Generation of PHYHIPL Ser19Stop Knock-in Mice

We generated the PHYHIPL Ser19Stop knock-in mice using the electroporation method according to our previous report with some modifications [20 (link)]. In this study, we used synthetic crRNA (Alt-R® CRISPR-Cas9 crRNA; IDT, Coralville, IA, USA) and tracrRNA (Alt-R® CRISPR-Cas9 tracrRNA, IDT) instead of single guide RNA (sgRNA). We also designed the corresponding donor single-stranded oligodeoxynucleotides (ssODNs) with 5′- and 3′-homology arms. Target sequence: CATATCCCATGAGATCTTGAAGG. Equal volumes of crRNA and tracrRNA were combined in a duplex buffer (IDT) in a thermal cycler at 95 °C for 5 min following the manufacturer’s protocol. crRNA/tracrRNA (3 μM), recombinant Cas9 protein (100 ng/μL; GeneArt Platinum™ Cas9 Nuclease, Thermo Fisher Scientific, Waltham, MA, USA) and ssODN (400 ng/μL; Ultramer®, IDT) were mixed in Opti-MEM I (Thermo Fisher Scientific) before electroporation. Fertilized eggs were isolated from the superovulated C57BL/6 J female mice 21 h after administering human chorionic gonadotropin (hCG). The electroporation was conducted at the 1-cell stage (24–26 h after hCG), and the 2-cell stage embryos were transferred to the oviduct of pseudopregnant ICR females (Charles River Japan, Yokohama, Japan). We confirmed the mutant alleles of the obtained mice (Fig. 1d) by sequencing PCR products.