Kuliyev, E. | Search bioRxiv

bioRxiv2022

One-step generation of a conditional allele in mice using a short artificial intron

Despite tremendous advances in genome editing technologies, generation of conditional alleles in mice has remained challenging. Recent studies in cells have successfully made use of short artificial introns to engineer conditional alleles. The approach consists of inserting intronic sequences flanked by two loxP sites within an exon of a gene using CRISPR-Cas9 technology. Under normal conditions, the artificial intron is removed by the splicing machinery, allowing for expression of the gene product. Following Cre-mediated recombination of the two loxP sites, the intron is disabled, and splicing can no longer occur. The remaining intronic sequences create a frameshift and early translational termination. Here we describe the application of this technology to engineer conditional alleles in mice using Scyl1 as a model gene. Insertion of the cassette occurred in 17% of edited mice obtained from pronuclear stage zygote microinjection. Mice homozygous for the insertion expressed SCYL1 at levels comparable to wild-type mice and showed no overt abnormalities associated with the loss of Scyl1 function, indicating the proper removal of the artificial intron. Deletion of the cassette via Cre-mediated recombination in vivo occurred at high frequency, abrogated SCYL1 protein expression, and resulted in loss-of-function phenotypes. Our results broaden the applicability of this approach to engineering conditional alleles in mice.

genetics↗

bioRxiv2020

Structural defects caused by Acrodermatitis Enteropathica mutations in the extracellular domain account for mistrafficking and malfunction of ZIP4

ZIP4 is a representative member of the Zrt-/Irt-like protein (ZIP) transporter family and responsible for zinc uptake from diet. Loss-of-function mutations of human ZIP4 (hZIP4) drastically reduce zinc absorption, causing a life-threatening autosomal recessive disorder, Acrodermatitis Enteropathica (AE). Although the zinc transport machinery is located in the transmembrane domain conserved in the entire ZIP family, half of the missense mutations occur in the extracellular domain (ECD) of hZIP4, which is only present in a fraction of mammalian ZIPs. How the AE-causing mutations in the ECD lead to ZIP4 malfunction has not be fully clarified. In this work, we characterized all the seven confirmed AE-causing missense mutations in hZIP4-ECD and found that the variants exhibited completely abolished zinc transport activity measured in a cell-based transport assay. Although the variants were able to be expressed in HEK293T cells, they failed to traffic to cell surface and were largely retained in the ER with immature glycosylation. When the corresponding mutations were introduced in the ECD of ZIP4 from Pteropus Alecto, a close homolog of hZIP4, the variants exhibited impaired protein folding and reduced thermal stability, which likely account for intracellular mistrafficking of the AE-associated variants and as such a total loss of zinc uptake in cells. This work provides a molecular pathogenic mechanism for AE, which lays out a basis for potential therapy using small molecular chaperones.

biochemistry↗