Enhancement of Free Fatty Acids Production in Rhodotorula toruloides Using the CRISPR/Cas9-Based Base Editor

The CRISPR/Cas-based cytidine base editor system achieved single-gene disruption efficiencies of up to 90%.

• A gene was disrupted by introducing a premature stop codon through a C to T mutation.
• The system successfully disrupted four genes in parallel with a 5% efficiency.
• An inducible Cre-loxP system was integrated, achieving over 70% selection marker recycling efficiency.
• The CBE system was used to disrupt four genes related to lipid metabolism.
• The engineered strain produced 512.3 mg/L of free fatty acids.

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