Uncoupling gene expression noise along the central dogma using genome engineered human cell lines.

Eukaryotic protein synthesis is an inherently stochastic process. This stochasticity stems not only from variations in cell content between cells but also from thermodynamic fluctuations in a single cell. Ultimately, these inherently stochastic processes manifest as noise in gene expression, where even genetically identical cells in the same environment exhibit variation in their protein abundances. In order to elucidate the underlying sources that contribute to gene expression noise, we quantify the contribution of each step within the process of protein synthesis along the central dogma. We uncouple gene expression at the transcriptional, translational, and post-translational level using custom engineered circuits stably integrated in human cells using CRISPR. We provide a generalized framework to approximate intrinsic and extrinsic noise in a population of cells expressing an unbalanced two-reporter system. Our decomposition shows that the majority of intrinsic fluctuations stem from transcription and that coupling the two genes along the central dogma forces the fluctuations to propagate and accumulate along the same path, resulting in increased observed global correlation between the products.

Loss of function of the ALS-associated NEK1 kinase disrupts microtubule homeostasis and nuclear import.

Loss-of-function variants in NIMA-related kinase 1 (NEK1) constitute a major genetic cause of amyotrophic lateral sclerosis (ALS), accounting for 2 to 3% of all cases. However, how NEK1 mutations cause motor neuron (MN) dysfunction is unknown. Using mass spectrometry analyses for NEK1 interactors and NEK1-dependent expression changes, we find functional enrichment for proteins involved in the microtubule cytoskeleton and nucleocytoplasmic transport. We show that α-tubulin and importin-ß1, two key proteins involved in these processes, are phosphorylated by NEK1 in vitro. NEK1 is essential for motor control and survival in Drosophila models in vivo, while using several induced pluripotent stem cell (iPSC)-MN models, including NEK1 knockdown, kinase inhibition, and a patient mutation, we find evidence for disruptions in microtubule homeostasis and nuclear import. Notably, stabilizing microtubules with two distinct classes of drugs restored NEK1-dependent deficits in both pathways. The capacity of NEK1 to modulate these processes that are critically involved in ALS pathophysiology renders this kinase a formidable therapeutic candidate.

Artificial extracellular matrix scaffolds of mobile molecules enhance maturation of human stem cell-derived neurons.

Human induced pluripotent stem cell (hiPSC) technologies offer a unique resource for modeling neurological diseases. However, iPSC models are fraught with technical limitations including abnormal aggregation and inefficient maturation of differentiated neurons. These problems are in part due to the absence of synergistic cues of the native extracellular matrix (ECM). We report on the use of three artificial ECMs based on peptide amphiphile (PA) supramolecular nanofibers. All nanofibers display the laminin-derived IKVAV signal on their surface but differ in the nature of their non-bioactive domains. We find that nanofibers with greater intensity of internal supramolecular motion have enhanced bioactivity toward hiPSC-derived motor and cortical neurons. Proteomic, biochemical, and functional assays reveal that highly mobile PA scaffolds caused enhanced ß1-integrin pathway activation, reduced aggregation, increased arborization, and matured electrophysiological activity of neurons. Our work highlights the importance of designing biomimetic ECMs to study the development, function, and dysfunction of human neurons.

Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls.

The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.