A CRISPR/Cas9-based genome-editing platform enabling efficient and precise gene replacement in Lipomyces starkeyi

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Lipomyces starkeyi is an oleaginous yeast with significant industrial potential.
• Current genome engineering methods for L. starkeyi suffer from low efficiency and require long DNA sequences for targeting.

Purpose of the Study:

• To establish a robust CRISPR/Cas9 genome editing platform for Lipomyces starkeyi.
• To overcome limitations in gene targeting efficiency and homologous region length for L. starkeyi.

Main Methods:

• A codon-optimized Streptococcus pyogenes Cas9 was expressed in L. starkeyi.
• In vitro-transcribed single-guide RNAs (sgRNAs) were directly delivered, bypassing endogenous RNA polymerase III.
• CRISPR/Cas9 activity was validated using a GFP reporter and gene replacement at the LsURA3 locus.

Main Results:

• Cas9-induced mutations led to GFP disruption and fluorescence loss, confirming system activity.
• Precise gene replacement efficiency increased with homology arm length in wild-type strains.
• Suppression of non-homologous end joining (NHEJ) in a Δlslig4 strain enabled 100% accurate gene replacement with short homology arms.

Conclusions:

• A Pol III-independent CRISPR/Cas9 system combined with NHEJ suppression allows for precise genome editing in L. starkeyi.
• This optimized system provides a powerful tool for functional genomics and metabolic engineering in L. starkeyi.