protoSpaceJAM: an open-source, customizable and web-accessible design platform for CRISPR/Cas insertional knock-in.

CRISPR/Cas-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas insertional knock-in experiments, currently supporting SpCas9, SpCas9-VQR and enAsCas12a Cas enzymes. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce 'recoding' mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAM's design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for deep sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool at protospacejam.czbiohub.org or as a standalone Python package at github.com/czbiohub-sf/protoSpaceJAM.

An open-source FACS automation system for high-throughput cell biology.

Recent advances in gene editing are enabling the engineering of cells with an unprecedented level of scale. To capitalize on this opportunity, new methods are needed to accelerate the different steps required to manufacture and handle engineered cells. Here, we describe the development of an integrated software and hardware platform to automate Fluorescence-Activated Cell Sorting (FACS), a central step for the selection of cells displaying desired molecular attributes. Sorting large numbers of samples is laborious, and, to date, no automated system exists to sequentially manage FACS samples, likely owing to the need to tailor sorting conditions ("gating") to each individual sample. Our platform is built around a commercial instrument and integrates the handling and transfer of samples to and from the instrument, autonomous control of the instrument's software, and the algorithmic generation of sorting gates, resulting in walkaway functionality. Automation eliminates operator errors, standardizes gating conditions by eliminating operator-to-operator variations, and reduces hands-on labor by 93%. Moreover, our strategy for automating the operation of a commercial instrument control software in the absence of an Application Program Interface (API) exemplifies a universal solution for other instruments that lack an API. Our software and hardware designs are fully open-source and include step-by-step build documentation to contribute to a growing open ecosystem of tools for high-throughput cell biology.

Mantis: high-throughput 4D imaging and analysis of the molecular and physical architecture of cells.

High-throughput dynamic imaging of cells and organelles is essential for understanding complex cellular responses. We report Mantis, a high-throughput 4D microscope that integrates two complementary, gentle, live-cell imaging technologies: remote-refocus label-free microscopy and oblique light-sheet fluorescence microscopy. Additionally, we report shrimPy, an open-source software for high-throughput imaging, deconvolution, and single-cell phenotyping of 4D data. Using Mantis and shrimPy, we achieved high-content correlative imaging of molecular dynamics and the physical architecture of 20 cell lines every 15 minutes over 7.5 hours. This platform also facilitated detailed measurements of the impacts of viral infection on the architecture of host cells and host proteins. The Mantis platform can enable high-throughput profiling of intracellular dynamics, long-term imaging and analysis of cellular responses to perturbations, and live-cell optical screens to dissect gene regulatory networks.

Cell biology is .

50 years ago, cell biology was a nascent field. Today, it is a vast discipline whose principles and tools are also applied to other disciplines; vice versa, cell biologists are inspired by other fields. So, the question begs: what is cell biology? The answers are as diverse as the people who define it.

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq.

Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-κB inhibitor IκBα (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells.

Oxaliplatin disrupts nucleolar function through biophysical disintegration.

Platinum (Pt) compounds such as oxaliplatin are among the most commonly prescribed anti-cancer drugs. Despite their considerable clinical impact, the molecular basis of platinum cytotoxicity and cancer specificity remain unclear. Here we show that oxaliplatin, a backbone for the treatment of colorectal cancer, causes liquid-liquid demixing of nucleoli at clinically relevant concentrations. Our data suggest that this biophysical defect leads to cell-cycle arrest, shutdown of Pol I-mediated transcription, and ultimately cell death. We propose that instead of targeting a single molecule, oxaliplatin preferentially partitions into nucleoli, where it modifies nucleolar RNA and proteins. This mechanism provides a general approach for drugging the increasing number of cellular processes linked to biomolecular condensates.

Self-supervised deep learning encodes high-resolution features of protein subcellular localization.

Explaining the diversity and complexity of protein localization is essential to fully understand cellular architecture. Here we present cytoself, a deep-learning approach for fully self-supervised protein localization profiling and clustering. Cytoself leverages a self-supervised training scheme that does not require preexisting knowledge, categories or annotations. Training cytoself on images of 1,311 endogenously labeled proteins from the OpenCell database reveals a highly resolved protein localization atlas that recapitulates major scales of cellular organization, from coarse classes, such as nuclear and cytoplasmic, to the subtle localization signatures of individual protein complexes. We quantitatively validate cytoself's ability to cluster proteins into organelles and protein complexes, showing that cytoself outperforms previous self-supervised approaches. Moreover, to better understand the inner workings of our model, we dissect the emergent features from which our clustering is derived, interpret them in the context of the fluorescence images, and analyze the performance contributions of each component of our approach.

Correlative imaging of the spatio-angular dynamics of biological systems with multimodal instant polarization microscope.

The spatial and angular organization of biological macromolecules is a key determinant, as well as informative readout, of their function. Correlative imaging of the dynamic spatio-angular architecture of cells and organelles is valuable, but remains challenging with current methods. Correlative imaging of spatio-angular dynamics requires fast polarization-, depth-, and wavelength-diverse measurement of intrinsic optical properties and fluorescent labels. We report a multimodal instant polarization microscope (miPolScope) that combines a broadband polarization-resolved detector, automation, and reconstruction algorithms to enable label-free imaging of phase, retardance, and orientation, multiplexed with fluorescence imaging of concentration, anisotropy, and orientation of molecules at diffraction-limited resolution and high speed. miPolScope enabled multimodal imaging of myofibril architecture and contractile activity of beating cardiomyocytes, cell and organelle architecture of live HEK293T and U2OS cells, and density and anisotropy of white and grey matter of mouse brain tissue across the visible spectrum. We anticipate these developments in joint quantitative imaging of density and anisotropy to enable new studies in tissue pathology, mechanobiology, and imaging-based screens.

OpenCell: Endogenous tagging for the cartography of human cellular organization.

Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.

WASP integrates substrate topology and cell polarity to guide neutrophil migration.

To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation. Superresolution imaging reveals that WASP preferentially enriches to the necks of these substrate-induced invaginations, a distribution that could support substrate pinching. WASP facilitates recruitment of the Arp2/3 complex to these sites, stimulating local actin assembly that couples substrate features with the cytoskeleton. Surprisingly, WASP only enriches to membrane deformations in the front half of the cell, within a permissive zone set by WASP's front-biased regulator Cdc42. While WASP KO cells exhibit relatively normal migration on flat substrates, they are defective at topology-directed migration. Our data suggest that WASP integrates substrate topology with cell polarity by selectively polymerizing actin around substrate-induced membrane deformations in the front half of the cell.

Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing.

A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.

Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans.

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663.

Spatiotemporal dissection of the cell cycle with single-cell proteogenomics.

The cell cycle, over which cells grow and divide, is a fundamental process of life. Its dysregulation has devastating consequences, including cancer1-3. The cell cycle is driven by precise regulation of proteins in time and space, which creates variability between individual proliferating cells. To our knowledge, no systematic investigations of such cell-to-cell proteomic variability exist. Here we present a comprehensive, spatiotemporal map of human proteomic heterogeneity by integrating proteomics at subcellular resolution with single-cell transcriptomics and precise temporal measurements of individual cells in the cell cycle. We show that around one-fifth of the human proteome displays cell-to-cell variability, identify hundreds of proteins with previously unknown associations with mitosis and the cell cycle, and provide evidence that several of these proteins have oncogenic functions. Our results show that cell cycle progression explains less than half of all cell-to-cell variability, and that most cycling proteins are regulated post-translationally, rather than by transcriptomic cycling. These proteins are disproportionately phosphorylated by kinases that regulate cell fate, whereas non-cycling proteins that vary between cells are more likely to be modified by kinases that regulate metabolism. This spatially resolved proteomic map of the cell cycle is integrated into the Human Protein Atlas and will serve as a resource for accelerating molecular studies of the human cell cycle and cell proliferation.

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway.

Sphingolipids play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted human cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as glycosylphosphatidylinositol (GPI) anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway.

Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic protein disorder.

The nucleolus is essential for ribosome biogenesis and is involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human nucleolar proteome using confocal microscopy. In total, 1,318 nucleolar proteins were identified; 287 were localized to fibrillar components, and 157 were enriched along the nucleoplasmic border, indicating a potential fourth nucleolar subcompartment: the nucleoli rim. We found 65 nucleolar proteins (36 uncharacterized) to relocate to the chromosomal periphery during mitosis. Interestingly, we observed temporal partitioning into two recruitment phenotypes: early (prometaphase) and late (after metaphase), suggesting phase-specific functions. We further show that the expression of MKI67 is critical for this temporal partitioning. We provide the first proteome-wide analysis of intrinsic protein disorder for the human nucleolus and show that nucleolar proteins in general, and mitotic chromosome proteins in particular, have significantly higher intrinsic disorder level compared to cytosolic proteins. In summary, this study provides a comprehensive and essential resource of spatiotemporal expression data for the nucleolar proteome as part of the Human Protein Atlas.

Revealing architectural order with quantitative label-free imaging and deep learning.

We report quantitative label-free imaging with phase and polarization (QLIPP) for simultaneous measurement of density, anisotropy, and orientation of structures in unlabeled live cells and tissue slices. We combine QLIPP with deep neural networks to predict fluorescence images of diverse cell and tissue structures. QLIPP images reveal anatomical regions and axon tract orientation in prenatal human brain tissue sections that are not visible using brightfield imaging. We report a variant of U-Net architecture, multi-channel 2.5D U-Net, for computationally efficient prediction of fluorescence images in three dimensions and over large fields of view. Further, we develop data normalization methods for accurate prediction of myelin distribution over large brain regions. We show that experimental defects in labeling the human tissue can be rescued with quantitative label-free imaging and neural network model. We anticipate that the proposed method will enable new studies of architectural order at spatial scales ranging from organelles to tissue.

Microscopy is central to biological research and has enabled scientist to study the structure and dynamics of cells and their components within. Often, fluorescent dyes or trackers are used that can be detected under the microscope. However, this procedure can sometimes interfere with the biological processes being studied. Now, Guo, Yeh, Folkesson et al. have developed a new approach to examine structures within tissues and cells without the need for a fluorescent label. The technique, called QLIPP, uses the phase and polarization of the light passing through the sample to get information about its makeup. A computational model was used to decode the characteristics of the light and to provide information about the density and orientation of molecules in live cells and brain tissue samples of mice and human. This way, Guo et al. were able to reveal details that conventional microscopy would have missed. Then, a type of machine learning, known as 'deep learning', was used to translate the density and orientation images into fluorescence images, which enabled the researchers to predict specific structures in human brain tissue sections. QLIPP can be added as a module to a microscope and its software is available open source. Guo et al. hope that this approach can be used across many fields of biology, for example, to map the connectivity of nerve cells in the human brain or to identify how cells respond to infection. However, further work in automating other aspects, such as sample preparation and analysis, will be needed to realize the full benefits.

Pervasive functional translation of noncanonical human open reading frames.

Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.

Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation.

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia (FTD). However, we lack a systematic understanding of the cellular pathways controlling prion-like propagation of aggregation. To uncover such pathways, here we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation monitored by FRET. Our screens uncovered that knockdown of several components of the endosomal sorting complexes required for transport (ESCRT) machinery, including charged multivesicular body protein 6 (CHMP6), or CHMP2A in combination with CHMP2B (whose gene is linked to familial FTD), promote propagation of tau aggregation. We found that knocking down the genes encoding these proteins also causes damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. Together, these findings suggest that endolysosomal escape is a critical step in tau propagation in neurodegenerative diseases.

Large dataset enables prediction of repair after CRISPR-Cas9 editing in primary T cells.

Understanding of repair outcomes after Cas9-induced DNA cleavage is still limited, especially in primary human cells. We sequence repair outcomes at 1,656 on-target genomic sites in primary human T cells and use these data to train a machine learning model, which we have called CRISPR Repair Outcome (SPROUT). SPROUT accurately predicts the length, probability and sequence of nucleotide insertions and deletions, and will facilitate design of SpCas9 guide RNAs in therapeutically important primary human cells.