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Browsing Rice Research Data by Subject "CRISPRa"
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Item Activating natural product synthesis using CRISPR interference and activation systems in Streptomyces(Rice University, 2021-10-27) Ameruoso, Andrea; Department of BiosciencesThe rise of antibiotic-resistant bacteria represents a major threat to global health, creating an urgent need to discover new antibiotics. Natural products derived from the genus Streptomyces represent a rich and diverse repertoire of chemical molecules from which new antibiotics are likely to be found. However, a major challenge is that the biosynthetic gene clusters (BGCs) responsible for natural product synthesis are often poorly expressed under laboratory culturing conditions, thus preventing isolation and screening of novel chemicals. To address this, we describe a novel approach to activate silent BGCs through rewiring endogenous regulation using synthetic gene regulators based upon CRISPR-Cas. First, we create CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems that allow for highly programmable and effective gene repression and activation in Streptomyces. We then harness these tools to activate a silent BGC through perturbing its endogenous regulatory network. Together, this work advances the synthetic regulatory toolbox for Streptomyces and facilitates the programmable activation of silent BGCs for novel chemical discovery.Item Rational engineering of a modular bacterial CRISPR-Cas activation platform with expanded target range(Rice University, 2020) Chappell, James; Department of BiosciencesCRISPR-Cas activator (CRISPRa) systems that selectively turn on transcription of a target gene are a potentially transformative technology for programming cellular function. While in eukaryotes versatile CRISPRa systems exist, in bacteria these systems suffer from a limited ability to activate different genes due to strict distance-dependent requirements of functional target binding sites, and require greater customization to optimize performance in different genetic and cellular contexts. To address this, we apply a rational protein engineering approach to create a new CRISPRa platform that is highly modular to allow for easy customization and has increased targeting flexibility through harnessing engineered Cas proteins. We first demonstrate that transcription activation domains can be recruited by CRISPR-Cas through noncovalent protein-protein interactions, which allows each component to be encoded on separate and easily interchangeable plasmid elements. We then exploit this modularity to rapidly screen a library of different activation domains, creating new systems with distinct regulatory properties. Furthermore, we demonstrate that by harnessing a library of circularly permuted Cas proteins, we can create CRISPRa systems that have different target binding site requirements, which together, allow for expanded target range.