Exploiting CRISPR/Cas9 to engineer precise segmental deletions in mouse embryonic stem cells

Abstract

Summary: In this protocol, we use CRISPR/Cas9 to generate large deletions of the entire coding region of a gene of interest, generating a hemizygous cell line. Next, we systematically engineer precise in-frame deletions within the intact wild-type allele, facilitating study of multi-domain proteins. The optimized protocol described here allows us to rapidly screen for effective sgRNA pairs and to engineer either an in-frame deletion or a frameshift mutation in high frequencies in mouse embryonic stem cells.For complete details on the use and execution of this protocol, please refer to Panday et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Genetics
• Molecular Biology
• CRISPR
• Stem Cells