Generation of knockout mouse models of cyclin-dependent kinase inhibitors by engineered nuclease-mediated genome editing

Abstract

Abstract Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer. Mutations in cyclin-dependent kinase inhibitors controlling the G1 phase of the cell cycle are prevalent in various cancers. Mice lacking the tumor suppressors p16Ink4a (Cdkn2a, cyclin-dependent kinase inhibitor 2a), p19Arf (an alternative reading frame product of Cdkn2a,), and p27Kip1 (Cdkn1b, cyclin-dependent kinase inhibitor 1b) result in malignant progression of epithelial cancers, sarcomas, and melanomas, respectively. Here, we generated knockout mouse models for each of these three cyclin-dependent kinase inhibitors using engineered nucleases. The p16Ink4a and p19Arf knockout mice were generated via transcription activator-like effector nucleases (TALENs), and p27Kip1 knockout mice via clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9). These gene editing technologies were targeted to the first exon of each gene, to induce frameshifts producing premature termination codons. Unlike preexisting embryonic stem cell-based knockout mice, our mouse models are free from selectable markers or other external gene insertions, permitting more precise study of cell cycle-related diseases without confounding influences of foreign DNA.

Keywords

• TALEN
• CRISPR/Cas9
• cyclin-dependent kinase inhibitor