LZTR1 deficiency exerts high metastatic potential by enhancing sensitivity to EMT induction and controlling KLHL12-mediated collagen secretion.

Leucine zipper-like transcriptional regulator 1 (LZTR1), a substrate adaptor of Cullin 3 (CUL3)-based E3 ubiquitin ligase, regulates proteostasis of the RAS subfamily. Mutations in LZTR1 have been identified in patients with several types of cancer. However, the role of LZTR1 in tumor metastasis and the target molecules of LZTR1, excluding the RAS subfamily, are not clearly understood. Here, we show that LZTR1 deficiency increases tumor growth and metastasis. In lung adenocarcinoma cells, LZTR1 deficiency induced the accumulation of the RAS subfamily and enhanced cell proliferation, invasion, and xenograft tumor growth. Multi-omics analysis to clarify the pathways related to tumor progression showed that MAPK signaling, epithelial-mesenchymal transition (EMT), and extracellular matrix (ECM) remodeling-related gene ontology terms were enriched in LZTR1 knockout cells. Indeed, LZTR1 deficiency induced high expression of EMT markers under TGF-ß1 treatment. Our search for novel substrates that interact with LZTR1 resulted in the discovery of a Kelch-like protein 12 (KLHL12), which is involved in collagen secretion. LZTR1 could inhibit KLHL12-mediated ubiquitination of SEC31A, a component of coat protein complex II (COPII), whereas LZTR1 deficiency promoted collagen secretion. LZTR1-RIT1 and LZTR1-KLHL12 worked independently regarding molecular interactions and did not directly interfere with each other. Further, we found that LZTR1 deficiency significantly increases lung metastasis and promotes ECM deposition around metastatic tumors. Since collagen-rich extracellular matrix act as pathways for migration and facilitate metastasis, increased expression of RAS and collagen deposition may exert synergistic or additive effects leading to tumor progression and metastasis. In conclusion, LZTR1 deficiency exerts high metastatic potential by enhancing sensitivity to EMT induction and promoting collagen secretion. The functional inhibition of KLHL12 by LZTR1 provides important evidence that LZTR1 may be a repressor of BTB-Kelch family members. These results provide clues to the mechanism of LZTR1-deficiency carcinogenesis.

Chromosomal microdeletion leading to pituitary gigantism through hormone-gene overexpression.

Pituitary gigantism is a rare endocrinopathy characterized by tall stature due to growth hormone (GH) hypersecretion. This condition is generally linked to a genetic predisposition to tumors that produce GH or GH-releasing hormone (GHRH). Here, we report a Japanese woman who exhibited prominent body growth from infancy to reach an adult height of 197.4 cm (+7.4 standard deviation). Her blood GH levels were markedly elevated. She carried no pathogenic variants in known growth-controlling genes but had a hitherto unreported 752 kb heterozygous deletion at 20q11.23. The microdeletion was located 8.9 kb upstream of GHRH and encompassed exons 2-9 of a ubiquitously expressed gene TTI1 together with 12 other genes, pseudogenes and non-coding RNAs. Transcript analyses of the patient's leukocytes showed that the microdeletion produced chimeric mRNAs consisting of exon 1 of TTI1 and all coding exons of GHRH. In silico analysis detected promoter-associated genomic features around TTI1 exon 1. Genome-edited mice carrying the same microdeletion recapitulated accelerated body growth from a few weeks after birth. The mutant mice developed pituitary hyperplasia and exhibited ectopic Ghrh expression in all tissues examined. Thus, the extreme phenotype of pituitary gigantism in the patient likely reflects GHRH overexpression driven by an acquired promoter. The results of this study indicate that germline submicroscopic deletions have the potential to cause conspicuous developmental abnormalities due to gene overexpression. Furthermore, this study provides evidence that constitutive expression of a hormone-encoding gene can result in congenital disease.

Integrator complex subunit 15 controls mRNA splicing and is critical for eye development.

The eye and brain are composed of elaborately organized tissues, development of which is supported by spatiotemporally precise expression of a number of transcription factors and developmental regulators. Here we report the molecular and genetic characterization of Integrator complex subunit 15 (INTS15). INTS15 was identified in search for the causative gene(s) for an autosomal-dominant eye disease with variable individual manifestation found in a large pedigree. While homozygous Ints15 knockout mice are embryonic lethal, mutant mice lacking a small C-terminal region of Ints15 show ocular malformations similar to the human patients. INTS15 is highly expressed in the eye and brain during embryogenesis and stably interacts with the Integrator complex to support small nuclear RNA 3' end processing. Its knockdown resulted in missplicing of a large number of genes, probably as a secondary consequence, and substantially affected genes associated with eye and brain development. Moreover, studies using human iPS cells-derived neural progenitor cells showed that INTS15 is critical for axonal outgrowth in retinal ganglion cells. This study suggests a new link between general transcription machinery and a highly specific hereditary disease.

SOX9 and SRY binding sites on mouse mXYSRa/Enh13 enhancer redundantly regulate Sox9 expression to varying degrees.

Sox9 plays an essential role in mammalian testis formation. It has been reported that gene expression in the testes is regulated by enhancers. Among them, mXYSRa/Enh13-which is located at far upstream of the transcription start site-plays a critical role, wherein its deletion causes complete male-to-female sex reversal in mice. It has been proposed that the binding sites (BSs) of SOX9 and SRY, the latter of which is the sex determining gene on the Y chromosome, are associated with mXYSRa/Enh13. They function as an enhancer, whereby the sequences are evolutionarily conserved and in vivo binding of SOX9 and SRY to mXYSRa/Enh13 has been demonstrated previously. However, their precise in vivo functions have not been examined to date. To this end, this study generated mice with substitutions on the SOX9 and SRY BSs to reveal their in vivo functions. Homozygous mutants of SOX9 and SRY BS were indistinguishable from XY males, whereas double mutants had small testes, suggesting that these functions are redundant and that there is another functional sequence on mXYSRa/Enh13, since mXYSRa/Enh13 deletion mice are XY females. In addition, the majority of hemizygous mice with substitutions in SOX9 BS and SRY BS were female and male, respectively, suggesting that SOX9 BS contributes more to SRY BS for mXYSRa/Enh13 to function. The additive effect of SOX9 and SRY via these BSs was verified using an in vitro assay. In conclusion, SOX9 BS and SRY BS function redundantly in vivo, and at least one more functional sequence should exist in mXYSRa/Enh13.

Establishment and visual analysis of CBA/J-Pde6bY347Y/Y347X and C3H/HeJ-Pde6bY347Y/Y347X mice.

In CBA/J and C3H/HeJ mice, retinitis pigmentosa is inherited as an autosomal-recessive trait due to a mutation in Pde6b, which encodes cGMP phosphodiesterase subunit b. In these strains, the Y347X mutation in Pde6b leads to the upregulation of cGMP levels, increased Ca2+ influx induces rod death, and the outer segment and rod cells entirely disappeared by 35 days after birth. In the present study, we utilized the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas)9-mediated gene editing to repair the Y347X mutation in CBA/J and C3H/HeJ mice. Evaluation of the established CBA/J-Pde6b Y347Y/Y347X and C3H/HeJ-Pde6b Y347Y/Y347X mice, which were confirmed to have normal retinal layers by live fundoscopic imaging and histopathological analysis, revealed improved visual acuity based on the visual cliff and light/dark transition tests. Furthermore, our analyses revealed that the visible platform test was a more effective tool for testing visual behavior in these mice. The results suggest that the established strains can serve as control groups for CBA/J and C3H/HeJ in ophthalmology studies involving retinitis pigmentosa.

POU1F1/Pou1f1 c.143-83A > G Variant Disrupts the Branch Site in Pre-mRNA and Leads to Dwarfism.

POU Class 1 Homeobox1 (POU1F1/Pou1f1) is a well-established pituitary-specific transcription factor, and causes, when mutated, combined pituitary hormone deficiency in humans and mice. POU1F1/Pou1f1 has 2 isoforms: the alpha and beta isoforms. Recently, pathogenic variants in the unique coding region of the beta isoform (beta domain) and the intron near the exon-intron boundary for the beta domain were reported, although their functional consequences remain obscure. In this study, we generated mice carrying the Pou1f1 c.143-83A>G substitution that recapitulates the human intronic variant near the exon-intron boundary for the beta domain. Homozygous mice showed postnatal growth failure, with an average body weight that was 35% of wild-type littermates at 12 weeks, which was accompanied by anterior pituitary hypoplasia and deficiency of circulating insulin-like growth factor 1 and thyroxine. The results of RNA-seq analysis of the pituitary gland were consistent with reduction of somatotrophs, and this was confirmed immunohistochemically. Reverse transcription polymerase chain reaction of pituitary Pou1f1 mRNA showed abnormal splicing in homozygous mice, with a decrease in the alpha isoform, an increase in the beta isoform, and the emergence of the exon-skipped transcript. We further characterized artificial variants in or near the beta domain, which were candidate positions of the branch site in pre-mRNA, using cultured cell-basis analysis and found that only c.143-83A>G produced transcripts similar to the mice model. Our report is the first to show that the c.143-83A>G variant leads to splicing disruption and causes morphological and functional abnormalities in the pituitary gland. Furthermore, our mice will contribute understanding the role of POU1F1/Pou1f1 transcripts in pituitary development.

Turnover of mammal sex chromosomes in the Sry-deficient Amami spiny rat is due to male-specific upregulation of Sox9.

Mammalian sex chromosomes are highly conserved, and sex is determined by SRY on the Y chromosome. Two exceptional rodent groups in which some species lack a Y chromosome and Sry offer insights into how novel sex genes can arise and replace Sry, leading to sex chromosome turnover. However, intensive study over three decades has failed to reveal the identity of novel sex genes in either of these lineages. We here report our discovery of a male-specific duplication of an enhancer of Sox9 in the Amami spiny rat Tokudaia osimensis, in which males and females have only a single X chromosome (XO/XO) and the Y chromosome and Sry are completely lost. We performed a comprehensive survey to detect sex-specific genomic regions in the spiny rat. Sex-related genomic differences were limited to a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on an autosome. Hi-C analysis using male spiny rat cells showed the duplicated region has potential chromatin interaction with Sox9. The duplicated unit harbored a 1,262-bp element homologous to mouse enhancer 14 (Enh14), a candidate Sox9 enhancer that is functionally redundant in mice. Transgenic reporter mice showed that the spiny rat Enh14 can function as an embryonic testis enhancer in mice. Embryonic gonads of XX mice in which Enh14 was replaced by the duplicated spiny rat Enh14 showed increased Sox9 expression and decreased Foxl2 expression. We propose that male-specific duplication of this Sox9 enhancer substituted for Sry function, defining a novel Y chromosome in the spiny rat.

Tmsb10 triggers fetal Leydig differentiation by suppressing the RAS/ERK pathway.

Leydig cells in fetal testes play crucial roles in masculinizing fetuses through androgen production. Gene knockout studies have revealed that growth factors are implicated in fetal Leydig cell (FLC) differentiation, but little is known about the mechanisms regulating this process. We investigate this issue by characterizing FLC progenitor cells using single-cell RNA sequencing. The sequence datasets suggest that thymosin ß10 (Tmsb10) is transiently upregulated in the progenitors. While studying the function of Tmsb10, we reveal that platelet-derived growth factor (PDGF) regulates ciliogenesis through the RAS/ERK and PI3K/AKT pathways, and thereby promotes desert hedgehog (DHH)-dependent FLC differentiation. Tmsb10 expressed in the progenitor cells induces their differentiation into FLCs by suppressing the RAS/ERK pathway. Through characterizing the transiently expressed Tmsb10 in the FLC progenitors, this study unveils the molecular process of FLC differentiation and shows that it is cooperatively induced by DHH and PDGF.

Human SRY Expression at the Sex-determining Period is Insufficient to Drive Testis Development in Mice.

The sex-determining region of the Y chromosome, Sry/SRY, is an initiation factor for testis development in both humans and mice. Although the functional compatibility between murine SRY and human SRY was previously examined in transgenic mice, their equivalency remains inconclusive. Because molecular interaction and timeline of mammalian sex determination were mostly described in murine experiments, we generated a mouse model in which Sry was substituted with human SRY to verify the compatibility. The mouse model had the human SRY open reading frame at the locus of murine Sry exon 1-Sry(SRY) mice-and was generated using the CRISPR/Cas9 system. The reproductive system of the mice was analyzed. The expression of human SRY in the fetal gonadal ridge of Sry(SRY) mice was detected. The external and internal genitalia of adult Sry(SRY) mice were similar to those of wild-type females, without any significant difference in anogenital distance. Sry(SRY) mice obtained gonads, which were morphologically considered as ovaries. Histological analysis revealed that the cortical regions of gonads from adult Sry(SRY) mice contained few follicles. We successfully replaced genes on the Y chromosome with targeted genome editing using the CRISPR/Cas9 system. Because the Sry(SRY) XY mice did not develop testis, we concluded that human SRY was insufficient to drive testis development in mouse embryos. The difference in response elements and lack of glutamine-rich domains may have invalidated human SRY function in mice. Signal transduction between Sry/SRY expression and Sox9/SOX9 activation is possibly organized in a species-specific manner.

A Testis-Specific Long Noncoding RNA, Start, Is a Regulator of Steroidogenesis in Mouse Leydig Cells.

The testis expresses many long noncoding RNAs (lncRNAs), but their functions and overview of lncRNA variety are not well understood. The mouse Prss/Tessp locus contains six serine protease genes and two lncRNAs that have been suggested to play important roles in spermatogenesis. Here, we found a novel testis-specific lncRNA, Start (Steroidogenesis activating lncRNA in testis), in this locus. Start is 1822 nucleotides in length and was found to be localized mostly in the cytosol of germ cells and Leydig cells, although nuclear localization was also observed. Start-knockout (KO) mice generated by the CRISPR/Cas9 system were fertile and showed no morphological abnormality in adults. However, in adult Start-KO testes, RNA-seq and qRT-PCR analyses revealed an increase in the expression of steroidogenic genes such as Star and Hsd3b1, while ELISA analysis revealed that the testosterone levels in serum and testis were significantly low. Interestingly, at 8 days postpartum, both steroidogenic gene expression and testosterone level were decreased in Start-KO mice. Since overexpression of Start in two Leydig-derived cell lines resulted in elevation of the expression of steroidogenic genes including Star and Hsd3b1, Start is likely to be involved in their upregulation. The increase in expression of steroidogenic genes in adult Start-KO testes might be caused by a secondary effect via the androgen receptor autocrine pathway or the hypothalamus-pituitary-gonadal axis. Additionally, we observed a reduced number of Leydig cells at 8 days postpartum. Collectively, our results strongly suggest that Start is a regulator of steroidogenesis in Leydig cells. The current study provides an insight into the overall picture of the function of testis lncRNAs.

Humanization of a tandem repeat in IG-DMR causes stochastic restoration of paternal imprinting at mouse Dlk1-Dio3 domain.

The Dlk1-Dio3 imprinted domain, regulated by an intergenic differentially methylated region (IG-DMR), is important for mammalian embryonic development. Although previous studies have reported that DNA methylation of a tandem repeated array sequence in paternal IG-DMR (IG-DMR-Rep) plays an essential role in the maintenance of DNA methylation in mice, the function of a tandem repeated array sequence in human IG-DMR (hRep) is unknown. Here, we generated mice with a human tandem repeated sequence, which replaced the mouse IG-DMR-Rep. Mice that transmitted the humanized allele paternally exhibited variable methylation status at the IG-DMR and were stochastically rescued from the lethality of IG-DMR-Rep deficiency, suggesting that hRep plays a role in human IG-DMR for the regulation of imprinted expression. Moreover, chromatin immunoprecipitation analysis showed that TRIM28 was enriched in hypermethylated paternal hRep without ZFP57. Our results suggest that hRep contributes to the maintenance of human IG-DMR methylation imprints via the recruitment of TRIM28.

Rnd2 differentially regulates oligodendrocyte myelination at different developmental periods.

In the CNS, oligodendrocyte precursor cells differentiate into oligodendrocytes to wrap their plasma membranes around neuronal axons, generating mature neural networks with myelin sheaths according to spatial and temporal patterns. While myelination is known to be one of the most dynamic cell morphological changes, the overall intrinsic and extrinsic molecular cues controlling myelination remain to be fully clarified. Here, we describe the biphasic roles of Rnd2, an atypical branch of the Rho family GTPase, in oligodendrocyte myelination during development and after maturation in mice. Compared with littermate controls, oligodendrocyte-specific Rnd2 knockout mice exhibit decreased myelin thickness at the onset of myelination but increased myelin thickness in the later period. Larger proportions of Rho kinase and its substrate Mbs, the signaling unit that negatively regulates oligodendrocyte myelination, are phosphorylated at the onset of myelination, while their smaller proportions are phosphorylated in the later period. In addition, we confirm the biphasic role of Rnd2 through experiments with oligodendrocyte-specific Rnd2 transgenic mice. We conclude that Rnd2 positively regulates myelination in the early myelinating period and negatively regulates myelination in the later period. This unique modulator thus plays different roles depending on the myelination period.

Identification of the first promoter-specific gain-of-function SOX9 missense variant (p.E50K) in a patient with 46,XX ovotesticular disorder of sex development.

SOX9, a transcription factor, is expressed in the undifferentiated XX and XY gonads. SRY induces significant upregulation of SOX9 expression in XY gonads. Loss-of-function SOX9 variants cause testicular dysgenesis in 46,XY patients, while duplication of the total gene or the upstream regulatory region results in testicular development in 46,XX patients. However, gain-of-function (GoF) SOX9 variants have not been reported previously. We report the case of a 16-year-old female patient with a 46,XX karyotype who had masculinized external genitalia and unilateral ovotestis. Next-generation sequencing-based genetic screening for disorders of sex development led to the identification of a novel SOX9 variant (p.Glu50Lys), transmitted from the phenotypically normal father. Expression analysis showed that E50K-SOX9 enhanced transactivation of the luciferase reporter containing the testis enhancer sequence core element compared with that containing the wildtype-SOX9. This GoF activity was not observed in the luciferase reporter containing Amh, the gene for anti-Müllerian hormone. We genetically engineered female mice (Sox9E50K/E50K ), and they showed no abnormalities in the external genitalia or ovaries. In conclusion, a novel SOX9 variant with a promoter-specific GoF activity was identified in vitro; however, the disease phenotype was not recapitulated by the mouse model. At present, the association between the GoF SOX9 variant and the ovotestis phenotype remains unclear. Future studies are needed to verify the possible association.

Exploring trophoblast-specific Tead4 enhancers through chromatin conformation capture assays followed by functional screening.

Tead4 is critical for blastocyst development and trophoblast differentiation. We assayed long-range chromosomal interactions on the Tead4 promoter in mouse embryonic stem (ES) cells and trophoblast stem (TS) cells. Using luciferase reporter assays with ES and TS cells for 34 candidate enhancer regions, we identified five genomic fragments that increased Tead4 promoter activity in a TS-specific manner. The five loci consisted of three intra- and two inter-chromosomal loci relative to Tead4 on chromosome 6. We established five mouse lines with one of the five enhancer elements deleted and evaluated the effect of each deletion on Tead4 expression in blastocysts. By quantitative RT-PCR, we measured a 42% decrease in Tead4 expression in the blastocysts with a homozygous deletion with a 1.5 kb genomic interval on chromosome 19 (n = 14) than in wild-type blastocysts. By conducting RNA-seq analysis, we confirmed the trans effect of this enhancer deletion on Tead4 without significant cis effects on its neighbor genes at least within a 1.7 Mb distance. Our results demonstrated that the genomic interval on chromosome 19 is required for the appropriate level of Tead4 expression in blastocysts and suggested that an inter-chromosomal enhancer-promoter interaction may be the underlying mechanism.

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.

Renal medullary tonicity regulates RNF183 expression in the collecting ducts via NFAT5.

Nuclear factor of activated T-cells 5 (NFAT5) directly binds to the promoter of the RING finger protein 183 (RNF183) gene and induces its transcription under hypertonic conditions in mouse inner-medullary collecting duct (mIMCD-3) cells. However, there is no specific anti-RNF183 antibody for immunostaining; therefore, it is unclear whether NFAT5 regulates RNF183 expression in vivo and where RNF183 is localized in the kidney. This study investigated NFAT5-regulated in vivo RNF183 expression and localization using CRISPR/Cas9-mediated RNF183-green fluorescent protein (RNF183-GFP) knock-in mice. GFP with linker sequences was introduced upstream of an RNF183 open reading frame in exon 3 by homologous recombination through a donor plasmid. Immunofluorescence staining using GFP antibody revealed that GFP signals gradually increase from the outer medulla down to the inner medulla and colocalize with aquaporin-2. Furosemide treatment dramatically decreased RNF183 expression in the renal medulla, consistent with the decrease in NFAT5 protein and target gene mRNA expression. Furosemide treatment of mIMCD-3 cells did not affect mRNA expression and RNF183 promoter activities. These results indicated that RNF183 is predominantly expressed in the renal medullary collecting ducts, and that decreased renal medullary tonicity by furosemide treatment decreases RNF183 expression by NFAT5 downregulation.

Germline-Derived Gain-of-Function Variants of Gsα-Coding GNAS Gene Identified in Nephrogenic Syndrome of Inappropriate Antidiuresis.

BACKGROUND: The stimulatory G-protein α-subunit encoded by GNAS exons 1-13 (GNAS-Gsα) mediates signal transduction of multiple G protein-coupled receptors, including arginine vasopressin receptor 2 (AVPR2). Various germline-derived loss-of-function GNAS-Gsα variants of maternal and paternal origin have been found in pseudohypoparathyroidism type Ia and pseudopseudohypoparathyroidism, respectively. Specific somatic gain-of-function GNAS-Gsα variants have been detected in McCune-Albright syndrome and may result in phosphate wasting. However, no germline-derived gain-of-function variant has been identified, implying that such a variant causes embryonic lethality. METHODS: We performed whole-exome sequencing in two families with dominantly inherited nephrogenic syndrome of inappropriate antidiuresis (NSIAD) as a salient phenotype after excluding a gain-of-function variant of AVPR2 and functional studies for identified variants. RESULTS: Whole-exome sequencing revealed two GNAS-Gsα candidate variants for NSIAD: GNAS-Gsα p.(F68_G70del) in one family and GNAS-Gsα p.(M255V) in one family. Both variants were absent from public and in-house databases. Of genes with rare variants, GNAS-Gsα alone was involved in AVPR2 signaling and shared by the families. Protein structural analyses revealed a gain-of-function-compatible conformational property for p.M255V-Gsα, although such assessment was not possible for p.F68_G70del-Gsα. Both variants had gain-of-function effects that were significantly milder than those of McCune-Albright syndrome-specific somatic Gsα variants. Model mice for p.F68_G70del-Gsα showed normal survivability and NSIAD-compatible phenotype, whereas those for p.M255V-Gsα exhibited severe failure to thrive. CONCLUSIONS: This study shows that germline-derived gain-of-function rare variants of GNAS-Gsα exist and cause NSIAD as a novel Gsα-mediated genetic disease. It is likely that AVPR2 signaling is most sensitive to GNAS-Gsα's gain-of-function effects.

Mapping of a responsible region for sex reversal upstream of Sox9 by production of mice with serial deletion in a genomic locus.

Sox9 plays critical roles in testis formation. By mapping four familial cases of disorders of sexual development, a 32.5 kb sequence located far upstream of SOX9 was previously identified as being a commonly deleted region and named the XY sex reversal region (XYSR). To narrow down a responsible sequence in XYSR, we generated mutant mice with a series of deletions in XYSR by application of the CRISPR/Cas9 system, using a mixture of sgRNAs targeting several kilobase (kb) intervals in the region. When the whole XYSR corresponding sequence in mice was deleted in XY karyotype individuals, the mutation resulted in female offspring, suggesting that an expression mechanism of SOX9/Sox9 through XYSR is conserved in human and mouse. Male-to-female sex reversal was found in mice with a 4.8 kb deletion. We identified a sequence conserved among humans, mice, and opossum, the deletion of which (783 bp) in mice resulted in male-to-female sex reversal. The sequence includes a recently reported critical gonad enhancer for Sox9. Although it cannot be concluded that the human sequence is responsible for XYSR, it is likely. This method is applicable for fine mapping of responsible sequences for disease-causing deletions especially with regard to rare diseases.

Creation of mutant mice with megabase-sized deletions containing custom-designed breakpoints by means of the CRISPR/Cas9 system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for creation of mutant mice with mutations mirroring those in human patients. Various methods have been developed for this purpose, including deletions, inversions, and translocations. So far, mutant mice with deletions of up to 1.2 megabases (Mb) have been generated by microinjection of the CRISPR/Cas9 system into fertilized eggs; however, a method for generation of mutant mice with a deletion of more than several Mb size is necessary because such deletions have often been identified as possible causes of human diseases. With an aim to enable the generation of disease models carrying large deletions with a breakpoint in custom-designed sequences, we developed a method for induction of an Mb-sized deletion by microinjection of a pair of sgRNAs, Cas9, and a donor plasmid into fertilized eggs. Using this method, we efficiently and rapidly generated mutant mice carrying deletions up to 5 Mb.

Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for genome editing. In this study, using a microinjection-based CRISPR/Cas9 system, we efficiently generated mouse lines carrying mutations at the Irx3 and Irx5 loci, which are located in close proximity on a chromosome and are functionally redundant. During the generation of Irx3/Irx5 double mutant mice, a deletion of ~0.5 Mb between the Irx3 and Irx5 loci was unintentionally identified in 6 out of 27 living pups by PCR based genotyping analysis. This deletion was confirmed by DNA fluorescence in situ hybridization analysis of fibroblasts. These results indicate that the mutant mice with a deletion of at least 0.5 Mb in their genome can be generated by the CRISPR/Cas9 system through microinjection into fertilized eggs. Our findings expand the utility of the CRISPR/Cas9 system in production of disease model animals with large deletions.