Efficient production and transmission of CRISPR/Cas9-mediated mutant alleles at the IG-DMR via generation of mosaic mice using a modified 2CC method.

Generation of mutant imprinting control region (ICR) mice using genome editing is an important approach for elucidating ICR functions. IG-DMR is an ICR in the Dlk1-Dio3 imprinted domain that contains functional regions-in both parental alleles-that are essential for embryonic development. One drawback of this approach is that embryonic lethality can occur from aberrant expression of the imprinted genes if IG-DMR gets mutated in either the paternal or maternal allele. To overcome this problem, we generated mosaic mice that contained cells with modified IG-DMR alleles and wild-type cells using the 2CC method that allowed for microinjection of the CRISPR/Cas9 constructs into a blastomere of 2-cell embryos. This method improved the birth rate of the founder pups relative to that obtained using the standard protocol. We also successfully produced mosaic mice in which the tandem repeat array sequence in the IG-DMR had been replaced by homology directed repair. Additionally, paternal transmission of the replaced allele caused aberrant expression of the imprinted genes due to hypomethylation of the IG-DMR, indicating that the replaced allele recapitulated our deletion model. Our results indicate that this method is useful for the generation of mutant mice in which a genomic locus essential for normal development has been genetically edited.

A tandem repeat array in IG-DMR is essential for imprinting of paternal allele at the Dlk1-Dio3 domain during embryonic development.

Genomic imprinting is a phenomenon that causes parent-origin-specific monoallelic expression of a small subset of genes, known as imprinted genes, by parentally inherited epigenetic marks. Imprinted genes at the delta-like homolog 1 gene (Dlk1)-type III iodothyronine deiodinase gene (Dio3) imprinted domain, regulated by intergenic differentially methylated region (IG-DMR), are essential for normal development of late embryonic stages. Although the functions of IG-DMR have been reported by generating knockout mice, molecular details of the regulatory mechanisms are not fully understood as the specific sequence(s) of IG-DMR have not been identified. Here, we generated mutant mice by deleting a 216 bp tandem repeated sequence in IG-DMR, which comprised seven repeats of 24 bp motifs, by genome editing technologies. The mutant mice showed that paternal transmission of the deletion allele, but not maternal transmission, induces severe growth retardation and perinatal lethality, possibly due to placental defects. Embryos with a paternally transmitted deletion allele showed biallelic expression of maternally expressed genes and repression of paternally expressed genes. DNA methylation status also showed loss of methylation at IG-DMR and Gtl2-DMR, indicating that the tandem repeat sequence of IG-DMR is one of the functional sequences of IG-DMR, which is required for maintaining DNA methylation imprints of paternal allele at IG-DMR.