Search

Extrachromosomal DNA (ecDNA) is a hallmark of aggressive cancer, contributing to both oncogene amplification and tumor heterogeneity. Here, we used Hi-C, super-resolution imaging, and long-read sequencing to explore the nuclear architecture of MYC -amplified ecDNA in colorectal cancer cells. Intriguingly, we observed frequent spatial proximity between ecDNA and 68 repetitive elements which we called ecDNA-interacting elements or EIEs. To characterize a potential regulatory role of EIEs, we focused on a fragment of the L1M4a1#LINE/L1 which we found to be co-amplified with MYC on ecDNA, gaining enhancer-associated chromatin marks in contrast to its normally silenced state. This EIE, in particular, existed as a naturally occurring structural variant upstream of MYC , gaining oncogenic potential in the transcriptionally permissive ecDNA environment. This EIE sequence is sufficient to enhance MYC expression and is required for cancer cell fitness. These findings suggest that silent repetitive genomic elements can be reactivated on ecDNA, leading to functional cooption and amplification. Repeat element activation on ecDNA represents a mechanism of accelerated evolution and tumor heterogeneity and may have diagnostic and therapeutic potential.

DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis . Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9's targeting activity at specific DNA sites may be engineered without modifying Cas9's core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis -acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells., Competing Interests: Jef Boeke is a Founder and Director of CDI Labs, Inc., a Founder of and consultant to Neochromosome, Inc, a Founder, SAB member of and consultant to ReOpen Diagnostics, LLC and serves or served on the Scientific Advisory Board of the following: Logomix, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Sangamo, Inc., Tessera Therapeutics, Inc. and the Wyss Institute. Marcus Noyes is a founder of TBG Therapeutics, Inc. and consultant to Tessera Therapeutics, Inc. New York University filed a provisional patent application for findings described in this work, with Gregory Goldberg, Marcus Noyes, and Jef Boeke listed as inventors. The other authors declare no competing interests. Correspondence and requests for materials should be addressed to Jef.Boeke@nyulangone.org, Gregory.Goldberg@nyulangone.org, or Marcus.Noyes@nyulangone.org.

Background: Xenotransplantation of genetically engineered porcine organs has the potential to address the challenge of organ donor shortage. Two cases of porcine-to-human kidney xenotransplantation were performed, yet the physiological effects on the xenografts and the recipients' immune responses remain largely uncharacterized., Methods: We performed single-cell RNA sequencing (scRNA-seq) and longitudinal RNA-seq analyses of the porcine kidneys to dissect xenotransplantation-associated cellular dynamics and xenograft-recipient interactions. We additionally performed longitudinal scRNA-seq of the peripheral blood mononuclear cells (PBMCs) to detect recipient immune responses across time., Findings: Although no hyperacute rejection signals were detected, scRNA-seq analyses of the xenografts found evidence of endothelial cell and immune response activation, indicating early signs of antibody-mediated rejection. Tracing the cells' species origin, we found human immune cell infiltration in both xenografts. Human transcripts in the longitudinal bulk RNA-seq revealed that human immune cell infiltration and the activation of interferon-gamma-induced chemokine expression occurred by 12 and 48 h post-xenotransplantation, respectively. Concordantly, longitudinal scRNA-seq of PBMCs also revealed two phases of the recipients' immune responses at 12 and 48-53 h. Lastly, we observed global expression signatures of xenotransplantation-associated kidney tissue damage in the xenografts. Surprisingly, we detected a rapid increase of proliferative cells in both xenografts, indicating the activation of the porcine tissue repair program., Conclusions: Longitudinal and single-cell transcriptomic analyses of porcine kidneys and the recipient's PBMCs revealed time-resolved cellular dynamics of xenograft-recipient interactions during xenotransplantation. These cues can be leveraged for designing gene edits and immunosuppression regimens to optimize xenotransplantation outcomes., Funding: This work was supported by NIH RM1HG009491 and DP5OD033430., Competing Interests: Declaration of interests J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Opentrons LabWorks/Neochromosome, Inc.; and serves or served on the scientific advisory boards of the following: CZ Biohub New York, LLC; Logomix, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sangamo, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute. R.A.M. is on scientific advisory boards for eGenesis, Sanofi, Regeneron, CareDx, and Hansa Biopharma; is a consultant to Recombinetics; reports consulting fees from Hansa Medical, Regeneron, Thermo Fisher Scientific, Genentech, CareDx, One Lambda, ITB Med, Sanofi, and PPD Development; and reports grant support from Hansa Biopharma, all unrelated to the present work. R.A.M. also reports grant support from United Therapeutics Corporation, PBC. All other authors have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In addition to replicative histones, eukaryotic genomes encode a repertoire of non-replicative variant histones, providing additional layers of structural and epigenetic regulation. Here, we systematically replace individual replicative human histones with non-replicative human variant histones using a histone replacement system in yeast. We show that variants H2A.J, TsH2B, and H3.5 complement their respective replicative counterparts. However, macroH2A1 fails to complement, and its overexpression is toxic in yeast, negatively interacting with yeast's native histones and kinetochore genes. To isolate yeast with macroH2A1 chromatin, we uncouple the effects of its macro and histone fold domains, revealing that both domains suffice to override native nucleosome positioning. Furthermore, both uncoupled constructs of macroH2A1 exhibit lower nucleosome occupancy, decreased short-range chromatin interactions (<20 kb), disrupted centromeric clustering, and increased chromosome instability. Our observations demonstrate that lack of a canonical histone H2A dramatically alters chromatin organization in yeast, leading to genome instability and substantial fitness defects., Competing Interests: Declaration of interests J.D.B. is a founder and director of CDI Labs; a founder of and consultant to Opentrons Labworks/Neochromosome; and serves or served on the scientific advisory board of the following: CZ BioHub NY LLC, Logomix, Sangamo, Modern Meadow, Rome Therapeutics, SeaHub, Tessera Therapeutics, and the Wyss Institute., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Genomic context critically modulates regulatory function but is difficult to manipulate systematically. The murine insulin-like growth factor 2 (Igf2)/H19 locus is a paradigmatic model of enhancer selectivity, whereby CTCF occupancy at an imprinting control region directs downstream enhancers to activate either H19 or Igf2. We used synthetic regulatory genomics to repeatedly replace the native locus with 157-kb payloads, and we systematically dissected its architecture. Enhancer deletion and ectopic delivery revealed previously uncharacterized long-range regulatory dependencies at the native locus. Exchanging the H19 enhancer cluster with the Sox2 locus control region (LCR) showed that the H19 enhancers relied on their native surroundings while the Sox2 LCR functioned autonomously. Analysis of regulatory DNA actuation across cell types revealed that these enhancer clusters typify broader classes of context sensitivity genome wide. These results show that unexpected dependencies influence even well-studied loci, and our approach permits large-scale manipulation of complete loci to investigate the relationship between regulatory architecture and function., Competing Interests: Declaration of interests R.B., J.D.B., and M.T.M. are listed as inventors on a patent application describing Big-IN. Jef Boeke is a founder and director of CDI Labs, Inc.; a founder of and consultant to Opentrons LabWorks/Neochromosome, Inc.; and serves (or served) on the scientific advisory board of the following: CZ Biohub New York, LLC; Logomix, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sangamo, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Pervasive transcriptional activity is observed across diverse species. The genomes of extant organisms have undergone billions of years of evolution, making it unclear whether these genomic activities represent effects of selection or 'noise' 1-4 . Characterizing default genome states could help understand whether pervasive transcriptional activity has biological meaning. Here we addressed this question by introducing a synthetic 101-kb locus into the genomes of Saccharomyces cerevisiae and Mus musculus and characterizing genomic activity. The locus was designed by reversing but not complementing human HPRT1, including its flanking regions, thus retaining basic features of the natural sequence but ablating evolved coding or regulatory information. We observed widespread activity of both reversed and native HPRT1 loci in yeast, despite the lack of evolved yeast promoters. By contrast, the reversed locus displayed no activity at all in mouse embryonic stem cells, and instead exhibited repressive chromatin signatures. The repressive signature was alleviated in a locus variant lacking CpG dinucleotides; nevertheless, this variant was also transcriptionally inactive. These results show that synthetic genomic sequences that lack coding information are active in yeast, but inactive in mouse embryonic stem cells, consistent with a major difference in 'default genomic states' between these two divergent eukaryotic cell types, with implications for understanding pervasive transcription, horizontal transfer of genetic information and the birth of new genes., (© 2024. The Author(s).)

Core histone genes display a remarkable diversity of cis-regulatory mechanisms despite their protein sequence conservation. However, the dynamics and significance of this regulatory turnover are not well understood. Here, we describe the evolutionary history of core histone gene regulation across 400 million years in budding yeasts. We find that canonical mode of core histone regulation-mediated by the trans-regulator Spt10-is ancient, likely emerging between 320 and 380 million years ago and is fixed in the majority of extant species. Unexpectedly, we uncovered the emergence of a novel core histone regulatory mode in the Hanseniaspora genus, from its fast-evolving lineage, which coincided with the loss of 1 copy of its paralogous core histone genes. We show that the ancestral Spt10 histone regulatory mode was replaced, via cis-regulatory changes in the histone control regions, by a derived Mcm1 histone regulatory mode and that this rewiring event occurred with no changes to the trans-regulator, Mcm1, itself. Finally, we studied the growth dynamics of the cell cycle and histone synthesis in genetically modified Hanseniaspora uvarum. We find that H. uvarum divides rapidly, with most cells completing a cell cycle within 60 minutes. Interestingly, we observed that the regulatory coupling between histone and DNA synthesis was lost in H. uvarum. Our results demonstrate that core histone gene regulation was fixed anciently in budding yeasts, however it has greatly diverged in the Hanseniaspora fast-evolving lineage., Competing Interests: Conflicts of interest JDB is a Founder and Director of CDI Labs, Inc., a Founder of and consultant to Neochromosome, Inc., a Founder, SAB member of, and consultant to ReOpen Diagnostics, LLC, and serves or served on the Scientific Advisory Board of Logomix, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Sangamo, Inc., Tessera Therapeutics, Inc., and the Wyss Institute. JLS is a scientific advisor for WittGen Biotechnologies. JLS is an advisor for ForensisGroup, Inc., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the 'anthropomorphous apes' 1-3 , with a proposed role in contributing to human bipedalism 4-6 . Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element-inserted into an intron of the TBXT gene 7-9 -pairs with a neighbouring 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 multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt, mimicking the expression pattern of its hominoid orthologue TBXT. Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans 10 . Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today., (© 2024. The Author(s).)

Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we construct SparLox83R, a yeast strain containing 83 loxPsym sites distributed across all 16 chromosomes. SCRaMbLE of SparLox83R produces versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, when combined with synthetic chromosomes, SCRaMbLE of hetero-diploids with SparLox83R leads to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to hetero-diploids only with wild-type chromosomes. Analysis of the SCRaMbLEd strain with increased tolerance to nocodazole demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool for studying the consequence of genomic rearrangements and accelerating strain engineering in Saccharomyces cerevisiae., (© 2024. The Author(s).)

Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast), marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful; however, co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast; however, this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4-CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell cycle progression and kinetochore function in wild-type (WT) cells. Suppressors of CENP-A toxicity included gene deletions of subunits of 3 conserved chromatin remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation., Competing Interests: Conflicts of interest J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc.; and a founder, SAB member of, and consultant to ReOpen Diagnostics, LLC, and serves or served on the scientific advisory board of the following: LogoMix Inc.; Modern Meadow Inc.; ROME Therapeutics, Inc.; Sample6 Inc.; Sangamo, Inc.; Tessera Therapeutics Inc., and the Wyss Institute. G.O. and M.A.B.H. declare no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes., Competing Interests: Declaration of interests J.D.B. is a Founder and Director of CDI Labs, Inc.; a Founder of and consultant to Neochromosome, Inc.; a Founder, SAB member of, and consultant to ReOpen Diagnostics and Logomix, Inc., LLC; and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute. All other authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes., Competing Interests: Declaration of interests J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc; and a founder, Scientific Advisory Board (SAB) member of, and consultant to ReOpen Diagnostics, LLC. J.D.B. also serves or served on the SABs of the following: Logomix, Inc.; Sangamo, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute. Y.A. is a Founder and CSO of Logomix, Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The genome of an organism is inherited from its ancestor and continues to evolve over time, however, the extent to which the current version could be altered remains unknown. To probe the genome plasticity of Saccharomyces cerevisiae, here we replace the native left arm of chromosome XII (chrXIIL) with a linear artificial chromosome harboring small sets of reconstructed genes. We find that as few as 12 genes are sufficient for cell viability, whereas 25 genes are required to recover the partial fitness defects observed in the 12-gene strain. Next, we demonstrate that these genes can be reconstructed individually using synthetic regulatory sequences and recoded open-reading frames with a "one-amino-acid-one-codon" strategy to remain functional. Finally, a synthetic neochromsome with the reconstructed genes is assembled which could substitute chrXIIL for viability. Together, our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional eukaryotic chromosomes from entirely artificial sequences., (© 2023. The Author(s).)

The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNA Ser CGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain., Competing Interests: Declaration of interests J.D.B. is a founder and director of CDI Labs, Inc., a founder of and consultant to Neochromosome, Inc., and a founder, SAB member of, and consultant to ReOpen Diagnostics, LLC and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc., Logomix Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs., Competing Interests: Declaration of interests J.D.B. is a Founder and Director of CDI Labs, Inc.; a Founder of and consultant to Neochromosome, Inc.; a Founder, SAB member of, and consultant to ReOpen Diagnostics, LLC; and serves or served on the Scientific Advisory Board of the following: Logomix, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Sangamo, Inc., Tessera Therapeutics, Inc., and the Wyss Institute., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In November 2022 the first Dark Genome Symposium was held in Boston, USA. The meeting was hosted by Rome Therapeutics and Enara Bio, two biotechnology companies working on translating our growing understanding of this vast genetic landscape into therapies for human disease. The spirit and ambition of the meeting was one of shared knowledge, looking to strengthen the network of researchers engaged in the field. The meeting opened with a welcome from Rosana Kapeller and Kevin Pojasek followed by a first session of field defining talks from key academics in the space. A series of panels, bringing together academia and industry views, were then convened covering a wide range of pertinent topics. Finally, Richard Young and David Ting gave their views on the future direction and promise for patient impact inherent in the growing understanding of the Dark Genome., (© 2023. The Author(s).)

We describe construction of the synthetic yeast chromosome XI ( synXI ) and reveal the effects of redesign at non-coding DNA elements. The 660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled from synthesized DNA fragments before CRISPR-based methods were used in a process of bug discovery, redesign, and chromosome repair, including precise compaction of 200 kb of repeat sequence. Repaired defects were related to poor centromere function and mitochondrial health and were associated with modifications to non-coding regions. As part of the Sc2.0 design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show that these sites can facilitate induced extrachromosomal circular DNA (eccDNA) formation, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI contributes to our understanding of non-coding DNA elements, provides a useful tool for eccDNA study, and will inform future synthetic genome design., Competing Interests: B.A.B. is a scientific advisory board (SAB) member of Eden Bio Ltd. T.E. is a consultant to Replay Holdings, LLC and SAB member of Modern Synthesis, Inc. J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc.; a consultant to Opentrons Labworks, Inc.; a founder and SAB member of and consultant to ReOpen Diagnostics, LLC; and serves or served on the SAB of the following: Sangamo, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute. L.A.M. is a founder of Neochromosome, Inc. and an employee of Opentrons Labworks, Inc., (© 2023 The Author(s).)

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxP sym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications., Competing Interests: T.C.W. and A.C.C. are founders and shareholders of Number 8 Bio Pty Ltd. I.T.P. is an advisor of Number 8 Bio Pty Ltd. J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc; a founder, SAB member of, and consultant to ReOpen Diagnostics, LLC; and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc. and the Wyss Institute. J.S.B. is a founder of Neochromosome, Inc. and a consultant to Opentrons Labworks, Inc. L.A.M. is a founder of Neochromosome, Inc. and an employee of Opentrons Labworks, Inc., (© 2023 The Author(s).)

Pioneering advances in genome engineering, and specifically in genome writing, have revolutionized the field of synthetic biology, propelling us toward the creation of synthetic genomes. The Sc2.0 project aims to build the first fully synthetic eukaryotic organism by assembling the genome of Saccharomyces cerevisiae . With the completion of synthetic chromosome VIII ( synVIII ) described here, this goal is within reach. In addition to writing the yeast genome, we sought to manipulate an essential functional element: the point centromere. By relocating the native centromere sequence to various positions along chromosome VIII , we discovered that the minimal 118-bp CEN8 sequence is insufficient for conferring chromosomal stability at ectopic locations. Expanding the transplanted sequence to include a small segment (∼500 bp) of the CDEIII -proximal pericentromere improved chromosome stability, demonstrating that minimal centromeres display context-dependent functionality., Competing Interests: J.B. is a founder and director of CDI Labs, Inc., a founder of and consultant to Neochromosome, Inc., a founder, SAB member of, and consultant to ReOpen Diagnostics, LLC, and serves or served on the Scientific Advisory Board of the following: Logomix, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Sangamo, Inc., Tessera Therapeutics, Inc., and the Wyss Institute. J.S.B. is a founder of and consultant to Neochromosome. G.S. is a consultant to Neochromosome Inc. and ZenithAI., (© 2023 The Authors.)

Aneuploidy compromises genomic stability, often leading to embryo inviability, and is frequently associated with tumorigenesis and aging. Different aneuploid chromosome stoichiometries lead to distinct transcriptomic and phenotypic changes, making it helpful to study aneuploidy in tightly controlled genetic backgrounds. By deploying the engineered SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) system to the newly synthesized megabase Sc2.0 chromosome VII ( synVII ), we constructed a synthetic disomic yeast and screened hundreds of SCRaMbLEd derivatives with diverse chromosomal rearrangements. Phenotypic characterization and multi-omics analysis revealed that fitness defects associated with aneuploidy could be restored by (1) removing most of the chromosome content or (2) modifying specific regions in the duplicated chromosome. These findings indicate that both chromosome copy number and specific chromosomal regions contribute to the aneuploidy-related phenotypes, and the synthetic chromosome resource opens new paradigms in studying aneuploidy., Competing Interests: J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc.; and a founder and scientific advisory board member of and consultant to ReOpen Diagnostics, LLC. J.D.B. serves or served on the scientific advisory board of the following: Logomix, Inc., Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute. J.S.B. is a founder of Neochromsome, Inc.; is a consultant to Opentrons Labworks, Inc.; and serves on the scientific advisory board of Reflexion Pharmaceuticals, Inc., (© 2023 The Authors.)

Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a redesigned synthetic Saccharomyces cerevisiae chromosome XV , synXV . We designed and built synXV to harbor strategically inserted features, modified elements, and synonymously recoded genes throughout the chromosome. Based on the recoded chromosome, we developed a method to enable chrDBTL: CRISPR-Cas9-mediated mitotic recombination with endoreduplication (CRIMiRE). CRIMiRE allowed the creation of customized wild-type/synthetic combinations, accelerating genotype-phenotype mapping and synthetic chromosome redesign. We also leveraged synXV as a "build-to-learn" model organism for translation studies by ribosome profiling. We conducted a locus-to-locus comparison of ribosome occupancy between synXV and the wild-type chromosome, providing insight into the effects of codon changes and redesigned features on translation dynamics in vivo . Overall, we established synXV as a versatile reconfigurable system that advances chrDBTL for understanding biological mechanisms and engineering strains., Competing Interests: J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc.; and a founder of, scientific advisory board member of, and consultant to ReOpen Diagnostics, LLC and serves or served on the scientific advisory boards of the following: Sangamo, Inc.; Logomix, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute., (© 2023 The Authors.)

We designed and synthesized synI , which is ∼21.6% shorter than native chrI , the smallest chromosome in Saccharomyces cerevisiae . SynI was designed for attachment to another synthetic chromosome due to concerns surrounding potential instability and karyotype imbalance and is now attached to synIII , yielding the first synthetic yeast fusion chromosome. Additional fusion chromosomes were constructed to study nuclear function. ChrIII-I and chrIX-III-I fusion chromosomes have twisted structures, which depend on silencing protein Sir3. As a smaller chromosome, chrI also faces special challenges in assuring meiotic crossovers required for efficient homolog disjunction. Centromere deletions into fusion chromosomes revealed opposing effects of core centromeres and pericentromeres in modulating deposition of the crossover-promoting protein Red1. These effects extend over 100 kb and promote disproportionate Red1 enrichment, and thus crossover potential, on small chromosomes like chrI . These findings reveal the power of synthetic genomics to uncover new biology and deconvolute complex biological systems., Competing Interests: J.D.B, L.A.M., and J.S.B. are founders of Neochromosome, Inc. J.D.B. is also a consultant of Neochromosome; a Founder and Director of CDI Labs, Inc,; a Founder of, Scientific Advisory Board member of, and consultant to ReOpen Diagnostics, LLC; and serves or served on the Scientific Advisory Board of the following: Logomix, Inc.; Sangamo, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute. N.A. is a synthetic biology specialist at Alagene., (© 2023 The Authors.)

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX ). Combined chromosome construction methods were used to synthesize and integrate its left arm ( synIXL ) into a strain containing previously described synIXR . We identified and resolved a bug affecting expression of EST3 , a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX , suggesting a potential gene dosage compensation relationship between these chromosomes., Competing Interests: J.D.B. is a founder and director of CDI Labs, Inc., a founder of and consultant to Neochromosome, Inc, a founder, SAB member of, and consultant to ReOpen Diagnostics, LLC, and serves or served on the scientific advisory board of the following: Logomix, Inc., Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute. J.S.B. is a founder of Neochromosome, Inc., consultant to Opentrons Labworks, Inc., and advisor to Reflexion Pharmaceuticals, Inc., (© 2023 The Authors.)

Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases 1,2 . Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe 'mammalian switching antibiotic resistance markers progressively for integration' (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing., (© 2023. The Author(s).)

Macroglia (astrocytes and oligodendrocytes) are required for normal development and function of the central nervous system, yet many questions remain about their emergence during the development of the brain and spinal cord. Here we used single-cell/single-nucleus RNA sequencing (scRNA-seq/snRNA-seq) 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 the fate specification of glia in both species and report heterogeneous expression of astrocyte surface markers across differentiating cells. We then used our transcriptomic data to optimize a previous mouse astrocyte differentiation protocol, decreasing the overall protocol length and complexity. Finally, we used multi-omic, dual single-nuclei (sn)RNA-seq/snATAC-seq analysis to uncover potential genomic regulatory sites mediating glial differentiation. These datasets will enable future optimization of glial differentiation protocols and provide insight into human glial differentiation., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Genetically modified xenografts are one of the most promising solutions to the discrepancy between the numbers of available human organs for transplantation and potential recipients. To date, a porcine heart has been implanted into only one human recipient. Here, using 10-gene-edited pigs, we transplanted porcine hearts into two brain-dead human recipients and monitored xenograft function, hemodynamics and systemic responses over the course of 66 hours. Although both xenografts demonstrated excellent cardiac function immediately after transplantation and continued to function for the duration of the study, cardiac function declined postoperatively in one case, attributed to a size mismatch between the donor pig and the recipient. For both hearts, we confirmed transgene expression and found no evidence of cellular or antibody-mediated rejection, as assessed using histology, flow cytometry and a cytotoxic crossmatch assay. Moreover, we found no evidence of zoonotic transmission from the donor pigs to the human recipients. While substantial additional work will be needed to advance this technology to human trials, these results indicate that pig-to-human heart xenotransplantation can be performed successfully without hyperacute rejection or zoonosis., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Purpose: Long interspersed nuclear element-1 (LINE-1 or L1) comprises 17% of the human genome. Retrotransposons may perturb gene integrity or alter gene expression by altering regulatory regions in the genome. The germline employs a number of mechanisms, including cytosine methylation, to repress retrotransposon transcription throughout most of life. Demethylation during germ cell and early embryo development de-represses retrotransposons. Intriguingly, de novo genetic variation appearing in sperm has been implicated in a number of disorders in offspring, including autism spectrum disorder, schizophrenia, and bipolar disorder. We hypothesize that human sperm exhibit de novo retrotransposition and employ a new sequencing method, single cell transposon insertion profiling by sequencing (scTIPseq) to map them in small amounts of human sperm., Methods: Cross-sectional case-control study of sperm samples (n=10 men; ages 32-55 years old) from consenting men undergoing IVF at NYU Langone Fertility Center. scTIPseq identified novel LINE-1 insertions in individual sperm and TIPseqHunter, a custom bioinformatics pipeline, compared the architecture of sperm LINE-1 to known LINE-1 insertions from the European database of Human specific LINE-1 (L1Hs) retrotransposon insertions (euL1db)., Results: scTIPseq identified 17 novel insertions in sperm. New insertions were mainly intergenic or intronic. Only one sample did not exhibit new insertions. The location or number of novel insertions did not differ by paternal age., Conclusion: This study for the first time reports novel LINE-1 insertions in human sperm, demonstrating the feasibility of scTIPseq, and identifies new contributors to genetic diversity in the human germ line., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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. Saccharomyces 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, introducing designer variations to episomal assemblies with high efficiency and fidelity remains challenging. Here we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, a method for rapid engineering of large synthetic episomal DNA constructs. We demonstrate that CRISPR editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes. We optimize CREEPY for efficient and precise multiplex editing of >100 kb yeast episomes, providing an expanded toolkit for synthetic genomics., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A synthetic biology approach toward constructing an RNA-based genome expands our understanding of living things and opens avenues for technological advancement. For the precise design of an artificial RNA replicon either from scratch or based on a natural RNA replicon, understanding structure-function relationships of RNA sequences is critical. However, our knowledge remains limited to a few particular structural elements intensively studied so far. Here, we conducted a series of site-directed mutagenesis studies of yeast narnaviruses ScNV20S and ScNV23S, perhaps the simplest natural autonomous RNA replicons, to identify RNA elements required for maintenance and replication. RNA structure disruption corresponding to various portions of the entire narnavirus genome suggests that pervasive RNA folding, in addition to the precise secondary structure of genome termini, is essential for maintenance of the RNA replicon in vivo. Computational RNA structure analyses suggest that this scenario likely applies to other "narna-like" viruses. This finding implies selective pressure on these simplest autonomous natural RNA replicons to fold into a unique structure that acquires both thermodynamic and biological stability. We propose the importance of pervasive RNA folding for the design of RNA replicons that could serve as a platform for in vivo continuous evolution as well as an interesting model to study the origin of life.

LINE-1 (L1) is the only autonomously active retrotransposon in the human genome, and accounts for 17% of the human genome. The L1 mRNA encodes two proteins, ORF1p and ORF2p, both essential for retrotransposition. ORF2p has reverse transcriptase and endonuclease activities, while ORF1p is a homotrimeric RNA-binding protein with poorly understood function. Here, we show that condensation of ORF1p is critical for L1 retrotransposition. Using a combination of biochemical reconstitution and live-cell imaging, we demonstrate that electrostatic interactions and trimer conformational dynamics together tune the properties of ORF1p assemblies to allow for efficient L1 ribonucleoprotein (RNP) complex formation in cells. Furthermore, we relate the dynamics of ORF1p assembly and RNP condensate material properties to the ability to complete the entire retrotransposon life-cycle. Mutations that prevented ORF1p condensation led to loss of retrotransposition activity, while orthogonal restoration of coiled-coil conformational flexibility rescued both condensation and retrotransposition. Based on these observations, we propose that dynamic ORF1p oligomerization on L1 RNA drives the formation of an L1 RNP condensate that is essential for retrotransposition., Competing Interests: SS, SK, FE, LD, LH No competing interests declared, JB is a Founder and Director of CDI Labs, Inc, a Founder of and consultant to Neochromosome, Inc, a Founder, SAB member of and consultant to ReOpen Diagnostics, LLC and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc, Modern Meadow, Inc, Rome Therapeutics, Inc, Sample6, Inc, Tessera Therapeutics, Inc and the Wyss Institute, (© 2023, Sil et al.)

Forcing budding yeast to chromatinize their DNA with human histones manifests an abrupt fitness cost. We previously proposed chromosomal aneuploidy and missense mutations as two potential modes of adaptation to histone humanization. Here, we show that aneuploidy in histone-humanized yeasts is specific to a subset of chromosomes that are defined by their centromeric evolutionary origins but that these aneuploidies are not adaptive. Instead, we find that a set of missense mutations in outer kinetochore proteins drives adaptation to human histones. Furthermore, we characterize the molecular mechanism underlying adaptation in two mutants of the outer kinetochore DASH/Dam1 complex, which reduce aneuploidy by suppression of chromosome instability. Molecular modeling and biochemical experiments show that these two mutants likely disrupt a conserved oligomerization interface thereby weakening microtubule attachments. We propose a model through which weakened microtubule attachments promote increased kinetochore-microtubule turnover and thus suppress chromosome instability. In sum, our data show how a set of point mutations evolved in histone-humanized yeasts to counterbalance human histone-induced chromosomal instability through weakening microtubule interactions, eventually promoting a return to euploidy., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Sox2 expression in mouse embryonic stem cells (mESCs) depends on a distal cluster of DNase I hypersensitive sites (DHSs), but their individual contributions and degree of interdependence remain a mystery. We analyzed the endogenous Sox2 locus using Big-IN to scarlessly integrate large DNA 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 for Sox2 expression. We found that two DHSs comprising a handful of key TF recognition sequences were each sufficient for long-range activation of Sox2 expression. By contrast, three nearby DHSs were entirely context dependent, showing no activity alone but dramatically augmenting the activity of the autonomous DHSs. Our results highlight the role of context in modulating genomic regulatory element function, and our synthetic regulatory genomics approach provides a roadmap for the dissection of other genomic loci., Competing Interests: Declaration of interests R.B., J.D.B., and M.T.M. are listed as inventors on a patent application describing Big-IN. J.D.B. is a founder and Director of CDI Labs, Inc., a founder of and consultant to Neochromosome, Inc., a founder of, SAB member of, and consultant to ReOpen Diagnostics, LLC, and serves or served on the Scientific Advisory Board of the following: Logomix, Inc., Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute., (Copyright © 2023 Elsevier Inc. All rights reserved.)

LINE-1 retrotransposons are sequences capable of copying themselves to new genomic loci via an RNA intermediate. New studies implicate LINE-1 in a range of diseases, especially in the context of aging, but without an accurate understanding of where and when LINE-1 is expressed, a full accounting of its role in health and disease is not possible. We therefore developed a method-5' scL1seq-that makes use of a widely available library preparation method (10x Genomics 5' single cell RNA-seq) to measure LINE-1 expression in tens of thousands of single cells. We recapitulated the known pattern of LINE-1 expression in tumors-present in cancer cells, absent from immune cells-and identified hitherto undescribed LINE-1 expression in human epithelial cells and mouse hippocampal neurons. In both cases, we saw a modest increase with age, supporting recent research connecting LINE-1 to age related diseases., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The nucleolus is the most prominent membraneless compartment within the nucleus-dedicated to the metabolism of ribosomal RNA. Nucleoli are composed of hundreds of ribosomal DNA (rDNA) repeated genes that form large chromosomal clusters, whose high recombination rates can cause nucleolar dysfunction and promote genome instability. Intriguingly, the evolving architecture of eukaryotic genomes appears to have favored two strategic rDNA locations-where a single locus per chromosome is situated either near the centromere ( CEN ) or the telomere. Here, we deployed an innovative genome engineering approach to cut and paste to an ectopic chromosomal location-the ~1.5 mega-base rDNA locus in a single step using CRISPR technology. This "megablock" rDNA engineering was performed in a fused-karyotype strain of Saccharomyces cerevisiae . The strategic repositioning of this locus within the megachromosome allowed experimentally mimicking and monitoring the outcome of an rDNA migratory event, in which twin rDNA loci coexist on the same chromosomal arm. We showed that the twin-rDNA yeast readily adapts, exhibiting wild-type growth and maintaining rRNA homeostasis, and that the twin loci form a single nucleolus throughout the cell cycle. Unexpectedly, the size of each rDNA array appears to depend on its position relative to the CEN , in that the locus that is CEN -distal undergoes size reduction at a higher frequency compared to the CEN -proximal counterpart. Finally, we provided molecular evidence supporting a mechanism called paralogous cis -rDNA interference, which potentially explains why placing two identical repeated arrays on the same chromosome may negatively affect their function and structural stability.

Technologies to profoundly engineer biology are becoming increasingly affordable, powerful, and accessible to a widening group of actors. While offering tremendous potential to fuel biological research and the bioeconomy, this development also increases the risk of inadvertent or deliberate creation and dissemination of pathogens. Effective regulatory and technological frameworks need to be developed and deployed to manage these emerging biosafety and biosecurity risks. Here, we review digital and biological approaches of a range of technology readiness levels suited to address these challenges. Digital sequence screening technologies already are used to control access to synthetic DNA of concern. We examine the current state of the art of sequence screening, challenges and future directions, and environmental surveillance for the presence of engineered organisms. As biosafety layer on the organism level, we discuss genetic biocontainment systems that can be used to created host organisms with an intrinsic barrier against unchecked environmental proliferation., Competing Interests: D.D. is a co-founder of Lattice Automation, Inc. and Asimov Inc. Both companies create engineered biological systems using software and automation. J.D.B. is a Founder and Director of CDI Labs, Inc., a Founder of and consultant to Neochromosome, Inc, a Founder, SAB member of and consultant to Re-Open Diagnostics, LLC, and serves or served on the Scientific Advisory Board of the following: Sangamo Therapeutics, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc. and the Wyss Institute. T.K. is co-founder of Ginkgo Bioworks, Inc. EL is CEO and co-founder of Twist Bioscience, Inc., and J.D. is an employee of Twist Bioscience, Inc., (© 2023 The Author(s).)

Genomic studies in age-related macular degeneration (AMD) have identified genetic variants that account for the majority of AMD risk. An important next step is to understand the functional consequences and downstream effects of the identified AMD-associated genetic variants. Instrumental for this next step are 'omics' technologies, which enable high-throughput characterization and quantification of biological molecules, and subsequent integration of genomics with these omics datasets, a field referred to as systems genomics. Single cell sequencing studies of the retina and choroid demonstrated that the majority of candidate AMD genes identified through genomic studies are expressed in non-neuronal cells, such as the retinal pigment epithelium (RPE), glia, myeloid and choroidal cells, highlighting that many different retinal and choroidal cell types contribute to the pathogenesis of AMD. Expression quantitative trait locus (eQTL) studies in retinal tissue have identified putative causal genes by demonstrating a genetic overlap between gene regulation and AMD risk. Linking genetic data to complement measurements in the systemic circulation has aided in understanding the effect of AMD-associated genetic variants in the complement system, and supports that protein QTL (pQTL) studies in plasma or serum samples may aid in understanding the effect of genetic variants and pinpointing causal genes in AMD. A recent epigenomic study fine-mapped AMD causal variants by determing regulatory regions in RPE cells differentiated from induced pluripotent stem cells (iPSC-RPE). Another approach that is being employed to pinpoint causal AMD genes is to produce synthetic DNA assemblons representing risk and protective haplotypes, which are then delivered to cellular or animal model systems. Pinpointing causal genes and understanding disease mechanisms is crucial for the next step towards clinical translation. Clinical trials targeting proteins encoded by the AMD-associated genomic loci C3, CFB, CFI, CFH, and ARMS2/HTRA1 are currently ongoing, and a phase III clinical trial for C3 inhibition recently showed a modest reduction of lesion growth in geographic atrophy. The EYERISK consortium recently developed a genetic test for AMD that allows genotyping of common and rare variants in AMD-associated genes. Polygenic risk scores (PRS) were applied to quantify AMD genetic risk, and may aid in predicting AMD progression. In conclusion, genomic studies represent a turning point in our exploration of AMD. The results of those studies now serve as a driving force for several clinical trials. Expanding to omics and systems genomics will further decipher function and causality from the associations that have been reported, and will enable the development of therapies that will lessen the burden of AMD., Competing Interests: Declaration of competing interest AIdH is currently an employee of AbbVie; LDO, HHC, HC and LH are employees of Genentech; SK is an employee of Gemini Therapeutics. GSH is a shareholder, consultant, and co-founder of Perceive Biotherapeutics, Inc. and an inventor on patents and patent applications owned by the University of Iowa and the University of Utah. RFM, APV, TS, FG, JLH, JJW, SJT, RA, DS, CCWK, JDB, KAF, BHFW and MBG have no competing interests to declare., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly, raising the possibility that these pathways were lost from organisms with access to abundant EAAs. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in mammalian cells. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli . These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese hamster ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation. 13 C-tracing verified de novo biosynthesis of valine and further revealed build-up of pathway intermediate 2,3-dihydroxy-3-isovalerate. Increasing the dosage of downstream ilvD boosted pathway performance and allowed for long-term propagation of second-generation cells in valine-free medium at 3.2 days per doubling. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage., Competing Interests: JT, RM, AK, SP, HB, SG, LL, MS, XG, JM, MP, DJ No competing interests declared, JB is a Founder and Director of CDI Labs, Inc, a Founder of Neochromosome, Inc, a Founder and SAB member of ReOpen Diagnostics, and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc, Modern Meadow, Inc, Rome Therapeutics, Sample6, Inc, Tessera Therapeutics, Inc and the Wyss Institute, HW H.H.W. is a scientific advisor to SNIPR Biome, Arranta Bio, Genus plc, Kingdom Supercultures, Fitibomics, and VecX Biomedicine and a scientific cofounder of Aclid, (© 2022, Trolle, McBee et al.)

Eukaryotic genomes vary in terms of size, chromosome number, and genetic complexity. Their temporal organization is complex, reflecting coordination between DNA folding and function. Here, we used fused karyotypes of budding yeast to characterize the effects of chromosome length on nuclear architecture. We found that size-matched megachromosomes expand to occupy a larger fraction of the enlarged nucleus. Hi-C maps reveal changes in the three-dimensional structure corresponding to inactivated centromeres and telomeres. De-clustering of inactive centromeres results in their loss of early replication, highlighting a functional correlation between genome organization and replication timing. Repositioning of former telomere-proximal regions on chromosome arms exposed a subset of contacts between flocculin genes. Chromatin reorganization of megachromosomes during cell division remained unperturbed, and it revealed that centromere-rDNA contacts in anaphase, extending over 0.3 Mb on wild-type chromosome, cannot exceed ∼1.7 Mb. Our results highlight the relevance of engineered karyotypes to unveiling relationships between genome organization and function., Competing Interests: J.D.B. is a founder and director of CDI Labs, a founder of Neochromosome, Inc, a founder of and consultant to Re-Open Diagnostics, and serves or served on the Scientific Advisory Boards of Modern Meadow, Rome Therapeutics, Sample6, Sangamo, Tessera Therapeutics, and the Wyss Institute. The remaining authors declare no competing interests., (© 2022 The Authors.)

Precise Hox gene expression is crucial for embryonic patterning. Intra- Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.

Overwriting counterselectable markers is an efficient strategy for removing wild-type DNA or replacing it with payload DNA of interest. Currently, one bottleneck of efficient genome engineering in mammals is the shortage of counterselectable (negative selection) markers that work robustly without affecting organismal developmental potential. Here, we report a conditional Piga knockout strategy that enables efficient proaerolysin-based counterselection in mouse embryonic stem cells. The conditional Piga knockout cells show similar proaerolysin resistance as full (non-conditional) Piga deletion cells, which enables the use of a PIGA transgene as a counterselectable marker for genome engineering purposes. Native Piga function is readily restored in conditional Piga knockout cells to facilitate subsequent mouse development. We also demonstrate the generality of our strategy by engineering a conditional knockout of endogenous Hprt . Taken together, our work provides a new tool for advanced mouse genome writing and mouse model establishment., Competing Interests: Jef Boeke is a Founder and Director of CDI Labs, Inc., a Founder of and consultant to Neochromosome, Inc, a Founder, SAB member of and consultant to Re-Open Diagnostics, LLC and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute., (© 2022 The Authors.)

Background: Xenografts from genetically modified pigs have become one of the most promising solutions to the dearth of human organs available for transplantation. The challenge in this model has been hyperacute rejection. To avoid this, pigs have been bred with a knockout of the alpha-1,3-galactosyltransferase gene and with subcapsular autologous thymic tissue., Methods: We transplanted kidneys from these genetically modified pigs into two brain-dead human recipients whose circulatory and respiratory activity was maintained on ventilators for the duration of the study. We performed serial biopsies and monitored the urine output and kinetic estimated glomerular filtration rate (eGFR) to assess renal function and xenograft rejection., Results: The xenograft in both recipients began to make urine within moments after reperfusion. Over the 54-hour study, the kinetic eGFR increased from 23 ml per minute per 1.73 m 2 of body-surface area before transplantation to 62 ml per minute per 1.73 m 2 after transplantation in Recipient 1 and from 55 to 109 ml per minute per 1.73 m 2 in Recipient 2. In both recipients, the creatinine level, which had been at a steady state, decreased after implantation of the xenograft, from 1.97 to 0.82 mg per deciliter in Recipient 1 and from 1.10 to 0.57 mg per deciliter in Recipient 2. The transplanted kidneys remained pink and well-perfused, continuing to make urine throughout the study. Biopsies that were performed at 6, 24, 48, and 54 hours revealed no signs of hyperacute or antibody-mediated rejection. Hourly urine output with the xenograft was more than double the output with the native kidneys., Conclusions: Genetically modified kidney xenografts from pigs remained viable and functioning in brain-dead human recipients for 54 hours, without signs of hyperacute rejection. (Funded by Lung Biotechnology.)., (Copyright © 2022 Massachusetts Medical Society.)

Sequence features of genes and their flanking regulatory regions are determinants of RNA transcript isoform expression and have been used as context-independent plug-and-play modules in synthetic biology. However, genetic context-including the adjacent transcriptional environment-also influences transcript isoform expression levels and boundaries. We used synthetic yeast strains with stochastically repositioned genes to systematically disentangle the effects of sequence and context. Profiling 120 million full-length transcript molecules across 612 genomic perturbations, we observed sequence-independent alterations to gene expression levels and transcript isoform boundaries that were influenced by neighboring transcription. We identified features of transcriptional context that could predict these alterations and used these features to engineer a synthetic circuit where transcript length was controlled by neighboring transcription. This demonstrates how positional context can be leveraged in synthetic genome engineering.

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway., Competing Interests: Competing interest statement: D.F. is a founder and president of The Informatics Factory and serves or served on the scientific advisory board (SAB) or consults for Spectragen Informatics, Protein Metrics, and Preverna. J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc.; a founder SAB member of and consultant to ReOpen Diagnostics, LLC; and serves or served on the SAB of the following: Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute. K.H.B. contributed a review with one of the reviewers of this manuscript within the last three years., (Copyright © 2022 the Author(s). Published by PNAS.)