ABSTRACT
Programmable nuclease-based genome editing technologies, including the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system, are becoming an essential component of many applications ranging from agriculture to medicine. However, fundamental limitations currently prevent the widespread, safe, and practical use of genome editors, especially for human disease interventions. These limitations include off-target effects, a lack of control over editing activity, suboptimal DNA repair outcomes, insufficient target conversion, and inadequate delivery performance. This perspective focuses on the potential for biological chemistry to address these limitations such that newly developed genome editing technologies can enable the broadest range of potential future applications. Equally important will be the development of these powerful technologies within a relevant ethical framework that emphasizes safety and responsible innovation.
Subject(s)
Biology/methods , CRISPR-Cas Systems/genetics , Gene Editing/methods , Genome , Animals , Biology/ethics , CRISPR-Associated Proteins/metabolism , DNA/genetics , DNA Breaks, Double-Stranded , DNA End-Joining Repair/genetics , Endonucleases/metabolism , Gene Editing/ethics , Gene Transfer Techniques , Humans , RNA, Guide, Kinetoplastida/geneticsABSTRACT
A transposon insertional mutagenesis spore library of the pathogen Bacillus anthracis was screened to identify mutants altered in germination kinetics. One mutant exhibited an accelerated rate of germination in association with disruption of benK. This gene encodes a putative protein with high homology to membrane transporters that facilitate benzoate transport. We hypothesized that BenK may not be only spore associated, but also have a vegetative cell role. A reporter strain with a translational fusion of benK to green fluorescent protein demonstrated that full-length BenK was present in vegetative cells and that a BenK degradation product was present in spores by detecting the reporter using fluorescence and Western blot analysis. A minimum inhibitory concentration assay indicated that vegetative cells of a benK::Kan mutant were more susceptible to the antimicrobial effects of Na-benzoate. The mutant spores germinated to a greater extent within 1 h than the wild type in an in vitro fluorescence assay. The disruption of benK also resulted in spores that were less readily phagocytosed in a macrophage assay. Despite these altered in vitro phenotypes, no apparent effect of the BenK protein on virulence in the intranasal mouse model or the guinea pig competitive assay was observed. This work shows that, although the BenK protein does not impact fitness or virulence in an infection model, it is involved in other aspects of both the spore and vegetative forms of the organism.
Subject(s)
Bacillus anthracis/physiology , Bacterial Proteins/genetics , Phenotype , Spores, Bacterial , Anti-Bacterial Agents/pharmacology , Bacillus anthracis/drug effects , Bacillus anthracis/pathogenicity , Genetic Fitness , Macrophages/immunology , Macrophages/microbiology , Microbial Sensitivity Tests , Mutation , Phagocytosis/genetics , Phagocytosis/immunology , VirulenceABSTRACT
Pathogenesis of Bacillus anthracis is associated with the production of lethal toxin (LT), which activates the murine Nalp1b/Nlrp1b inflammasome and induces caspase-1-dependent pyroptotic death in macrophages and dendritic cells. In this study, we investigated the effect of allelic variation of Nlrp1b on the outcome of LT challenge and infection by B. anthracis spores. Nlrp1b allelic variation did not alter the kinetics or pathology of end-stage disease induced by purified LT, suggesting that, in contrast to previous reports, macrophage lysis does not contribute directly to LT-mediated pathology. However, animals expressing a LT-sensitive allele of Nlrp1b showed an early inflammatory response to LT and increased resistance to infection by B. anthracis. Data presented here support a model whereby LT-mediated activation of Nlrp1b and subsequent lysis of macrophages is not a mechanism used by B. anthracis to promote virulence, but rather a protective host-mediated innate immune response.
Subject(s)
Anthrax/genetics , Anthrax/immunology , Apoptosis Regulatory Proteins/genetics , Genetic Predisposition to Disease , Animals , Antigens, Bacterial/toxicity , Bacillus anthracis/immunology , Bacillus anthracis/pathogenicity , Bacterial Toxins/toxicity , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
Yersinia pestis is a Department of Health and Human Services select agent as defined in federal regulations. Certain attenuated strains of Y. pestis, such as the pgm(-) strain, are exempt from these regulations. Herein we describe a strategy to verify the absence of the pgm locus in Y. pestis strains being considered as candidates for select agent exemption by PCR analysis of virulence-associated genes.
Subject(s)
Pigments, Biological/genetics , Plague/microbiology , Polymerase Chain Reaction/methods , United States Dept. of Health and Human Services/legislation & jurisprudence , Yersinia pestis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA Primers/genetics , Humans , Mutation , Pigments, Biological/metabolism , United States , Virulence Factors/genetics , Virulence Factors/metabolism , Yersinia pestis/metabolismABSTRACT
TenA catalyzes the hydrolysis of 4-amino-5-aminomethyl-2-methylpyrimidine and participates in the salvage of base-degraded thiamin. Here, we describe mutagenesis of the active site of TenA guided by structures of the enzyme complexed to a substrate analog and to the product. Catalytic roles for each of the active site residues are identified and a mechanism for the reaction is described.
