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).

Automated summary

In this study, CRISPR/Cas9 was employed to generate large deletions in the coding region of a gene of interest, producing a hemizygous cell line. Precise in-frame deletions were then engineered within the intact wild type allele, enabling the study of multi-domain proteins. The optimized protocol allowed for efficient screening of effective sgRNA pairs and the engineering of either in-frame deletions or frameshift mutations in mouse embryonic stem cells.