Bacterial genome engineering using CRISPR-associated transposases.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposases have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in Escherichia coli at efficiencies approaching ~100%. Moreover, they generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species.

Small RNA-Sequencing Library Preparation for the Halophilic Archaeon Haloferax volcanii.

Posttranscriptional regulation actuated by small RNAs (sRNAs) plays essential roles in a wide variety of cellular processes, especially in stress responses and environmental signaling. Hundreds of sRNAs have recently been discovered in archaea using genome-wide approaches but the molecular mechanisms of only a few have been characterized experimentally. Here, we describe how to build sRNA sequencing libraries using size-selected total RNA in the model archaeon, Haloferax volcanii , to provide a tool to further characterize sRNAs in archaea.

Conversion of Ribosome-Protected mRNA to DNA and Deep Sequencing for Ribosome Profiling in Haloferax volcanii.

The translation of messenger RNA (mRNA) into protein is an essential process for all forms of life. The ability to monitor this process in a quantitative way by ribosome profiling-based approaches has revolutionized our ability to monitor protein synthesis in vivo and to explore and model complex cellular processes. Ribosome profiling is a high-throughput technique that globally analyzes the full set of ribosomes engaged in translation, providing insights into important aspects of the mechanism of protein synthesis and its regulation.

Post-transcriptional regulation of redox homeostasis by the small RNA SHOxi in haloarchaea.

While haloarchaea are highly resistant to oxidative stress, a comprehensive understanding of the processes regulating this remarkable response is lacking. Oxidative stress-responsive small non-coding RNAs (sRNAs) have been reported in the model archaeon, , but targets and mechanisms have not been elucidated. Using a combination of high throughput and reverse molecular genetic approaches, we elucidated the functional role of the most up-regulated intergenic sRNA during oxidative stress in , named mall RNA in aloferax dative Stress ().

Ribosome profiling in archaea reveals leaderless translation, novel translational initiation sites, and ribosome pausing at single codon resolution.

High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life.

The Non-Coding Regulatory RNA Revolution in Archaea.

Small non-coding RNAs (sRNAs) are ubiquitously found in the three domains of life playing large-scale roles in gene regulation, transposable element silencing and defense against foreign elements. While a substantial body of experimental work has been done to uncover function of sRNAs in Bacteria and Eukarya, the functional roles of sRNAs in Archaea are still poorly understood. Recently, high throughput studies using RNA-sequencing revealed that sRNAs are broadly expressed in the Archaea, comprising thousands of transcripts within the transcriptome during non-challenged and stressed conditions.

Transcriptional Landscape and Regulatory Roles of Small Noncoding RNAs in the Oxidative Stress Response of the Haloarchaeon Haloferax volcanii.

Haloarchaea in their natural environment are exposed to hypersalinity, intense solar radiation, and desiccation, all of which generate high levels of oxidative stress. Previous work has shown that haloarchaea are an order of magnitude more resistant to oxidative stress than most mesophilic organisms. Despite this resistance, the pathways haloarchaea use to respond to oxidative stress damage are similar to those of nonresistant organisms, suggesting that regulatory processes might be key to their robustness.