], integrated into the genome via lentivirus or piggybac transposase[21,22]
Here we present a facile approach for generating genome-wide genetic interaction maps for individual genes in cell types of interest using CRISPR technology
Summary
Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. 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. RNAi5 and CRISPR technology[6,7,8,9,10] have been used to study pairwise interactions for up to hundreds of genes[11]; screening all combinations of protein coding genes in the human genome would require, at bare minimum, approximately 400 million perturbations and 200 billion cells, which is equivalent to 5000 concurrent genome-wide screens This scale is exacerbated by the diversity of cell types in which to study such interactions. The rich set of resulting genetic interactions shown here coupled with the ease of conducting such screens illustrate the power of this technology
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