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We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery ( i -GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i -GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i- GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i -GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.

Meiosis is a hallmark event in germ cell development that accompanies sequential events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 knockout (KO) spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome. Furthermore, mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 (ΔRING-TRIM41) and found that ΔRING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result suggested that TRIM41 exerts its function on the chromosome axes. Our study revealed that Trim41 is essential for preventing SYCP3 overloading, suggesting a TRIM41-mediated mechanism for regulating chromosome axis protein dynamics during male meiotic progression.

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 ( Kctd19 ) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.

Alport syndrome is an inherited chronic human kidney disease, characterized by glomerular basement membrane abnormalities. This disease is caused by mutations in COL4A3 , COL4A4 , or COL4A5 gene. The knockout mice for Col4 α 3 , Col4 α 4 , and Col4 α 5 are developed and well characterized for the study of Alport syndrome. However, disease progression and effects of pharmacological therapy depend on the genetic variability. This model was reliable only to mouse. In this study, we created a novel Alport syndrome rat model utilizing the rGONAD technology, which generated rat with a deletion of the Col4 α 5 gene. Col4 α 5 deficient rats showed hematuria, proteinuria, high levels of BUN, Cre, and then died at 18 to 28 weeks of age (Hemizygous mutant males). Histological and ultrastructural analyses displayed the abnormalities including parietal cell hyperplasia, mesangial sclerosis, and interstitial fibrosis. Then, we demonstrated that α3/α4/α5 (IV) and α5/α5/α6 (IV) chains of type IV collagen disrupted in Col4α5 deficient rats. Thus, Col4 α 5 mutant rat is a reliable candidate for the Alport syndrome model for underlying the mechanism of kidney diseases and further identifying potential therapeutic targets for human renal diseases.

Background Recent progress in development of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology in various organisms. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) in mice; a novel in vivo genome editing system that does not require ex vivo handling of embryos, and this technology is newly developed and renamed as “ i mproved GONAD” ( i -GONAD). However, this technology has been limited only to mice. Therefore in this study, we challenge to apply this technology to rats. Results Here, we determine the most suitable condition for in vivo gene delivery towards rat preimplantation embryos using tetramethylrhodamine-labelled dextran, termed as R at improved GONAD (rGONAD). Then, to investigate whether this method is feasible to generate genome-edited rats by delivery of CRISPR/Cas9 components, the tyrosinase ( Tyr ) gene was used as a target. Some pups showed albino -colored coat, indicating disruption of wild-type Tyr gene allele. Furthermore, we confirm that rGONAD method can be used to introduce genetic changes in rat genome by the ssODN-based knock-in. Conclusions We first establish the rGONAD method for generating genome-edited rats. We demonstrate high efficiency of the rGONAD method to produce knock-out and knock-in rats, which will facilitate the production of rat genome engineering experiment. The rGONAD method can also be readily applicable in mammals such as guinea pig, hamster, cow, pig, and other mammals.

Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective sperm motility) are known causes of male infertility in human patients. Despite many studies, the molecular details of the globozoospermia etiology are still poorly understood. Serine-rich single-pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene in mammals. In this study, we generated Ssmem1 knockout (KO) mice using the CRISPR/Cas9 system, demonstrated that Ssmem1 is essential for male fertility in mice, and found that SSMEM1 protein is expressed during spermatogenesis but not in mature sperm. The sterility of the Ssmem1 KO (null) mice is associated with globozoospermia and loss of sperm motility. To decipher the mechanism causing the phenotype, we analyzed testes with transmission electron microscopy and discovered that Ssmem1-disrupted spermatids have abnormal localization of Golgi at steps eight and nine of spermatid development. Immunofluorescence analysis with anti-Golgin-97 to label the trans-Golgi network, also showed delayed movement of the Golgi to the spermatid posterior region, which causes failure of sperm head shaping, disorganization of the cell organelles, and entrapped tails in the cytoplasmic droplet. In summary, SSMEM1 is crucial for intracellular Golgi movement to ensure proper spatiotemporal formation of the sperm head that is required for fertilization. These studies and the pathway in which SSMEM1 functions have implications for human male infertility and identifying potential targets for nonhormonal contraception. Summary sentence Absence of serine-rich single-pass membrane protein 1 (SSMEM1) leads to male infertility because of globozoospermia in mice.

Microtubule organization and reorganization during the cell cycle are achieved by regulation of the number, distribution and activity of microtubule-organizing centres (MTOCs). In fission yeast, the Mto1/2 complex determines the activity and distribution of cytoplasmic MTOCs. Upon mitosis, cytoplasmic microtubule nucleation ceases; inactivation of the Mto1/2 complex is triggered by Mto2 hyperphosphorylation. However, the protein kinase(s) that phosphorylates Mto2 remains elusive. Here we show that a conserved signalling network, called MOR (morphogenesis Orb6 network) in fission yeast, negatively regulates cytoplasmic MTOCs through Mto2 phosphorylation to ensure proper microtubule organization. Inactivation of Orb6 kinase, the most downstream MOR component, by attenuation of MOR signalling leads to reduced Mto2 phosphorylation, coincident with increased number of both Mto2 puncta and cytoplasmic microtubules. These defects cause the emergence of uncoordinated mitotic cells with cytoplasmic microtubules, resulting in reduced spindle assembly. Thus, the regulation of Mto2 by the MOR is crucial for cytoplasmic microtubule organization and contributes to reorganization of the microtubule cytoskeletons during the cell cycle.

Tubulointerstitial fibrosis is a progressive process affecting the kidneys, causing renal failure that can be life-threatening. Thus, renal fibrosis has become a serious concern in the ageing population; however, fibrotic development cannot be diagnosed early and assessed noninvasively in both patients and experimental animal models. Here, we found that serum amyloid A3 (Saa3) expression is a potent indicator of early renal fibrosis; we also established in vivo Saa3/C/EBPβ-promoter bioluminescence imaging as a sensitive and specific tool for early detection and visualization of tubulointerstitial fibrosis. Saa3 promoter activity is specifically upregulated in parallel with tumor necrosis factor α (TNF-α) and fibrotic marker collagen I in injured kidneys. C/EBPβ, upregulated in injured kidneys and expressed in tubular epithelial cells, is essential for the increased Saa3 promoter activity in response to TNF-α, suggesting that C/EBPβ plays a crucial role in renal fibrosis development. Our model successfully enabled visualization of the suppressive effects of a citrus flavonoid derivative, glucosyl-hesperidin, on inflammation and fibrosis in kidney disease, indicating that this model could be widely used in exploring therapeutic agents for fibrotic diseases.

Successful completion of the S phase in fission yeast is needed for the switch from monopolar to bipolar growth. The DNA replication checkpoint kinase Cds1 is found to regulate this process by modulating the activity of microtubule plus-end binding proteins that are responsible for cell growth. Microtubules are central to eukaryotic cell morphogenesis. Microtubule plus-end tracking proteins (+TIPs) transport polarity factors to the cell cortex, thereby playing a key role in both microtubule dynamics and cell polarity. However, the signalling pathway linking +TIPs to cell polarity control remains elusive. Here we show that the fission yeast checkpoint kinase Cds1 (Chk2 homologue) delays the transition of growth polarity from monopolar to bipolar (termed NETO; new-end take-off). The +TIPs CLIP170 homologue Tip1 and kinesin Tea2 are responsible for this delay, which is accompanied by a reduction in microtubule dynamics at the cell tip. Remarkably, microtubule stabilization occurs asymmetrically, prominently at the non-growing cell end, which induces abnormal accumulation of the polarity factor Tea1. Importantly, NETO delay requires activation of calcineurin, which is carried out by Cds1, resulting in Tip1 dephosphorylation. Thus, our study establishes a critical link between calcineurin and checkpoint-dependent cell morphogenesis.