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. Furthermore, we highlight the need for understanding the baseline cell genotype and phenotype to appropriately understand the starting cell material and interpret and attribute the impact of engineering on cells. We emphasize that heterogeneity within a cell pool and the inherent variability in the cellular materials used for genome engineering are complex, but of high value to characterize and account for where possible, to move toward the potential of generating desired and predictable engineered products. Provided is a framework cause-and-effect diagram for CRISPR/Cas9 genome editing toward identifying and mitigating potential sources of variability. We encourage researchers to consider the variability of source materials and undertake strategies, which may include those described here, for detecting, attributing and minimizing additional sources of variability where possible toward the aim of fostering greater reliability, confidence and reproducibility in genome-engineering studies. Graphical Abstract.

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. Achieving the correct understanding may be hindered by the way units, quantities, and stoichiometries are reported in the field. We highlight the variability in how editing formulations are reported in the literature and examine how a reference molecule could be used to verify the delivery of a reagent into cells. We provide recommendations on how more accurate reporting of editing formulations and more careful verification of the steps in an editing experiment can help set baseline expectations of reagent performance, toward the aim of enabling genome editing studies to be more reproducible. We conclude with a future outlook on technologies that can further our control and enable our understanding of genome editing outcomes at the single-cell level.

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. We find that the immediate consequence of MYC overexpression is an increase in the duration rather than in the frequency of bursts, a functional role that is different from the majority of human transcription factors. We further propose that the mechanism by which MYC exerts global effects on the active period of genes is by altering the binding dynamics of transcription factors involved in RNA polymerase II complex assembly and productive elongation.

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. In this review we highlight studies that characterize the temporal and spatial characteristics of these spots, examine possible pitfalls in interpreting these kind of imaging data, and outline directions where single-cell imaging may advance in ways to further our understanding of transcription regulation.

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. Much of the variability may arise from random effects occurring in the processes of gene expression (transcription, RNA processing and decay, translation). The molecular basis of these effects is largely unknown. Likewise, a functional role of this variability in growth, differentiation and disease has only been elucidated in a few cases. In this review, we highlight recent experimental and theoretical advances for measuring and analyzing stochastic variation.

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. Phosphorylated TFE3 was retained in the cytosol through its interaction with the cytosolic chaperone 14-3-3. After starvation, TFE3 rapidly translocated to the nucleus and bound to the CLEAR elements present in the promoter region of many lysosomal genes, thereby inducing lysosomal biogenesis. Depletion of endogenous TFE3 entirely abolished the response of ARPE-19 cells to starvation, suggesting that TFE3 plays a critical role in nutrient sensing and regulation of energy metabolism. Furthermore, overexpression of TFE3 triggered lysosomal exocytosis and resulted in efficient cellular clearance in a cellular model of a lysosomal storage disorder, Pompe disease, thus identifying TFE3 as a potential therapeutic target for the treatment of lysosomal disorders.