Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Area of Science:

• Genomics
• Molecular Biology
• Developmental Biology

Background:

• Enhancers regulate tissue-specific gene expression and cell identity.
• Enhancer-gene matching is challenging due to their distal and variable locations.
• CRISPR/Cas9 technology offers precise genome editing for functional studies.

Purpose of the Study:

• To present a protocol for CRISPR/Cas9-mediated monoallelic enhancer deletion in mouse embryonic stem cells.
• To enable the study of cis-regulation of gene expression by transcriptional enhancers.
• To overcome challenges in enhancer-gene association and functional validation.

Main Methods:

• CRISPR/Cas9 genome editing was employed for targeted deletion of enhancer regions.
• The protocol was optimized for F1 hybrid mouse embryonic stem cells (Mus musculus(129) x Mus castaneus).
• Monoallelic deletion facilitated analysis of enhancer function without lethality, aided by single nucleotide polymorphisms (SNPs) for screening.

Main Results:

• A reliable protocol for CRISPR/Cas9 mediated monoallelic enhancer deletion in mouse ES cells was established.
• The method allows for efficient screening and expression analysis due to inherent genetic variation (SNPs).
• This approach enables direct functional assessment of enhancer activity in endogenous gene regulation.

Conclusions:

• CRISPR/Cas9 mediated monoallelic deletion is a powerful tool for studying enhancer function and cis-regulation.
• This protocol provides a robust method for investigating gene expression control and cell identity.
• The use of F1 hybrid cells with SNPs simplifies the analysis of enhancer effects on gene transcription.