Biotechnology and Bioengineering (BB) | Microbial Genetics, Physiology, and Metabolism
Effective Blocking of Microbial Transcriptional Initiation by dCas9-NG-Mediated CRISPR Interference
Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of KoreaCorrespondence to:
Received: August 28, 2020; Revised: September 19, 2020; Accepted: September 20, 2020
J. Microbiol. Biotechnol. 2020; 30(12): 1919-1926
Published December 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence has been constructed by removing the dependence on the last base G of PAM through protein engineering of Cas9. In this study, a dCas9-NG protein was engineered by introducing two active site mutations in Cas9-NG, and its ability to regulate transcription was evaluated in the gal promoter in E. coli. Analysis of cell growth rate, D-galactose consumption rate, and gal transcripts confirmed that dCas9-NG can completely repress the promoter by recognizing DNA targets with PAM of 5'-NGG, NGA, NGC, NGT, and NAG. Our study showed possible PAM sequences for dCas9-NG and provided information on target-specific sgRNA design for regulation of both gene expression and cellular metabolism.
CRISPR interference, PAM sequence, dCas9-NG, gal promoter, D-galactose
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