RESUMO
The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Vetores Genéticos/genéticaRESUMO
The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly-described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.
RESUMO
BACKGROUND: Next-generation sequencing (NGS) is widely used for genome-wide identification and quantification of DNA elements involved in the regulation of gene transcription. Studies that generate multiple high-throughput NGS datasets require data integration methods for two general tasks: 1) generation of genome-wide data tracks representing an aggregate of multiple replicates of the same experiment; and 2) combination of tracks from different experimental types that provide complementary information regarding the location of genomic features such as enhancers. RESULTS: NGS-Integrator is a Java-based command line application, facilitating efficient integration of multiple genome-wide NGS datasets. NGS-Integrator first transforms all input data tracks using the complement of the minimum Bayes' factor so that all values are expressed in the range [0,1] representing the probability of a true signal given the background noise. Then, NGS-Integrator calculates the joint probability for every genomic position to create an integrated track. We provide examples using real NGS data generated in our laboratory and from the mouse ENCODE database. CONCLUSIONS: Our results show that NGS-Integrator is both time- and memory-efficient. Our examples show that NGS-Integrator can integrate information to facilitate downstream analyses that identify functional regulatory domains along the genome.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Software , Animais , Teorema de Bayes , Genoma , Genômica , CamundongosRESUMO
A versatile biomimetic theranostic agent based on magnetic melanin nanoparticles is developed for positron-emission tomography/magnetic resonance/photoacoustic/photothermal multimodal-imaging-guided cancer photothermal therapy and UV and γ-irradiation protection.
Assuntos
Materiais Biocompatíveis/uso terapêutico , Raios gama , Neoplasias/terapia , Fototerapia , Nanomedicina Teranóstica , Raios Ultravioleta , Animais , Materiais Biocompatíveis/química , Linhagem Celular Tumoral , Sobrevivência Celular , Cobre/química , Raios gama/uso terapêutico , Humanos , Imageamento por Ressonância Magnética , Melaninas/química , Camundongos , Camundongos Nus , Nanopartículas/química , Nanopartículas/uso terapêutico , Nanopartículas/toxicidade , Neoplasias/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Transplante HeterólogoRESUMO
Anisotropic gold nanostructures with unique plasmonic properties, specifically the strong absorption of light in the near-infrared region (650-900 nm) due to the excitation of plasmon oscillations, have been widely employed as photothermal conversion agents (PTCAs) for cancer photothermal therapy (PTT). However, the reported PTCAs show suboptimal photothermal conversion efficiency (η), even gold nanocages (η = 63%), which limits their biomedical applications. Herein, we fabricated gold bellflowers (GBFs) with an ultrahigh photothermal conversion efficiency (η = 74%) via a novel liquid/liquid/gas triphasic interface system, using chloroauric acid as a gold source, and o-phenetidine as a reducing agent. The well-defined GBFs with multiple-branched petals show adjustable localized surface plasmon resonance (LSPR) from 760 to 1100 nm by tuning the petal length and circular bottom diameter. Originating from the monophasic and biphasic systems used in the creation of gold nanourchins (GNUs) and gold microspheres (GMPs) respectively, the triphasic interface system successfully produced GBFs. The possible formation mechanisms of GNUs, GMPs, and GBFs in the different systems were also investigated and discussed. We found that the formation mechanism of GNUs and GBFs followed classical crystallization, while the formation of GMPs followed non-classical crystallization.