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

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).