Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq.

Pooled CRISPR screens coupled with single-cell RNA-sequencing have enabled systematic interrogation of gene function and regulatory networks. Here, we introduce Cas13 RNA Perturb-seq (CaRPool-seq), which leverages the RNA-targeting CRISPR-Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable CRISPR array that is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes. We compared CaRPool-seq to existing Cas9-based methods, highlighting its unique strength to efficiently profile combinatorially perturbed cells. Finally, we apply CaRPool-seq to perform multiplexed combinatorial perturbations of myeloid differentiation regulators in an acute myeloid leukemia (AML) model system and identify extensive interactions between different chromatin regulators that can enhance or suppress AML differentiation phenotypes.

Phylogenomic Analysis of the Parrots of the World Distinguishes Artifactual from Biological Sources of Gene Tree Discordance.

Gene tree discordance is expected in phylogenomic trees and biological processes are often invoked to explain it. However, heterogeneous levels of phylogenetic signal among individuals within data sets may cause artifactual sources of topological discordance. We examined how the information content in tips and subclades impacts topological discordance in the parrots (Order: Psittaciformes), a diverse and highly threatened clade of nearly 400 species. Using ultraconserved elements from 96% of the clade's species-level diversity, we estimated concatenated and species trees for 382 ingroup taxa. We found that discordance among tree topologies was most common at nodes dating between the late Miocene and Pliocene, and often at the taxonomic level of the genus. Accordingly, we used two metrics to characterize information content in tips and assess the degree to which conflict between trees was being driven by lower-quality samples. Most instances of topological conflict and nonmonophyletic genera in the species tree could be objectively identified using these metrics. For subclades still discordant after tip-based filtering, we used a machine learning approach to determine whether phylogenetic signal or noise was the more important predictor of metrics supporting the alternative topologies. We found that when signal favored one of the topologies, the noise was the most important variable in poorly performing models that favored the alternative topology. In sum, we show that artifactual sources of gene tree discordance, which are likely a common phenomenon in many data sets, can be distinguished from biological sources by quantifying the information content in each tip and modeling which factors support each topology. [Historical DNA; machine learning; museomics; Psittaciformes; species tree.].

Evolutionary systems biology reveals patterns of rice adaptation to drought-prone agro-ecosystems.

Rice (Oryza sativa) was domesticated around 10,000 years ago and has developed into a staple for half of humanity. The crop evolved and is currently grown in stably wet and intermittently dry agro-ecosystems, but patterns of adaptation to differences in water availability remain poorly understood. While previous field studies have evaluated plant developmental adaptations to water deficit, adaptive variation in functional and hydraulic components, particularly in relation to gene expression, has received less attention. Here, we take an evolutionary systems biology approach to characterize adaptive drought resistance traits across roots and shoots. We find that rice harbors heritable variation in molecular, physiological, and morphological traits that is linked to higher fitness under drought. We identify modules of co-expressed genes that are associated with adaptive drought avoidance and tolerance mechanisms. These expression modules showed evidence of polygenic adaptation in rice subgroups harboring accessions that evolved in drought-prone agro-ecosystems. Fitness-linked expression patterns allowed us to identify the drought-adaptive nature of optimizing photosynthesis and interactions with arbuscular mycorrhizal fungi. Taken together, our study provides an unprecedented, integrative view of rice adaptation to water-limited field conditions.

Integrated analysis of multimodal single-cell data.

The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce "weighted-nearest neighbor" analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.

Characterizing the molecular regulation of inhibitory immune checkpoints with multimodal single-cell screens.

The expression of inhibitory immune checkpoint molecules, such as programmed death-ligand (PD-L)1, is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune responses. Here, we apply expanded CRISPR-compatible (EC)CITE-seq, a technology that combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape, that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Applying these tools, we identify and validate regulators of PD-L1 and leverage our multimodal data to identify both transcriptional and post-transcriptional modes of regulation. Specifically, we discover that the Kelch-like protein KEAP1 and the transcriptional activator NRF2 mediate the upregulation of PD-L1 after interferon (IFN)-γ stimulation. Our results identify a new mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multimodal single-cell perturbation screens.

Uneven Missing Data Skew Phylogenomic Relationships within the Lories and Lorikeets.

The resolution of the Tree of Life has accelerated with advances in DNA sequencing technology. To achieve dense taxon sampling, it is often necessary to obtain DNA from historical museum specimens to supplement modern genetic samples. However, DNA from historical material is generally degraded, which presents various challenges. In this study, we evaluated how the coverage at variant sites and missing data among historical and modern samples impacts phylogenomic inference. We explored these patterns in the brush-tongued parrots (lories and lorikeets) of Australasia by sampling ultraconserved elements in 105 taxa. Trees estimated with low coverage characters had several clades where relationships appeared to be influenced by whether the sample came from historical or modern specimens, which were not observed when more stringent filtering was applied. To assess if the topologies were affected by missing data, we performed an outlier analysis of sites and loci, and a data reduction approach where we excluded sites based on data completeness. Depending on the outlier test, 0.15% of total sites or 38% of loci were driving the topological differences among trees, and at these sites, historical samples had 10.9× more missing data than modern ones. In contrast, 70% data completeness was necessary to avoid spurious relationships. Predictive modeling found that outlier analysis scores were correlated with parsimony informative sites in the clades whose topologies changed the most by filtering. After accounting for biased loci and understanding the stability of relationships, we inferred a more robust phylogenetic hypothesis for lories and lorikeets.

The strength and pattern of natural selection on gene expression in rice.

Levels of gene expression underpin organismal phenotypes1,2, but the nature of selection that acts on gene expression and its role in adaptive evolution remain unknown1,2. Here we assayed gene expression in rice (Oryza sativa)3, and used phenotypic selection analysis to estimate the type and strength of selection on the levels of more than 15,000 transcripts4,5. Variation in most transcripts appears (nearly) neutral or under very weak stabilizing selection in wet paddy conditions (with median standardized selection differentials near zero), but selection is stronger under drought conditions. Overall, more transcripts are conditionally neutral (2.83%) than are antagonistically pleiotropic6 (0.04%), and transcripts that display lower levels of expression and stochastic noise7-9 and higher levels of plasticity9 are under stronger selection. Selection strength was further weakly negatively associated with levels of cis-regulation and network connectivity9. Our multivariate analysis suggests that selection acts on the expression of photosynthesis genes4,5, but that the efficacy of selection is genetically constrained under drought conditions10. Drought selected for earlier flowering11,12 and a higher expression of OsMADS18 (Os07g0605200), which encodes a MADS-box transcription factor and is a known regulator of early flowering13-marking this gene as a drought-escape gene11,12. The ability to estimate selection strengths provides insights into how selection can shape molecular traits at the core of gene action.

Comprehensive Integration of Single-Cell Data.

Single-cell transcriptomics has transformed our ability to characterize cell states, but deep biological understanding requires more than a taxonomic listing of clusters. As new methods arise to measure distinct cellular modalities, a key analytical challenge is to integrate these datasets to better understand cellular identity and function. Here, we develop a strategy to "anchor" diverse datasets together, enabling us to integrate single-cell measurements not only across scRNA-seq technologies, but also across different modalities. After demonstrating improvement over existing methods for integrating scRNA-seq data, we anchor scRNA-seq experiments with scATAC-seq to explore chromatin differences in closely related interneuron subsets and project protein expression measurements onto a bone marrow atlas to characterize lymphocyte populations. Lastly, we harmonize in situ gene expression and scRNA-seq datasets, allowing transcriptome-wide imputation of spatial gene expression patterns. Our work presents a strategy for the assembly of harmonized references and transfer of information across datasets.

Phylogeography and species limits in the red-shouldered hawk (Buteo lineatus): Characterization of the Northern Florida Suture Zone in birds.

The North American red-shouldered hawk, Buteo lineatus, is comprised of two widely allopatric eastern and western populations with an additional well-marked subspecies in the Florida peninsula. The two eastern populations meet in northern Florida, the location of a well-known suture zone in many nonavian organisms. We sequenced the complete mitochondrial ND2 gene and two nuclear introns to investigate its genetic population structure and species status. No mitochondrial haplotypes were shared between the eastern and western populations, and genetic variance among 14 populations was 0.42; almost all of this (0.40) was distributed among the three regions. A clade of haplotypes very common in the Florida peninsula decreased in frequency elsewhere and, when modeled as a hybrid zone, had an estimated width of 1,158 km with a center near Ocala, FL. Ecological niche modeling suggests the western, eastern, and Florida peninsula populations were geographically isolated during the last glacial maximum. We consider these to represent three phylogenetic species. A coalescent analysis incorporating incomplete lineage sorting and gene tree uncertainty also suggested the divergence between the western and eastern populations is consistent with species-level divergence. With the addition of this hawk, four avian species are now known to hybridize along the Gulf Coast of the United States in or near the Northern Florida Suture Zone. The widths of these avian zones vary substantially (176-1,158 km) and appear to reflect magnitude of gene flow, rather than extent of genetic differentiation. None of these birds was suggested as possible exemplars in the original description of the suture zone. Of the six species that were so identified, three have been surveyed to date, but none of those was found to be genetically differentiated.

A single-cell transcriptional roadmap for cardiopharyngeal fate diversification.

In vertebrates, multipotent progenitors located in the pharyngeal mesoderm form cardiomyocytes and branchiomeric head muscles, but the dynamic gene expression programmes and mechanisms underlying cardiopharyngeal multipotency and heart versus head muscle fate choices remain elusive. Here, we used single-cell genomics in the simple chordate model Ciona to reconstruct developmental trajectories forming first and second heart lineages and pharyngeal muscle precursors and characterize the molecular underpinnings of cardiopharyngeal fate choices. We show that FGF-MAPK signalling maintains multipotency and promotes the pharyngeal muscle fate, whereas signal termination permits the deployment of a pan-cardiac programme, shared by the first and second heart lineages, to define heart identity. In the second heart lineage, a Tbx1/10-Dach pathway actively suppresses the first heart lineage programme, conditioning later cell diversity in the beating heart. Finally, cross-species comparisons between Ciona and the mouse evoke the deep evolutionary origins of cardiopharyngeal networks in chordates.

Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics.

Despite rapid developments in single cell sequencing, sample-specific batch effects, detection of cell multiplets, and experimental costs remain outstanding challenges. Here, we introduce Cell Hashing, where oligo-tagged antibodies against ubiquitously expressed surface proteins uniquely label cells from distinct samples, which can be subsequently pooled. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its original sample, robustly identify cross-sample multiplets, and "super-load" commercial droplet-based systems for significant cost reduction. We validate our approach using a complementary genetic approach and demonstrate how hashing can generalize the benefits of single cell multiplexing to diverse samples and experimental designs.

Species delimitation and biogeography of the gnatcatchers and gnatwrens (Aves: Polioptilidae).

The New World avian family Polioptilidae (gnatcatchers and gnatwrens) is distributed from Argentina to Canada and includes 15 species and more than 60 subspecies. No study to date has evaluated phylogenetic relationships within this family and the historical pattern of diversification within the group remains unknown. Moreover, species limits, particularly in widespread taxa that show geographic variation, remain unclear. In this study, we delimited species and estimated phylogenetic relationships using multilocus data for the entire family. We then used the inferred diversity along with alternative taxonomic classification schemes to evaluate how lumping and splitting of both taxa and geographical areas influenced biogeographic inference. Species-tree analyses grouped Polioptilidae into four main clades: Microbates, Ramphocaenus, a Polioptila guianensis complex, and the remaining members of Polioptila. Ramphocaenus melanurus was sister to the clade containing M. cinereiventris and M. collaris, which formed a clade sister to all species within Polioptila. Polioptila was composed of two clades, the first of which included the P. guianensis complex; the other contained all remaining species in the genus. Using multispecies coalescent modeling, we inferred a more than 3-fold increase in species diversity, of which 87% represent currently recognized species or subspecies. Much of this diversity corresponded to subspecies that occur in the Neotropics. We identified three polyphyletic species, and delimited 4-6 previously undescribed candidate taxa. Probabilistic modeling of geographic ranges on the species tree indicated that the family likely had an ancestral origin in South America, with all three genera independently colonizing North America. Support for this hypothesis, however, was sensitive to the taxonomic classification scheme used and the number of geographical areas allowed. Our study proposes the first phylogenetic hypothesis for Polioptilidae and provides genealogical support for the reclassification of species limits. Species limits and the resolution of geographical areas that taxa inhabit influence the inferred spatial diversification history.

Genomic divergence in allopatric Northern Cardinals of the North American warm deserts is linked to behavioral differentiation.

Biogeographic barriers are considered important in initiating speciation through geographic isolation, but they rarely indiscriminately and completely reduce gene flow across entire communities. Explicitly demonstrating which factors are associated with gene-flow levels across barriers would help elucidate how speciation is initiated and isolation maintained. Here, we investigated the association of behavioral isolation on population differentiation in Northern Cardinals (Cardinalis cardinalis) distributed across the Cochise Filter Barrier, a region of transitional habitat which separates the Sonoran and Chihuahuan deserts of North America. Using genomewide markers, we modeled demographic history by fitting the data to isolation and isolation-with-migration models. The best-fit model indicated that desert populations diverged in the Pleistocene with low, historic, and asymmetric gene flow across the barrier. We then tested behavioral isolation using reciprocal call-broadcast experiments to compare song recognition between deserts, controlling for song dialect changes within deserts. We found that male Northern Cardinals in both deserts were most aggressive to local songs and failed to recognize across-barrier songs. A correlation of genomic differentiation and strong song discrimination is consistent with a model where speciation is initiated across a barrier and maintained by behavioral isolation.

Isolation with asymmetric gene flow during the nonsynchronous divergence of dry forest birds.

Dry forest bird communities in South America are often fragmented by intervening mountains and rainforests, generating high local endemism. The historical assembly of dry forest communities likely results from dynamic processes linked to numerous population histories among codistributed species. Nevertheless, species may diversify in the same way through time if landscape and environmental features, or species ecologies, similarly structure populations. Here we tested whether six co-distributed taxon pairs that occur in the dry forests of the Tumbes and Marañón Valley of northwestern South America show concordant patterns and modes of diversification. We employed a genome reduction technique, double-digest restriction site-associated DNA sequencing, and obtained 4407-7186 genomewide SNPs. We estimated demographic history in each taxon pair and inferred that all pairs had the same best-fit demographic model: isolation with asymmetric gene flow from the Tumbes into the Marañón Valley, suggesting a common diversification mode. Overall, we also observed congruence in effective population size (Ne ) patterns where ancestral Ne were 2.9-11.0× larger than present-day Marañón Valley populations and 0.3-2.0× larger than Tumbesian populations. Present-day Marañón Valley Ne was smaller than Tumbes. In contrast, we found simultaneous population isolation due to a single event to be unlikely as taxon pairs diverged over an extended period of time (0.1-2.9 Ma) with multiple nonoverlapping divergence periods. Our results show that even when populations of codistributed species asynchronously diverge, the mode of their differentiation can remain conserved over millions of years. Divergence by allopatric isolation due to barrier formation does not explain the mode of differentiation between these two bird assemblages; rather, migration of individuals occurred before and after geographic isolation.

Heterologous mitochondrial DNA recombination in human cells.

Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches. First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events. Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes. This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.

Increased plaque burden in brains of APP mutant MnSOD heterozygous knockout mice.

A growing body of evidence suggests a relationship between oxidative stress and beta-amyloid (Abeta) peptide accumulation, a hallmark in the pathogenesis of Alzheimer's disease (AD). However, a direct causal relationship between oxidative stress and Abeta pathology has not been established in vivo. Therefore, we crossed mice with a knockout of one allele of manganese superoxide dismutase (MnSOD), a critical antioxidant enzyme, with Tg19959 mice, which overexpress a doubly mutated human beta-amyloid precursor protein (APP). Partial deficiency of MnSOD, which is well established to cause elevated oxidative stress, significantly increased brain Abeta levels and Abeta plaque burden in Tg19959 mice. These results indicate that oxidative stress can promote the pathogenesis of AD and further support the feasibility of antioxidant approaches for AD therapy.

Somatic mitochondrial DNA mutations in cortex and substantia nigra in aging and Parkinson's disease.

Oxidative damage to mitochondrial DNA (mtDNA) increases with age in the brain and can induce G:C to T:A and T:A to G:C point mutations. Though rare at any particular site, multiple somatic mtDNA mutations induced by oxidative damage or by other mechanisms may accumulate with age in the brain and thus could play a role in aging and neurodegenerative diseases. However, no prior study has quantified the total burden of mtDNA point mutation subtypes in the brain. Using a highly sensitive cloning and sequencing strategy, we find that the aggregate levels of G:C to T:A and T:A to G:C transversions and of all point mutations increase with age in the frontal cortex (FCtx). In the substantia nigra (SN), the aggregate levels of point mutations in young controls are similar to the levels in the SN or FCtx of elderly subjects. Extrapolation from our data suggests an average of 2.7 (FCtx) to 3.2 (SN) somatic point mutations per mitochondrial genome in elderly subjects. There were no significant differences between Parkinson's disease (PD) patients and age-matched controls in somatic mutation levels. These results indicate that individually rare mtDNA point mutations reach a high aggregate burden in FCtx and SN of elderly subjects.