Effective Blocking of Microbial Transcriptional Initiation by dCas9-NG-Mediated CRISPR Interference

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.