Biology subjects

Brosh, R.

Publications and source records attributed to Brosh, R..

8 recordsLinked to original sources

bioRxiv2022

Mouse genomic rewriting and tailoring: synthetic Trp53 and humanized ACE2

Genetically Engineered Mouse Models (GEMMs) aid in understanding human pathologies and developing new therapeutics, yet recapitulating human diseases authentically in mice is challenging to design and execute. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences controlling spatiotemporal gene expression patterns and splicing to human diseases. It is thus apparent that including regulatory genomic regions during the engineering of GEMMs is highly preferable for disease modeling, with the prerequisite of large-scale genome engineering ability. Existing genome engineering methods have limits on the size and efficiency of DNA delivery, hampering routine creation of highly informative GEMMs. Here, we describe mSwAP-In (mammalian Switching Antibiotic resistance markers Progressively for Integration), a method for efficient genome rewriting in mouse embryonic stem cells. We first demonstrated the use of mSwAP-In for iterative genome rewriting of up to 115 kb of the Trp53 locus, as well as for genomic humanization of up to 180 kb ACE2 locus in response to the COVID-19 pandemic. Second, we showed the hACE2 GEMM authentically recapitulated human ACE2 expression patterns and splicing, and importantly, presented milder symptoms without mortality when challenged with SARS-CoV-2 compared to the K18-ACE2 model, thus representing a more authentic model of infection.

genomics↗

bioRxiv2022

Episomal editing of synthetic constructs in yeast using CRISPR

Use of synthetic genomics to design and build "big" DNA has revolutionized our ability to answer fundamental biological questions by employing a bottom-up approach. S. cerevisiae, or budding yeast, has become the major platform to assemble large synthetic constructs thanks to its powerful homologous recombination machinery and the availability of well-established molecular biology techniques. However, efficiently and precisely introducing designer variations to episomal assemblies remains challenging. Here, we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, for rapid engineering of mammalian DNA constructs larger than 100 kb. We demonstrate that editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes with CRISPR. After optimizing CREEPY for episomal editing, we achieve efficient simplex and multiplex editing as demonstrated by engineering a mouse Sox2-harboring episome.

synthetic biology↗

bioRxiv2022

Germline Transmission of a Circular Human Artificial Chromosome in the Mouse

Although the structure and function of the alphoid-tetO Human Artificial Chromosome (tetO-HAC) has been previously described in cell culture models and somatically in the mouse, in vivo persistence and stability throughout meiosis and across generations were not evaluated. Here we report germline transmission of a circular tetO-HAC across three mouse generations without observable health or reproductive deficiencies. Furthermore, we show that the tetO-HAC is maintained without selection as an episome and can be efficiently transmitted by both ova and sperm.

synthetic biology↗

bioRxiv2022

Genomic analysis of a synthetic reversed sequence reveals default chromatin states in yeast and mammalian cells

Up to 93% of the human genome may show evidence of transcription, yet annotated transcripts account for less than 5%. It is unclear what makes up this major discrepancy, and to what extent the excess transcription has a definable biological function, or is just a pervasive byproduct of non-specific RNA polymerase binding and transcription initiation. Understanding the default state of the genome would be informative in determining whether the observed pervasive activity has a definable function. The genome of any modern organism has undergone billions of years of evolution, making it unclear whether any observed genomic activity, or lack thereof, has been selected for. We sought to address this question by introducing a completely novel 100-kb locus into the genomes of two eukaryotic organisms, S. cerevisiae and M. musculus, and characterizing its genomic activity based on chromatin accessibility and transcription. The locus was designed by reversing (but not complementing) the sequence of the human HPRT1 locus, including [~]30-kb of both upstream and downstream regulatory regions, allowing retention of sequence features like repeat frequency and GC content but ablating coding information and transcription factor binding sites. We also compared this reversed locus with a synthetic version of the normal human HPRT1 locus in both organismal contexts. Despite neither the synthetic HPRT1 locus nor its reverse version coding for any promoters evolved for gene expression in yeast, we observed widespread transcriptional activity of both loci. This activity was observed both when the loci were present as episomes and when chromosomally integrated, although it did not correspond to any of the known HPRT1 functional regulatory elements. In contrast, when integrated in the mouse genome, the synthetic HPRT1 locus showed transcriptional activity corresponding precisely to the HPRT1 coding sequence, while the reverse locus displayed no activity at all. Together, these results show that genomic sequences with no coding information are active in yeast, but relatively inactive in mouse, indicating a potentially major difference in "default genomic states" between these two divergent eukaryotes.

molecular biology↗

bioRxiv2022

Dissection of a complex enhancer cluster at the Sox2 locus

Expression of Sox2 in mouse embryonic stem cells (mESCs) depends on a distal regulatory cluster of DNase I hypersensitive sites (DHSs), but their individual contributions and degree of independence remain a mystery. Here, we comprehensively analyze the regulatory architecture of Sox2 at its endogenous locus using Big-IN to scarlessly integrate DNA payloads ranging up to 143 kb. We analyzed 83 payloads incorporating deletions, rearrangements, and inversions affecting single or multiple DHSs, as well as surgical alterations to transcription factor (TF) recognition sequences. Multiple mESC clones were derived for each payload, sequence-verified, and analyzed to establish the necessity and sufficiency of genomic features for Sox2 expression. We found that two LCR DHSs comprising a handful of key TF recognition sequences were each sufficient to autonomously sustain significant expression in mESCs. However, three additional LCR DHSs were entirely context-dependent, in that they showed no activity alone but could dramatically augment activity of the core DHSs. Our synthetic regulatory genomics approach demonstrates that composite regulatory elements can be reduced to a tractable set of essential sequence features, and is readily scalable to investigate regulatory architecture at other key loci genome-wide.

genomics↗

bioRxiv2021

Early stage differentiation of glia in human and mice.

Macroglia (astrocytes and oligodendrocytes) are required for normal development and function of the central nervous system, yet many questions remain about their emergence in the brain and spinal cord. Here we used single-cell RNA sequencing (scRNAseq) to analyze over 298,000 cells and nuclei during macroglia differentiation from mouse embryonic and human induced pluripotent stem cells. We computationally identify candidate genes involved in fate specification of glia in both species, and report heterogeneous expression of astrocyte surface markers across differentiating cells. We then used our scRNAseq data to optimize a previous mouse astrocyte differentiation protocol, decreasing the overall protocol length and complexity. Finally, we used multiomic, dual single nuclei (sn)RNAseq/snATACseq analysis to uncover potential genomic regulatory sites mediating glial differentiation. These datasets enable future optimization of glial differentiation protocols and provide insight into human glial differentiation.

neuroscience↗

bioRxiv2021

The genetic basis of tail-loss evolution in humans and apes

The loss of the tail is one of the main anatomical evolutionary changes to have occurred along the lineage leading to humans and to the "anthropomorphous apes"1,2. This morphological reprogramming in the ancestral hominoids has been long considered to have accommodated a characteristic style of locomotion and contributed to the evolution of bipedalism in humans3-5. Yet, the precise genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Primate genome sequencing projects have made possible the identification of causal links between genotypic and phenotypic changes6-8, and enable the search for hominoid-specific genetic elements controlling tail development9. Here, we present evidence that tail-loss evolution was mediated by the insertion of an individual Alu element into the genome of the hominoid ancestor. We demonstrate that this Alu element - inserted into an intron of the TBXT gene (also called T or Brachyury10-12) - pairs with a neighboring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated a mouse model that mimics the expression of human TBXT products by expressing both full-length and exon-skipped isoforms of the mouse TBXT ortholog. We found that mice with this genotype exhibit the complete absence of a tail or a shortened tail, supporting the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype, albeit with incomplete penetrance. We further noted that mice homozygous for the exon-skipped isoforms exhibited embryonic spinal cord malformations, resembling a neural tube defect condition, which affects [~]1/1000 human neonates13. We propose that selection for the loss of the tail along the hominoid lineage was associated with an adaptive cost of potential neural tube defects and that this ancient evolutionary trade-off may thus continue to affect human health today.

evolutionary biology↗

bioRxiv2021

Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modes controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce synthetic regulatory reconstitution, a novel conceptual framework for studying gene regulation and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130-170 kilobases each) to an ectopic location in the mouse genome. We find that a HoxA cluster lacking distal enhancers recapitulates correct patterns of chromatin remodeling and transcription in response to patterning signals, while distal enhancers are required for full transcriptional output. Synthetic regulatory reconstitution is a generalizable strategy to decipher the regulatory logic of gene expression in complex genomes. One-Sentence SummaryReconstitution of gene regulation using large DNA constructs unravels the regulatory logic of a developmental gene locus.

synthetic biology↗