(P-069) Knock-in on the Y chromosome

Abstract Authors: Yuzuki Goto¹; Takuto Yamamoto¹; Satoshi Mashiko¹; Masahiro Sakata¹; Daiki Shikata¹; Shinnosuke Honda¹; Shuntaro Ikeda¹; Naojiro Minami¹
1. Reproductive Biology, Graduate School of Agriculture, Kyoto University, Japan

Abstract Text: The Rosa26 locus is commonly used for autosomal knock-in, as Rosa26-targeted knock-in is easy to be implemented with stable gene expressions and has little effect on other endogenous genes. For sex chromosomal knock-in, the housekeeping genes Hprt and Pgk1 are generally used for the X chromosome as targeting loci. On the other hand, no commonly used targeting loci have been established for the Y chromosome unlike above cases. However, in a previous study, knock-in mice with tdTomato in the Ddx3y (DEAD-Box Helicase 3 Y-Linked, Dby) locus on the Y chromosome were generated because Ddx3y knock-out mice are non-lethal and normally fertile (Hirata et al., 2022). Therefore, in this study, we followed up the Y chromosomal knock-in using the Dby locus with the GFP gene and a gene cassette longer than the GFP gene. In addition, we aimed to increase the knock-in efficiency. In order to perform knock-in only on the Y chromosome, it was favorable to microinject genome editing tools into the male pronuclei before pronuclear fusion. To increase knock-in efficiency, we employed the co-injection of a plasmid with homologous arms at both ends of the gene cassette and Cas9 ribonucleoprotein (Cas9 RNP), which is Cas9 protein and gRNA complex, into the S-phase pronucleus in early embryos (Abe et al., 2020).
After knocking in the GFP gene into the Dby locus, we verified the knock-in efficiency and the presence of nonspecific insertions. We performed in vitro fertilization in ICR mice and microinjected the GFP-gene containing plasmid and Cas9 RNP into the male pronuclei 6～8 hours after insemination. After culturing the embryos to blastocysts, the presence or absence of GFP fluorescence was examined under a fluorescence microscope and then embryos were genotyped. Next, a gene cassette of 2887 bp, which is even longer than the GFP gene, was knocked-in into the Dby locus. In vitro fertilization was performed to obtain zygotes from hybrid mice (B6D2F1), and after microinjection into the male pronuclei as described above, embryos were cultured to the 2-cell or blastocyst stage, and then transferred to pseudopregnant ICR mice by oviductal transfer or Non-Surgical Embryo Transfer (NSET) using the mNSET Device for Mice 60010 (ParaTechs), respectively, and the resulting pups were genotyped.
For knock-in of the GFP gene, we observed a 4.5% (1 out of 22 injected embryos） efficiency of knock-in in the appropriate locus. For knock-in of the 2887 bp gene cassette, the efficiency of obtaining knock-in mice was 21.4% (3 out of 14 pups). In a previous study, knock-in mice were obtained at a 2.4% efficiency, in which the GFP gene was inserted into the intergenic region sequence of the Ddx3y and Uty genes by microinjection in the cytoplasm (Zhao et al., 2019). Compared with the report, the efficiency of acquiring knock-in mice in the present study is relatively high. The knock-in tool injection in the S-phase pronucleus (Abe et al., 2020), which was effective for knock-in into the Rosa26 locus, was also effective for that on the Y chromosome. The knock-in technique used in this study should be applicable for knocking in other genes on sex chromosomes in the future.