Journal
SCIENTIFIC REPORTS
Volume 6, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/srep39076
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Funding
- Paul G. Allen Foundation
- Pew Charitable Trusts
- National Science Foundation CAREER Award
- NIH [R01 GM118565-01]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1253843] Funding Source: National Science Foundation
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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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