Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters.

The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.

Chromatin context-dependent regulation and epigenetic manipulation of prime editing.

We set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing, a precise genome engineering tool. Using a highly sensitive method for mapping the genomic locations of randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% to 94% for an identical target site and edit. Position effects on prime editing efficiency are well predicted by chromatin marks, e.g., positively by H3K79me2 and negatively by H3K9me3. Next, we developed a multiplex perturbational framework to assess the interaction of trans-acting factors with the cis-chromatin environment on editing outcomes. Applying this framework to DNA repair factors, we identify HLTF as a context-dependent repressor of prime editing. Finally, several lines of evidence suggest that active transcriptional elongation enhances prime editing. Consistent with this, we show we can robustly decrease or increase the efficiency of prime editing by preceding it with CRISPR-mediated silencing or activation, respectively.

A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

Induction and in silico staging of human gastruloids with neural tube, segmented somites & advanced cell types.

Embryonic organoids are emerging as powerful models for studying early mammalian development. For example, stem cell-derived 'gastruloids' form elongating structures containing all three germ layers1-4. However, although elongated, human gastruloids do not morphologically resemble post-implantation embryos. Here we show that a specific, discontinuous regimen of retinoic acid (RA) robustly induces human gastruloids with embryo-like morphological structures, including a neural tube and segmented somites. Single cell RNA-seq (sc-RNA-seq) further reveals that these human 'RA-gastruloids' contain more advanced cell types than conventional gastruloids, including neural crest cells, renal progenitor cells, skeletal muscle cells, and, rarely, neural progenitor cells. We apply a new approach to computationally stage human RA-gastruloids relative to somite-resolved mouse embryos, early human embryos and other gastruloid models, and find that the developmental stage of human RA-gastruloids is comparable to that of E9.5 mouse embryos, although some cell types show greater or lesser progression. We chemically perturb WNT and BMP signaling in human RA-gastruloids and find that these signaling pathways regulate somite patterning and neural tube length, respectively, while genetic perturbation of the transcription factors PAX3 and TBX6 markedly compromises the formation of neural crest and somites/renal cells, respectively. Human RA-gastruloids complement other embryonic organoids in serving as a simple, robust and screenable model for decoding early human embryogenesis.

Chromatin context-dependent regulation and epigenetic manipulation of prime editing.

Prime editing is a powerful means of introducing precise changes to specific locations in mammalian genomes. However, the widely varying efficiency of prime editing across target sites of interest has limited its adoption in the context of both basic research and clinical settings. Here, we set out to exhaustively characterize the impact of the cis- chromatin environment on prime editing efficiency. Using a newly developed and highly sensitive method for mapping the genomic locations of a randomly integrated "sensor", we identify specific epigenetic features that strongly correlate with the highly variable efficiency of prime editing across different genomic locations. Next, to assess the interaction of trans -acting factors with the cis -chromatin environment, we develop and apply a pooled genetic screening approach with which the impact of knocking down various DNA repair factors on prime editing efficiency can be stratified by cis -chromatin context. Finally, we demonstrate that we can dramatically modulate the efficiency of prime editing through epigenome editing, i.e. altering chromatin state in a locus-specific manner in order to increase or decrease the efficiency of prime editing at a target site. Looking forward, we envision that the insights and tools described here will broaden the range of both basic research and therapeutic contexts in which prime editing is useful.

A single-cell transcriptional timelapse of mouse embryonic development, from gastrula to pup.

The house mouse, Mus musculus, is an exceptional model system, combining genetic tractability with close homology to human biology. Gestation in mouse development lasts just under three weeks, a period during which its genome orchestrates the astonishing transformation of a single cell zygote into a free-living pup composed of >500 million cells. Towards a global framework for exploring mammalian development, we applied single cell combinatorial indexing (sci-*) to profile the transcriptional states of 12.4 million nuclei from 83 precisely staged embryos spanning late gastrulation (embryonic day 8 or E8) to birth (postnatal day 0 or P0), with 2-hr temporal resolution during somitogenesis, 6-hr resolution through to birth, and 20-min resolution during the immediate postpartum period. From these data (E8 to P0), we annotate dozens of trajectories and hundreds of cell types and perform deeper analyses of the unfolding of the posterior embryo during somitogenesis as well as the ontogenesis of the kidney, mesenchyme, retina, and early neurons. Finally, we leverage the depth and temporal resolution of these whole embryo snapshots, together with other published data, to construct and curate a rooted tree of cell type relationships that spans mouse development from zygote to pup. Throughout this tree, we systematically nominate sets of transcription factors (TFs) and other genes as candidate drivers of the in vivo differentiation of hundreds of mammalian cell types. Remarkably, the most dramatic shifts in transcriptional state are observed in a restricted set of cell types in the hours immediately following birth, and presumably underlie the massive changes in physiology that must accompany the successful transition of a placental mammal to extrauterine life.

Massively parallel characterization of transcriptional regulatory elements in three diverse human cell types.

The human genome contains millions of candidate cis-regulatory elements (CREs) with cell-type-specific activities that shape both health and myriad disease states. However, we lack a functional understanding of the sequence features that control the activity and cell-type-specific features of these CREs. Here, we used lentivirus-based massively parallel reporter assays (lentiMPRAs) to test the regulatory activity of over 680,000 sequences, representing a nearly comprehensive set of all annotated CREs among three cell types (HepG2, K562, and WTC11), finding 41.7% to be functional. By testing sequences in both orientations, we find promoters to have significant strand orientation effects. We also observe that their 200 nucleotide cores function as non-cell-type-specific 'on switches' providing similar expression levels to their associated gene. In contrast, enhancers have weaker orientation effects, but increased tissue-specific characteristics. Utilizing our lentiMPRA data, we develop sequence-based models to predict CRE function with high accuracy and delineate regulatory motifs. Testing an additional lentiMPRA library encompassing 60,000 CREs in all three cell types, we further identified factors that determine cell-type specificity. Collectively, our work provides an exhaustive catalog of functional CREs in three widely used cell lines, and showcases how large-scale functional measurements can be used to dissect regulatory grammar.

Optimized single-nucleus transcriptional profiling by combinatorial indexing.

Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) is a powerful method for recovering gene expression data from an exponentially scalable number of individual cells or nuclei. However, sci-RNA-seq is a complex protocol that has historically exhibited variable performance on different tissues, as well as lower sensitivity than alternative methods. Here, we report a simplified, optimized version of the sci-RNA-seq protocol with three rounds of split-pool indexing that is faster, more robust and more sensitive and has a higher yield than the original protocol, with reagent costs on the order of 1 cent per cell or less. The total hands-on time from nuclei isolation to final library preparation takes 2-3 d, depending on the number of samples sharing the experiment. The improvements also allow RNA profiling from tissues rich in RNases like older mouse embryos or adult tissues that were problematic for the original method. We showcase the optimized protocol via whole-organism analysis of an E16.5 mouse embryo, profiling ~380,000 nuclei in a single experiment. Finally, we introduce a 'Tiny-Sci' protocol for experiments in which input material is very limited.

Embryo model completes gastrulation to neurulation and organogenesis.

Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro1-5, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells6-11. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.

Results of genetic analysis of 11 341 participants enrolled in the My Life, Our Future hemophilia genotyping initiative in the United States.

BACKGROUND: Hemophilia A (HA) and hemophilia B (HB) are rare inherited bleeding disorders. Although causative genetic variants are clinically relevant, in 2012 only 20% of US patients had been genotyped. OBJECTIVES: My Life, Our Future (MLOF) was a multisector cross-sectional US initiative to improve our understanding of hemophilia through widespread genotyping. METHODS: Subjects and potential genetic carriers were enrolled at US hemophilia treatment centers (HTCs). Bloodworks performed genotyping and returned results to providers. Clinical data were abstracted from the American Thrombosis and Hemostasis Network dataset. Community education was provided by the National Hemophilia Foundation. RESULTS: From 2013 to 2017, 107 HTCs enrolled 11 341 subjects (68.8% male, 31.2% female) for testing for HA (n = 8976), HB (n = 2358), HA/HB (n = 3), and hemophilia not otherwise specified (n = 4). Variants were detected in most male patients (98.2%% HA, 98.1% HB). 1914 unique variants were found (1482 F8, 431 F9); 744 were novel (610 F8, 134 F9). Inhibitor data were available for 6986 subjects (5583 HA; 1403 HB). In severe HA, genotypes with the highest inhibitor rates were large deletions (77/80), complex intron 22 inversions (9/17), and no variant found (7/14). In severe HB, the highest rates were large deletions (24/42). Inhibitors were reported in 27.3% of Black versus 16.2% of White patients. CONCLUSIONS: The findings of MLOF are reported, the largest hemophilia genotyping project performed to date. The results support the need for comprehensive genetic approaches in hemophilia. This effort has contributed significantly towards better understanding variation in the F8 and F9 genes in hemophilia and risks of inhibitor formation.

Systematic reconstruction of cellular trajectories across mouse embryogenesis.

Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single-cell zygote gives rise to millions of cells expressing a panoply of molecular programs. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here, we set out to integrate several single-cell RNA-sequencing (scRNA-seq) datasets that collectively span mouse gastrulation and organogenesis, supplemented with new profiling of ~150,000 nuclei from approximately embryonic day 8.5 (E8.5) embryos staged in one-somite increments. Overall, we define cell states at each of 19 successive stages spanning E3.5 to E13.5 and heuristically connect them to their pseudoancestors and pseudodescendants. Although constructed through automated procedures, the resulting directed acyclic graph (TOME (trajectories of mammalian embryogenesis)) is largely consistent with our contemporary understanding of mammalian development. We leverage TOME to systematically nominate transcription factors (TFs) as candidate regulators of each cell type's specification, as well as 'cell-type homologs' across vertebrate evolution.

SwabExpress: An End-to-End Protocol for Extraction-Free COVID-19 Testing.

BACKGROUND: The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse-transcription PCR (RT-qPCR). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce. To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction. METHODS: We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral inactivation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance. RESULTS: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/µL, 100% sensitivity, and 99.4% specificity when compared side by side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time. CONCLUSION: SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.

Genome-wide strand asymmetry in massively parallel reporter activity favors genic strands.

Massively parallel reporter assays (MPRAs) are useful tools to characterize regulatory elements in human genomes. An aspect of MPRAs that is not typically the focus of analysis is their intrinsic ability to differentiate activity levels for a given sequence element when placed in both of its possible orientations relative to the reporter construct. Here, we describe pervasive strand asymmetry of MPRA signals in data sets from multiple reporter configurations in both published and newly reported data. These effects are reproducible across different cell types and in different treatments within a cell type and are observed both within and outside of annotated regulatory elements. From elements in gene bodies, MPRA strand asymmetry favors the sense strand, suggesting that function related to endogenous transcription is driving the phenomenon. Similarly, we find that within Alu mobile element insertions, strand asymmetry favors the transcribed strand of the ancestral retrotransposon. The effect is consistent across the multiplicity of Alu elements in human genomes and is more pronounced in less diverged Alu elements. We find sequence features driving MPRA strand asymmetry and show its prediction from sequence alone. We see some evidence for RNA stabilization and transcriptional activation mechanisms and hypothesize that the effect is driven by natural selection favoring efficient transcription. Our results indicate that strand asymmetry is a pervasive and reproducible feature in MPRA data. More importantly, the fact that MPRA asymmetry favors naturally transcribed strands suggests that it stems from preserved biological functions that have a substantial, global impact on gene and genome evolution.

SwabExpress: An end-to-end protocol for extraction-free COVID-19 testing.

BACKGROUND: The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse transcription PCR (RT-qPCR) (1). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce (2). To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction. METHODS: We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral activation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance. RESULTS: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/uL, 100% sensitivity, and 99.4% specificity when compared side-by-side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time. CONCLUSION: SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.

Caspase-8, association with Alzheimer's Disease and functional analysis of rare variants.

The accumulation of amyloid beta (Aß) peptide (Amyloid cascade hypothesis), an APP protein cleavage product, is a leading hypothesis in the etiology of Alzheimer's disease (AD). In order to identify additional AD risk genes, we performed targeted sequencing and rare variant burden association study for nine candidate genes involved in the amyloid metabolism in 1886 AD cases and 1700 controls. We identified a significant variant burden association for the gene encoding caspase-8, CASP8 (p = 8.6x10-5). For two CASP8 variants, p.K148R and p.I298V, the association remained significant in a combined sample of 10,820 cases and 8,881 controls. For both variants we performed bioinformatics structural, expression and enzymatic activity studies and obtained evidence for loss of function effects. In addition to their role in amyloid processing, caspase-8 and its downstream effector caspase-3 are involved in synaptic plasticity, learning, memory and control of microglia pro-inflammatory activation and associated neurotoxicity, indicating additional mechanisms that might contribute to AD. As caspase inhibition has been proposed as a mechanism for AD treatment, our finding that AD-associated CASP8 variants reduce caspase function calls for caution and is an impetus for further studies on the role of caspases in AD and other neurodegenerative diseases.

Novel approach to genetic analysis and results in 3000 hemophilia patients enrolled in the My Life, Our Future initiative.

Hemophilia A and B are rare, X-linked bleeding disorders. My Life, Our Future (MLOF) is a collaborative project established to genotype and study hemophilia. Patients were enrolled at US hemophilia treatment centers (HTCs). Genotyping was performed centrally using next-generation sequencing (NGS) with an approach that detected common F8 gene inversions simultaneously with F8 and F9 gene sequencing followed by confirmation using standard genotyping methods. Sixty-nine HTCs enrolled the first 3000 patients in under 3 years. Clinically reportable DNA variants were detected in 98.1% (2357/2401) of hemophilia A and 99.3% (595/599) of hemophilia B patients. Of the 924 unique variants found, 285 were novel. Predicted gene-disrupting variants were common in severe disease; missense variants predominated in mild-moderate disease. Novel DNA variants accounted for ∼30% of variants found and were detected continuously throughout the project, indicating that additional variation likely remains undiscovered. The NGS approach detected >1 reportable variants in 36 patients (10 females), a finding with potential clinical implications. NGS also detected incidental variants unlikely to cause disease, including 11 variants previously reported in hemophilia. Although these genes are thought to be conserved, our findings support caution in interpretation of new variants. In summary, MLOF has contributed significantly toward variant annotation in the F8 and F9 genes. In the near future, investigators will be able to access MLOF data and repository samples for research to advance our understanding of hemophilia.

MIPgen: optimized modeling and design of molecular inversion probes for targeted resequencing.

UNLABELLED: Molecular inversion probes (MIPs) enable cost-effective multiplex targeted gene resequencing in large cohorts. However, the design of individual MIPs is a critical parameter governing the performance of this technology with respect to capture uniformity and specificity. MIPgen is a user-friendly package that simplifies the process of designing custom MIP assays to arbitrary targets. New logistic and SVM-derived models enable in silico predictions of assay success, and assay redesign exhibits improved coverage uniformity relative to previous methods, which in turn improves the utility of MIPs for cost-effective targeted sequencing for candidate gene validation and for diagnostic sequencing in a clinical setting. AVAILABILITY AND IMPLEMENTATION: MIPgen is implemented in C++. Source code and accompanying Python scripts are available at http://shendurelab.github.io/MIPGEN/.

The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line.

The HeLa cell line was established in 1951 from cervical cancer cells taken from a patient, Henrietta Lacks. This was the first successful attempt to immortalize human-derived cells in vitro. The robust growth and unrestricted distribution of HeLa cells resulted in its broad adoption--both intentionally and through widespread cross-contamination--and for the past 60 years it has served a role analogous to that of a model organism. The cumulative impact of the HeLa cell line on research is demonstrated by its occurrence in more than 74,000 PubMed abstracts (approximately 0.3%). The genomic architecture of HeLa remains largely unexplored beyond its karyotype, partly because like many cancers, its extensive aneuploidy renders such analyses challenging. We carried out haplotype-resolved whole-genome sequencing of the HeLa CCL-2 strain, examined point- and indel-mutation variations, mapped copy-number variations and loss of heterozygosity regions, and phased variants across full chromosome arms. We also investigated variation and copy-number profiles for HeLa S3 and eight additional strains. We find that HeLa is relatively stable in terms of point variation, with few new mutations accumulating after early passaging. Haplotype resolution facilitated reconstruction of an amplified, highly rearranged region of chromosome 8q24.21 at which integration of the human papilloma virus type 18 (HPV-18) genome occurred and that is likely to be the event that initiated tumorigenesis. We combined these maps with RNA-seq and ENCODE Project data sets to phase the HeLa epigenome. This revealed strong, haplotype-specific activation of the proto-oncogene MYC by the integrated HPV-18 genome approximately 500 kilobases upstream, and enabled global analyses of the relationship between gene dosage and expression. These data provide an extensively phased, high-quality reference genome for past and future experiments relying on HeLa, and demonstrate the value of haplotype resolution for characterizing cancer genomes and epigenomes.

Multiplex targeted sequencing identifies recurrently mutated genes in autism spectrum disorders.

Exome sequencing studies of autism spectrum disorders (ASDs) have identified many de novo mutations but few recurrently disrupted genes. We therefore developed a modified molecular inversion probe method enabling ultra-low-cost candidate gene resequencing in very large cohorts. To demonstrate the power of this approach, we captured and sequenced 44 candidate genes in 2446 ASD probands. We discovered 27 de novo events in 16 genes, 59% of which are predicted to truncate proteins or disrupt splicing. We estimate that recurrent disruptive mutations in six genes-CHD8, DYRK1A, GRIN2B, TBR1, PTEN, and TBL1XR1-may contribute to 1% of sporadic ASDs. Our data support associations between specific genes and reciprocal subphenotypes (CHD8-macrocephaly and DYRK1A-microcephaly) and replicate the importance of a ß-catenin-chromatin-remodeling network to ASD etiology.