Abstract
Clustered regularly interspersed short palindromic repeats (CRISPR) interference (CRISPRi) is a powerful technology for sequence-specifically repressing gene expression in bacterial cells. CRISPRi requires only a single protein and a custom-designed guide RNA for specific gene targeting. In Escherichia coli, CRISPRi repression efficiency is high (~300-fold), and there are no observable off-target effects. The method can be scaled up as a general strategy for the repression of many genes simultaneously using multiple designed guide RNAs. Here we provide a protocol for efficient guide RNA design, cloning, and assay of the CRISPRi system in E. coli. In principle, this protocol can be used to construct CRISPRi systems for gene repression in other species of bacteria.
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Acknowledgements
We thank the Lei Qi lab, Carol Gross lab, and Wendell Lim lab for their support. J.S.H. acknowledges the support from Biophysics Graduate Program at UCSF. Spencer Wong acknowledges the support from Summer Research Training Program (SRTP) at UCSF. This work was supported by NIH P50 (grant GM081879, L.S.Q.), NIH Director’s Early Independence Award (grant OD017887, L.S.Q.), and a Ruth L. Kirschstein National Research Service Award (F32GM108222-01, J.M.P.).
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Hawkins, J.S., Wong, S., Peters, J.M., Almeida, R., Qi, L.S. (2015). Targeted Transcriptional Repression in Bacteria Using CRISPR Interference (CRISPRi). In: Lundgren, M., Charpentier, E., Fineran, P. (eds) CRISPR. Methods in Molecular Biology, vol 1311. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2687-9_23
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DOI: https://doi.org/10.1007/978-1-4939-2687-9_23
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2686-2
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