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tCRISPRi: tunable and reversible, one-step control of gene expression

  1. Author:
    Li, Xintian
    Jun, Yonggun
    Erickstad, Michael J.
    Brown, Steven D.
    Parks, Adam
    Court, Donald
    Jun, Suckjoon

  2. Author Address

    Univ Calif San Diego, Sect Mol Biol, Div Biol Sci, La Jolla, CA 92093 USA.Univ Calif San Diego, Dept Phys, La Jolla, CA 92093 USA.NCI, RNA Biol Lab, Ctr Canc Res, Ft Detrick, MD 21702 USA.Leidos Biomed Res Inc, Basic Sci Program, GRCBL Mol Control & Genet Sect, Frederick Natl Lab Canc Res, Frederick, MD 21702 USA.Natl Cent Univ, Dept Phys, Taoyuan, Taiwan.
    1. Year: 2016
    2. Date: Dec 20
  2. Journal: SCIENTIFIC REPORTS
  3. NATURE PUBLISHING GROUP,
    1. 6
    2. Pages: 39076
  5. Type of Article: Article
  6. Article Number: ARTN 39076
  7. ISSN: 2045-2322
  1. 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 P-BAD to 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.

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External Sources

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  2. DOI: 10.1038/srep39076
  3. View record in Web of Science®
  4. WOS: 000390062800001

Library Notes

  1. Fiscal Year: FY2016-2017
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