Protocol to study sufficiency of cis-regulatory elements in mouse embryonic stem cells using a CRISPR-mediated knockin approach

Abstract

Summary: 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). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Genetics
• Molecular Biology
• Gene Expression
• CRISPR
• Stem Cells