title: tCRISPRi: tunable and reversible, one-step control of gene expression.
abstract: The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, "tCRISPRi", for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBADto quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.
authors.affiliation: Section of Molecular Biology, Division of Biological Sciences and Department of Physics, UC San Diego, La Jolla, CA 92093, USA.
authors.forename: Xin-Tian
authors.initials: XT
authors.lastname: Li
authors.affiliation: Section of Molecular Biology, Division of Biological Sciences and Department of Physics, UC San Diego, La Jolla, CA 92093, USA.
authors.forename: Yonggun
authors.initials: Y
authors.lastname: Jun
authors.affiliation: Section of Molecular Biology, Division of Biological Sciences and Department of Physics, UC San Diego, La Jolla, CA 92093, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Erickstad
authors.affiliation: Section of Molecular Biology, Division of Biological Sciences and Department of Physics, UC San Diego, La Jolla, CA 92093, USA.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Brown
authors.affiliation: RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Parks
authors.affiliation: RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Court
authors.affiliation: Section of Molecular Biology, Division of Biological Sciences and Department of Physics, UC San Diego, La Jolla, CA 92093, USA.
authors.forename: Suckjoon
authors.initials: S
authors.lastname: Jun
mesh_terms: D001089:Arabinose / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D005818:Genetic Engineering / Q000379:methods*; D009876:Operon; D011401:Promoter Regions, Genetic
chemical_list: D001426:Bacterial Proteins; D001089:Arabinose