Genetic screens in isogenic mammalian cell lines without single cell cloning

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 laborintensive, 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 smallmolecule 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.

Drug mechanism‐of‐action discovery through the integration of pharmacological and CRISPR screens

“…Altogether, these results highlight a functional link between MARCH5 and sensitivity to MCL1 inhibitors. This is consistent with recent reports of a synthetic‐lethal interaction between MARCH5 and the MCL1 negative regulator BCL2L1 (DeWeirdt et al , ), and MARCH5‐dependent degradation of the MCL1/NOXA complex (Djajawi et al , ; Haschka et al , ). With further investigation, the link between MCL1 and MARCH5 could shed light on the mechanism‐of‐action of MCL1 inhibitors and the development of stratification approaches in solid tumours, such as breast carcinomas.…”

Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors

“…This finding is in contrast to a number of recent studies that performed genome-scale CRISPR-Cas9 screens in VeroE6, Huh-7, Huh-7.5.1, Huh-7.5, and ACE2-overexpressing A549 cells, which failed to identify significant enrichment of the same PPI network ( Baggen et al., 2020 ; Daniloski et al., 2020 ; Heaton et al., 2020 ; Schneider et al, 2020 ; Wang et al., 2020 ; Wei et al., 2020 ; Zhu et al., 2020 ). We speculate that this difference might be due in part to well-established advantages of focused screens, including (1) improved signal-to-noise ratios in the absence of sgRNAs targeting genes that are likely to dominate the screen (e.g., the SARS-CoV-2 cellular receptor, ACE2), (2) increased coverage by including more sgRNAs per gene (ten versus four), (3) feasibility of exploring multiple screening conditions (e.g., different viruses and temperatures), and (4) higher screen representation (i.e., >1,500-fold versus < 750-fold), as well as the lower propensity of Huh-7.5 cells to undergo syncytia formation in relation to ACE2-overexpressing A549 cells, which potentially confounds pooled screening results ( DeWeirdt et al., 2020 ; Doench, 2018 ; Parnas et al., 2015 ; Schneider et al, 2020 ). Thus, deeply probing individual interactome proteins by using a focused sgRNA library coupled with functional readouts across multiple conditions allowed us to identify many host factors required for SARS-CoV-2 infection that were missed in genome-scale CRISPR screens.…”

“…CRISPR-Cas9 forward genetics and data from IP-MS studies can be combined in a high-coverage hypothesis-driven single guide RNA (sgRNA) screening campaign ( Kelly et al., 2020 ). Focused libraries offer several advantages: (1) increased coverage (smaller libraries allow interrogation of many sgRNAs per gene, thereby lowering the rate of false negatives), (2) increased resolution (smaller libraries ensure higher screen representation at every point of the screen), and (3) a wider range of screening conditions (smaller libraries are more compatible with highly parallel testing of multiple technical and biological conditions) ( DeWeirdt et al., 2020 ; Doench, 2018 ; Parnas et al., 2015 ).…”

An integrative oncogene-dependency map identifies unique vulnerabilities of oncogenic EGFR, KRAS, and RIT1 in lung cancer

“…Indeed, only one lung adenocarcinoma cell line has been identified with a canonical RIT1 M90I mutation 6 . An alternative strategy is CRISPR screens of isogenic cell models 20,23 in which introduced oncogenes confer a "selectable" phenotype to the cells. Because RIT1 variants can confer resistance to erlotinib in PC9 cells, an EGFR-mutant lung cancer cell line 10 , this drug resistance phenotype could provide a powerful screening system to probe the requirements for RIT1 function in a highly controlled and robust system.…”