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Reposted by Will Allen

Xiaowei Zhuang's Lab @zhuanglab.bsky.social · 25/11/2024

Check out this preprint on Perturb-multi (doi.org/10.1101/2024...), a multi-year collaborative effort that enabled multi-modal in vivo genotype-phenotype mapping by pooled genetic perturbations and high-dimensionality phenotype readout by imaging and sequencing. bsky.app/profile/weal...

doi.org

A platform for multimodal in vivo pooled genetic screens reveals regulators of liver function

Organ function requires coordinated activities of thousands of genes in distinct, spatially organized cell types. Understanding the basis of emergent tissue function requires approaches to dissect the...

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Will Allen @weallen1.bsky.social · 21/11/2024

My lab at @devbiostanford.bsky.social (www.allenlabstanford.org) is looking for motivated postdocs and graduate students who are excited about developing new technology and applying in vivo genetic screens to reverse-engineer tissue function. Contact me at wallen@stanford.edu if you are interested!

allenlabstanford.org

Allen Lab

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Will Allen @weallen1.bsky.social · 21/11/2024

This was an amazing team effort over multiple years, bringing together diverse expertise from Harvard and @whiteheadinstitute.bsky.social @mitofficial.bsky.social @hhmi.bsky.social . We look forward to expanding to the genome-wide scale and extending to other organs!

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Will Allen @weallen1.bsky.social · 21/11/2024

Along with recent spatial developments – e.g. Perturb-FISH, Perturb-View and CRISPR-Map – and fantastic in vivo work from Xin Jin and Randall Platt, this is an extremely exciting time for pooled screening, particularly with large-scale AI efforts (Virtual Cells) on the horizon.

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Will Allen @weallen1.bsky.social · 21/11/2024

his work builds on foundational Perturb-Seq dating back to 2016 from the Weissman @jswlab.bsky.social and Regev Labs, and pioneering work on imaging-based pooled genetic screening using MERFISH dating back to 2017 from the Zhuang and Elf labs, and using ISS from 2019 from the Blainey Lab.

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Will Allen @weallen1.bsky.social · 21/11/2024

By integrating sequencing and imaging, we can achieve insights that would be difficult from each modality alone, such as convergent effects of mechanistically distinct pathways that all produce cellular steatosis.

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Will Allen @weallen1.bsky.social · 21/11/2024

A core aspect of liver organization and function is hepatocyte zonation. Our data revealed both expected regulators of Zonation (Wnt signaling, O2 sensing) as well as potentially novel pathways, in a cell autonomous manner. This work reveals the highly dynamic nature of zonation state.

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Will Allen @weallen1.bsky.social · 21/11/2024

Because these genetic perturbations are introduced into a living mouse, we can look at the interactions between gene function and physiological state. We can identify functional re-wiring following overnight fasting, which leads to dramatic metabolic state-dependent responses to some perturbations

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Will Allen @weallen1.bsky.social · 21/11/2024

We can then zoom in to identify the specific functional effects of perturbing different genes in an automated and unsupervised way. Combining subcellular imaging with deep learning-based embeddings identified Npc1 as a regulator of lysosome structure (measured through Cathepsin B staining)

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Will Allen @weallen1.bsky.social · 21/11/2024

This rich, multimodal data enables multiple analyses of how genetic perturbations affect multiple aspects of tissue structure and function from the same animal. At a high level, global multimodal analysis of different perturbations allows us to group them into shared pathways.

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Will Allen @weallen1.bsky.social · 21/11/2024

In mosaic perturbed mouse livers, we detected small clumps of cells with the same perturbation and measured potent transcriptional phenotypes.

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Will Allen @weallen1.bsky.social · 21/11/2024

We apply this approach in the mouse liver, using mosaic CRISPR KO via neonatal lenti delivery (developed by Kristin Knouse) followed by AAV-mediated induction of Cas9 activity in the adult animal to test how perturbation of hundreds of genes impact diverse aspects of hepatocyte structure + function

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Will Allen @weallen1.bsky.social · 21/11/2024

Applying this approach in wild type livers revealed rich complexity of single-cell morphologies that can be related to heterogeneity in transcriptional state in the same cells. We believe this will be broadly useful for mapping tissue structure even in un-perturbed tissue.

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Will Allen @weallen1.bsky.social · 21/11/2024

Multiplexed protein and RNA imaging produces incredibly rich datasets of diverse aspects of subcellular morphology. XingJie Pan developed a deep learning approach, building on Loic Royer's CytoSelf, that allows us to quantitatively analyze millions of pictures of cells.

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Will Allen @weallen1.bsky.social · 21/11/2024

Through extensive optimization we improved the efficiency of RCA-MERFISH in heavily fixed tissue by orders of magnitude, and made it compatible with multiplexed immunostaining – allowing spatially-resolved protein+RNA readout in the same cells w/ fully automated fluidics and imaging.

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Will Allen @weallen1.bsky.social · 21/11/2024

To enable RCA-MERFISH, we developed new methods for large-scale probe synthesis from femtomolar Twist pools of oligos. This solves a major problem of using long (>100 mer) probes and enables genome-scale CRISPR library readout.

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Will Allen @weallen1.bsky.social · 21/11/2024

Combining RCA-MERFISH for imaging cellular RNAs and perturbation barcodes with multiplexed immunofluorescence for imaging proteins and cell morphology, our approach enables robust in vivo imaging-based pooled genetic screening of complex tissues.

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Will Allen @weallen1.bsky.social · 21/11/2024

Building on MERFISH developed by Xiaowei Zhuang’s lab and RCA and related methods developed by Mats Nilsson, Karl Deisseroth, and Xiao Wang, we develop RCA-MERFISH to amplify and readout endogenous gene expression and short RNA barcodes in tissue sections.

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Will Allen @weallen1.bsky.social · 21/11/2024

Leveraging @10xgenomics.bsky.social Flex technology, we develop fixed-cell Perturb-Seq with direct hybridization to sgRNAs. The ability to use fixed cells makes large-scale in vivo Perturb-seq drastically easier and cheaper, while accurately measuring cellular gene expression.

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Will Allen @weallen1.bsky.social · 21/11/2024

This preprint is the result of years of work developing and optimizing powerful new tools for large-scale in vivo pooled genetic screens in heavily fixed tissue, with sequencing- and imaging-based readouts of genome-wide gene expression and multiplexed subcellular morphology.

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Will Allen @weallen1.bsky.social · 21/11/2024

Organ function requires coordinated activities of 1000s of genes in spatially organized cell types under conditions not replicable in vitro. Understanding the molecular basis of emergent tissue function requires new approaches to dissecting diverse cellular+tissue phenotypes in live animals

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