Journal of visualized experiments : JoVEApr 15, 2016

Using Gene Editing to Delete One Copy of DNA Control Regions in Mouse Embryonic Stem Cells to Study Their Function

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Mar 11, 2026

Abstract

Homozygous deletion of essential gene regulatory elements may lead to lethality, while monoallelic deletion allows for the study of gene expression regulation.

Enhancers are crucial for controlling cell identity by regulating gene expression in a position and orientation independent manner.
Deleting enhancer regions provides direct evidence of their role in gene transcription.
CRISPR/Cas9 technology can efficiently edit genomes in various model organisms, enabling precise modifications.
Monoallelic deletion of transcriptional enhancers helps avoid complications associated with essential gene deletions.
In F1 mouse embryonic stem cells, enhanced screening and expression analysis can be achieved through the use of single nucleotide polymorphisms.

Simplified

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus(129) x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

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Using Gene Editing to Delete One Copy of DNA Control Regions in Mouse Embryonic Stem Cells to Study Their Function

Abstract

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