Biology subjects

Agmon, N.

Publications and source records attributed to Agmon, N..

5 recordsLinked to original sources

bioRxiv2025

Genomic REWRITE of Immune Interfaces Reveals Heritable Epigenetic Memory in Human Stem Cells

Human leukocyte antigen (HLA) polymorphism underlies antigen presentation, immune recognition, and central tolerance, yet the complex, multigenic structure of native HLA loci has precluded systematic engineering in human pluripotent stem cells (hPSCs). Existing genome engineering demonstrations in hPSCs focus on smaller integrations or at ectopic sites, leaving scar-minimized rewriting of native human loci at >100 kb scale a major technical challenge. Here, we present REWRITE, a modular platform for scar-minimized genome writing that enables engineering of large human genomic loci exceeding 100 kb at their native chromosomal sites. Using REWRITE, we deleted 105 kb spanning the dispersed HLA class-I locus and installed either compact or full-length refactored synthetic HLA haplotypes. Both refactored architectures exhibited transient activity before re-acquiring the native inactive state; upon extended culture, engineered lines yielded global transcriptomes nearly indistinguishable from parental cells. The engineered loci were genomically stable, retained interferon-responsive HLA expression, and remained compatible with differentiation into endothelial-like and thymic epithelial-like cells. Together, these results establish REWRITE as a platform for locus-scale genome engineering in hPSCs and provides a system for studying HLA haplotype structure and function in an isogenic pluripotent cell background. One sentenceA practical platform for scar-minimized rewriting of native human loci exceeding 100 kb in hPSCs, demonstrated through synthetic refactoring of the class-I HLA region.

synthetic biology↗

bioRxiv2022

Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

The Sc2.0 project is building a eukaryotic synthetic genome from scratch, incorporating thousands of designer features. A major milestone has been achieved with the assembly of all individual Sc2.0 chromosomes. Here, we describe the consolidation of multiple synthetic chromosomes using endoreduplication intercross to generate a strain with 6.5 synthetic chromosomes. Genome-wide chromosome conformation capture and long-read direct RNA sequencing were performed on this strain to evaluate the effects of designer modifications, such as loxPsym site insertion, tRNA relocation, and intron deletion, on 3D chromosome organization and transcript isoform profiles. To precisely map "bugs", we developed a method, CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS", exploiting directed mitotic recombination in heterozygous diploids. Using this method, we first fine-mapped a synII defect resulting from two loxPsym sites in the 3 UTR of SHM1. This approach was also used to map a combinatorial bug associated with synIII and synX, revealing a highly unexpected genetic interaction that links transcriptional regulation, inositol metabolism and tRNASerCGA abundance. "Starvation" for tRNASerCGA leads to insufficient levels of the key positive inositol biosynthesis regulator, Swi3, which contains tandem UCG codons. Finally, to expedite consolidation, we employed a new method, chromosome swapping, to incorporate the largest chromosome (synIV), thereby consolidating more than half of the Sc2.0 genome in a single strain.

genomics↗

bioRxiv2020

Collective motion as a distinct behavioral state of the individual

The collective motion of swarms depends on adaptations at the individual level. We explored these and their effects on swarm formation and maintenance in locusts. The walking kinematics of individual insects were monitored under laboratory settings, before, as well as during collective motion in a group, and again after separation from the group. It was found that taking part in collective motion induced in the individual unique behavioural kinematics, suggesting the existence of a distinct behavioural mode that we term a "collective-motion-state". This state, characterized by behavioural adaptation to the social context, is long lasting, not induced by crowding per-se, but only by experiencing collective motion. Utilizing computational models, we show that this adaptability increases the robustness of the swarm. Overall, our findings suggest that collective-motion is not only an emergent property of the group, but also depends on a behavioural mode, rooted in endogenous mechanisms of the individual.

ecology↗

bioRxiv2018

Synthetic chromosome fusion: effects on genome structure and function

As part of the Synthetic Yeast 2.0 (Sc2.0) project, we designed and synthesized synthetic chromosome I. The total length of synI is [~]21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype imbalance. We used a variation of a previously developed, robust CRISPR-Cas9 method to fuse chromosome I to other chromosome arms of varying length: chrIXR (84kb), chrIIIR (202kb) and chrIVR (1Mb). All fusion chromosome strains grew like wild-type so we decided to attach synI to synIII. Through the investigation of three-dimensional structures of fusion chromosome strains, unexpected loops and twisted structures were formed in chrIII-I and chrIX-III-I fusion chromosomes, which depend on silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. We used these fusion chromosomes to show that axial element Red1 binding in meiosis is not strictly chromosome size dependent even though Red1 binding is enriched on the three smallest chromosomes in wild-type yeast, and we discovered an unexpected role for centromeres in Red1 binding patterns.

synthetic biology↗

bioRxiv2017

Human to yeast pathway transplantation: cross-species dissection of the adenine de novo pathway regulatory node

Pathway transplantation from one organism to another represents a means to a more complete understanding of a biochemical or regulatory process. The purine biosynthesis pathway, a core metabolic function, was transplanted from human to yeast. We replaced the entire Saccharomyces cerevisiae adenine de novo pathway with the cognate human pathway components. A yeast strain was \"humanized\" for the full pathway by deleting all relevant yeast genes completely and then providing the human pathway in trans using a neochromosome expressing the human protein coding regions under the transcriptional control of their cognate yeast promoters and terminators. The \"humanized\" yeast strain grows in the absence of adenine, indicating complementation of the yeast pathway by the full set of human proteins. While the strain with the neochromosome is indeed prototrophic, it grows slowly in the absence of adenine. Dissection of the phenotype revealed that the human ortholog of ADE4, PPAT, shows only partial complementation. We have used several strategies to understand this phenotype, that point to PPAT/ADE4 as the central regulatory node. Pathway metabolites are responsible for regulating PPATs protein abundance through transcription and proteolysis as well as its enzymatic activity by allosteric regulation in these yeast cells. Extensive phylogenetic analysis of PPATs from diverse organisms hints at adaptations of the enzyme-level regulation to the metabolite levels in the organism. Finally, we isolated specific mutations in PPAT as well as in other genes involved in the purine metabolic network that alleviate incomplete complementation by PPAT and provide further insight into the complex regulation of this critical metabolic pathway.

synthetic biology↗