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.

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.