title: Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs.
abstract: CRISPR-based transcription regulators (CRISPR-TRs) have transformed the current synthetic biology landscape by allowing specific activation or repression of any target gene. Here we report a modular and versatile framework enabling rapid implementation of inducible CRISPR-TRs in mammalian cells. This strategy relies on the design of a spacer-blocking hairpin (SBH) structure at the 5' end of the single guide RNA (sgRNA), which abrogates the function of CRISPR-transcriptional activators. By replacing the SBH loop with ligand-controlled RNA-cleaving units, we demonstrate conditional activation of quiescent sgRNAs programmed to respond to genetically encoded or externally delivered triggers. We use this system to couple multiple synthetic and endogenous target genes with specific inducers, and assemble gene regulatory modules demonstrating parallel and orthogonal transcriptional programs. We anticipate that this 'plug and play' approach will be a valuable addition to the synthetic biology toolkit, facilitating the understanding of natural gene circuits and the design of cell-based therapeutic strategies.
authors.affiliation: Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Quentin R V
authors.initials: QR
authors.lastname: Ferry
authors.affiliation: Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Radostina
authors.initials: R
authors.lastname: Lyutova
authors.affiliation: Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Tudor A
authors.initials: TA
authors.lastname: Fulga
mesh_terms: D000465:Algorithms; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D005434:Flow Cytometry; D053263:Gene Regulatory Networks / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection
chemical_list: D008024:Ligands; D000094704:RNA, Guide, CRISPR-Cas Systems