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Updated: Sep 6, 2025
Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells
Published on: April 2, 2016
Protocol to study sufficiency of cis-regulatory elements in mouse embryonic stem cells using a CRISPR-mediated
Tomas Pachano1, Alvaro Rada-Iglesias2
1Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/Universidad de Cantabria, Albert Einstein 22, 39011 Santander, Spain; Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Campus-Vienna-Biocenter 1, 1030 Vienna, Austria.
Abstract:
cis-regulatory elements (CREs) orchestrate the spatiotemporal control of gene expression. The regulatory activity of CREs is typically assessed by reporter assays, in which CREs are studied outside their endogenous context. To circumvent this problem, we developed a CRISPR-Cas9 knockin approach to study CREs in a scar-free genomic context. Here, we describe the design, transfection, and screening protocol to insert CREs in mouse embryonic stem cells. Our strategy can provide important insights into the sufficiency of CREs for gene expression control. For complete details on the use and execution of this protocol, please refer to Pachano et al. (2021).
