Abstract
Genome editing technologies such as CRISPR–Cas9 are widely used to establish causal associations between mutations and phenotypes. However, CRISPR–Cas9 is rarely used to analyze promoter regions. The insulin promoter region (approximately 1,000 bp) directs β cell-specific expression of insulin, which in vitro studies show is regulated by ubiquitous, as well as pancreatic, β cell-specific transcription factors. However, we are unaware of any confirmatory in vivo studies. Here, we used CRISPR–Cas9 technology to generate mice with mutations in the promoter regions of the insulin I (Ins1) and II (Ins2) genes. We generated 4 homozygous diabetic mice with 2 distinct mutations in the highly conserved C1 elements in each of the Ins1 and Ins2 promoters (3 deletions and 1 replacement in total). Remarkably, all mice with homozygous or heterozygous mutations in other loci were not diabetic. Thus, the C1 element in mice is required for Ins transcription in vivo.
Highlights
Genome editing technologies such as clustered regularly interspaced short palindromic repeat (CRISPR)–Cas[9] are widely used to establish causal associations between mutations and phenotypes
Our detailed analysis of published mammalian promoter sequences revealed that most of the critical promoter sequence elements are well conserved[25], bases –151 to –103 of the mouse insulin gene (Ins)[1] and Ins[2] promoters and bases –149 to –102 of the human insulin promoter (Fig. 1a, Supplementary Fig. 1). These sequences comprise the GG2–A2, C1 and E1 elements (Fig. 1a, Supplementary Fig. 1), which was previously known as the rat insulin promoter element 3 (RIPE3), to which cell-specific and ubiquitous factors bind[13]
The 1–3 deleted bases were located between the C1 and E1 elements or within the sequences bordering the C1 element (Fig. 1b)
Summary
Genome editing technologies such as CRISPR–Cas[9] are widely used to establish causal associations between mutations and phenotypes. Certain transcription factors that govern β cell differentiation (pancreatic and duodenal homeobox[1]; Pdx[1], neuronal differentiation; NeuroD and v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A; MafA) interact directly with insulin promoter regulatory elements[9,10,11,12,13,14,15] Most of these proteins bind to specific sites and form multiprotein transcriptional complexes situated on the insulin promoter[16,17]. We used CRISPR–Cas[9] technology to generate mice with partial deletions of the Ins[1] and Ins[2] promoters, which showed that only homozygous mice with mutations in the highly conserved C1 elements of the Ins[1] and Ins[2] promoters developed diabetes, indicating the value of genome editing in studying the regulation of insulin synthesis in vivo
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