title: Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells.
abstract: 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.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto.
authors.forename: Sakthi D
authors.initials: SD
authors.lastname: Moorthy
authors.affiliation: Department of Cell and Systems Biology, University of Toronto; ja.mitchell@utoronto.ca.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Mitchell
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression / Q000502:physiology; D060005:Genotyping Techniques; D059765:Homologous Recombination / Q000235:genetics*; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000378:metabolism*; D010957:Plasmids; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014162:Transfection
chemical_list: D004247:DNA