High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast

Area of Science:

• Genetics
• Molecular Biology
• Yeast Genetics

Background:

• Understanding genome function requires large genetic variant libraries.
• Current methods for library construction are challenging and inefficient.

Purpose of the Study:

• To introduce a novel Cas9-based approach for generating precise genetic variant libraries in yeast.
• To demonstrate the utility of this method for high-throughput functional genomics studies.

Main Methods:

• Utilized Cas9 nuclease for targeted genetic alterations (deletions, substitutions, insertions) in yeast.
• Developed methods for pooled mutant fitness tracking.
• Constructed tiling deletion and point mutation libraries for SGS1 DNA helicase.
• Created a genome-wide library targeting small open reading frames (smORFs).

Main Results:

• Achieved 80-100% efficiency in generating defined genetic variants.
• Characterized the DNA helicase SGS1 using comprehensive deletion and point mutation libraries.
• Identified essential smORFs crucial for yeast growth under diverse conditions.

Conclusions:

• The Cas9-based strategy enables efficient and precise construction of large genetic variant libraries.
• This approach facilitates high-throughput investigation of fundamental biological questions and gene function.
• The method is valuable for genome-wide screening and characterization of essential genes.