Even so, and despite amazing recent advances in this space, such as www.cell.com/cell/fulltex... from @weissmanlab.bsky.social, these studies can still be quite difficult to execute at genome-scale. So we turned to another approach: optical fluorescence imaging. - ThreadSky

jtneal.bsky.social • 30 days ago

Even so, and despite amazing recent advances in this space, such as https://www.cell.com/cell/fulltext/S0092-8674(22)00597-9 from @weissmanlab.bsky.social, these studies can still be quite difficult to execute at genome-scale. So we turned to another approach: optical fluorescence imaging.

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jtneal.bsky.social•30 days ago

Cell images, especially multicolor fluorescence images, are an immense source of phenotypic information, and thanks to approaches like Cell Painting from @drannecarpenter.bsky.social , we can extract this information as quantitative features (size, shape, texture, etc.) in automated fashion.

jtneal.bsky.social•30 days ago

Such data can be generated easily & cheaply, but historically hasn’t been ideal for large-scale functional genomics because imaging experiments could not use pooled genetic perturbation libraries (no way to optically determine which cell received which perturbation).

jtneal.bsky.social•30 days ago

Fortunately, the Blainey Lab and others solved this problem by developing methods to sequence individual perturbation barcodes using in situ sequencing, so we can now assign quantitative cell phenotypes AND barcodes to individual cells, enabling optical pooled screens. https://www.cell.com/cell/fulltext/S0092-8674(19)31067-0

jtneal.bsky.social•30 days ago

So, we teamed up with Anne and Paul’s lab, along with Calvin Jan at Calico, with the aim of building an accessible & unbiased high-content cell profiling platform that could be applied to genome-scale CRISPR screens (Project PERISCOPE).

jtneal.bsky.social•30 days ago

To do this, we first built a variant of the Cell Painting panel that allowed destaining of fluorophores from a subset of markers (via cleavable disulfide or azidomethyl linker) so that we could perform four color in situ sequencing-by-synthesis without fluorescence overlap.

jtneal.bsky.social•30 days ago

We ( @erinweisbart.bsky.social , @bethcimini.bsky.social , Greg Way) also built scalable, distributed open source analysis pipelines for alignment/cropping/background correction/barcode calling/etc. allowing us to turn millions of fluorescence images into sgRNA-assigned single cell profiles

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