The Malaria Cell Atlas a comprehensive reference of single parasite transcriptomes across the complete Plasmodium life cycle file S1, bioRxiv, 2019-01-23
Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. Here we profile the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire Plasmodium berghei life cycle. We then use our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a complex eukaryotic parasite and an open access reference data set for the study of malaria parasites.
biorxiv genomics 100-200-users 2019Accurate detection of m6A RNA modifications in native RNA sequences, bioRxiv, 2019-01-21
The field of epitranscriptomics has undergone an enormous expansion in the last few years; however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here we show that using Oxford Nanopore Technologies, N6-methyladenosine (m6A) RNA modifications can be detected with high accuracy, in the form of systematic errors and decreased base-calling qualities. Our results open new avenues to investigate the universe of RNA modifications with single nucleotide resolution, in individual RNA molecules.
biorxiv molecular-biology 100-200-users 2019Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin, bioRxiv, 2019-01-21
AbstractMembraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct “territories.” Strikingly, H3K9me23-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me23 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.Author summaryThe three dimensional (3D) organization of genomes in cell nuclei can influence a wide variety of genome functions. However, most of our understanding of this critical architecture has been limited to the gene-rich euchromatin, and largely ignores the gene-poor and repeat-rich pericentromeric heterochromatin, or PCH. PCH comprises large part of most eukaryotic genomes, forms 3D PCH domains in nuclei, and plays vital role in chromosome dynamics and genome stability. In this study, we developed a new method that overcomes the technical challenges imposed by the highly repetitive PCH DNA, and generated a comprehensive picture of its 3D organization. Combined with image analyses, we revealed a hierarchical organization of the PCH domains. Surprisingly, we showed that distant euchromatic regions enriched for repressive epigenetic marks also dynamically interact with the main PCH domains. These 3D interactions are mediated by liquid-liquid phase separation mechanisms, similar to how oil and vinegar separate in salad dressing, and can influence the fitness of individuals. Our discoveries have strong implications for how seemingly “junk” DNA could impact functions in the gene-rich euchromatin.
biorxiv genomics 100-200-users 2019Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me23 islands located in euchromatic genome, bioRxiv, 2019-01-21
Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct 'territories,' in which 'intra-arm' interactions are the most prevalent, followed by 3D contacts between specific PCH regions on different chromosomes. Strikingly, H3K9me23-enriched regions embedded in euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me23 enrichment, are likely mediated by liquid-liquid phase separation, and influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.
biorxiv genomics 100-200-users 2019Identification and mitigation of pervasive off-target activity in CRISPR-Cas9 screens for essential non-coding elements Supplementary Information, bioRxiv, 2019-01-19
Pooled CRISPR-Cas9 screens have recently emerged as a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we conducted a genome-scale screen for essential CTCF loop anchors in the K562 leukemia cell line. Surprisingly, the primary drivers of signal in this screen were single guide RNAs (sgRNAs) with low specificity scores. After removing these guides, we found that there were no CTCF loop anchors critical for cell growth. We also observed this effect in an independent screen fine-mapping the core motifs in enhancers of the GATA1 gene. We then conducted screens in parallel with CRISPRi and CRISPRa, which do not induce DNA damage, and found that an unexpected and distinct set of off-targets also caused strong confounding growth effects with these epigenome-editing platforms. Promisingly, strict filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and allowed for the identification of essential enhancers, which we validated extensively. Together, our results show off-target activity can severely limit identification of essential functional motifs by active Cas9, while strictly filtered CRISPRi screens can be reliably used for assaying larger regulatory elements.
biorxiv genomics 100-200-users 2019Direct synthesis of EM-visible gold nanoparticles on genetically encoded tags for single-molecule visualization in cells, bioRxiv, 2019-01-18
Single-molecule visualization in cells with genetically encoded tags for electron microscopy (EM) has been a long-awaited but unimplemented tool for cell biologists. Here, we report an approach for directly synthesizing EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags for single-molecule visualization in cells. We first uncovered an auto-nucleation suppression mechanism that allows specific synthesis of AuNPs on isolated cysteine-rich tags. We next exploited this mechanism to develop an approach for single-molecule detection of proteins in prokaryotic cells and achieved an unprecedented labeling efficiency. We then expanded it to more complicated eukaryotic cells and successfully detected the proteins targeted to various organelles, including the membranes of endoplasmic reticulum (ER) and nuclear envelope, ER lumen, nuclear pores, spindle pole bodies, and mitochondrial matrix. Thus, our implementation of genetically encoded tags for EM should allow cell biologists to address an enormous range of biological questions at single-molecule level in diverse cellular ultrastructural contexts without using antibodies.
biorxiv biophysics 100-200-users 2019