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. 2020 Feb 28;11:282. doi: 10.3389/fmicb.2020.00282

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Copyright © 2020 Kim, Kim and Lee.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Differential repression of galK structural gene by dCas9-crRNAs recognizing modified PAM sequences. (A) Precise target recognition and proper PAM sequences are required for the blocking of transcriptional elongation in the galK gene by CRISPRi. The galK-specific crRNA was transcribed from plasmid pHK459. (B) Design of target DNA sequences with modified PAM sequences in galK gene. The modified PAM sequences of NNG (top) and NGN (bottom) were evaluated by growth profiles (C), utilization of D-galactose (D), and comparison of D-galactose consumption rates (E). D-galactose was added at 6 h, and the concentration changes of residual D-galactose between 9 and 12 h were used to calculate D-galactose consumption rates. Arrows indicate when L-arabinose (if needed) and D-galactose were added, and when samples were taken for RNA extraction.