Dyes illuminate live human embryogenesis.

Assisted reproduction is on the rise globally. Cell morphology is commonly used for embryo selection, but the cell biology of early preimplantation development remains poorly understood. In this issue of Cell, Domingo-Muelas et al. reveal novel features of human embryos with critical implications for preimplantation genetic testing.

Human embryo live imaging reveals nuclear DNA shedding during blastocyst expansion and biopsy.

Proper preimplantation development is essential to assemble a blastocyst capable of implantation. Live imaging has uncovered major events driving early development in mouse embryos; yet, studies in humans have been limited by restrictions on genetic manipulation and lack of imaging approaches. We have overcome this barrier by combining fluorescent dyes with live imaging to reveal the dynamics of chromosome segregation, compaction, polarization, blastocyst formation, and hatching in the human embryo. We also show that blastocyst expansion mechanically constrains trophectoderm cells, causing nuclear budding and DNA shedding into the cytoplasm. Furthermore, cells with lower perinuclear keratin levels are more prone to undergo DNA loss. Moreover, applying trophectoderm biopsy, a mechanical procedure performed clinically for genetic testing, increases DNA shedding. Thus, our work reveals distinct processes underlying human development compared with mouse and suggests that aneuploidies in human embryos may not only originate from chromosome segregation errors during mitosis but also from nuclear DNA shedding.

Functional interrogation of DNA damage response variants with base editing screens.

Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.

Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway.

The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).

Genetic Screen for Cell Fitness in High or Low Oxygen Highlights Mitochondrial and Lipid Metabolism.

Human cells are able to sense and adapt to variations in oxygen levels. Historically, much research in this field has focused on hypoxia-inducible factor (HIF) signaling and reactive oxygen species (ROS). Here, we perform genome-wide CRISPR growth screens at 21%, 5%, and 1% oxygen to systematically identify gene knockouts with relative fitness defects in high oxygen (213 genes) or low oxygen (109 genes), most without known connection to HIF or ROS. Knockouts of many mitochondrial pathways thought to be essential, including complex I and enzymes in Fe-S biosynthesis, grow relatively well at low oxygen and thus are buffered by hypoxia. In contrast, in certain cell types, knockout of lipid biosynthetic and peroxisomal genes causes fitness defects only in low oxygen. Our resource nominates genetic diseases whose severity may be modulated by oxygen and links hundreds of genes to oxygen homeostasis.

A New Wave of Genomics for All.

Millions of people have taken direct-to-consumer DNA tests, but not everyone is happy with the status quo. Several startups in the genetic testing space are aiming to empower individuals and build communities in order to boost research and, ultimately, public health.

Screening Human Embryos for Polygenic Traits Has Limited Utility.

The increasing proportion of variance in human complex traits explained by polygenic scores, along with progress in preimplantation genetic diagnosis, suggests the possibility of screening embryos for traits such as height or cognitive ability. However, the expected outcomes of embryo screening are unclear, which undermines discussion of associated ethical concerns. Here, we use theory, simulations, and real data to evaluate the potential gain of embryo screening, defined as the difference in trait value between the top-scoring embryo and the average embryo. The gain increases very slowly with the number of embryos but more rapidly with the variance explained by the score. Given current technology, the average gain due to screening would be ≈2.5 cm for height and ≈2.5 IQ points for cognitive ability. These mean values are accompanied by wide prediction intervals, and indeed, in large nuclear families, the majority of children top-scoring for height are not the tallest.

Genomic Medicine-Progress, Pitfalls, and Promise.

In the wake of the Human Genome Project (HGP), strong expectations were set for the timeline and impact of genomics on medicine-an anticipated transformation in the diagnosis, treatment, and prevention of disease. In this Perspective, we take stock of the nascent field of genomic medicine. In what areas, if any, is genomics delivering on this promise, or is the path to success clear? Where are we falling short, and why? What have been the unanticipated developments? Overall, we argue that the optimism surrounding the transformational potential of genomics on medicine remains justified, albeit with a considerably different form and timescale than originally projected. We also argue that the field needs to pivot back to basics, as understanding the entirety of the genotype-to-phenotype equation is a likely prerequisite for delivering on the full potential of the human genome to advance the human condition.

Personalized Medicine and the Power of Electronic Health Records.

Personalized medicine has largely been enabled by the integration of genomic and other data with electronic health records (EHRs) in the United States and elsewhere. Increased EHR adoption across various clinical settings and the establishment of EHR-linked population-based biobanks provide unprecedented opportunities for the types of translational and implementation research that drive personalized medicine. We review advances in the digitization of health information and the proliferation of genomic research in health systems and provide insights into emerging paths for the widespread implementation of personalized medicine.

Diseases Have No Borders, Neither Should Research!

In response to recent anti-Chinese sentiment in the US, Sunney Xie uses his own experiences to assert that American ideals should not be replaced by nationalism and populism and that everybody wins in Sino-US scientific collaborations, contrary to what Americans are led to believe: that China is the sole beneficiary.

A Diagnosis for All Rare Genetic Diseases: The Horizon and the Next Frontiers.

The introduction of exome sequencing in the clinic has sparked tremendous optimism for the future of rare disease diagnosis, and there is exciting opportunity to further leverage these advances. To provide diagnostic clarity to all of these patients, however, there is a critical need for the field to develop and implement strategies to understand the mechanisms underlying all rare diseases and translate these to clinical care.

Optical Pooled Screens in Human Cells.

Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.

Genomic Analysis in the Age of Human Genome Sequencing.

Affordable genome sequencing technologies promise to revolutionize the field of human genetics by enabling comprehensive studies that interrogate all classes of genome variation, genome-wide, across the entire allele frequency spectrum. Ongoing projects worldwide are sequencing many thousands-and soon millions-of human genomes as part of various gene mapping studies, biobanking efforts, and clinical programs. However, while genome sequencing data production has become routine, genome analysis and interpretation remain challenging endeavors with many limitations and caveats. Here, we review the current state of technologies for genetic variant discovery, genotyping, and functional interpretation and discuss the prospects for future advances. We focus on germline variants discovered by whole-genome sequencing, genome-wide functional genomic approaches for predicting and measuring variant functional effects, and implications for studies of common and rare human disease.

Genomic Analyses from Non-invasive Prenatal Testing Reveal Genetic Associations, Patterns of Viral Infections, and Chinese Population History.

We analyze whole-genome sequencing data from 141,431 Chinese women generated for non-invasive prenatal testing (NIPT). We use these data to characterize the population genetic structure and to investigate genetic associations with maternal and infectious traits. We show that the present day distribution of alleles is a function of both ancient migration and very recent population movements. We reveal novel phenotype-genotype associations, including several replicated associations with height and BMI, an association between maternal age and EMB, and between twin pregnancy and NRG1. Finally, we identify a unique pattern of circulating viral DNA in plasma with high prevalence of hepatitis B and other clinically relevant maternal infections. A GWAS for viral infections identifies an exceptionally strong association between integrated herpesvirus 6 and MOV10L1, which affects piwi-interacting RNA (piRNA) processing and PIWI protein function. These findings demonstrate the great value and potential of accumulating NIPT data for worldwide medical and genetic analyses.

Pooled Genome-Scale CRISPR Screens in Single Cells.

Assigning functions to genes and learning how to control their expression are part of the foundation of cell biology and therapeutic development. An efficient and unbiased method to accomplish this is genetic screening, which historically required laborious clone generation and phenotyping and is still limited by scale today. The rapid technological progress on modulating gene function with CRISPR-Cas and measuring it in individual cells has now relaxed the major experimental constraints and enabled pooled screening with complex readouts from single cells. Here, we review the principles and practical considerations for pooled single-cell CRISPR screening. We discuss perturbation strategies, experimental model systems, matching the perturbation to the individual cells, reading out cell phenotypes, and data analysis. Our focus is on single-cell RNA sequencing and cell sorting-based readouts, including image-enabled cell sorting. We expect this transformative approach to fuel biomedical research for the next several decades.

Interpreting and integrating genomic tests results in clinical cancer care: Overview and practical guidance.

The last decade has seen rapid progress in the use of genomic tests, including gene panels, whole-exome sequencing, and whole-genome sequencing, in research and clinical cancer care. These advances have created expansive opportunities to characterize the molecular attributes of cancer, revealing a subset of cancer-associated aberrations called driver mutations. The identification of these driver mutations can unearth vulnerabilities of cancer cells to targeted therapeutics, which has led to the development and approval of novel diagnostics and personalized interventions in various malignancies. The applications of this modern approach, often referred to as precision oncology or precision cancer medicine, are already becoming a staple in cancer care and will expand exponentially over the coming years. Although genomic tests can lead to better outcomes by informing cancer risk, prognosis, and therapeutic selection, they remain underutilized in routine cancer care. A contributing factor is a lack of understanding of their clinical utility and the difficulty of results interpretation by the broad oncology community. Practical guidelines on how to interpret and integrate genomic information in the clinical setting, addressed to clinicians without expertise in cancer genomics, are currently limited. Building upon the genomic foundations of cancer and the concept of precision oncology, the authors have developed practical guidance to aid the interpretation of genomic test results that help inform clinical decision making for patients with cancer. They also discuss the challenges that prevent the wider implementation of precision oncology.

In vivo screens using a selective CRISPR antigen removal lentiviral vector system reveal immune dependencies in renal cell carcinoma.

CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.

Genome-wide Screens Identify Lineage- and Tumor-Specific Genes Modulating MHC-I- and MHC-II-Restricted Immunosurveillance of Human Lymphomas.

Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.

The expanding diagnostic toolbox for rare genetic diseases.

Genomic technologies, such as targeted, exome and short-read genome sequencing approaches, have revolutionized the care of patients with rare genetic diseases. However, more than half of patients remain without a diagnosis. Emerging approaches from research-based settings such as long-read genome sequencing and optical genome mapping hold promise for improving the identification of disease-causal genetic variants. In addition, new omic technologies that measure the transcriptome, epigenome, proteome or metabolome are showing great potential for variant interpretation. As genetic testing options rapidly expand, the clinical community needs to be mindful of their individual strengths and limitations, as well as remaining challenges, to select the appropriate diagnostic test, correctly interpret results and drive innovation to address insufficiencies. If used effectively - through truly integrative multi-omics approaches and data sharing - the resulting large quantities of data from these established and emerging technologies will greatly improve the interpretative power of genetic and genomic diagnostics for rare diseases.

The Heidelberg Screen for Pattern Mutants of Drosophila: A Personal Account.

In large-scale mutagenesis screens performed in 1979-1980 at the EMBL in Heidelberg, we isolated mutations affecting the pattern or structure of the larval cuticle in Drosophila. The 600 mutants we characterized could be assigned to 120 genes and represent the majority of such genes in the genome. These mutants subsequently provided a rich resource for understanding many fundamental developmental processes, such as the transcriptional hierarchies controlling segmentation, the establishment of cell states by signaling pathways, and the differentiation of epithelial cells. Most of the Heidelberg genes are now molecularly known, and many of them are conserved in other animals, including humans. Although the screens were initially driven entirely by curiosity, the mutants now serve as models for many human diseases. In this review, we describe the rationale of the screening procedures and provide a classification of the genes on the basis of their initial phenotypes and the subsequent molecular analyses.