Variability in genome-engineering source materials: consider your starting point.

The presence and impact of variability in cells as the source material for genome engineering are important to consider for the design, execution and interpretation of outcomes of a genome-engineering process. Variability may be present at the genotype and phenotype level, yet the impact of these sources of variability on a genome-engineering experiment may not be regularly considered by researchers. In this perspective, we use clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) genome editing of mammalian cells to provide examples of how variation within or across cell samples may mislead a researcher in their expectations about the cells they are engineering.

Fire Burn and Cauldron Bubble: What Is in Your Genome Editing Brew?

Genome editing is a rapidly evolving biotechnology with the potential to transform many sectors of industry such as agriculture, biomanufacturing, and medicine. This technology is enabled by an ever-growing portfolio of biomolecular reagents that span the central dogma, from DNA to RNA to protein. In this paper, we draw from our unique perspective as the National Metrology Institute of the United States to bring attention to the importance of understanding and reporting genome editing formulations accurately and promoting concepts to verify successful delivery into cells.

MYC amplifies gene expression through global changes in transcription factor dynamics.

The MYC oncogene has been studied for decades, yet there is still intense debate over how this transcription factor controls gene expression. Here, we seek to answer these questions with an in vivo readout of discrete events of gene expression in single cells. We engineered an optogenetic variant of MYC (Pi-MYC) and combined this tool with single-molecule RNA and protein imaging techniques to investigate the role of MYC in modulating transcriptional bursting and transcription factor binding dynamics in human cells.

Towards a 'Spot On' Understanding of Transcription in the Nucleus.

Regulation of transcription by RNA Polymerase II (RNAPII) is a rapidly evolving area of research. Technological developments in microscopy have revealed insight into the dynamics, structure, and localization of transcription components within single cells. A frequent observation in many studies is the appearance of 'spots' in cell nuclei associated with the transcription process.

Single-cell systems biology: probing the basic unit of information flow.

Gene expression varies across cells in a population or a tissue. This heterogeneity has come into sharp focus in recent years through developments in new imaging and sequencing technologies. However, our ability to measure variation has outpaced our ability to interpret it.

The nutrient-responsive transcription factor TFE3 promotes autophagy, lysosomal biogenesis, and clearance of cellular debris.

The discovery of a gene network regulating lysosomal biogenesis and its transcriptional regulator transcription factor EB (TFEB) revealed that cells monitor lysosomal function and respond to degradation requirements and environmental cues. We report the identification of transcription factor E3 (TFE3) as another regulator of lysosomal homeostasis that induced expression of genes encoding proteins involved in autophagy and lysosomal biogenesis in ARPE-19 cells in response to starvation and lysosomal stress. We found that in nutrient-replete cells, TFE3 was recruited to lysosomes through interaction with active Rag guanosine triphosphatases (GTPases) and exhibited mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1)-dependent phosphorylation.