Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated...

Area of Science:

• Genomics and Molecular Biology
• Gene Regulation and Function Analysis
• CRISPR-Cas9 Technology Applications

Background:

• Genome-wide association studies (GWAS) identify numerous disease- and trait-associated variants.
• These variants often reside in regulatory DNA regions, but their precise functions remain largely uncharacterized.
• High-resolution methods are needed to dissect the functional roles of these regulatory elements.

Purpose of the Study:

• To develop and demonstrate a high-throughput, high-resolution methodology for interrogating trait-associated regulatory DNA.
• To identify minimal functional sequences within large genomic regions linked to specific traits.
• To apply this method to the HBS1L-MYB intergenic region associated with red blood cell traits.

Main Methods:

• Development of a computational tool for creating saturating-mutagenesis libraries using single or multiple designer nucleases.
• Incorporation of variant information into library design for precise interrogation.
• Application of the methodology to the HBS1L-MYB locus and analysis of genomic copy number for off-target effects.

Main Results:

• Successful application of the high-throughput, saturating mutagenesis approach to map regulatory elements.
• Identification of putative regulatory elements controlling MYB expression in the HBS1L-MYB region.
• Demonstration of the importance of off-target analysis, revealing potential false-positive regions due to copy number variations.

Conclusions:

• The developed methodology enables high-throughput and high-resolution functional mapping of regulatory DNA.
• This approach is widely applicable for dissecting complex disease- and trait-associated genomic regions.
• Precise identification of minimal functional sequences within regulatory loci is achievable, advancing our understanding of gene regulation.