dCas9 regulator to neutralize competition in CRISPRi circuits

Area of Science:

• Synthetic Biology
• Molecular Biology
• Gene Regulation

Background:

• CRISPR interference (CRISPRi) enables simultaneous gene regulation.
• Transcriptional regulation of multiple targets is hindered by competition for dCas9, leading to interdependent sgRNA responses.
• This competition significantly alters repression efficiency when multiple sgRNAs are expressed.

Purpose of the Study:

• To develop a dCas9 concentration regulator to decouple sgRNA-mediated regulatory paths.
• To enable independent transcriptional regulation of multiple genes using CRISPRi.
• To facilitate predictable composition of CRISPRi-based genetic modules for larger synthetic circuits.

Main Methods:

• Implementation of a negative feedback loop to regulate dCas9 concentration.
• Demonstration of the regulator's performance in single-stage and layered CRISPRi genetic circuits.
• Quantification of the decoupling effect on sgRNA dose-response curves.

Main Results:

• The dCas9 regulator maintains approximately constant sgRNA dose-response curves, independent of other sgRNAs.
• Competition effects, causing up to 15-fold changes in circuit I/O response, were eliminated.
• Decoupling of regulatory paths was achieved, enabling concurrent and independent gene regulation.

Conclusions:

• The dCas9 regulator effectively decouples sgRNA-mediated regulatory paths.
• This technology is essential for the predictable design of complex synthetic genetic circuits.
• Enables robust and scalable applications of CRISPRi in synthetic biology.