Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9

Area of Science:

• Genomics
• Molecular Biology
• Gene Regulation

Background:

• Traditional methods for identifying noncoding regulatory elements, like reporter assays, often fail to replicate endogenous cellular conditions.
• Understanding the function of the vast noncoding genome is a significant challenge in biology.

Purpose of the Study:

• To develop and apply CRISPR-Cas9 genetic screens for the systematic identification and characterization of functional enhancers within their native genomic context.
• To investigate the role of identified enhancers in regulating key genes such as p53 (TP53) and ERα (ESR1).

Main Methods:

• Utilized two distinct CRISPR-Cas9 genetic screening strategies targeting transcription factor binding sites within enhancer regions.
• Employed a genomic CRISPR-Cas9 tiling screen for high-resolution mapping of functional domains within enhancers.

Main Results:

• Successfully identified several functional enhancer elements using the developed CRISPR-Cas9 screening approaches.
• Characterized the specific roles of two identified enhancers in the regulation of p53 (TP53) and ERα (ESR1) gene expression.
• Demonstrated the precision of genomic CRISPR-Cas9 tiling screens in delineating functional enhancer sub-regions.

Conclusions:

• CRISPR-Cas9 genetic screens provide a powerful tool for identifying and functionally characterizing noncoding regulatory elements in their endogenous settings.
• This approach overcomes limitations of traditional reporter assays and enhances our ability to study the noncoding genome.
• The findings contribute to a deeper understanding of gene regulation by enhancers and their impact on cellular processes.