Regulation of Microbial Metabolic Rates Using CRISPR Interference With Expanded PAM Sequences

Area of Science:

• Molecular Biology
• Microbiology
• Synthetic Biology

Background:

• CRISPR interference (CRISPRi) utilizes deactivated Cas9 (dCas9) and guide RNA (crRNA) to bind target DNA, repressing transcription without cleavage.
• The protospacer adjacent motif (PAM) sequence requirement for dCas9 binding can limit CRISPRi applications.
• Developing controllable CRISPRi systems in bacteria is crucial for understanding gene regulation and synthetic biology.

Purpose of the Study:

• To construct and evaluate a chromosomally integrated CRISPRi system in Escherichia coli.
• To investigate the efficacy of CRISPRi in repressing target gene expression by targeting transcriptional initiation and elongation.
• To explore the impact of modified PAM sequences on CRISPRi efficiency and expand its targeting range.

Main Methods:

• A chromosomally integrated dCas9 expression system in E. coli was established under L-arabinose induction.
• Plasmids encoding crRNAs targeting the gal promoter and galETK genes were introduced into dCas9-expressing E. coli.
• Cellular growth and galactose metabolic rates were measured, alongside RT-qPCR analysis of target gene transcription.

Main Results:

• CRISPRi targeting transcriptional initiation of the gal operon fully inhibited cell growth and galactose metabolism.
• Targeting transcriptional elongation partially retarded growth and metabolism.
• RT-qPCR confirmed transcriptional repression by CRISPRi, even with modified PAM sequences, demonstrating expanded targeting capabilities.

Conclusions:

• CRISPRi is an effective tool for controlling bacterial metabolic rates and cell growth by targeting specific genes or regulatory regions.
• The observed loose PAM sequence dependence in CRISPRi broadens the scope of its potential DNA targets.
• This engineered CRISPRi system offers a versatile platform for gene regulation in E. coli and other microorganisms.