High-resolution genome-wide mapping of chromosome-arm-scale truncations induced by CRISPR-Cas9 editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a powerful tool for introducing targeted mutations in DNA, but recent studies have shown that it can have unintended effects such as structural changes. However, these studies have not yet looked genome wide or across data types. Here we performed a phenotypic CRISPR-Cas9 scan targeting 17,065 genes in primary human cells, revealing a 'proximity bias' in which CRISPR knockouts show unexpected similarities to unrelated genes on the same chromosome arm. This bias was found to be consistent across cell types, laboratories, Cas9 delivery methods and assay modalities, and the data suggest that it is caused by telomeric truncations of chromosome arms, with cell cycle and apoptotic pathways playing a mediating role. Additionally, a simple correction is demonstrated to mitigate this pervasive bias while preserving biological relationships. This previously uncharacterized effect has implications for functional genomic studies using CRISPR-Cas9, with applications in discovery biology, drug-target identification, cell therapies and genetic therapeutics.

Single-cell sequencing of primate preimplantation embryos reveals chromosome elimination via cellular fragmentation and blastomere exclusion.

Aneuploidy that arises during meiosis and/or mitosis is a major contributor to early embryo loss. We previously showed that human preimplantation embryos encapsulate missegregated chromosomes into micronuclei while undergoing cellular fragmentation and that fragments can contain chromosomal material, but the source of this DNA was unknown. Here, we leveraged the use of a nonhuman primate model and single-cell DNA-sequencing (scDNA-seq) to examine the chromosomal content of 471 individual samples comprising 254 blastomeres, 42 polar bodies, and 175 cellular fragments from a large number (N = 50) of disassembled rhesus cleavage-stage embryos. Our analysis revealed that the aneuploidy and micronucleation frequency is conserved between humans and macaques, and that fragments encapsulate whole and/or partial chromosomes lost from blastomeres. Single-cell/fragment genotyping showed that these chromosome-containing cellular fragments (CCFs) can be maternally or paternally derived and display double-stranded DNA breaks. DNA breakage was further indicated by reciprocal subchromosomal losses/gains between blastomeres and large segmental errors primarily detected at the terminal ends of chromosomes. By combining time-lapse imaging with scDNA-seq, we determined that multipolar divisions at the zygote or two-cell stage were associated with CCFs and generated a random mixture of chromosomally normal and abnormal blastomeres with uniparental or biparental origins. Despite frequent chromosome missegregation at the cleavage-stage, we show that CCFs and nondividing aneuploid blastomeres showing extensive DNA damage are prevented from incorporation into blastocysts. These findings suggest that embryos respond to chromosomal errors by encapsulation into micronuclei, elimination via cellular fragmentation, and selection against highly aneuploid blastomeres to overcome chromosome instability during preimplantation development.

Stable Histone Methylation Changes at Proteoglycan Network Genes Following Ethanol Exposure.

Alcohol use disorder (AUD) is a chronic mental illness in which patients often achieve protracted periods of abstinence prior to relapse. Epigenetic mechanisms may provide an explanation for the persisting gene expression changes that can be observed even after long periods of abstinence and may contribute to relapse. In this study, we examined two histone modifications, histone 3 lysine 4 tri-methylation (H3K4me3) and histone 3 lysine 27 tri-methylation (H3K27me3), in the prefrontal cortex of Withdrawal Seizure Resistant (WSR) mice 21 days after 72 h of ethanol vapor exposure. These histone modifications were selected because they are associated with active promoters (H3K4me3) and repressed gene expression in a euchromatic environment (H3K27me3). We performed a genome-wide analysis to identify differences in H3K4me3 and H3K27me3 levels in post-ethanol exposure vs. control mice by ChIP-seq. We detected a global reduction in H3K4me3 peaks and increase in H3K27me3 peaks in post-ethanol exposure mice compared to controls, these changes are consistent with persistent reductions in gene expression. Pathway analysis of genes displaying changes in H3K4me3 and H3K27me3 revealed enrichment for genes involved in proteoglycan and calcium signaling pathways, respectively. Microarray analysis of 7,683 genes and qPCR analysis identified eight genes displaying concordant regulation of gene expression and H3K4me3/H3K27me3. We also compared changes in H3K4me3 and/or H3K27me3 from our study with changes in gene expression in response to ethanol from published literature and we found that the expression of 52% of the genes with altered H3K4me3 binding and 40% of genes with H3K27me3 differences are altered by ethanol exposure. The chromatin changes associated with the 21-day post-exposure period suggest that this period is a unique state in the addiction cycle that differs from ethanol intoxication and acute withdrawal. These results provide insights into the enduring effects of ethanol on proteoglycan and calcium signaling genes in the brain.

Epigenetic maintenance of topological domains in the highly rearranged gibbon genome.

The relationship between evolutionary genome remodeling and the three-dimensional structure of the genome remain largely unexplored. Here, we use the heavily rearranged gibbon genome to examine how evolutionary chromosomal rearrangements impact genome-wide chromatin interactions, topologically associating domains (TADs), and their epigenetic landscape. We use high-resolution maps of gibbon-human breaks of synteny (BOS), apply Hi-C in gibbon, measure an array of epigenetic features, and perform cross-species comparisons. We find that gibbon rearrangements occur at TAD boundaries, independent of the parameters used to identify TADs. This overlap is supported by a remarkable genetic and epigenetic similarity between BOS and TAD boundaries, namely presence of CpG islands and SINE elements, and enrichment in CTCF and H3K4me3 binding. Cross-species comparisons reveal that regions orthologous to BOS also correspond with boundaries of large (400-600 kb) TADs in human and other mammalian species. The colocalization of rearrangement breakpoints and TAD boundaries may be due to higher chromatin fragility at these locations and/or increased selective pressure against rearrangements that disrupt TAD integrity. We also examine the small portion of BOS that did not overlap with TAD boundaries and gave rise to novel TADs in the gibbon genome. We postulate that these new TADs generally lack deleterious consequences. Last, we show that limited epigenetic homogenization occurs across breakpoints, irrespective of their time of occurrence in the gibbon lineage. Overall, our findings demonstrate remarkable conservation of chromatin interactions and epigenetic landscape in gibbons, in spite of extensive genomic shuffling.

Conserved syntenic clusters of protein coding genes are missing in birds.

BACKGROUND: Birds are one of the most highly successful and diverse groups of vertebrates, having evolved a number of distinct characteristics, including feathers and wings, a sturdy lightweight skeleton and unique respiratory and urinary/excretion systems. However, the genetic basis of these traits is poorly understood. RESULTS: Using comparative genomics based on extensive searches of 60 avian genomes, we have found that birds lack approximately 274 protein coding genes that are present in the genomes of most vertebrate lineages and are for the most part organized in conserved syntenic clusters in non-avian sauropsids and in humans. These genes are located in regions associated with chromosomal rearrangements, and are largely present in crocodiles, suggesting that their loss occurred subsequent to the split of dinosaurs/birds from crocodilians. Many of these genes are associated with lethality in rodents, human genetic disorders, or biological functions targeting various tissues. Functional enrichment analysis combined with orthogroup analysis and paralog searches revealed enrichments that were shared by non-avian species, present only in birds, or shared between all species. CONCLUSIONS: Together these results provide a clearer definition of the genetic background of extant birds, extend the findings of previous studies on missing avian genes, and provide clues about molecular events that shaped avian evolution. They also have implications for fields that largely benefit from avian studies, including development, immune system, oncogenesis, and brain function and cognition. With regards to the missing genes, birds can be considered 'natural knockouts' that may become invaluable model organisms for several human diseases.

Gibbon genome and the fast karyotype evolution of small apes.

Gibbons are small arboreal apes that display an accelerated rate of evolutionary chromosomal rearrangement and occupy a key node in the primate phylogeny between Old World monkeys and great apes. Here we present the assembly and analysis of a northern white-cheeked gibbon (Nomascus leucogenys) genome. We describe the propensity for a gibbon-specific retrotransposon (LAVA) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage. We further show that the gibbon genera (Nomascus, Hylobates, Hoolock and Symphalangus) experienced a near-instantaneous radiation ∼5 million years ago, coincident with major geographical changes in southeast Asia that caused cycles of habitat compression and expansion. Finally, we identify signatures of positive selection in genes important for forelimb development (TBX5) and connective tissues (COL1A1) that may have been involved in the adaptation of gibbons to their arboreal habitat.