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

Abstract

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).

Keywords: CRISPR; Genetics; Molecular Biology; Stem Cells.

Graphical abstract

Figure 1

Schematic showing CRISPR/Cas9 design in mES cells (A) Representative mouse gene (gene of interest) containing 14 exons (black rectangles) and representative mouse protein (protein of interest). Exons 1–6 code for domain 1 that binds protein 1 (pink oval). Exon 14 codes for domain 2 that binds protein 2 (blue oval). (B) Schematic showing stepwise domain deletions and corresponding predicted protein structures. (1) Schematic showing both alleles of a mouse gene of interest (gene +/+), with positions of sgRNAs (red arrows) to engineer a hemizygote. (2) Generation of a hemizygote (gene +/–) using sgRNAs. Primer pairs (red half arrows) used to validate the large deletion, and the intact left and right junctions are detected by PCR as shown. Representative gel (right) showing expected PCR products in clones. (3) Deletion of domain 1 (Δdomain1/–) using sgRNA pairs 3+4 (red arrows) to generate an in-frame mutation (top) and a gel image showing the expected PCR outcome using primers shown (red half arrows). The predicted protein structure (Protein of Interest Δdomain 1) is also shown (bottom). (4) Deletion of domain 2 (Δdomain2/–) using sgRNA pairs 5+6 (red arrows) to generate an in-frame mutation (top) and gel image showing the desired deletion outcome using primers shown (red half arrows). The predicted protein structure (Protein of Interest Δdomain 2) is also shown (bottom).