Biology subjects

Goldstein, J. M.

Publications and source records attributed to Goldstein, J. M..

6 recordsLinked to original sources

bioRxiv2026

In vivo gene disruption and homology-directed repair in muscles and muscle stem cells using CRISPR/Cas9

Programmable endonucleases such as CRISPR/Cas9 provide powerful tools to edit mammalian genomes by engaging cellular mechanisms of DNA double-strand break (DSB) repair. CRISPR-catalysed homology-directed repair (CRISPR-HDR), though generally less efficient than other modes of DNA repair, holds particular promise to enable precise sequence replacement by targeted insertion of a homologous DNA template1,2. While recent studies have reported appreciable levels of HDR in cardiomyocytes in vivo3, skeletal muscle myofibres have historically been considered refractory to HDR-mediated genome editing4. Furthermore, how repair outcomes differ across tissues after systemic delivery of CRISPR/Cas9 editors, whether precise HDR editing can be achieved in regenerative tissue stem cells, and how developmental timing influences accessibility to CRISPR-induced repair remain unclear. Here, we use an adeno-associated virus (AAV)-delivered in vivo GFP-to-BFP colour-switching reporter system (AAV-GFP-to-BFP) to examine in vivo CRISPR-HDR with cellular- and tissue-level resolution. We find that postnatal cardiac muscle, skeletal muscle, and muscle stem cells undergo templated HDR at different rates across discrete developmental stages in mice. While HDR-edited muscle stem cells and myofibres were readily detectable after in vivo editing in juvenile mice, editing in neonatal mice yielded more efficient HDR in cardiac tissue. Based on these results, we adapted the CRISPR-HDR approach to rescue the therapeutically relevant Dmd mutation in mdx mice, demonstrating recoding to the wild-type protein sequence in both skeletal and cardiac muscles. These results provide a framework for advancing donor-templated DNA repair in living postnatal animals, and reveal unexpected cellular, developmental, and disease-related constraints on precise, therapeutic in vivo gene correction.

cell biology↗

bioRxiv2022

Functional substitutions of amino acids that differ between GDF11 and GDF8 impact skeletal development and skeletal muscle.

Growth differentiation factor 11 (GDF11) and GDF8 (MSTN) are closely related TGF{beta} family proteins that interact with nearly identical signaling receptors and antagonist proteins. However, GDF11 appears to activate SMAD2/3 in vitro and in vivo more potently than GDF8. The ligands possess divergent structural properties, whereby substituting unique GDF11 amino acids into GDF8 enhanced activity of the resulting chimeric GDF8. We investigated potentially distinct endogenous activities of GDF11 and GDF8 in vivo by genetically modifying their mature signaling domains. Full recoding of the GDF8 mature domain to that of GDF11 yielded mice lacking GDF8, with GDF11 levels [~]50-fold higher than normal, and exhibiting modestly decreased muscle mass, with no apparent negative impacts on health or survival to adulthood. Substitution of two specific amino acids in the fingertip region of GDF11 with the corresponding GDF8 residues resulted in prenatal axial skeletal transformations, consistent with Gdf11-deficient mice, without apparent perturbation of skeletal or cardiac muscle development or homeostasis. These experiments uncover distinctive features between the GDF11 and GDF8 mature domains in vivo and identify specific requirement for GDF11 in early-stage skeletal development. Summary StatementReplacement of amino acids unique to GDF11 and GDF8 into the native locus of the other ligand yields measurable, differential skeletal and muscle phenotypes, revealing distinct features between the ligands.

developmental biology↗

bioRxiv2022

Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types

Aging is a complex process involving transcriptomic changes associated with deterioration across multiple tissues and organs, including the brain. Recent studies using heterochronic parabiosis have shown that various aspects of aging-associated decline are modifiable or even reversible. To better understand how this occurs, we performed single-cell transcriptomic profiling of young and old mouse brains following parabiosis. For each cell type, we catalogued alterations in gene expression, molecular pathways, transcriptional networks, ligand-receptor interactions, and senescence status. Our analyses identified gene signatures demonstrating that heterochronic parabiosis regulates several hallmarks of aging in a cell-type-specific manner. Brain endothelial cells were found to be especially malleable to this intervention, exhibiting dynamic transcriptional changes that affect vascular structure and function. These findings suggest novel strategies for slowing deterioration and driving regeneration in the aging brain through approaches that do not rely on disease-specific mechanisms or actions of individual circulating factors.

neuroscience↗

bioRxiv2020

Rapid Development of Neutralizing and Diagnostic SARS-COV-2 Mouse Monoclonal Antibodies

The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the development and functional characterization of 29 nanomolar-affinity mouse SARS-CoV-2 mAbs created by an accelerated immunization and hybridoma screening process. Differing functions, including binding of diverse protein epitopes, viral neutralization, impact on RBD-hACE2 binding, and immunohistochemical staining of infected lung tissue, were correlated with variable gene usage and sequence.

immunology↗

bioRxiv2020

Examining sex-differentiated genetic effects across neuropsychiatric and behavioral traits

BackgroundThe origin of sex differences in prevalence and presentation of neuropsychiatric and behavioral traits is largely unknown. Given established genetic contributions and correlations across these traits, we tested for a sex-differentiated genetic architecture within and between traits. MethodsUsing genome-wide association study (GWAS) summary statistics for 20 neuropsychiatric and behavioral traits, we tested for differences in SNP-based heritability (h2) and genetic correlation (rg<1) between sexes. For each trait, we computed z-scores from sex-stratified GWAS regression coefficients and identified genes with sex-differentiated effects. We calculated Pearson correlation coefficients between z-scores for each trait pair, to assess whether specific pairs share variants with sex-differentiated effects. Finally, we tested for sex differences in between-trait genetic correlations. ResultsWith current sample sizes (and power), we found no significant, consistent sex differences in SNP-based h2. Between-sex, within-trait genetic correlations were consistently high, although significantly less than 1 for educational attainment and risk-taking behavior. We identified genome-wide significant genes with sex-differentiated effects for eight traits. Several trait pairs shared sex-differentiated effects. The top 0.1% of genes with sex-differentiated effects across traits overlapped with neuron- and synapse-related gene sets. Most between-trait genetic correlation estimates were similar across sex, with several exceptions (e.g. educational attainment & risk-taking behavior). ConclusionsSex differences in the common autosomal genetic architecture of neuropsychiatric and behavioral phenotypes are small and polygenic, requiring large sample sizes. Genes with sex-differentiated effects are enriched for neuron-related gene sets. This work motivates further investigation of genetic, as well as environmental, influences on sex differences.

genetics↗

bioRxiv2019

The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis

RNA has been classically known to play central roles in biology, including maintaining telomeres1, protein synthesis2, and in sex chromosome compensation in certain species3,4. At the center of these important biological systems are noncoding RNAs. While thousands of long noncoding RNAs (lncRNAs) have been identified in mammalian genomes5-8, attributing RNA-based roles to lncRNA loci requires an assessment of whether the observed effect could be due to DNA regulatory elements, the act of transcription, or the lncRNA transcript. Here, we use the syntenically conserved lncRNA locus, Functional intergenic repeating RNA element (Firre), that is located on the X chromosome as a model to discriminate between DNA- and RNA-mediated effects in vivo. To this end, we generated genetically defined loss-of-function, gain-of-function, and rescue mouse models for Firre and provide genetic evidence that the Firre locus produces a trans-acting RNA. We report that: (i) Firre mutant mice have cell-specific defects during hematopoiesis and changes in gene expression that can be rescued by induction of Firre RNA from a transgene in the Firre knockout background, (ii) mice overexpressing Firre from a transgene exhibit increased levels of pro-inflammatory cytokines and impaired survival upon exposure to lipopolysaccharide, and (iii) deletion of the Firre locus did not result in changes in local gene expression on the X chromosome in 9 different biological contexts, suggesting that Firre does not function by cis-acting RNA or DNA elements. Together, our results provide genetic evidence that the Firre locus produces a trans-acting lncRNA that has physiological roles in hematopoiesis and immune function.

immunology↗