Uncovering the dynamics of precise repair at CRISPR/Cas9-induced double-strand breaks.

CRISPR/Cas9 is widely used for precise mutagenesis through targeted DNA double-strand breaks (DSBs) induction followed by error-prone repair. A better understanding of this process requires measuring the rates of cutting, error-prone, and precise repair, which have remained elusive so far. Here, we present a molecular and computational toolkit for multiplexed quantification of DSB intermediates and repair products by single-molecule sequencing. Using this approach, we characterize the dynamics of DSB induction, processing and repair at endogenous loci along a 72 h time-course in tomato protoplasts. Combining this data with kinetic modeling reveals that indel accumulation is determined by the combined effect of the rates of DSB induction processing of broken ends, and precise versus error repair. In this study, 64-88% of the molecules were cleaved in the three targets analyzed, while indels ranged between 15-41%. Precise repair accounts for most of the gap between cleavage and error repair, representing up to 70% of all repair events. Altogether, this system exposes flux in the DSB repair process, decoupling induction and repair dynamics, and suggesting an essential role of high-fidelity repair in limiting the efficiency of CRISPR-mediated mutagenesis.

CRISPR/Cas9-induced DNA breaks trigger crossover, chromosomal loss, and chromothripsis-like rearrangements.

DNA double-stranded breaks (DSBs) generated by the Cas9 nuclease are commonly repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR). However, little is known about unrepaired DSBs and the type of damage they trigger in plants. We designed an assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay, we detected a tomato (Solanum lycopersicum) flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole-genome sequencing and molecular and cytological analyses supports a model in which a breakage-fusion-bridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a tool for precise breeding via targeted crossover detection. It also uncovers CRISPR-mediated chromothripsis-like events in plants.

Shared community effects and the non-genetic maternal environment shape cortisol levels in wild chimpanzees.

Mechanisms of inheritance remain poorly defined for many fitness-mediating traits, especially in long-lived animals with protracted development. Using 6,123 urinary samples from 170 wild chimpanzees, we examined the contributions of genetics, non-genetic maternal effects, and shared community effects on variation in cortisol levels, an established predictor of survival in long-lived primates. Despite evidence for consistent individual variation in cortisol levels across years, between-group effects were more influential and made an overwhelming contribution to variation in this trait. Focusing on within-group variation, non-genetic maternal effects accounted for 8% of the individual differences in average cortisol levels, significantly more than that attributable to genetic factors, which was indistinguishable from zero. These maternal effects are consistent with a primary role of a shared environment in shaping physiology. For chimpanzees, and perhaps other species with long life histories, community and maternal effects appear more relevant than genetic inheritance in shaping key physiological traits.

Genome sequences of five Sitopsis species of Aegilops and the origin of polyploid wheat B subgenome.

Common wheat (Triticum aestivum, BBAADD) is a major staple food crop worldwide. The diploid progenitors of the A and D subgenomes have been unequivocally identified; that of B, however, remains ambiguous and controversial but is suspected to be related to species of Aegilops, section Sitopsis. Here, we report the assembly of chromosome-level genome sequences of all five Sitopsis species, namely Aegilops bicornis, Ae. longissima, Ae. searsii, Ae. sharonensis, and Ae. speltoides, as well as the partial assembly of the Amblyopyrum muticum (synonym Aegilops mutica) genome for phylogenetic analysis. Our results reveal that the donor of the common wheat B subgenome is a distinct, and most probably extinct, diploid species that diverged from an ancestral progenitor of the B lineage to which the still extant Ae. speltoides and Am. muticum belong. In addition, we identified interspecific genetic introgressions throughout the evolution of the Triticum/Aegilops species complex. The five Sitopsis species have various assembled genome sizes (4.11-5.89 Gb) with high proportions of repetitive sequences (85.99%-89.81%); nonetheless, they retain high collinearity with other genomes or subgenomes of species in the Triticum/Aegilops complex. Differences in genome size were primarily due to independent post-speciation amplification of transposons. We also identified a set of Sitopsis genes pertinent to important agronomic traits that can be harnessed for wheat breeding. These newly assembled genome resources provide a new roadmap for evolutionary and genetic studies of the Triticum/Aegilops complex, as well as for wheat improvement.

On the fitness of informative cues in complex environments.

To be able to deal with uncertainty is of primary importance to most living organisms. When cues provide information about the state of the environment, organisms can use them to respond flexibly. Life forms have evolved complex adaptations and sensory mechanisms to use these environmental cues and extract valuable information about the environment. Previous work has shown a theoretical limit to the amount of fitness benefit possible to be extracted from the cues. We show that the previously used information theoretical approaches can be generalised to scenarios involving any potential relationship between the number of possible phenotypes and environmental states. Such cases are relevant when physiological constraints or complex ecological scenarios lead to the number of environmental states exceeding potential phenotypes. We illustrate cases in which these scenarios can emerge: along environmental gradients, such as geographical transects or complex environments, where organisms adopt different bet-hedging strategies, switching stochastically between phenotypes or developing intermediate ones. In conclusion, we develop an information-theoretic extensible approach for investigating and quantifying fitness in ecological studies.

Unearthing Neanderthal population history using nuclear and mitochondrial DNA from cave sediments.

Bones and teeth are important sources of Pleistocene hominin DNA, but are rarely recovered at archaeological sites. Mitochondrial DNA (mtDNA) has been retrieved from cave sediments but provides limited value for studying population relationships. We therefore developed methods for the enrichment and analysis of nuclear DNA from sediments and applied them to cave deposits in western Europe and southern Siberia dated to between 200,000 and 50,000 years ago. We detected a population replacement in northern Spain about 100,000 years ago, which was accompanied by a turnover of mtDNA. We also identified two radiation events in Neanderthal history during the early part of the Late Pleistocene. Our work lays the ground for studying the population history of ancient hominins from trace amounts of nuclear DNA in sediments.

Initial Upper Palaeolithic humans in Europe had recent Neanderthal ancestry.

Modern humans appeared in Europe by at least 45,000 years ago1-5, but the extent of their interactions with Neanderthals, who disappeared by about 40,000 years ago6, and their relationship to the broader expansion of modern humans outside Africa are poorly understood. Here we present genome-wide data from three individuals dated to between 45,930 and 42,580 years ago from Bacho Kiro Cave, Bulgaria1,2. They are the earliest Late Pleistocene modern humans known to have been recovered in Europe so far, and were found in association with an Initial Upper Palaeolithic artefact assemblage. Unlike two previously studied individuals of similar ages from Romania7 and Siberia8 who did not contribute detectably to later populations, these individuals are more closely related to present-day and ancient populations in East Asia and the Americas than to later west Eurasian populations. This indicates that they belonged to a modern human migration into Europe that was not previously known from the genetic record, and provides evidence that there was at least some continuity between the earliest modern humans in Europe and later people in Eurasia. Moreover, we find that all three individuals had Neanderthal ancestors a few generations back in their family history, confirming that the first European modern humans mixed with Neanderthals and suggesting that such mixing could have been common.

Neandertal introgression and accumulation of hypomorphic mutations in the neuropeptide S (NPS) system promote attenuated functionality.

The neuropeptide S (NPS) system plays an important role in fear and fear memory processing but has also been associated with allergic and inflammatory diseases. Genes for NPS and its receptor NPSR1 are found in all tetrapods. Compared to non-human primates, several non-synonymous single-nucleotide polymorphisms (SNPs) occur in both human genes that collectively result in functional attenuation, suggesting adaptive mechanisms in a human context. To investigate historic and geographic origins of these hypomorphic mutations and explore genetic signs of selection, we analyzed ancient genomes and worldwide genotype frequencies of four prototypic SNPs in the NPS system. Neandertal and Denisovan genomes contain exclusively ancestral alleles for NPSR1 while all derived alleles occur in ancient genomes of anatomically modern humans, indicating that they arose in modern Homo sapiens. Worldwide genotype frequencies for three hypomorphic NPSR1 SNPs show significant regional homogeneity but follow a gradient towards increasing derived allele frequencies that supports an out-of-Africa scenario. Increased density of high-frequency polymorphisms around the three NPSR1 loci suggests weak or possibly balancing selection. A hypomorphic mutation in the NPS precursor, however, was detected at high frequency in Eurasian Neandertal genomes and shows genetic signatures indicating that it was introgressed into the human gene pool, particularly in Southern Europe, by interbreeding with Neandertals. We discuss potential evolutionary scenarios including behavior and immune-based natural selection.

Population Dynamics and Structural Effects at Short and Long Range Support the Hypothesis of the Selective Advantage of the G614 SARS-CoV-2 Spike Variant.

SARS-CoV-2 epidemics quickly propagated worldwide, sorting virus genomic variants in newly established propagules of infections. Stochasticity in transmission within and between countries or an actual selective advantage could explain the global high frequency reached by some genomic variants. Using statistical analyses, demographic reconstructions, and molecular dynamics simulations, we show that the globally invasive G614 spike variant 1) underwent a significant demographic expansion in most countries explained neither by stochastic effects nor by overrepresentation in clinical samples, 2) increases the spike S1/S2 furin-like site conformational plasticity (short-range effect), and 3) modifies the internal motion of the receptor-binding domain affecting its cross-connection with other functional domains (long-range effect). Our results support the hypothesis of a selective advantage at the basis of the spread of the G614 variant, which we suggest may be due to structural modification of the spike protein at the S1/S2 proteolytic site, and provide structural information to guide the design of variant-specific drugs.

A high-coverage Neandertal genome from Chagyrskaya Cave.

We sequenced the genome of a Neandertal from Chagyrskaya Cave in the Altai Mountains, Russia, to 27-fold genomic coverage. We show that this Neandertal was a female and that she was more related to Neandertals in western Eurasia [Prüfer et al., Science 358, 655-658 (2017); Hajdinjak et al., Nature 555, 652-656 (2018)] than to Neandertals who lived earlier in Denisova Cave [Prüfer et al., Nature 505, 43-49 (2014)], which is located about 100 km away. About 12.9% of the Chagyrskaya genome is spanned by homozygous regions that are between 2.5 and 10 centiMorgans (cM) long. This is consistent with the fact that Siberian Neandertals lived in relatively isolated populations of less than 60 individuals. In contrast, a Neandertal from Europe, a Denisovan from the Altai Mountains, and ancient modern humans seem to have lived in populations of larger sizes. The availability of three Neandertal genomes of high quality allows a view of genetic features that were unique to Neandertals and that are likely to have been at high frequency among them. We find that genes highly expressed in the striatum in the basal ganglia of the brain carry more amino-acid-changing substitutions than genes expressed elsewhere in the brain, suggesting that the striatum may have evolved unique functions in Neandertals.

The nature of Neanderthal introgression revealed by 27,566 Icelandic genomes.

Human evolutionary history is rich with the interbreeding of divergent populations. Most humans outside of Africa trace about 2% of their genomes to admixture from Neanderthals, which occurred 50-60 thousand years ago1. Here we examine the effect of this event using 14.4 million putative archaic chromosome fragments that were detected in fully phased whole-genome sequences from 27,566 Icelanders, corresponding to a range of 56,388-112,709 unique archaic fragments that cover 38.0-48.2% of the callable genome. On the basis of the similarity with known archaic genomes, we assign 84.5% of fragments to an Altai or Vindija Neanderthal origin and 3.3% to Denisovan origin; 12.2% of fragments are of unknown origin. We find that Icelanders have more Denisovan-like fragments than expected through incomplete lineage sorting. This is best explained by Denisovan gene flow, either into ancestors of the introgressing Neanderthals or directly into humans. A within-individual, paired comparison of archaic fragments with syntenic non-archaic fragments revealed that, although the overall rate of mutation was similar in humans and Neanderthals during the 500 thousand years that their lineages were separate, there were differences in the relative frequencies of mutation types-perhaps due to different generation intervals for males and females. Finally, we assessed 271 phenotypes, report 5 associations driven by variants in archaic fragments and show that the majority of previously reported associations are better explained by non-archaic variants.

A genetic analysis of the Gibraltar Neanderthals.

The Forbes' Quarry and Devil's Tower partial crania from Gibraltar are among the first Neanderthal remains ever found. Here, we show that small amounts of ancient DNA are preserved in the petrous bones of the 2 individuals despite unfavorable climatic conditions. However, the endogenous Neanderthal DNA is present among an overwhelming excess of recent human DNA. Using improved DNA library construction methods that enrich for DNA fragments carrying deaminated cytosine residues, we were able to sequence 70 and 0.4 megabase pairs (Mbp) nuclear DNA of the Forbes' Quarry and Devil's Tower specimens, respectively, as well as large parts of the mitochondrial genome of the Forbes' Quarry individual. We confirm that the Forbes' Quarry individual was a female and the Devil's Tower individual a male. We also show that the Forbes' Quarry individual is genetically more similar to the ∼120,000-y-old Neanderthals from Scladina Cave in Belgium (Scladina I-4A) and Hohlenstein-Stadel Cave in Germany, as well as to a ∼60,000- to 70,000-y-old Neanderthal from Russia (Mezmaiskaya 1), than to a ∼49,000-y-old Neanderthal from El Sidrón (El Sidrón 1253) in northern Spain and other younger Neanderthals from Europe and western Asia. This suggests that the Forbes' Quarry fossil predates the latter Neanderthals. The preservation of archaic human DNA in the warm coastal climate of Gibraltar, close to the shores of Africa, raises hopes for the future recovery of archaic human DNA from regions in which climatic conditions are less than optimal for DNA preservation.

The Current Genomic Landscape of Western South America: Andes, Amazonia, and Pacific Coast.

Studies of Native South American genetic diversity have helped to shed light on the peopling and differentiation of the continent, but available data are sparse for the major ecogeographic domains. These include the Pacific Coast, a potential early migration route; the Andes, home to the most expansive complex societies and to one of the most widely spoken indigenous language families of the continent (Quechua); and Amazonia, with its understudied population structure and rich cultural diversity. Here, we explore the genetic structure of 176 individuals from these three domains, genotyped with the Affymetrix Human Origins array. We infer multiple sources of ancestry within the Native American ancestry component; one with clear predominance on the Coast and in the Andes, and at least two distinct substrates in neighboring Amazonia, including a previously undetected ancestry characteristic of northern Ecuador and Colombia. Amazonian populations are also involved in recent gene-flow with each other and across ecogeographic domains, which does not accord with the traditional view of small, isolated groups. Long-distance genetic connections between speakers of the same language family suggest that indigenous languages here were spread not by cultural contact alone. Finally, Native American populations admixed with post-Columbian European and African sources at different times, with few cases of prolonged isolation. With our results we emphasize the importance of including understudied regions of the continent in high-resolution genetic studies, and we illustrate the potential of SNP chip arrays for informative regional-scale analysis.

Nuclear DNA from two early Neandertals reveals 80,000 years of genetic continuity in Europe.

Little is known about the population history of Neandertals over the hundreds of thousands of years of their existence. We retrieved nuclear genomic sequences from two Neandertals, one from Hohlenstein-Stadel Cave in Germany and the other from Scladina Cave in Belgium, who lived around 120,000 years ago. Despite the deeply divergent mitochondrial lineage present in the former individual, both Neandertals are genetically closer to later Neandertals from Europe than to a roughly contemporaneous individual from Siberia. That the Hohlenstein-Stadel and Scladina individuals lived around the time of their most recent common ancestor with later Neandertals suggests that all later Neandertals trace at least part of their ancestry back to these early European Neandertals.

The complexity of understanding others as the evolutionary origin of empathy and emotional contagion.

Contagious yawning, emotional contagion and empathy are characterized by the activation of similar neurophysiological states or responses in an observed individual and an observer. For example, it is hard to keep one's mouth closed when imagining someone yawning, or not feeling distressed while observing other individuals perceiving pain. The evolutionary origin of these widespread phenomena is unclear, since a direct benefit is not always apparent. We explore a game theoretical model for the evolution of mind-reading strategies, used to predict and respond to others' behavior. In particular we explore the evolutionary scenarios favoring simulative strategies, which recruit overlapping neural circuits when performing as well as when observing a specific behavior. We show that these mechanisms are advantageous in complex environments, by allowing an observer to use information about its own behavior to interpret that of others. However, without inhibition of the recruited neural circuits, the observer would perform the corresponding downstream action, rather than produce the appropriate social response. We identify evolutionary trade-offs that could hinder this inhibition, leading to emotional contagion as a by-product of mind-reading. The interaction of this model with kinship is complex. We show that empathy likely evolved in a scenario where kin- and other indirect benefits co-opt strategies originally evolved for mind-reading, and that this model explains observed patterns of emotional contagion with kin or group members.

Age estimates for hominin fossils and the onset of the Upper Palaeolithic at Denisova Cave.

Denisova Cave in the Siberian Altai (Russia) is a key site for understanding the complex relationships between hominin groups that inhabited Eurasia in the Middle and Late Pleistocene epoch. DNA sequenced from human remains found at this site has revealed the presence of a hitherto unknown hominin group, the Denisovans1,2, and high-coverage genomes from both Neanderthal and Denisovan fossils provide evidence for admixture between these two populations3. Determining the age of these fossils is important if we are to understand the nature of hominin interaction, and aspects of their cultural and subsistence adaptations. Here we present 50 radiocarbon determinations from the late Middle and Upper Palaeolithic layers of the site. We also report three direct dates for hominin fragments and obtain a mitochondrial DNA sequence for one of them. We apply a Bayesian age modelling approach that combines chronometric (radiocarbon, uranium series and optical ages), stratigraphic and genetic data to calculate probabilistically the age of the human fossils at the site. Our modelled estimate for the age of the oldest Denisovan fossil suggests that this group was present at the site as early as 195,000 years ago (at 95.4% probability). All Neanderthal fossils-as well as Denisova 11, the daughter of a Neanderthal and a Denisovan4-date to between 80,000 and 140,000 years ago. The youngest Denisovan dates to 52,000-76,000 years ago. Direct radiocarbon dating of Upper Palaeolithic tooth pendants and bone points yielded the earliest evidence for the production of these artefacts in northern Eurasia, between 43,000 and 49,000 calibrated years before present (taken as AD 1950). On the basis of current archaeological evidence, it may be assumed that these artefacts are associated with the Denisovan population. It is not currently possible to determine whether anatomically modern humans were involved in their production, as modern-human fossil and genetic evidence of such antiquity has not yet been identified in the Altai region.

Turning Vice into Virtue: Using Batch-Effects to Detect Errors in Large Genomic Data Sets.

It is often unavoidable to combine data from different sequencing centers or sequencing platforms when compiling data sets with a large number of individuals. However, the different data are likely to contain specific systematic errors that will appear as SNPs. Here, we devise a method to detect systematic errors in combined data sets. To measure quality differences between individual genomes, we study pairs of variants that reside on different chromosomes and co-occur in individuals. The abundance of these pairs of variants in different genomes is then used to detect systematic errors due to batch effects. Applying our method to the 1000 Genomes data set, we find that coding regions are enriched for errors, where ∼1% of the higher frequency variants are predicted to be erroneous, whereas errors outside of coding regions are much rarer (<0.001%). As expected, predicted errors are found less often than other variants in a data set that was generated with a different sequencing technology, indicating that many of the candidates are indeed errors. However, predicted 1000 Genomes errors are also found in other large data sets; our observation is thus not specific to the 1000 Genomes data set. Our results show that batch effects can be turned into a virtue by using the resulting variation in large scale data sets to detect systematic errors.

The genome of the offspring of a Neanderthal mother and a Denisovan father.

Neanderthals and Denisovans are extinct groups of hominins that separated from each other more than 390,000 years ago1,2. Here we present the genome of 'Denisova 11', a bone fragment from Denisova Cave (Russia)3 and show that it comes from an individual who had a Neanderthal mother and a Denisovan father. The father, whose genome bears traces of Neanderthal ancestry, came from a population related to a later Denisovan found in the cave4-6. The mother came from a population more closely related to Neanderthals who lived later in Europe2,7 than to an earlier Neanderthal found in Denisova Cave8, suggesting that migrations of Neanderthals between eastern and western Eurasia occurred sometime after 120,000 years ago. The finding of a first-generation Neanderthal-Denisovan offspring among the small number of archaic specimens sequenced to date suggests that mixing between Late Pleistocene hominin groups was common when they met.

Reconstructing the genetic history of late Neanderthals.

Although it has previously been shown that Neanderthals contributed DNA to modern humans, not much is known about the genetic diversity of Neanderthals or the relationship between late Neanderthal populations at the time at which their last interactions with early modern humans occurred and before they eventually disappeared. Our ability to retrieve DNA from a larger number of Neanderthal individuals has been limited by poor preservation of endogenous DNA and contamination of Neanderthal skeletal remains by large amounts of microbial and present-day human DNA. Here we use hypochlorite treatment of as little as 9 mg of bone or tooth powder to generate between 1- and 2.7-fold genomic coverage of five Neanderthals who lived around 39,000 to 47,000 years ago (that is, late Neanderthals), thereby doubling the number of Neanderthals for which genome sequences are available. Genetic similarity among late Neanderthals is well predicted by their geographical location, and comparison to the genome of an older Neanderthal from the Caucasus indicates that a population turnover is likely to have occurred, either in the Caucasus or throughout Europe, towards the end of Neanderthal history. We find that the bulk of Neanderthal gene flow into early modern humans originated from one or more source populations that diverged from the Neanderthals that were studied here at least 70,000 years ago, but after they split from a previously sequenced Neanderthal from Siberia around 150,000 years ago. Although four of the Neanderthals studied here post-date the putative arrival of early modern humans into Europe, we do not detect any recent gene flow from early modern humans in their ancestry.