CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications.

In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.

CRISPR-Cas tools for simultaneous transcription & translation control in bacteria.

Robust control over gene translation at arbitrary mRNA targets is an outstanding challenge in microbial synthetic biology. The development of tools that can regulate translation will greatly expand our ability to precisely control genes across the genome. In Escherichia coli, most genes are contained in multi-gene operons, which are subject to polar effects where targeting one gene for repression leads to silencing of other genes in the same operon. These effects pose a challenge for independently regulating individual genes in multi-gene operons. Here, we use CRISPR-dCas13 to address this challenge. We find dCas13-mediated repression exhibits up to 6-fold lower polar effects compared to dCas9. We then show that we can selectively activate single genes in a synthetic multi-gene operon by coupling dCas9 transcriptional activation of an operon with dCas13 translational repression of individual genes within the operon. We also show that dCas13 and dCas9 can be multiplexed for improved biosynthesis of a medically-relevant human milk oligosaccharide. Taken together, our findings suggest that combining transcriptional and translational control can access effects that are difficult to achieve with either mode independently. These combined tools for gene regulation will expand our abilities to precisely engineer bacteria for biotechnology and perform systematic genetic screens.

Open-source workflow design and management software to interrogate duckweed growth conditions and stress responses.

Duckweeds, a family of floating aquatic plants, are ideal model plants for laboratory experiments because they are small, easy to cultivate, and reproduce quickly. Duckweed cultivation, for the purposes of scientific research, requires that lineages are maintained as continuous populations of asexually propagating fronds, so research teams need to develop optimized cultivation conditions and coordinate maintenance tasks for duckweed stocks. Additionally, computational image analysis is proving to be a powerful duckweed research tool, but researchers lack software tools to assist with data collection and storage in a way that can feed into scripted data analysis. We set out to support these processes using a laboratory management software called Aquarium, an open-source application developed to manage laboratory inventory and plan experiments. We developed a suite of duckweed cultivation and experimentation operation types in Aquarium, which we then integrated with novel data analysis scripts. We then demonstrated the efficacy of our system with a series of image-based growth assays, and explored how our framework could be used to develop optimized cultivation protocols. We discuss the unexpected advantages and the limitations of this approach, suggesting areas for future software tool development. In its current state, our approach helps to bridge the gap between laboratory implementation and data analytical software for duckweed biologists and builds a foundation for future development of end-to-end computational tools in plant science.

Engineering activatable promoters for scalable and multi-input CRISPRa/i circuits.

Dynamic, multi-input gene regulatory networks (GRNs) are ubiquitous in nature. Multilayer CRISPR-based genetic circuits hold great promise for building GRNs akin to those found in naturally occurring biological systems. We develop an approach for creating high-performing activatable promoters that can be assembled into deep, wide, and multi-input CRISPR-activation and -interference (CRISPRa/i) GRNs. By integrating sequence-based design and in vivo screening, we engineer activatable promoters that achieve up to 1,000-fold dynamic range in an Escherichia coli-based cell-free system. These components enable CRISPRa GRNs that are six layers deep and four branches wide. We show the generalizability of the promoter engineering workflow by improving the dynamic range of the light-dependent EL222 optogenetic system from 6-fold to 34-fold. Additionally, high dynamic range promoters enable CRISPRa systems mediated by small molecules and protein-protein interactions. We apply these tools to build input-responsive CRISPRa/i GRNs, including feedback loops, logic gates, multilayer cascades, and dynamic pulse modulators. Our work provides a generalizable approach for the design of high dynamic range activatable promoters and enables classes of gene regulatory functions in cell-free systems.

Promotion of tobacco products on Facebook: policy versus practice.

BACKGROUND: Facebook has a comprehensive set of policies intended to inhibit promotion and sales of tobacco products. Their effectiveness has yet to be studied. METHODS: Leading tobacco brands (388) were identified via Nielsen and Ranker databases and 108 were found to maintain brand-sponsored Facebook pages. Key indicators of alignment with Facebook policy were evaluated. RESULTS: Purchase links (eg, 'shop now' button) on brand-sponsored pages were found for hookah tobaccos (41%), e-cigarettes (74%), smokeless (50%) and cigars (31%). Sales promotions (eg, discount coupons) were present in hookah tobacco (48%), e-cigarette (76%) and cigar (69%) brand-sponsored pages. While conventional cigarettes did not maintain brand-sponsored pages, they were featured in 80% of online tobacco vendors' Facebook pages. The requirement for age gating, to exclude those <18 from viewing tobacco promotion, was absent in hookah tobacco (78%), e-cigarette (62%) and cigar (21%) brand-sponsored pages and for 90% of online tobacco stores which promote leading cigarette brands (eg, Marlboro, Camel). Many of the brand-sponsored tobacco product pages had thousands of 'likes'. CONCLUSIONS: It is laudable that Facebook has policies intended to interdict tobacco promotion throughout its platform. Nevertheless, widespread tobacco promotion and sales were found at variance with the company's policies governing advertising, commerce, page content and under age access. Vetting could be improved by automated screening in partnership with human reviewers.