Biology subjects

Garrity, A. J.

Publications and source records attributed to Garrity, A. J..

2 recordsLinked to original sources

bioRxiv2018

Pooled optical screens in human cells

Large-scale genetic screens play a key role in the systematic discovery of genes underlying cellular phenotypes. Pooling of genetic perturbations greatly increases screening throughput, but has so far been limited to screens of enrichments defined by cell fitness and flow cytometry, or to comparatively low-throughput single cell gene expression profiles. Although microscopy is a rich source of spatial and temporal information about mammalian cells, high-content imaging screens have been restricted to much less efficient arrayed formats. Here, we introduce an optical method to link perturbations and their phenotypic outcomes at the singlecell level in a pooled setting. Barcoded perturbations are read out by targeted in situ sequencing following image-based phenotyping. We apply this technology to screen a focused set of 952 genes across >3 million cells for involvement in NF-{kappa}B activation by imaging the translocation of RelA (p65) to the nucleus, recovering 20 known pathway components and 3 novel candidate positive regulators of IL-1{beta} and TNF-stimulated immune responses.

genomics↗

bioRxiv2018

Lentiviral co-packaging mitigates the effects of intermolecular recombination and multiple integrations in pooled genetic screens

Lentiviral vectors are widely used for functional genomic screens, enabling efficient and stable transduction of target cells with libraries of genetic elements. Unfortunately, designs that rely on integrating multiple variable sequences, such as combinatorial perturbations or perturbations linked to barcodes, may be compromised by unintended consequences of lentiviral packaging. Intermolecular recombination between library elements and integration of multiple perturbations (even at limiting virus dilution) can negatively impact the sensitivity of pooled screens. Here, we describe a simple approach to prevent recombination between lentiviral vectors containing multiple linked variable elements, such as the recently reported CRISP-seq, Perturb-seq, and Mosaic-seq designs. We show that modifying the packaging protocol by diluting the perturbation library with a carrier plasmid increases the fraction of correct, single integrations from <60% to >90%, at the cost of reducing titer by 100-fold.

genomics↗