Tools, Strains, and Strategies To Effectively Conduct Anaerobic and Aerobic Transcriptional Reporter Screens and Assays in Staphylococcus aureus

Staphylococcus aureus is a human pathogen and a leading cause of infectious disease-related illness and death worldwide. For S. aureus to successfully colonize and invade host tissues, it must tightly control the expression of genes encoding virulence factors.

A ratiometric dual color luciferase reporter for fast characterization of transcriptional regulatory elements

Plant synthetic biology requires precise characterization of genetic elements to construct complex genetic circuits that can improve plant traits or confer them new characteristics. Transcriptional reporter assays are essential to quantify the effect of gene expression regulator elements. Therefore, transcriptional reporter systems are a key tool in understanding control of gene expression in biology. In this work we construct and characterize a dual-color luciferase ratiometric reporter system that possesses several advantages over currently used reporters. It is ratiometric, reducing variability and increasing consistency between experiments; it is fast, as both reporters can be measured at the same time in a single reaction, and it is cheaper to perform than current dual-luciferase reporter assays. We have validated our system quantifying the transcriptional capability of a panel of promoters and terminators commonly used in synthetic biology with a broad range of expression magnitudes, and in a biologically relevant system, nitrate response.

Inhibition of mTOR decreases insoluble protein burden by reducing translation in C. elegans

Aging animals accumulate insoluble protein as a consequence of a decline of proteostatic maintenance with age. In Caenorhabditis elegans, for instance, levels of detergent-insoluble protein increase with age. In longer-lived strains of C. elegans, this accumulation occurs more slowly, implying a link to lifespan determination. We further explored this link, and found that detergent-insoluble protein accumulates more rapidly at higher temperatures, a condition where lifespan is short. We employed a C. elegans strain carrying a GFP transcriptional reporter under the control of a heat shock (hsp-16.2) promoter to investigate the dynamics of proteostatic failure in individual nematodes. We found that early, sporadic activation of hsp-16.2 was predictive of shorter remaining lifespan in individual nematodes. Exposure to rapamycin, resulting in reduced mTOR signaling, delayed spurious expression, extended lifespan, and delayed accumulation of insoluble protein, suggesting that targets downstream of the mTOR pathway regulate the accumulation of insoluble protein. We specifically explored ribosomal S6 kinase (rsks-1) as one such candidate and found that RNAi against rsks-1 also resulted in less age-dependent accumulation of insoluble protein and extended lifespan. Our results demonstrate that inhibition of protein translation via reduced mTOR signaling resulted in slower accumulation of insoluble protein, delayed proteostatic crisis and extended lifespan in C. elegans.

Efficient generation of endogenous fluorescent reporters by Nested CRISPR in Caenorhabditis elegans

CRISPR-based genome editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is still inefficient. The use of plasmids with selection markers is an effective methodology, but often requires laborious and complicated cloning steps. We have established a cloning-free ribonucleoprotein-driven Nested CRISPR method that robustly produces endogenous fluorescent reporters. This methodology is based on the division of the GFP and mCherry sequences in three fragments. In the first step we use ssDNA donors (≤200 bp) to insert 5’ and 3’ fragments in the place of interest. In the second step, we use these sequences as homology regions for Homology Directed Repair (HDR) with a dsDNA donor (PCR product, ≈700 bp) including the middle fragment, thus completing the fluorescent protein sequence. This method is advantageous because the first step with ssDNA donors is known to be very efficient, and the second step, uses universal reagents, including validated PCR products and crRNAs, to create fluorescent reporters reaching reliable editing efficiencies as high as 40%. We have also used Nested CRISPR in a non-essential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step.In the search of modifications to optimize the method, we tested synthetic sgRNAs, but we did not observe a significant increase in the efficacy compared to independently adding tracrRNA and crRNA to the injection mix. Conveniently, we also found that both steps of Nested CRISPR could be performed in a single injection. Finally, we discuss the utility of Nested CRISPR for targeted insertion of long DNA fragments in other systems and prospects of this method in the future.

Chemical control of a CRISPR-Cas9 acetyltransferase

Lysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Towards this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators, and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype, and reveal a role for p300’s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tuneable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role, and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci.