Esther Mintzer

Proboscideans have developed a suite of genetic changes including those responsible for tumor suppression and environmental adaptation. Here we examine a handful of woolly mammoth-specific deletions, and their potential contributions to arctic adaptation, as well as the expanded TP53 genetic repertoire in elephants. We use CRISPR-Cas9 to introduce mammoth-specific noncoding deletions as well as knockouts of TP53, all 29 TP53 retrogenes, or both in combination in Asian elephant cell lines, and examine the transcriptomic response. We find that many of the mammoth-specific deletions likely contribute to various arctic phenotypes including vascular development, metabolism and thermogenesis, and hair and skin adaptations. We also find that while there is considerable overlap in DNA damage responses of the TP53 and retrogene knockouts, retrogenes knockouts also exhibit strong enrichment of many extracellular pathways suggesting they may play a role in the tumor microenvironment and mitigating metastatic growth.

Acne vulgaris, rosacea, and hidradenitis suppurativa are enduring inflammatory skin conditions that frequently manifest with akin clinical attributes, posing a considerable challenge for their distinctive diagnosis. While these conditions do exhibit certain resemblances, they also demonstrate distinct underlying pathophysiological mechanisms and treatment modalities. Delving into both the molecular parallels and disparities among these three disorders can yield invaluable insights for refined diagnostics, effective management, and targeted therapeutic interventions. In this report, we present a comparative analysis of transcriptomic data across these three diseases, elucidating differentially expressed genes and enriched pathways specific to each ailment, as well as those shared among them. We also identified high dose dietary zinc as a potential therapeutic agent and validated its efficacy in an acne mouse model.

While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. In this study, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified traffic light reporter (TLR) system (TLR-multi-Cas variant 1 [MCV1]) that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knockins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knockins in mammalian cell lines.

The interrelatedness of human blood innate lymphoid cell (ILC) subsets, and how they are perturbed by HIV-1, remains unclear. Transcriptional and chromatin profiling separated blood ILCs into ILC2s, ILCPs, one cluster that included CD56 ^dim and CD56 ⁻ NK cells, and CD56 ^hi NK cells that have features of both CD56 ^dim/– NK cells and ILCs. In contrast to mice, human NK cells expressed tissue repair protein amphiregulin (AREG), with greater production by CD56 ^hi NK cells than by ILCs. AREG was induced by TCF7/WNT signaling, IL-2, or IL-15, but not by inflammatory cytokines, and was inhibited by TGFB1, a cytokine elevated in people living with HIV-1. NK cell knockout of the TGFB1-stimulated WNT antagonist RUNX3 increased AREG production. In people living with HIV-1, AREG ⁺ NK cell percentage correlated with numbers of ILCs and CD4 ⁺ T cells, and correlated inversely with inflammatory cytokine IL-6. RNA-Seq showed increased antiviral gene expression in all ILC subsets from people who were HIV-1 viremic, and increased expression of anti-inflammatory gene MYDGF in CD56 ^hi NK cells from elite controllers. Functionally-defective CD56 ⁻ NK cells were increased in people living with HIV-1 in inverse correlation with CD56 ^dim NK cells, ILCs, and CD4 ⁺ T cells. Experiments with human PBMCs ex vivo and in humanized mice revealed that CD4 ⁺ T cells and their production of IL-2 prevented CD56 ^dim transition to CD56 ⁻ NK cells by activating mTOR, and, in people living with HIV-1, plasma IL-2 correlated with CD4 ⁺ T cell number but not with CD8 ⁺ T cells. These studies clarify how ILC subsets are interrelated and provide insight into how HIV-1 infection disrupts NK cells, including homeostatic functions of NK cells discovered here. Graphical