Integrating CRISPR-Enabled Trackable Genome Engineering and Transcriptomic Analysis of Global Regulators for Antibiotic Resistance Selection and Identification in Escherichia coli.

It is important to expedite our understanding of antibiotic resistance to address the increasing numbers of fatalities and environmental pollution due to the emergence of antibiotic resistance and multidrug-resistant strains. Here, we combined the CRISPR-enabled trackable genome engineering (CREATE) technology and transcriptomic analysis to investigate antibiotic tolerance in We developed rationally designed site saturation mutagenesis libraries targeting 23 global regulators to identify fitness-conferring mutations in response to diverse antibiotic stresses. We identified seven novel mutations that confer resistance to the ribosome-targeting antibiotics doxycycline, thiamphenicol, and gentamicin in To the best of our knowledge, these mutations that we identified have not been reported previously during treatment with the indicated antibiotics.

Small molecule regulated sgRNAs enable control of genome editing in E. coli by Cas9.

CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To further the utility of Cas9 there have been efforts to achieve temporal control over its nuclease activity. While different approaches have focused on regulation of CRISPR interference or editing in mammalian cells, none of the reported methods enable control of the nuclease activity in bacteria.

Deep scanning lysine metabolism in .

Our limited ability to predict genotype-phenotype relationships has called for strategies that allow testing of thousands of hypotheses in parallel. Deep scanning mutagenesis has been successfully implemented to map genotype-phenotype relationships at a single-protein scale, allowing scientists to elucidate properties that are difficult to predict. However, most phenotypes are dictated by several proteins that are interconnected through complex and robust regulatory and metabolic networks.

Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to the Antimalarial Drug Fosmidomycin in E. coli.

Sequence to activity mapping technologies are rapidly developing, enabling the generation and isolation of mutations conferring novel phenotypes. Here we used the CRISPR enabled trackable genome engineering (CREATE) technology to investigate the inhibition of the essential ispC gene in its native genomic context in Escherichia coli. We created a full saturation library of 33 sites proximal to the ligand binding pocket and challenged this library with the antimalarial drug fosmidomycin, which targets the ispC gene product, DXR.

Multiplex navigation of global regulatory networks (MINR) in yeast for improved ethanol tolerance and production.

Multiplex navigation of global regulatory networks (MINR) is an approach for combinatorially reprogramming gene expression to manipulate complex phenotypes. We designed, constructed, and mapped MINR libraries containing 43,020 specific mutations in 25 regulatory genes expected to perturb the yeast regulatory network. We selected growth competition experiments for library mutants conferring increased ethanol and/or glucose tolerance.

Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production.

Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in Escherichia coli for improved production of the platform chemical 3-hydroxypropionic acid (3HP).

Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering.

Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli.

CRISPR EnAbled Trackable genome Engineering for isopropanol production in Escherichia coli.

Isopropanol is an important target molecule for sustainable production of fuels and chemicals. Increases in DNA synthesis and synthetic biology capabilities have resulted in the development of a range of new strategies for the more rapid design, construction, and testing of production strains. Here, we report on the use of such capabilities to construct and test 903 different variants of the isopropanol production pathway in Escherichia coli.

Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.

Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale.

Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli.

Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in Escherichia coli.

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E.

The emergence of commodity-scale genetic manipulation.

Since the 1970s technological advancements in the fields of synthetic biology and metabolic engineering have led to a dramatic reduction in both time and cost required for generating genomic mutations in a variety of organisms. The union of genomic editing machinery, DNA inkjet printers, and bioinformatics algorithms allows engineers to design a library of thousands of unique oligos as well as build and test these designs on a ∼2 months time-scale and at a cost of roughly ∼0.3 cents per base pair.

Multiplexed tracking of combinatorial genomic mutations in engineered cell populations.

Multiplexed genome engineering approaches can be used to generate targeted genetic diversity in cell populations on laboratory timescales, but methods to track mutations and link them to phenotypes have been lacking. We present an approach for tracking combinatorial engineered libraries (TRACE) through the simultaneous mapping of millions of combinatorially engineered genomes at single-cell resolution. Distal genomic sites are assembled into individual DNA constructs that are compatible with next-generation sequencing strategies.

Codon compression algorithms for saturation mutagenesis.

Saturation mutagenesis is employed in protein engineering and genome-editing efforts to generate libraries that span amino acid design space. Traditionally, this is accomplished by using degenerate/compressed codons such as NNK (N = A/C/G/T, K = G/T), which covers all amino acids and one stop codon. These solutions suffer from two types of redundancy: (a) different codons for the same amino acid lead to bias, and (b) wild type amino acid is included within the library.

Modularity of select riboswitch expression platforms enables facile engineering of novel genetic regulatory devices.

RNA-based biosensors and regulatory devices have received significant attention for their potential in a broad array of synthetic biology applications. One of the primary difficulties in engineering these molecules is the lack of facile methods to link sensory modules, or aptamers, to readout domains. Such efforts typically require extensive screening or selection of sequences that facilitate interdomain communication.

Single-molecule studies of the lysine riboswitch reveal effector-dependent conformational dynamics of the aptamer domain.

The lysine riboswitch is a cis-acting RNA genetic regulatory element found in the leader sequence of bacterial mRNAs coding for proteins related to biosynthesis or transport of lysine. Structural analysis of the lysine-binding aptamer domain of this RNA has revealed that it completely encapsulates the ligand and therefore must undergo a structural opening/closing upon interaction with lysine. In this work, single-molecule fluorescence resonance energy transfer (FRET) methods are used to monitor these ligand-induced structural transitions that are central to lysine riboswitch function.

Insights into the regulatory landscape of the lysine riboswitch.

A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors, these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main-chain atoms of the amino acid.

Riboswitches: structures and mechanisms.

A critical feature of the hypothesized RNA world would have been the ability to control chemical processes in response to environmental cues. Riboswitches present themselves as viable candidates for a sophisticated mechanism of regulatory control in RNA-based life. These regulatory elements in the modern world are most commonly found in the 5'-untranslated regions of bacterial mRNAs, directly interacting with metabolites as a means of regulating expression of the coding region via a secondary structural switch.

Discrimination between closely related cellular metabolites by the SAM-I riboswitch.

The SAM-I riboswitch is a cis-acting element of genetic control found in bacterial mRNAs that specifically binds S-adenosylmethionine (SAM). We previously determined the 2.9-A X-ray crystal structure of the effector-binding domain of this RNA element, revealing details of RNA-ligand recognition.

A switch in time: detailing the life of a riboswitch.

Riboswitches are non-protein coding RNA elements typically found in the 5' untranslated region (5'-UTR) of mRNAs that utilize metabolite binding to control expression of their own transcript. The RNA-ligand interaction causes conformational changes in the RNA that direct the cotranscriptional folding of a downstream secondary structural switch that interfaces with the expression machinery. This review describes the structural themes common to the different RNA-metabolite complexes studied to date and conclusions that can be made regarding how these RNAs efficiently couple metabolite binding to gene regulation.

Determining structures of RNA aptamers and riboswitches by X-ray crystallography.

Structural biology plays a central role in gaining a full understanding of the myriad roles of RNA in biology. In recent years, innovative approaches in RNA purification and crystallographic methods have lead to the visualization of an increasing number of unique structures, providing new insights into its function at the atomic level. This article presents general protocols which have streamlined the process of obtaining a homogeneous sample of properly folded and active RNA in high concentrations that crystallizes well in the presence of a suitable heavy-atom for phasing.

Strategies in RNA crystallography.

A number of RNAs ranging from small helices to large megadalton ribonucleoprotein complexes have been solved to atomic resolution using X-ray crystallography. As with proteins, RNA crystallography involves a number of screening trials in which the concentration of macromolecule, precipitant, salt, and temperature are varied, an approach known as searching "condition space." In contrast to proteins, the nature of base pairing in nucleic acids creates predictable secondary structure that facilitates the rational design of RNA variants, allowing "sequence space" to be screened in parallel.

Crystal structure of the lysine riboswitch regulatory mRNA element.

Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.