Development of a multi-locus CRISPR gene drive system in budding yeast

Area of Science:

• Biotechnology
• Molecular Biology
• Synthetic Biology

Background:

• CRISPR/Cas gene editing has revolutionized biomedical research.
• Gene drives, a CRISPR/Cas arrangement, can rapidly spread genetic elements through populations.
• Current gene drive systems face challenges in biosafety, containment, and control.

Purpose of the Study:

• To develop an advanced, multi-locus gene drive system using budding yeast.
• To create a minimal system requiring only Cas9 and three guide RNAs for propagating multiple gene drives.
• To demonstrate the system's utility for targeted allele replacement and suppression of DNA repair pathways.

Main Methods:

• Utilized budding yeast (Saccharomyces cerevisiae) as a model organism.
• Engineered a minimal CRISPR/Cas9 system with three guide RNAs to drive three genetic elements.
• Demonstrated targeted allele replacement of native genes and modification of DNA Ligase IV to suppress NHEJ repair.

Main Results:

• Successfully developed and demonstrated a functional artificial multi-locus gene drive system in yeast.
• Showcased the system's capability for precise gene editing, including allele replacement.
• Validated the suppression of non-homologous end joining (NHEJ) repair pathways by targeting DNA Ligase IV.

Conclusions:

• Multi-locus gene drives offer expanded capabilities for complex genetic manipulations.
• This system provides enhanced control, redundancy, and resistance management for gene drive applications.
• The developed system advances the design and implementation of safer and more effective gene drives.