RESUMEN
Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health1. Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells2, we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle to deliver a base editor and modify Escherichia coli colonizing the mouse gut. Editing of a ß-lactamase gene in a model E. coli strain resulted in a median editing efficiency of 93% of the target bacterial population with a single dose. Edited bacteria were stably maintained in the mouse gut for at least 42 days following treatment. This was achieved using a non-replicative DNA vector, preventing maintenance and dissemination of the payload. We then leveraged this approach to edit several genes of therapeutic relevance in E. coli and Klebsiella pneumoniae strains in vitro and demonstrate in situ editing of a gene involved in the production of curli in a pathogenic E. coli strain. Our work demonstrates the feasibility of modifying bacteria directly in the gut, offering a new avenue to investigate the function of bacterial genes and opening the door to the design of new microbiome-targeted therapies.
Asunto(s)
Escherichia coli , Microbioma Gastrointestinal , Edición Génica , Klebsiella pneumoniae , beta-Lactamasas , Animales , Ratones , Escherichia coli/genética , Edición Génica/métodos , Klebsiella pneumoniae/genética , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Femenino , Bacteriófagos/genética , Sistemas CRISPR-Cas/genética , Masculino , Tracto Gastrointestinal/microbiología , Genes Bacterianos/genética , Vectores Genéticos/genéticaRESUMEN
Phage-derived integrases can catalyze irreversible, site-specific integration of transgenic payloads into a chromosomal locus, resulting in mammalian cells that stably express transgenes or circuits of interest. Previous studies have demonstrated high-efficiency integration by the Bxb1 integrase in mammalian cells. Here, we show that a point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus in Chinese hamster ovary cells, resulting in the highest integration efficiency reported with a site-specific integrase in mammalian cells. Bxb1-GA point mutant sites do not cross-react with Bxb1 wild-type sites, enabling their use in applications that require orthogonal pairs of target sites. In comparison, we test the efficiency and orthogonality of ÏC31 and Wß integrases, and show that Wß has an integration efficiency between those of Bxb1-GA and wild-type Bxb1. Our data present a toolbox of integrases for inserting payloads such as gene circuits or therapeutic transgenes into mammalian cell lines.
Asunto(s)
Integrasas/metabolismo , Animales , Células CHO , Cricetulus , Citometría de Flujo , Genética , Genómica/métodos , Integrasas/genética , Mutación Puntual/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras GenéticasRESUMEN
Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines.
Asunto(s)
Ingeniería Celular/métodos , Redes Reguladoras de Genes , Animales , Línea Celular , Clonación Molecular , Biblioteca de Genes , HumanosRESUMEN
Antibiotics target conserved bacterial cellular pathways or growth functions and therefore cannot selectively kill specific members of a complex microbial population. Here, we develop programmable, sequence-specific antimicrobials using the RNA-guided nuclease Cas9 (refs.1,2) delivered by a bacteriophage. We show that Cas9, reprogrammed to target virulence genes, kills virulent, but not avirulent, Staphylococcus aureus. Reprogramming the nuclease to target antibiotic resistance genes destroys staphylococcal plasmids that harbor antibiotic resistance genes and immunizes avirulent staphylococci to prevent the spread of plasmid-borne resistance genes. We also show that CRISPR-Cas9 antimicrobials function in vivo to kill S. aureus in a mouse skin colonization model. This technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner.
Asunto(s)
Antiinfecciosos/administración & dosificación , Sistemas CRISPR-Cas , Farmacorresistencia Microbiana/genética , Animales , Antiinfecciosos/síntesis química , Bacteriófagos , Secuencia de Bases , Ratones , Plásmidos/genética , Staphylococcus aureus/efectos de los fármacos , Virulencia/efectos de los fármacos , Virulencia/genéticaRESUMEN
We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.
Asunto(s)
Redes Reguladoras de Genes , Ingeniería Genética/métodos , Células HEK293 , HumanosRESUMEN
Phenotypic robustness is a highly sought after goal for synthetic biology. There are many well-studied examples of robust systems in biology, and for the advancement of synthetic biology, particularly in performance-critical applications, fundamental understanding of how robustness is both achieved and maintained is very important. A synthetic circuit may fail to behave as expected for a multitude of reasons, and since many of these failures are difficult to predict a priori, a better understanding of a circuit's behavior as well as its possible failures are needed. In this chapter, we outline work that has been done in developing design principles for robust synthetic circuits, as well as sharing our experiences designing and constructing gene circuits.