Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines.

Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species.

Engineering transcriptional regulation of pentose metabolism in Rhodosporidium toruloides for improved conversion of xylose to bioproducts.

Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. To increase the rate of pentose assimilation in R.

Engineering Rhodosporidium toruloides for production of 3-hydroxypropionic acid from lignocellulosic hydrolysate.

Microbial production of valuable bioproducts is a promising route towards green and sustainable manufacturing. The oleaginous yeast, Rhodosporidium toruloides, has emerged as an attractive host for the production of biofuels and bioproducts from lignocellulosic hydrolysates. 3-hydroxypropionic acid (3HP) is an attractive platform molecule that can be used to produce a wide range of commodity chemicals.

Advanced one-pot deconstruction and valorization of lignocellulosic biomass into triacetic acid lactone using Rhodosporidium toruloides.

Background: Rhodosporidium toruloides is capable of co-utilization of complex carbon sources and robust growth from lignocellulosic hydrolysates. This oleaginous yeast is therefore an attractive host for heterologous production of valuable bioproducts at high titers from low-cost, deconstructed biomass in an economically and environmentally sustainable manner. Here we demonstrate this by engineering R.

Bioconversion of cellulose into bisabolene using Ruminococcus flavefaciens and Rhodosporidium toruloides.

In this study, organic acids were demonstrated as a promising carbon source for bisabolene production by the non-conventional yeast, Rhodosporidium toruloides, at microscale with a maximum titre of 1055 ± 7 mg/L. A 125-fold scale-up of the optimal process, enhanced bisabolene titres 2.5-fold to 2606 mg/L.

CRISPR-RNAa: targeted activation of translation using dCas13 fusions to translation initiation factors.

Tools for synthetically controlling gene expression are a cornerstone of genetic engineering. CRISPRi and CRISPRa technologies have been applied extensively for programmable modulation of gene transcription, but there are few such tools for targeted modulation of protein translation rates. Here, we employ CRISPR-Cas13 as a programmable activator of translation.

Potentiating antibiotic efficacy via perturbation of non-essential gene expression.

Proliferation of multidrug-resistant (MDR) bacteria poses a threat to human health, requiring new strategies. Here we propose using fitness neutral gene expression perturbations to potentiate antibiotics. We systematically explored 270 gene knockout-antibiotic combinations in Escherichia coli, identifying 90 synergistic interactions.

Facile accelerated specific therapeutic (FAST) platform develops antisense therapies to counter multidrug-resistant bacteria.

Multidrug-resistant (MDR) bacteria pose a grave concern to global health, which is perpetuated by a lack of new treatments and countermeasure platforms to combat outbreaks or antibiotic resistance. To address this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform that can develop effective peptide nucleic acid (PNA) therapies against MDR bacteria within a week. Our FAST platform uses a bioinformatics toolbox to design sequence-specific PNAs targeting non-traditional pathways/genes of bacteria, then performs in-situ synthesis, validation, and efficacy testing of selected PNAs.

Conversion of poplar biomass into high-energy density tricyclic sesquiterpene jet fuel blendstocks.

Background: In an effort to ensure future energy security, reduce greenhouse gas emissions and create domestic jobs, the US has invested in technologies to develop sustainable biofuels and bioproducts from renewable carbon sources such as lignocellulosic biomass. Bio-derived jet fuel is of particular interest as aviation is less amenable to electrification compared to other modes of transportation and synthetic biology provides the ability to tailor fuel properties to enhance performance. Specific energy and energy density are important properties in determining the attractiveness of potential bio-derived jet fuels.

Multiplexed CRISPR-Cas9-Based Genome Editing of .

Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as and Significant advancements in the past few years have bolstered ' engineering capacity.

Nucleotide and structural label identification in single RNA molecules with quantum tunneling spectroscopy.

Although a number of advances have been made in RNA sequencing and structural characterization, the lack of a method for directly determining the sequence and structure of single RNA molecules has limited our ability to probe heterogeneity in gene expression at the level of single cells. Here we present a method for direct nucleotide identification and structural label mapping of single RNA molecules Quantum Molecular Sequencing (QMSeq). The method combines non-perturbative quantum tunneling spectroscopy to probe the molecular orbitals of ribonucleotides, new experimental biophysical parameters that fingerprint these molecular orbitals, and a machine learning classification algorithm to distinguish between the ribonucleotides.

Design of a De Novo Aggregating Antimicrobial Peptide and a Bacterial Conjugation-Based Delivery System.

Antibacterial resistance necessitates the development of novel treatment methods for infections. Protein aggregates have recently been applied as antimicrobials to disrupt bacterial homeostasis. Past work on protein aggregates has focused on genome mining for aggregation-prone sequences in bacterial genomes rather than on rational design of aggregating antimicrobial peptides.

Multiplexed deactivated CRISPR-Cas9 gene expression perturbations deter bacterial adaptation by inducing negative epistasis.

The ever-increasing threat of multi-drug resistant bacteria, a shrinking antibiotic pipeline, and the innate ability of microorganisms to adapt necessitates long-term strategies to slow the evolution of antibiotic resistance. Here we develop an approach, dubbed Controlled Hindrance of Adaptation of OrganismS or CHAOS, involving induction of epistasis between gene perturbations to deter adaption. We construct a combinatorial library of multiplexed, deactivated CRISPR-Cas9 devices to systematically perturb gene expression in .

CRISPR Gene Perturbations Provide Insights for Improving Bacterial Biofuel Tolerance.

Economically-viable biofuel production is often limited by low levels of microbial tolerance to high biofuel concentrations. Here we demonstrate the first application of deactivated CRISPR perturbations of gene expression to improve biofuel tolerance. We construct a library of 31 unique CRISPR inhibitions and activations of gene expression in and explore their impacts on growth during 10 days of exposure to n-butanol and n-hexane.

High-Throughput Block Optical DNA Sequence Identification.

Optical techniques for molecular diagnostics or DNA sequencing generally rely on small molecule fluorescent labels, which utilize light with a wavelength of several hundred nanometers for detection. Developing a label-free optical DNA sequencing technique will require nanoscale focusing of light, a high-throughput and multiplexed identification method, and a data compression technique to rapidly identify sequences and analyze genomic heterogeneity for big datasets. Such a method should identify characteristic molecular vibrations using optical spectroscopy, especially in the "fingerprinting region" from ≈400-1400 cm .

Transcriptome-Level Signatures in Gene Expression and Gene Expression Variability during Bacterial Adaptive Evolution.

Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation.

Gene Expression Variability Underlies Adaptive Resistance in Phenotypically Heterogeneous Bacterial Populations.

The root cause of the antibiotic resistance crisis is the ability of bacteria to evolve resistance to a multitude of antibiotics and other environmental toxins. The regulation of adaptation is difficult to pinpoint due to extensive phenotypic heterogeneity arising during evolution. Here, we investigate the mechanisms underlying general bacterial adaptation by evolving wild-type Escherichia coli populations to dissimilar chemical toxins.

CRISPR Perturbation of Gene Expression Alters Bacterial Fitness under Stress and Reveals Underlying Epistatic Constraints.

The evolution of antibiotic resistance has engendered an impending global health crisis that necessitates a greater understanding of how resistance emerges. The impact of nongenetic factors and how they influence the evolution of resistance is a largely unexplored area of research. Here we present a novel application of CRISPR-Cas9 technology for investigating how gene expression governs the adaptive pathways available to bacteria during the evolution of resistance.

Surveying the lipogenesis landscape in Yarrowia lipolytica through understanding the function of a Mga2p regulatory protein mutant.

Lipogenic organisms represent great starting points for metabolic engineering of oleochemical production. While previous engineering efforts were able to significantly improve lipid production in Yarrowia lipolytica, the lipogenesis landscape, especially with respect to regulatory elements, has not been fully explored. Through a comparative genomics and transcriptomics approach, we identified and validated a mutant mga2 protein that serves as a regulator of desaturase gene expression and potent lipogenesis factor.

Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function.

Manipulating gene expression using different types of plasmids (centromeric, 2-micron, and autonomously replicating sequence) can expand expression levels and enable a quick characterization of combinations, both of which are highly desired for metabolic engineering applications. However, for the powerful industrial workhorse Yarrowia lipolytica, only a low-copy CEN plasmid is available. In this study, we engineered the CEN plasmid from Y.

Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production.

Economic feasibility of biosynthetic fuel and chemical production hinges upon harnessing metabolism to achieve high titre and yield. Here we report a thorough genotypic and phenotypic optimization of an oleaginous organism to create a strain with significant lipogenesis capability. Specifically, we rewire Yarrowia lipolytica's native metabolism for superior de novo lipogenesis by coupling combinatorial multiplexing of lipogenesis targets with phenotypic induction.

Decaffeination and measurement of caffeine content by addicted Escherichia coli with a refactored N-demethylation operon from Pseudomonas putida CBB5.

The widespread use of caffeine (1,3,7-trimethylxanthine) and other methylxanthines in beverages and pharmaceuticals has led to significant environmental pollution. We have developed a portable caffeine degradation operon by refactoring the alkylxanthine degradation (Alx) gene cluster from Pseudomonas putida CBB5 to function in Escherichia coli. In the process, we discovered that adding a glutathione S-transferase from Janthinobacterium sp.

[Pathomorphological findings in irradiated cattle].

There is a description of the finding in whole-body irradiated young cattle exposed to 60Co in the doses of 3.7 and 2.0 J kg-1 (370 and 200 rad).

[Clinical changes in whole-body irradiated cattle].

Clinical manifestations of an acute radiation sickness in groups of heifers of the age of seven to nine months, irradiated with a multipoint spatial 60Co source with the doses of 3.7, 2.0, and 1.