Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genetic Engineering

Background:

• Large synthetic genetic circuits require numerous regulators for complex functions.
• Deactivated Cas9 (dCas9) fused with small guide RNA (sgRNA) is a programmable repressor, but its toxicity limits expression levels in bacteria.
• High sgRNA concentrations sequester dCas9, reducing its effectiveness.

Purpose of the Study:

• To engineer a non-toxic dCas9 variant for increased expression and utility in synthetic genetic circuits.
• To overcome the toxicity limitations of dCas9 in Escherichia coli.
• To assess the performance of the engineered dCas9 variant in large-scale genetic regulation.

Main Methods:

• Constructed a non-toxic dCas9 variant (dCas9*_PhlF) by mutating the PAM-binding site and fusing it to the PhlF repressor.
• Characterized dCas9*_PhlF toxicity and expression levels in Escherichia coli.
• Evaluated the repression capabilities and orthogonality of multiple sgRNA-promoter pairs using the engineered dCas9 variant.

Main Results:

• The dCas9*_PhlF variant exhibited significantly reduced toxicity, allowing up to 9600 molecules per cell compared to 530 for dCas9.
• PhlF multimerization increased cooperativity from n = 0.9 to 1.6.
• Co-expression of more than 15 sgRNAs led to a decline in repression, with a dynamic range below 10-fold.

Conclusions:

• Introduced a non-toxic dCas9 variant crucial for metabolic engineering and synthetic biology applications.
• Demonstrated that while dCas9 toxicity is mitigated, the number of simultaneously usable regulators is limited by shared cellular resources.