RESUMEN
Here, we present a generalized method of guide RNA "tuning" that enables Cas9 to discriminate between two target sites that differ by a single-nucleotide polymorphism. We employ our methodology to generate an in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.
Asunto(s)
Sistemas CRISPR-Cas , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Genómica/métodos , Mutación , ARN Guía de Kinetoplastida , Animales , Antibióticos Antituberculosos/farmacología , Escherichia coli/metabolismo , Genoma Bacteriano , Ratones , Rifampin/farmacologíaRESUMEN
Several programmable transcription factors exist based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems, and we assess the role of cooperativity in maximizing gene expression.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Drosophila melanogaster/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Drosophila melanogaster/genética , Genes prv , Ingeniería Genética , Humanos , Ratones , Factores de Transcripción/genéticaRESUMEN
We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.
Asunto(s)
Sistemas CRISPR-Cas/genética , ARN Guía de Kinetoplastida/análisis , Sitios de Unión , Proteínas Asociadas a CRISPR/genética , Citometría de Flujo , Colorantes Fluorescentes/análisis , Eliminación de Gen , Genes Reporteros , Ingeniería Genética/métodos , Vectores Genéticos , Genoma , Células HEK293 , Humanos , Microscopía Fluorescente , Mutagénesis , Mutación , Edición de ARN , Transcripción GenéticaRESUMEN
The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).
Asunto(s)
Endonucleasas , Técnicas Genéticas , ARN Guía de Kinetoplastida , Activación Transcripcional , Diferenciación Celular/genética , Endonucleasas/genética , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas , Neuronas/citología , Staphylococcus aureusRESUMEN
Streptococcus mutans is a commensal member of the healthy plaque biofilm and the primary causative agent of dental caries. The present study is an investigation of SloR, a 25-kDa metalloregulatory protein that modulates genes responsible for S. mutans-induced cariogenesis. Previous studies of SloR homologues in other bacterial pathogens have identified three domains critical to repressor functionality: an N-terminal DNA-binding domain, a central dimerization domain, and a C-terminal FeoA (previously SH3-like) domain. We used site-directed mutagenesis to identify critical amino acid residues within each of these domains of the SloR protein. Select residues were targeted for mutagenesis, and nonconservative amino acid substitutions were introduced by overlap extension PCR. Furthermore, three C-terminally truncated SloR variants were generated using conventional PCR. The repressor functionality and DNA-binding ability of each variant was assessed using CAT reporter gene assays, real-time semiquantitative reverse transcriptase (qRT)-PCR, and electrophoretic mobility shift assays. We identified 12 residues within SloR that cause significant derepression of sloABC promoter activity (P < 0.05) compared to the results for wild-type SloR. Derepression was particularly noteworthy in metal ion-binding site 1 mutants, consistent with the site's importance in gene repression by SloR. In addition, a hyperactive SloR(E169A/Q170A) mutant was identified as having significantly heightened repression of sloABC promoter activity, and experiments with C-terminal deletion mutants support involvement of the FeoA domain in SloR-mediated gene repression. Given these results, we describe the functional domains of the S. mutans SloR protein and propose that the hyperactive mutant could serve as a target for rational drug design aimed at repressing SloR-mediated virulence gene expression.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Manganeso/metabolismo , Streptococcus mutans/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dicroismo Circular , ADN Bacteriano , Ensayo de Cambio de Movilidad Electroforética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Streptococcus mutans/genética , Streptococcus mutans/patogenicidad , Relación Estructura-Actividad , Virulencia/genéticaRESUMEN
Recoding--the repurposing of genetic codons--is a powerful strategy for enhancing genomes with functions not commonly found in nature. Here, we report computational design, synthesis, and progress toward assembly of a 3.97-megabase, 57-codon Escherichia coli genome in which all 62,214 instances of seven codons were replaced with synonymous alternatives across all protein-coding genes. We have validated 63% of recoded genes by individually testing 55 segments of 50 kilobases each. We observed that 91% of tested essential genes retained functionality with limited fitness effect. We demonstrate identification and correction of lethal design exceptions, only 13 of which were found in 2229 genes. This work underscores the feasibility of rewriting genomes and establishes a framework for large-scale design, assembly, troubleshooting, and phenotypic analysis of synthetic organisms.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genes Sintéticos , Código Genético/fisiología , Genoma Bacteriano , Genes Esenciales , Genes Letales , Código Genético/genética , Ingeniería Genética , Fenotipo , Biosíntesis de Proteínas/genéticaRESUMEN
RNA interference (RNAi) is a powerful tool for functional genomics with the capacity to comprehensively analyze host-pathogen interactions. High-throughput RNAi screening is used to systematically perturb cellular pathways and discover therapeutic targets, but the method can be tedious and requires extensive capital equipment and expensive reagents. To aid in the development of an inexpensive miniaturized RNAi screening platform, we have developed a two part microfluidic system for patterning and screening gene targets on-chip to examine cellular pathways involved in virus entry and infection. First, a multilayer polydimethylsiloxane (PDMS)-based spotting device was used to array siRNA molecules into 96 microwells targeting markers of endocytosis, along with siRNA controls. By using a PDMS-based spotting device, we remove the need for a microarray printer necessary to perform previously described small scale (e.g. cellular microarrays) and microchip-based RNAi screening, while still minimizing reagent usage tenfold compared to conventional screening. Second, the siRNA spotted array was transferred to a reversibly sealed PDMS-based screening platform containing microchannels designed to enable efficient cell loading and transfection of mammalian cells while preventing cross-contamination between experimental conditions. Validation of the screening platform was examined using Vesicular stomatitis virus and emerging pathogen Rift Valley fever virus, which demonstrated virus entry pathways of clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively. The techniques here are adaptable to other well-characterized infection pathways with a potential for large scale screening in high containment biosafety laboratories.