Faculty Bibliography

The rapid pace of innovation in synthetic biology and genome engineering elicits a need to reevaluate systems of oversight to ensure that biosafety and biosecurity safeguards are keeping up. Accordingly, the regulation of nucleic acid synthesis, an enabling technology for synthetic biology and genome engineering, is a current focus of political debate in the United States. However, to develop appropriate governance that neither under- nor overregulates technological development, policy leaders must also appreciate how advances in synthetic biology and genome engineering are being employed in the interests of biosecurity to support human health, manufacturing, food security, ecosystems and the natural environment. Synthetic biology can support the development of new therapeutics and health technologies, alternative biomanufacturing methodologies, food and agricultural innovations, environmental biosensors for monitoring infectious and toxic agents, and interventions aimed at preserving and restoring our natural environment. This Perspective provides an overview of current and potential benefits of synthetic biology and genome engineering, aiming to balance the broader societal discussion of potential risks-particularly in this special issue of the journal-with potential social, economic, and environmental value to individuals and society at large.

Eukaryotic DNA wraps around histone octamers forming nucleosomes, which modulate genome function by defining chromatin environments with distinct accessibility. These well-conserved properties allowed "humanization" of the nucleosome core particle (NCP) in Saccharomyces cerevisiae at high fitness costs. Here, we studied histone-humanized yeast genomes to understand how species-specific chromatin affects nuclear organization and function. We found a size increase in human-NCP, linked to shorter free linker DNA, supporting decreased chromatin accessibility. Three-dimensional (3D) humanized genome maps showed increased chromatin compaction and defective centromere clustering, correlated with high chromosomal aneuploidy rate. Site-specific chromatin alterations were associated with lack of initiation of early origins of replication and dysregulation of the ribosomal (rDNA and rRNA) metabolism. This latter led to nucleolar fragmentation and rDNA-array instability, through a non-coding RNA-dependent mechanism, leading to its extraordinary, but entirely reversible, intra-chromosomal expansion. Overall, our results reveal species-specific properties of the NCP that define epigenome function across vast evolutionary distances.

Centromeres ensure accurate chromosome segregation, yet their DNA evolves rapidly across eukaryotes leaving the origins of new centromere architectures unclear^1-4. The brewer's yeast Saccharomyces cerevisiae exemplifies this long-standing puzzle. Its centromeres shifted ancestrally from large, repeat-rich, epigenetically specified forms to the compact, genetically defined 'point' centromeres^1,5. How this transition occurred has remained unresolved⁶. Here we identify evolutionarily related 'proto-point' centromeres that provide a resolution to the evolutionary origins of point centromeres. Proto-point centromeres contain a single centromeric nucleosome positioned over an AT-rich core, accompanied by relaxed organization and sequence variability of flanking cis-elements. In two species, these proto-point centromeres lie within retrotransposon-derived repeat clusters, linking ancestral repeat-rich centromeres to genetically encoded ones. Comparative and phylogenetic analyses indicate that proto-point and point centromeres evolved in an ancestor with retrotransposon-rich centromeres. These results identify long-terminal-repeat retrotransposons, specifically Ty5 sequences, as the genetic substrate for point-centromere evolution and provide a mechanistic route by which an epigenetic centromere can become genetically specified. More broadly, they show how selfish elements can be co-opted to perform essential chromosomal functions.

The ability to design and engineer mammalian genomes across arbitrary length scales would transform biology and medicine. Such capabilities would enable the systematic dissection of mechanisms governing gene regulation and the influence of complex haplotypes on human traits and disease. They would also facilitate the engineering of disease models that more faithfully recapitulate human physiology and of next-generation cell therapies harboring sophisticated genetic circuits. Over the past decade, advances in genome editing have made small, targeted modifications at single sites routine. However, achieving multiple coordinated alterations across long sequence windows (>10 kb) or installing large synthetic DNA segments in mammalian cells remains a major challenge. Recent advances in mammalian genome writing-the bottom-up design, assembly, and targeted integration of large custom DNA sequences, independent of any natural template-offer a potential solution. Here, we review key technological developments, highlight emerging applications, and discuss current bottlenecks and strategies for overcoming them.

Organ shortage remains a major challenge in transplantation, and gene-edited pig organs offer a promising solution^1-3. Despite gene-editing, the immune reactions following xenotransplantation can still cause transplant failure⁴. To understand the immunological response of a pig-to-human kidney xenotransplantation, we conducted large-scale multi-omics profiling of the xenograft and the host's blood over a 61-day procedure in a brain-dead human (decedent) recipient. Blood plasmablasts, natural killer (NK) cells, and dendritic cells increased between postoperative day (POD)10 and 28, concordant with expansion of IgG/IgA B-cell clonotypes, and subsequent biopsy-confirmed antibody-mediated rejection (AbMR) at POD33. Human T-cell frequencies increased from POD21 and peaked between POD33-49 in the blood and xenograft, coinciding with T-cell receptor diversification, expansion of a restricted TRBV2/J1 clonotype and histological evidence of a combined AbMR and cell-mediated rejection at POD49. At POD33, the most abundant human immune population in the graft was CXCL9+ macrophages, aligning with IFN-γ-driven inflammation and a Type I immune response. In addition, we see evidence of interactions between activated pig-resident macrophages and infiltrating human immune cells. Xenograft tissue showed pro-fibrotic tubular and interstitial injury, marked by S100A6⁵, SPP1⁶ (Osteopontin), and COLEC11⁷, at POD21-POD33. Proteomics profiling revealed human and pig complement activation, with decreased human component after AbMR therapy with complement inhibition. Collectively, these data delineate the molecular orchestration of human immune responses to a porcine kidney, revealing potential immunomodulatory targets for improving xenograft survival.

Xenotransplantation of genetically-modified pig kidneys offers a solution to the scarcity of organs for end-stage renal disease patients.¹ We performed a 61-day alpha-Gal knock-out pig kidney and thymic autograft transplant into a nephrectomized brain-dead human using clinically approved immunosuppression, without CD40 blockade or additional genetic modification. Hemodynamic and electrolyte stability and dialysis independence were achieved. Post-operative day (POD) 10 biopsies revealed glomerular IgM and IgA deposition, activation of early complement components and mesangiolysis with stable renal function without proteinuria, a phenotype not seen in allotransplantation. On POD 33, an abrupt increase in serum creatinine was associated with antibody-mediated rejection and increased donor-specific IgG. Plasma exchange, C3/C3b inhibition and rabbit anti-thymocyte globulin (rATG), completely reversed xenograft rejection. Pre-existing donor-reactive T cell clones expanded progressively in the circulation post-transplant, acquired an effector transcriptional profile and were detected in the POD 33 rejecting xenograft prior to rATG treatment. This study provides the first long-term physiologic, immunologic, and infectious disease monitoring of a pig-to-human kidney xenotransplant and indicates that pre-existing xenoreactive T cells and induced antibodies to unknown epitope(s) present a major challenge, despite significant immunosuppression. It also demonstrates that a minimally gene-edited pig kidney can support long-term life-sustaining physiologic functions in a human.