Quantitative and modularized CRISPR/dCas9-dCpf1 dual function system in Saccharomyces cerevisiae

Area of Science:

• Synthetic Biology
• Molecular Biology
• Metabolic Engineering

Background:

• CRISPR/dCas9 and CRISPR/dCpf1 systems offer potential for yeast metabolic pathway modulation but have limitations.
• A compensatory approach is needed to overcome individual system deficiencies.

Purpose of the Study:

• To construct and validate a dual functional CRISPR activation/inhibition (CRISPRa/i) system based on Sp-dCas9 and Fn-dCpf1.
• To achieve precise quantitative control over yeast gene expression for metabolic engineering.

Main Methods:

• Validated orthogonality and quantitative targeting of yeast promoters using various effector proteins and RNA scaffolds.
• Investigated CRISPR/dCas9 and CRISPR/dCpf1 systems for gene regulation rates and crRNA efficiency.
• Developed an orthogonal CRISPR/dCas9-dCpf1 inhibition system for simultaneous dual gene regulation.

Main Results:

• CRISPR/dCas9 system achieved 81.9% suppression to 627% activation.
• CRISPR/dCpf1 system demonstrated up to 530% transcriptional inhibition.
• Orthogonal system simultaneously regulated mCherry (54.6%) and eGFP (62.4%) without crosstalk.

Conclusions:

• Established an engineered yeast cell factory for β-carotene production using the bifunctional CRISPR/dCas9-dCpf1 system.
• The system enables targeted modulation of heterologous and endogenous metabolic pathways in Saccharomyces cerevisiae.
• Demonstrated superior quantitative effectiveness and expandability for simultaneous CRISPRa/i network control in yeast biotechnology.