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Cancer Genomics Many cancers are classified on the basis of the organ or tissue from which they originated. However, identifying the specific cells and conditions that precede tumorigenesis can help us understand and better treat the resulting disease. Nowicki-Osuch et al. used a single-cell approach to investigate the cell of origin for Barrett's esophagus (BE) and the mechanisms leading to the development of esophageal adenocarcinoma (EAC) (see the Perspective by Geboes and Hoorens). Analyses of healthy human esophageal tissues, mutational lineage tracing, and organoid models revealed that BE originates from the gastric cardia and that EAC arises from undifferentiated BE cells. This analysis provides a map of the transcriptional landscape of the healthy esophagus that can be compared with mouse models of disease. Science , abd1449, this issue p. [760][1]; see also abj9797, p. [737][2] [1]: /lookup/doi/10.1126/science.abd1449 [2]: /lookup/doi/10.1126/science.abj9797

Human Genomics Obesity is linked to many human diseases, including diabetes, cancer, and heart disease. There is thus great interest in understanding how genes predispose individuals to, or protect individuals from, obesity. Akbari et al. sequenced more than 600,000 exomes from the United Kingdom, the United States, and Mexico and identified 16 rare coding variants (see the Perspective by Yeo and O'Rahilly). Some of the alleles associated with body mass index (BMI) were brain-expressed G protein–coupled receptors. One variant allele was found in Mexican populations at low frequency and was associated with lower BMI. Deletion of this gene in mice resulted in a resistance to weight gain, suggesting that this gene provides an avenue of study for the prevention or treatment of obesity. Science , abf8683, this issue p. [eabf8683][1]; see also abh3556, p. [30][2] [1]: /lookup/doi/10.1126/science.abf8683 [2]: /lookup/doi/10.1126/science.abh3556

Human Genomics Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease with a genetic and environmental interaction. Lou Gehrig was a renowned professional American baseball player whose career was cut short by ALS, which has now also become known as Lou Gehrig's disease. Using Mendelian randomization (MR), which can help to identify genetic factors linked to phenotype, Julian et al. looked for genetic differences between individuals within the UK Biobank correlated with their activity level, including strenuous activity. From this, they identified genetic variants associated with ALS risk in individuals who regularly performed strenuous exercise. Of the validated ALS risk genes, 52% showed differential gene expression during exercise in individuals with such variants. Strenuous exercise is a highly heterogenous environmental factor, and more study is needed to determine how it becomes a risk factor for ALS. EBioMedicine 68 , 103397 (2021).

Human Evolution Humans are unique mammals that lack hair in many parts of their body and have an increased capacity for sweating, which assists in thermoregulation, through eccrine sweat glands. Using a comparative genomics approach to study the regulation of genes involved in the development of eccrine sweat glands, Aldea et al. investigated noncoding genomic elements to determine the differences underlying the increased eccrine sweat gland density of humans relative to other primates and mice. They found that human-specific evolution in a noncoding enhancer, hECE18, likely increases gene expression of the Engrailed1 gene, which is required for the development of eccrine sweat glands. Furthermore, humanized hECE18 knock-in mice have an increased number of these glands, supporting the idea that this human-specific adaptation is a result of changes in the noncoding regulatory regions of proteins. Proc. Natl. Acad. Sci. U.S.A. 118 , e2021722118 (2021).

Cancer Genomics Cells undergo many genomic changes as they progress toward metastatic cancer. One aspect of this change is to RNA expression and splicing isoforms, but how these differences affect tumor progression is not well characterized. Fish et al. developed a computational framework called pyTEISER that identifies structural cis-regulatory elements that control diverse types of RNA regulation. Applying pyTEISER to models of breast cancer metastasis, they discovered an RNA short-stem-loop element that forms a “structural splicing enhancer” that acts in cis to regulate alternative splicing of RNA transcripts. One of these interactions encompasses the RNA-binding protein SNRPA1 and results in alternative exon inclusion that affects metastatic capacity in xenograft models. Thus, RNA element binding may play a role in splicing regulation and is potentially an important component of the cis-splicing code. Science , this issue p. [eabc7531][1] [1]: /lookup/doi/10.1126/science.abc7531

Autoimmune Diversity Autoimmune diseases often show polygenic inheritance, making the identification of potential causal genetic variants difficult, especially across ancestrally divergent populations. Andreoletti et al. examined the transcriptomes of bulk immune cells from 120 systematic lupus erythematosus (SLE) patients of Asian and European ancestry. Disease-specific genetic signatures were revealed, as well as ancestrally associated differences in SLE molecular pathways and the role of gene expression variation in disease severity. Because SLE severity differs among populations, this study highlights the need to examine disease genetics in multiethnic cohorts for underlying differences and to explore the clinical treatment options for individuals of differing ancestries. Commun. Biol. 4 , 488 (2021).

Neurogenomics Humans have only a limited capacity to generate new neurons. These cells thus need to repair errors in the genome. To better understand this process, Reid et al. developed Repair-seq, a method to locate DNA repair within the genome of stem cell–derived neurons. DNA repair hotspots (DRHs) were more likely to occur within specific genomic features such as gene bodies as well as in genomic formations, open chromatin, and active regulatory regions. This method showed that repair was enriched at sites involved in neuronal function and identity. Furthermore, proteomic data indicated that genes in DRHs are enriched in Alzheimer's disease and that DRHs are more active in aging. These observations link neuronal DNA repair to aging and neurodegeneration. Science , this issue p. [91][1] [1]: /lookup/doi/10.1126/science.abb9032

Cancer Genomics The heterogeneity of mammalian tumors has been well documented, but it remains unknown how differences between individual cells lead to metastasis and spread throughout the body. Quinn et al. created a Cas9-based lineage tracer and used single-cell sequencing to generate phylogenies and follow the movement of metastatic human cancer cells implanted in the lung of a mouse xenograph model. Using this model, they found that within the same cell line, cancer cells exhibited diverse metastatic phenotypes. These subclones exhibited differential gene expression profiles, some of which were previously associated with metastasis. Science , this issue p. [eabc1944][1] [1]: /lookup/doi/10.1126/science.abc1944

Bacterial Genomics Single-cell genomics in bacteria has lagged relative to in eukaryotes because of their tough bacterial cell walls, low messenger RNA content, and lack of many posttranscriptional modifications. To tackle this challenge, Kuchina et al. developed microbial split-pool ligation transcriptomics, or microSPLiT, a single-cell sequencing method for both Gram-negative and Gram-positive bacteria. Sequencing both Escherichia coli and Bacillus subtilis showed differences in the heat shock response. Examining B. subtilis transcriptional patterns revealed that a small fraction of cells grown in laboratory medium express a myo-inositol catabolism pathway, which the cell could use in nonlaboratory environments, thus highlighting how microSPLiT can identify rare cellular states. Science , this issue p. [eaba5257][1] [1]: /lookup/doi/10.1126/science.aba5257

Antibiotic Resistance Antibiotic resistance arising from mutation is common among pathogenic bacteria. However, this process is not well understood, and most of the mutations that have been identified to confer resistance do so by modification of the intracellular target or enzymes that can disable the antibacterial compound within the cell. Screening for the evolution of resistance at different temperatures, Lopatkin et al. found that mutations that affect microbial metabolism can result in antibiotic resistance (see the Perspective by Zampieri). These mutations targeted central carbon and energy metabolism and revealed novel resistance mutations in core metabolic genes, expanding the known means by which pathogenic microbes can evolve resistance. Science , this issue p. [eaba0862][1]; see also p. [783][2] [1]: /lookup/doi/10.1126/science.aba0862 [2]: /lookup/doi/10.1126/science.abf7922

Cancer Genomics Mutations in the essential RUNX1 gene are associated with several diseases of the blood, including leukemias, familial platelet disorder, and other malignancies. How these mutations underly each phenotype is unknown. Using embryonic stem cells, Kellaway et al. examined the genomic effects of four different mutant RUNX1 transcription factor proteins. The mutations affect binding of RUNX1 to DNA or its transactivation domains in differentiated hematopoietic stem cells. Each mutation altered RUNX1 function by a different mechanism, leading to divergent trajectories for stem cell development for each disease phenotype. Thus, each malignancy will need a different therapeutic approach. Life Sci. Alliance 4 , e202000864 (2021).

In Situ Sequencing Identifying where specific RNAs occur within a cell or tissue has been limited by technology and imaging capabilities. Expansion microscopy has allowed for better visualization of small structures by expanding the tissues with a polymer- and hydrogel-based system. Alon et al. combined expansion microscopy with long-read in situ RNA sequencing, resulting in a more precise visualization of the location of specific transcripts. This method, termed “ExSeq” for expansion sequencing, was used to detect RNAs, both new transcripts and those previously demonstrated to localize to neuronal dendrites. Unlike other in situ sequencing methods, ExSeq does not target sets of genes. This technology thus unites spatial resolution, multiplexing, and an unbiased approach to reveal insights into RNA localization and its physiological roles in developing and active tissue. Science , this issue p. [eaax2656][1] [1]: /lookup/doi/10.1126/science.aax2656

Evolution Mutations generate variability that is either neutral or subject to natural selection. Robustness is a measure of the ability to withstand deleterious mutational effects. Zheng et al. exposed Escherichia coli populations expressing a yellow fluorescent protein to strong, weak, or no selection for yellow fluorescence for four generations. They then selected these populations to a related function, green fluorescence, for four more generations. The strong selection first for yellow and then green fluorescence resulted in the most green fluorescence and the accumulation of the most mutations. This outcome likely was due to the increased foldability of the protein. Selection thus provides a threshold for mutation accumulation, but robustness maintains a buffer necessary for protein evolution. Science , this issue p. [eabb5962][1] [1]: /lookup/doi/10.1126/science.abb5962

Neurogenomics CRISPR targeting in vivo, especially in mammals, can be difficult and time consuming when attempting to determine the effects of a single gene. However, such studies may be required to identify pathological gene variants with effects in specific cells along a developmental trajectory. To study the function of genes implicated in autism spectrum disorders (ASDs), Jin et al. applied a gene-editing and single-cell–sequencing system, Perturb-Seq, to knock out 35 ASD candidate genes in multiple mice embryos (see the Perspective by Treutlein and Camp). This method identified networks of gene expression in neuronal and glial cells that suggest new functions in ASD-related genes. Science , this issue p. [eaaz6063][1]; see also p. [1038][2] [1]: /lookup/doi/10.1126/science.aaz6063 [2]: /lookup/doi/10.1126/science.abf3661

Transposons Transposable element activity has been associated with cancer genomes, but whether this activity drives cancers or is incidental to tumorigenesis has been difficult to resolve. Tiwari et al. tested the function of the oncogene p53 on LINE1s, transposable elements found in human cells. Oncogene p53 stimulates repressive histone marks on LINE1s, which keeps them from replicating. If p53 becomes mutated in human cells, the epigenetic repressors on the LINE1s are not maintained. This allows LINE1s to become hyperactivate and replicate within the genome. In some cases, this causes chromosomal breakage, a hallmark of p53 loss-of-function cancers, and stimulates inflammation. Thus, when function is lost in p53 mutants and LINE1 activity increases, an acute oncogenic crisis looms. Genes Dev. 34 , 1439 (2020).

Coronavirus The immune system is complex and involves many genes, including those that encode cytokines known as interferons (IFNs). Individuals that lack specific IFNs can be more susceptible to infectious diseases. Furthermore, the autoantibody system dampens IFN response to prevent damage from pathogen-induced inflammation. Two studies now examine the likelihood that genetics affects the risk of severe coronavirus disease 2019 (COVID-19) through components of this system (see the Perspective by Beck and Aksentijevich). Q. Zhang et al. used a candidate gene approach and identified patients with severe COVID-19 who have mutations in genes involved in the regulation of type I and III IFN immunity. They found enrichment of these genes in patients and conclude that genetics may determine the clinical course of the infection. Bastard et al. identified individuals with high titers of neutralizing autoantibodies against type I IFN-α2 and IFN-ω in about 10% of patients with severe COVID-19 pneumonia. These autoantibodies were not found either in infected people who were asymptomatic or had milder phenotype or in healthy individuals. Together, these studies identify a means by which individuals at highest risk of life-threatening COVID-19 can be identified. Science , this issue p. [eabd4570][1], p. [eabd4585][2]; see also p. [404][3] [1]: /lookup/doi/10.1126/science.abd4570 [2]: /lookup/doi/10.1126/science.abd4585 [3]: /lookup/doi/10.1126/science.abe7591

Mutation Depending on the environment of the individual, the human bladder can be exposed to carcinogens as they are flushed through the body. Lawson et al. and Li et al. examined the genetic composition of laser-dissected microbiopsies from normal and cancer cells collected from the urothelium, a specialized epithelium lining the lower urinary tract (see the Perspective by Rozen). These complementary studies identified the mutational landscape of bladder urothelium through various sequencing strategies and identified high mutational heterogeneity within and between individuals and tumors. Both studies identified mutational profiles related to specific carcinogens such as aristolochic acid and the molecules found in tobacco. These studies present a comprehensive description of the diverse mutational landscape of the human bladder in health and disease, unraveling positive selection for cancer-causing mutations, a diversity of mutational processes, and large differences across individuals. Science , this issue p. [75][1], p. [82][2]; see also p. [34][3] [1]: /lookup/doi/10.1126/science.aba8347 [2]: /lookup/doi/10.1126/science.aba7300 [3]: /lookup/doi/10.1126/science.abe0955

Parasite Genetics Schistosomiasis is caused by a parasitic flatworm about which little is known. Therefore, options to combat human disease caused by schistosome infection are limited. To aid in our quest to develop treatments, two studies undertook molecular investigations of the parasite Schistosoma mansoni . By generating a single-cell atlas, Wendt et al. identified the developmental trajectory of the flatworm, including the blood-feeding gut required for its survival in the host. From these data, they found a gene required for gut development that, when knocked out through RNA interference, confers reduced pathology in infected mice. Wang et al. performed a large-scale RNA interference survey of S. mansoni and identified an essential pair of protein kinases that can be targeted by approved pharmacological intervention (see the Perspective by Anderson and Duraisingh). These molecular investigations add to our understanding of the schistosome parasite and provide biological information that may help to combat this neglected tropical disease. Science , this issue p. [1644][1], p. [1649][2]; see also p. [1562][3] [1]: /lookup/doi/10.1126/science.abb7709 [2]: /lookup/doi/10.1126/science.abb7699 [3]: /lookup/doi/10.1126/science.abe0710

Immunogenomics Microglia are the brain's immune cells, and they play important roles in health and neurodegenerative disease. Kracht et al. performed single-cell analysis of human microglial gene expression and chromatin accessibility and compared the results with those of other studies of human and mice microglial development. By using in situ validation, these data identify fetal microglial subsets that appear to be distinct from adult human microglia, suggesting functional differences between the developing and mature brain. Science , this issue p. [530][1] [1]: /lookup/doi/10.1126/science.aba5906

Gene Editing CRISPR-Cas DNA base editing typically requires a specific motif for targeting known as a protospacer-adjacent motif (PAM). This requirement limits the sequences within a genome that can be targeted. Walton et al. engineered specific variants of the Streptococcus pyogenes Cas9 enzyme named SpG and SpRY that could recognize and edit a wider array of PAMs. Using SpRY, the authors were able to correct previously uneditable mutations associated with human disease. Although off-target effects were observed for these engineered Cas enzymes at levels similar to those of the wild-type enzyme, depending on the context, these engineered enzymes widen the potential applications of precision genome editing. Science , this issue p. [290][1] [1]: /lookup/doi/10.1126/science.aba8853

Clonal Expansion Cells accumulate mutations as we age, and these mutations can be a source of diseases such as cancer. How cells containing mutations evolve, are maintained, and proliferate within the body has not been well characterized. Using a quantitative framework, Watson et al. applied population genetic theory to estimate mutation accumulation in cells in blood from sequencing data derived from nearly 50,000 healthy individuals (see the Perspective by Curtis). By evaluating how mutations differ between blood cell populations, a phenomenon known as clonal hematopoiesis, the researchers could observe how recurrent mutations can drive certain clonal lineages to high frequencies within an individual. The risk of specific mutations, some of which are associated with leukemias, rising to high frequencies may therefore be a function of cellular selection and the age at which the mutation originated. Science , this issue p. [1449][1]; see also p. [1426][2] [1]: /lookup/doi/10.1126/science.aay9333 [2]: /lookup/doi/10.1126/science.aba9891

Cortical Genetics The human cerebral cortex is important for cognition, and it is of interest to see how genetic variants affect its structure. Grasby et al. combined genetic data with brain magnetic resonance imaging from more than 50,000 people to generate a genome-wide analysis of how human genetic variation influences human cortical surface area and thickness. From this analysis, they identified variants associated with cortical structure, some of which affect signaling and gene expression. They observed overlap between genetic loci affecting cortical structure, brain development, and neuropsychiatric disease, and the correlation between these phenotypes is of interest for further study. Science , this issue p. [eaay6690][1] [1]: /lookup/doi/10.1126/science.aay6690

Transcription RNA transcripts are an easily accessed representation of gene expression, but we lack a comprehensive view of the life span of RNA within the single cell. Battich et al. developed a method to sequence messenger RNA labeled with 5-ethynyl-uridine (EU) in single cells (scEU-seq), which allows estimation of RNA transcription and degradation rates. When examining intestinal organoid cells, scEU-seq data can be used to discern between transcription and degradation during development, indicating that this method can be applied to better understand the relationship between gene expression and RNA degradation during development. Science , this issue p. [1151][1] [1]: /lookup/doi/10.1126/science.aax3072

Immunogenomics The human thymus is the organ responsible for the maturation of many types of T cells, which are immune cells that protect us from infection. However, it is not well known how these cells develop with a full immune complement that contains the necessary variation to protect us from a variety of pathogens. By performing single-cell RNA sequencing on more than 250,000 cells, Park et al. examined the changes that occur in the thymus over the course of a human life. They found that development occurs in a coordinated manner among immune cells and with their developmental microenvironment. These data allowed for the creation of models of how T cells with different specific immune functions develop in humans. Science , this issue p. [eaay3224][1] [1]: /lookup/doi/10.1126/science.aay3224

2543 BACKGROUND: The vascular endothelial growth factor (VEGF) is a mediator of angiogenesis and is associated with cancer progression and poor prognosis in patients with primary breast cancer. VEGF is a heparin-binding glycoprotein that is secreted as a homodimer of 45Kda. Most types of cells, but usually not endothelial cells themselves, secrete VEGF. VEGF, particularly VEGF-A, increases vascular permeability, vasodilatation in endothelial cells and can also stimulate cell migration and inhibit apoptosis. We previously reported that elderly women with advanced breast cancer have high levels of VEGF and that correction of anaemia in these patients results in a relevant decrease of VEGF’s plasmatic levels. AIMS: According to these outcomes we would now investigate if variation of VEGF may also affects time to progression’s disease and fatigue that is a frequent symptom in elderly people with cancer. METHODS: VEGF concentrations were analyzed by an enzyme-linked immunosorbent assay (ELISA) in 79 volunteers (mean age: 70 yrs) and 84 elderly breast cancer patients stage III in EDTA plasma specimens. A dosage of 400-500 IU/Kg/t.i.w. of rHuEPO s.c. was administered in order to reach at less 13-14g/dl Hb. Level of serum iron was also adjusted to 24±2 μmol/L if necessary. All patients were treated with liposomal doxorubicin (75 mg/sqm) and cyclophosphamide (600 mg/sqm) every 3 weeks for 6 months. Time-to-progression (TTP) of disease and Fatigue was evaluated with Kaplan-Meyer formula and FACT and FACIT questionary respectively. VEGF was quoted at baseline, after Hb adjustment (a), at the end of therapy (aa) and after 18 months follow-up (aaa). RESULTS: 52 pts (A group) showed: VEGF(baseline): 607±21 pg/ml - VEGF(a): 512±19 pg/ml - VEGF(aa): 319±18 pg/ml - VEGF(aaa): 185±10 pg/ml - Hb (baseline) Mean Values (MV) were: 8.7±2 g/dl. 32 pts (B group) showed: VEGF(baseline): 216±31 pg/ml - VEGF(a): 204±20 pg/ml - VEGF(aa): 185±18 pg/ml - VEGF(aaa): 107±9 pg/ml - Hb (baseline) Mean Values (MV) were: 10.4±1.5 g/dl. Volunteers: VEGF 94±17 pg/ml - Hb 12.6±1.7 g/dl. TTP (A group): 11±1.5 mths - TTP (B group): 14±1.5 mths. Fatigue’s evaluation was as following: 50±10 (A group baseline) 70±10 (A group after HB adjustment) - 80±10 (B group) - 90±10 (Volunteers). CONCLUSION: These findings support a role for hypoxia and anaemia induced VEGF in human breast cancer and also suggest that adjustment of low Hb levels may lead to better results in cancer management through the abrogation of VEGF induced functions. Scrutiny of GJIC (gap junction intercellular communication) activity in human breast cancer cell lines by the Scrape-Loading/Dye Transfer (SL/DT) method in basic condition and after treatment with a specific VEGF-Ab is ongoing in order to confirm these preliminary considerations.

Viral Evolution New species arise through the evolution of barriers to reproduction. This process is well understood where the emerging species are spatially isolated from one another. But the process of sympatric speciation—where the diverging species co-occur—is more enigmatic. Bacterial viruses make good models for addressing such questions because of their rapid generation times. Meyer et al. took bacteriophage λ and grew it on a mix of two strains of Escherichia coli. The virus duly split its host preferences into lineages with differing affinity for their respective E. coli hosts. In some experiments, the phages diverged to the extent that they became reproductively restricted to one strain of E. coli , even when the other was present. Science , this issue p. [1301][1] [1]: /lookup/doi/10.1126/science.aai8446

Cellular Dynamics The eukaryotic cell nucleus provides a home for the genetic material and accessory proteins. As a physical entity, the nucleus also plays an important role in cell dynamics. Bustin and Misteli review the impacts that the nucleus can have as a nongenetic force. For example, compacted DNA and the nuclear membrane affect nuclear morphology, the cellular response to mechanical force, cell migration, and cell signaling. Chromatin is not only affected by physical forces in cells but participates in a crosstalk of signaling inside cells. Science , this issue p. [10.1126/science.aad6933][1] [1]: /lookup/doi/10.1126/science.aad6933

Cancer Genomics Tumors harbor multiple cell types that are thought to play a role in the development of resistance to drug treatments. Tirosh et al. used single-cell sequencing to investigate the distribution of these differing genetic profiles within melanomas. Many cells harbored heterogeneous genetic programs that reflected two different states of genetic expression, one of which was linked to resistance development. Following drug treatment, the resistance-linked expression state was found at a much higher level. Furthermore, the environment of the melanoma cells affected their gene expression programs. Science , this issue p. [189][1] [1]: /lookup/doi/10.1126/science.aad0501

Neurogenomics Microglia are immune system cells that function in protecting and maintaining the brain. Gosselin et al. examined the epigenetics and RNA transcripts from single microglial cells and observed consistent profiles among samples despite differences in age, sex, and diagnosis. Mouse and

Immunogenomics Dendritic cells (DCs) are important components of the immune system that form from the bone marrow into two major cell lineages: plasmacytoid DCs and conventional DCs. See et al. applied single-cell RNA sequencing and cytometry by time-of-flight to characterize the developmental pathways of these cells. They identified blood DC precursors that shared surface markers with plasmacytoid DCs but that were functionally distinct. This unsuspected level of complexity in pre-DC populations reveals additional cell types and refines understanding of known cell types. Science , this issue p. [eaag3009][1] [1]: /lookup/doi/10.1126/science.aag3009

Immunogenomics Blood contains many types of cells, including many immune system components. Immune cells used to be characterized by marker-based assays, but now classification relies on the genes that cells express. Villani et al. used deep sequencing at the single-cell level and unbiased clustering to define six dendritic cell and four monocyte populations. This refined analysis has identified, among others, a previously unknown dendritic cell population that potently activates T cells. Further cell culture revealed possible differentiation progenitors within the different cell populations. Science , this issue p. [eaah4573][1] [1]: /lookup/doi/10.1126/science.aah4573

Cancer Genomics Glioma brain tumors that carry mutant copies of the IDH gene can be subdivided into two major classes. However, the development of and differences between these two classes are not well characterized. Venteicher et al. coupled bulk sequencing and publicly available data with single-cell RNA sequencing data on glioma patient tissue samples. They identified a common lineage program that is shared between glioma subtypes. This suggests that the observed differences between the two glioma classes originate from lineage-specific genetic changes and the tumor microenvironment. Science , this issue p. [eaai8478][1] [1]: /lookup/doi/10.1126/science.aai8478

Neurogenetics Gene expression changes and their control by accessible chromatin in the human brain during development is of great interest but limited accessibility. Trevino et al. avoided this problem by developing three-dimensional organoid models of human forebrain development and examining chromatin accessibility and gene expression at the single-cell level. From this analysis, they matched developmental profiles between the organoid and fetal samples, identified transcription factor binding profiles, and predicted how transcription factors are linked to cortical development. The researchers were able to correlate the expression of neurodevelopmental disease risk loci and genes with specific cell types during development. Science , this issue p. [eaay1645][1] [1]: /lookup/doi/10.1126/science.aay1645

Human Genetics The reduction in sequencing costs and the increase in prediction accuracy make individual assessment of genetic risk from mutations more attractive and valuable. Examining mutation rates in blood and sperm, Breuss et al. surveyed families in which a child has been diagnosed with autism spectrum disorder. The authors found a small set of individuals in which a mosaic of potentially causative mutations was observed in the father's blood or sperm. Differential mutational processes seem to govern when the genetic variants arise. Screening for paternal mosaicism might help determine the risk of autism in future children of fathers that carry a de novo mutation. Nat. Med. 26 , 143 (2020).

Multiplex Genomics Single-cell transcriptomic technologies have emerged as powerful tools to explore cellular heterogeneity at the resolution of individual cells. Srivatsan et al. now add another layer of information and complexity by combining single-cell transcriptomics with oligo hashing and small molecule screening in a method called sci-Plex. Because screening many chemical compounds requires the ability to profile many cells, and because screens perturb cells in many different ways, the authors demonstrate the effects of 188 compounds in three cancer lines. The sci-Plex method can capture gene expression profiles from thousands of experimental conditions in a single experiment. Science , this issue p. [45][1] [1]: /lookup/doi/10.1126/science.aax6234

DNA Damage Exposure to ultraviolet (UV) light can cause DNA damage and is a risk factor for cancer. For individuals, it would be valuable to be able to measure past UV exposure and thus future risk of melanoma. Premi et al. developed methods to sequence and analyze UV damage in the genome of human fibroblasts and melanocytes. In melanocytes, specific genetic loci are exquisitely sensitive to UV. These hyperhotspots of DNA damage associate with specific DNA motifs in sequences adjacent to protein coding genes and are expressed. This means that in every instance of UV-induced sunburn, about 20 cellular pathways affecting cellular growth and physiology are hit at least once. This is where the risk from UV may lie, rather than in rare oncogenic mutations. Proc. Natl. Acad. Sci. U.S.A. 116 , 24196 (2019).

Enhancer Genomics Enhancers are genomic regions that regulate gene expression, sometimes in a cell-dependent manner. However, most of our knowledge of human brain cell–type enhancers derives from studies of bulk human brain tissue. Nott et al. examined chromatin and promoter activity in cell nuclei isolated from human brains. Genetic variants associated with brain traits and disease showed cell-specific patterns of enhancer enrichment. These data indicate that Alzheimer's disease is regulated by genetic variants within microglial cells, whereas psychiatric diseases tend to affect neurons. Science , this issue p. [1134][1] [1]: /lookup/doi/10.1126/science.aay0793

Immunogenomics Single-cell RNA sequencing has begun to shed light on the full cellular diversity of specific organs. However, these studies rarely examine organ-specific immune cells. Stewart et al. sequenced healthy adult and fetal kidney samples at a single-cell level to define the heterogeneity in epithelial, myeloid, and lymphoid cells. From this dataset, they identified zonation of cells, with relevance to disease and the varied perturbations that occur in different tumor settings. This profiling of the human kidney generates a comprehensive census of existing cell populations that will help inform the diagnosis and treatment of kidney-related diseases. Science , this issue p. [1461][1] [1]: /lookup/doi/10.1126/science.aat5031

Human Genomics The genetics underlying who develops multiple sclerosis (MS) have been difficult to work out. Examining more than 47,000 cases and 68,000 controls with multiple genome-wide association studies, the International Multiple Sclerosis Genetics Consortium identified more than 200 risk loci in MS (see the Perspective by Briggs). Focusing on the best candidate genes, including a model of the major histocompatibility complex region, the authors identified statistically independent effects at the genome level. Gene expression studies detected that every major immune cell type is enriched for MS susceptibility genes and that MS risk variants are enriched in brain-resident immune cells, especially microglia. Up to 48% of the genetic contribution of MS can be explained through this analysis. Science , this issue p. [eaav7188][1]; see also p. [1383][2] [1]: /lookup/doi/10.1126/science.aav7188 [2]: /lookup/doi/10.1126/science.aay1439

Fungal Genetics Mutation can often occur as part of the process of cellular division and may have deleterious consequences for multicellular organisms. Through genomic sequencing of Marasmius oreades , a species of fairy ring mushroom, Hiltunen et al. found that in this relatively long-lived species, the accumulation of mutations is an order of magnitude less than previously discovered for any organism. This could not be attributed to purifying selection and indicates that this species has evolved high-fidelity replication and/or repair mechanisms to prevent mutation accumulation. Given that fungi, unlike mammals, do not sequester their reproductive germ cells, this is of interest in understanding how an organism can police its own cell division to maintain a low rate of mutation accumulation. Curr. Biol. 29 , 2758 (2019).

Cancer Genomics Genomic features are often examined at extremes to determine the impact of mutations. These genomic regions span from the trinucleotide context to megabases that underlie chromatin and chromosomal features. Examining mutational dynamics at the mesoscale, the intermediate span of the genome, Buisson et al. characterized the mutational dynamics of cancer (see the Perspective by Carter). They found that mutations caused by the APOBEC enzyme in DNA stem-loops, a mesoscale feature of the genome, could drive recurrent mutations. Many of these types of mutations have been identified as likely drivers of cancer. However, APOBEC-generated mutations outside of stem-loops were more likely to be cancer driver mutations, providing a genomic context for separating cancer driver from passenger mutations. Science , this issue p. [eaaw2872][1]; see also p. [1228][2] [1]: /lookup/doi/10.1126/science.aaw2872 [2]: /lookup/doi/10.1126/science.aax9108

Mutation Mutations in the sperm and eggs of humans have been attributed to errors in DNA replication. More than 75% of human germline mutations are paternal in origin. This is thought to be a result of male gametes undergoing more rounds of cell division than female gametes and thus having a greater probability of replication error. Gao et al. examined datasets of de novo mutations in the human germline and found that the mutation bias is not driven by spermatogenesis. They observed a surprising degree of C-to-G transversions and CpG transitions, indicative of DNA damage. The authors deduced that most mutations in early embryos are more likely to result from factors associated with maternal age at conception and accumulated damage in oocytes and embryos than from replication error. Proc. Natl. Acad. Sci. U.S.A. 116 , 9491 (2019).

Human Genomics Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease that affects nerve cells in the brain and the spinal cord. It has proven difficult to identify the early stages of disease and where it spreads within the body. Maniatis et al. used RNA sequencing to define transcriptomic changes over the course of disease in different regions of the spinal cord of a mouse ALS model and a postmortem human ALS spinal cord. From changes in gene expression, they identified disease-associated pathways and established the key steps in motor neuron degeneration observed in ALS. Science , this issue p. [89][1] [1]: /lookup/volpage/364/89?iss=6435

RNA Sequencing Mapping gene expression at the single-cell level within tissues remains a technical challenge. Rodriques et al. developed a method called Slide-seq, whereby RNA was spatially resolved from tissue sections by transfer onto a surface covered with DNA-barcoded beads. Applying Slide-seq to regions of a mouse brain revealed spatial gene expression patterns in the Purkinje layer of the cerebellum and axes of variation across Purkinje cell compartments. The authors used this method to dissect the temporal evolution of cell type–specific responses in a mouse model of traumatic brain injury. Science , this issue p. [1463][1] [1]: /lookup/volpage/363/1463?iss=6434

Synthetic Biology DNA and RNA are naturally composed of four nucleotide bases that form hydrogen bonds in order to pair. Hoshika et al. added an additional four synthetic nucleotides to produce an eight-letter genetic code and generate so-called hachimoji DNA. Coupled with an engineered T7 RNA polymerase, this expanded DNA alphabet could be transcribed into RNA. Thus, new forms of DNA that add information density to genetic biopolymers can be generated that may be useful for future synthetic biological applications. Science , this issue p. [884][1] [1]: /lookup/doi/10.1126/science.aat0971

Human Genetics Precision medicine promises the ability to identify risks and treat patients on the basis of pathogenic genetic variation. Two studies combined exome sequencing results for over 50,000 people with their electronic health records. Dewey et al. found that ∼3.5% of individuals in their cohort had clinically actionable genetic variants. Many of these variants affected blood lipid levels that could influence cardiovascular health. Abul-Husn et al. extended these findings to investigate the genetics and treatment of familial hypercholesterolemia, a risk factor for cardiovascular disease, within their patient pool. Genetic screening helped identify at-risk patients who could benefit from increased treatment. Science , this issue p. [10.1126/science.aaf6814][1], p. [10.1126/science.aaf7000][2] [1]: /lookup/doi/10.1126/science.aaf6814 [2]: /lookup/doi/10.1126/science.aaf7000

Cancer Genomics To better design treatments for cancer, it is important to understand the heterogeneity in tumors and how this contributes to metastasis. To examine this process, Bian et al. used a single-cell triple omics sequencing (scTrio-seq) technique to examine the mutations, transcriptome, and methylome within colorectal cancer tumors and metastases from 10 individual patients. The analysis provided insights into tumor evolution, linked DNA methylation to genetic lineages, and showed that DNA methylation levels are consistent within lineages but can differ substantially among clones. Science , this issue p. [1060][1] [1]: /lookup/doi/10.1126/science.aao3791

Neurogenomics Spatial transcriptomics can link molecularly described cell types to their anatomical positions and functional roles. Moffitt et al. used a combination of single-cell RNA-sequencing and MERFISH (multiplexed error-robust fluorescence in situ hybridization) to map the identity and location of specific cell types within the mouse preoptic hypothalamus and surrounding areas of the brain (see the Perspective by Tasic and Nicovich). They related these cell types to specific behaviors via gene activity. The approach provides an unbiased description of cell types of the preoptic area, which are important for sleep, thermoregulation, thirst, and social behavior. Science , this issue p. [eaau5324][1]; see also p. [749][2] [1]: /lookup/doi/10.1126/science.aau5324 [2]: /lookup/doi/10.1126/science.aav4841

Chromatin Structure The genome is organized within the nucleus as three-dimensional domains that modulate DNA-templated processes. Bintu et al. used high-throughput Oligopaint labeling and imaging to observe chromatin dynamics inside the nuclei of several different mammalian cell lines. After combining the datasets, single-cell matrices revealed chromatin arranged in topologically associating domains (TADs). Removing cohesin resulted in a loss of aggregate TADs among populations of cells, but specific TADs were still detected at the single-cell level. Furthermore, higher-order organization was detected, suggestive of cooperative interactions within the genome. Science , this issue p. [eaau1783][1] [1]: /lookup/doi/10.1126/science.aau1783