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.
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.
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•CRISPRoff is a single fusion protein that programs heritable epigenetic memory•CRISPRoff can heritably silence most genes, including genes without CpG islands•CRISPRoff is highly specific and has a broad targeting window across gene promoters•CRISPRoff epigenetic memory persists through differentiation of iPSCs into neurons
CRISPRoff programs heritable epigenetic memory and is able to heritably silence most genes, including genes without CpG islands. This heritable silencing is highly specific and persists through differentiation of iPSCs into neurons.
The unique photophysical properties of lanthanides, such as europium, terbium, and ytterbium, make them versatile molecular probes of biological systems. In particular, their long-lived ...photoluminescence, narrow bandwidth emissions, and large Stokes shifts enable experiments that are infeasible with organic fluorophores and fluorescent proteins. The ability of these metal ions to undergo luminescence resonance energy transfer, and photon upconversion further expands the capabilities of lanthanide probes. In this review, we describe recent advances in the design of lanthanide luminophores and their application in biological research. We also summarize the latest detection systems that have been developed to fully exploit the optical properties of lanthanide luminophores. We conclude with a discussion of remaining challenges and new frontiers in lanthanide technologies. The unprecedented levels of sensitivity and multiplexing afforded by rare-earth elements illustrate how chemistry can enable new approaches in biology.
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Lanthanide luminophores have unique photophysical properties that make them versatile reagents for optical imaging. In this review, Cho and Chen highlight advances that have unlocked the potential of these rare-earth metals as imaging tools and applications of lanthanide probes in cellular and organismal systems.
Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based ...single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.
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•Perturb-seq allows parallel screening with rich phenotypic output from single cells•Simultaneous delivery and identification of up to three CRISPR perturbations•Genome-scale screens dissect the mammalian unfolded protein response•Analytical methods separate perturbation responses from confounding effects
A strategy for barcoding CRISPR-mediated perturbations allows pooled expression profiling via single-cell RNA sequencing. Application to the mammalian unfolded protein response then enabled systematic delineation of the transcriptional arms of the response and functional clustering of genes affecting ER homeostasis.
Two major challenges of 3D bioprinting are the retention of structural fidelity and efficient endothelialization for tissue vascularization. Both of these issues are addressed by introducing a ...versatile 3D bioprinting strategy, in which a templating bioink is deposited layer‐by‐layer alongside a matrix bioink to establish void‐free multimaterial structures. After crosslinking the matrix phase, the templating phase is sacrificed to create a well‐defined 3D network of interconnected tubular channels. This void‐free 3D printing (VF‐3DP) approach circumvents the traditional concerns of structural collapse, deformation, and oxygen inhibition, moreover, it can be readily used to print materials that are widely considered “unprintable.” By preloading endothelial cells into the templating bioink, the inner surface of the channels can be efficiently cellularized with a confluent endothelial layer. This in situ endothelialization method can be used to produce endothelium with a far greater cell seeding uniformity than can be achieved using the conventional postseeding approach. This VF‐3DP approach can also be extended beyond tissue fabrication and toward customized hydrogel‐based microfluidics and self‐supported perfusable hydrogel constructs.
A void‐free 3D printing strategy is introduced that allows uniform and interconnected porous channels to be assembled, even using low‐concentration bioinks that cannot be printed directly. Moreover, preloading endothelial cells in the templating phase enable in situ endothelialization without the need for postseeding. This method can also be used to fabricate customized hydrogel‐based microfluidics and standalone perfusable 3D structures.
Developmental biology has been continually shaped by technological advances, evolving from a descriptive science into one immersed in molecular and cellular mechanisms. Most recently, genome ...sequencing and 'omics' profiling have provided developmental biologists with a wealth of genetic and biochemical information; however, fully translating this knowledge into functional understanding will require new experimental capabilities. Photoactivatable probes have emerged as particularly valuable tools for investigating developmental mechanisms, as they can enable rapid, specific manipulations of DNA, RNA, proteins, and cells with spatiotemporal precision. In this Perspective, we describe optochemical and optogenetic systems that have been applied in multicellular organisms, insights gained through the use of these probes, and their current limitations. We also suggest how chemical biologists can expand the reach of photoactivatable technologies and bring new depth to our understanding of organismal development.
Background: Increased DNA methylation is an epigenetic alteration that is common in human cancers and is often associated with transcriptional silencing. Aberrantly methylated DNA has also been ...proposed as a potential tumor marker. However, genes such as vimentin, which are transcriptionally silent in normal epithelium, have not until now been considered as targets for cancer-associated aberrant methylation and for use as cancer markers. Methods: We applied methylation-specific polymerase chain reaction to the vimentin gene, which is transcriptionally silent in normal colonocytes, and compared methylation of vimentin exon 1 in cancer tissues and in fecal DNA from colon cancer patients versus control samples from healthy subjects. Results: Vimentin exon-1 sequences were unmethylated in 45 of 46 normal colon tissues. In contrast, vimentin exon-1 sequences were methylated in 83% (38 of 46) and 53% (57 of 107) of tumors from two independently collected groups of colon cancer patients. When evaluated as a marker for colon cancer detection in fecal DNA from another set of colon cancer patients, aberrant vimentin methylation was detected in fecal DNA from 43 of 94 patients, for a sensitivity of 46% (95% confidence interval CI = 35% to 56%). The sensitivity for detecting stage I and II cancers was 43% (26 of 60 case patients) (95% CI = 31% to 57%). Only 10% (20 of 198 case patients) of control fecal DNA samples from cancer-free individuals tested positive for vimentin methylation, for a specificity of 90% (95% CI = 85% to 94%). Conclusions: Aberrant methylation of exon-1 sequences within the nontranscribed vimentin gene is a novel molecular biomarker of colon cancer and can be successfully detected in fecal DNA to identify nearly half of individuals with colon cancer.
Little is known regarding appropriate patient selection for and clinical outcomes with lung transplantation for respiratory failure due to Covid-19. This study analyzes lung transplantations reported ...in the United Network for Organ Sharing registry from August 2020 through September 2021.
Degenerative changes are commonly found in spine imaging but often occur in pain-free individuals as well as those with back pain. We sought to estimate the prevalence, by age, of common degenerative ...spine conditions by performing a systematic review studying the prevalence of spine degeneration on imaging in asymptomatic individuals.
We performed a systematic review of articles reporting the prevalence of imaging findings (CT or MR imaging) in asymptomatic individuals from published English literature through April 2014. Two reviewers evaluated each manuscript. We selected age groupings by decade (20, 30, 40, 50, 60, 70, 80 years), determining age-specific prevalence estimates. For each imaging finding, we fit a generalized linear mixed-effects model for the age-specific prevalence estimate clustering in the study, adjusting for the midpoint of the reported age interval.
Thirty-three articles reporting imaging findings for 3110 asymptomatic individuals met our study inclusion criteria. The prevalence of disk degeneration in asymptomatic individuals increased from 37% of 20-year-old individuals to 96% of 80-year-old individuals. Disk bulge prevalence increased from 30% of those 20 years of age to 84% of those 80 years of age. Disk protrusion prevalence increased from 29% of those 20 years of age to 43% of those 80 years of age. The prevalence of annular fissure increased from 19% of those 20 years of age to 29% of those 80 years of age.
Imaging findings of spine degeneration are present in high proportions of asymptomatic individuals, increasing with age. Many imaging-based degenerative features are likely part of normal aging and unassociated with pain. These imaging findings must be interpreted in the context of the patient's clinical condition.
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