title: Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.
abstract: A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, USA.
authors.forename: Young Je
authors.initials: YJ
authors.lastname: Lee
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, USA.
authors.forename: Allison
authors.initials: A
authors.lastname: Hoynes-O'Connor
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, USA.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Leong
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, USA tsmoon@wustl.edu.
authors.forename: Tae Seok
authors.identifier: http://orcid.org/0000-0001-8373-9051
authors.initials: TS
authors.lastname: Moon
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D016372:RNA, Antisense / Q000138:chemical synthesis / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000037:antagonists & inhibitors* / Q000235:genetics; D013297:Streptococcus pyogenes / Q000235:genetics* / Q000378:metabolism
chemical_list: D016372:RNA, Antisense; D000094704:RNA, Guide, CRISPR-Cas Systems; D049452:Green Fluorescent Proteins
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