iiinova — where your digital self keeps evolving

Also a big thank you goes to all other authors, the Austrian Academy of Sciences @oeaw.bsky.social , and all the friends at @imbavienna.bsky.social and beyond that supported this work in so many ways. We will always know who to be grateful to! Thanks for reading this absurdly long thread 🙈 18/

Last but not least a huge thanks to the heroes of the work, foremost Esther Uijttewaal for driving it home, but also the people that laid the critical ground work such as Naomi Botay and Annika Sell, as well as Joonsun Lee who developed a CRISPR-StAR validation system. 👏🏼👏🏼👏🏼🙇🏼 17/

This tread will never do justice to the wealth of results by years of dedicated work by so many wonderful scientists! Please read the paper, it’s open access! CRISPR-StAR truly has the potential open the door to high throughput genetics for so many disease models. 16/ www.nature.com/articles/s41...

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This allowed us to map the in vivo essentialome of therapy resistant melanoma next to the cell culture model and uncovered many genetic dependencies, that only hold true in vivo. Oxidative phosphorylation e.g. is a strong genetic dependency only in vivo. 15/

With this we dared a genome wide screen in a cell line with particularly poor engraftment ability. And sure enough conventional screening failed completely at a complexity of <2cells/sgRNA, but CRRISPR-StAR delivered and nicely separated positive and negative controls. 14/

POC experiments in vitro showed the power of this approach. At 1000 cells per sgRNA conventional screening paradigms work very well. Of course! But as you go down e.g. to 4 cells/sgRNA it’s a mess. 13/

This controls for intrinsic and extrinsic heterogeneity. It further samples only the desired cell type and time as editing is controlled by CreERT induction. That’s all possible by intercalated loxP sites and Stochastic Activation by Recombination: CRISPR-StAR 12/

For example upon engraftment in an experimental xenograft setting, half of the cells of a clonal population will activate the sgRNA while the other half will remain inactive. As phenotypes manifest, the salt-and-pepper control population will experience all the same complex biology. 11/

Now all ingredients were ready to build an entirely new paradigm of CRISPR screening. Instead of comparing sgRNA abundance to the historical control of before phenotypes manifest, we run an internal control population. 10/

Last piece in the puzzle: We needed a much better sgRNA prediction algorithm to empower CRISPR screening in vivo. We also developed that by implementing target protein features to sgRNA design. 9/ www.nature.com/articles/s41...

We then developed inducible sgRNAs to allow for controlling of gene editing activity. This system allowed for inducing as well as inactivating of sgRNAs to enable e.g. time-controlled sequential editing. 8/ www.nature.com/articles/s41...

CRISPR-UMI: single-cell lineage tracing of pooled CRISPR–Cas9 screens - Nature Methods

For pooled CRISPR screening we had also introduced the analysis of single cell derived clones by implementation of unique molecular identifiers (UMIs) to control for heterogeneity. 7/ www.nature.com/articles/nme...

nature.com

Combining each sgRNA with a unique molecular identifier in a genome-wide screen increases sensitivity and robustness in both positive and negative selection.

A reversible haploid mouse embryonic stem cell biobank resource for functional genomics - Nature

Step back in the endeavour: a few years ago already we developed a biobank of embryonic stem cell lines that allowed for studying gene function vis a vis the unperturbed internal control by conditional mutagenesis. 6/ www.nature.com/articles/nat...

nature.com

The Haplobank contains over 100,000 individually reversibly mutagenized, barcoded, mouse embryonic cell lines; proof-of-principle experiments were used to search for genes that are required for rhinov...

Yet if we want to truly understand biology, we need to embrace its complexity by studying it in it’s native environment. You will not understand the function of the parts of a sailboat unless you study it in it’s native environment. 5/

Moreover, single cell derived clones will grow at very heterogeneous rates reflective both of the cellular heterogeneity (e.g. stem cells in organoids) as well as the complexity of the environment. 4/