2020-12-172020-12-172020-07-012020Chappell, James (2020): Rational engineering of a modular bacterial CRISPR-Cas activation platform with expanded target range. [Dataset]. 1. Rice University. https://doi.org/10.25611/DG85-7Y09.https://hdl.handle.net/1911/109754Dataset is comprised of 11 excel (*.xlsx) files. Readme text is included.CRISPR-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.1.21 MBapplication/vnd.ms-excelengCC0 1.0 UniversalSynthetic biologygene regulationCRISPRaDatasethttps://doi.org/10.25611/DG85-7Y09