Introducing a point mutation is a fundamental method used to demonstrate the roles of particular nucleotides or amino acids in the genetic elements or proteins, and is widely used in in vitro experiments based on cultured cells and exogenously provided DNA. However, the in vivo application of this approach by modifying genomic loci is uncommon, partly due to its technical and temporal demands. This leaves many in vitro findings un-validated under in vivo conditions. We herein applied the CRISPR/Cas9 system to generate mice with point mutations in their genomes, which led to single amino acid substitutions in proteins of interest. By microinjecting gRNA, hCas9 mRNA and single-stranded donor oligonucleotides (ssODN) into mouse zygotes, we introduced defined genomic modifications in their genome with a low cost and in a short time. Both single gRNA/WT hCas9 and double nicking set-ups were effective. We also found that the distance between the modification site and gRNA target site was a significant parameter affecting the efficiency of the substitution. We believe that this is a powerful technique that can be used to examine the relevance of in vitro findings, as well as the mutations found in patients with genetic disorders, in an in vivo system.
Recently developed transcription activator-like effector nuclease (TALEN) technology has enabled the creation of knockout mice, even for genes on the Y chromosome. In this study, we generated a knockout mouse for Sry, a sex-determining gene on the Y chromosome, using microinjection of TALEN RNA into pronuclear stage oocytes. As expected, the knockout mouse had female external and internal genitalia, a female level of blood testosterone and a female sexually dimorphic nucleus in the brain. The knockout mouse exhibited an estrous cycle and performed copulatory behavior as females, although it was infertile or had reduced fertility. A histological analysis showed that the ovary of the knockout mouse displayed a reduced number of oocytes and luteinized unruptured follicles, implying that a reduced number of ovulated oocytes is a possible reason for infertility and/or reduced fertility in the KO mouse.I n most mammalian species, sex is determined by the presence or absence of the Y chromosome. In mice, the Sry gene locates to the minimum sex-determining region of the murine Y chromosome 1 , is expressed in the male genital ridge at the time of sex determination 2 and has been proven to be a sex-determining gene based on gain-of-function experiments, i.e., the overexpression of Sry in XX mice achieved with transgenic mouse technology reveals a male phenotype 3 . Also in humans, the SRY gene has been shown to play a pivotal role in sex determination: point mutations or deletions of the SRY gene are found in approximately 15% of XY females, and translocated SRY is found in the autosomes of most XX males 4 . Although there are a number of suggestive observations, it is important to confirm the function of Sry in vivo using loss-of-function analyses with targeted mutagenesis in order to examine whether Sry is the one and only sex-determining gene on the Y chromosome and to finally confirm the Sry gene as the sex-determining gene and provide an animal model of XY female syndrome. However, it is difficult to create knockout (KO) mice of Y-linked genes using conventional homologous recombination-based methods with embryonic stem (ES) cells, as the process requires an adequate length of specific sequences of homologous arms to construct a KO vector, and the Y chromosome is rich in repeats.In 2013, Sung et al. 5 first reported that KO mice can be produced using transcription activator-like effector nuclease (TALEN) technology without conventional homologous recombination-based methods. TALEN protein is an artificial sequence-specific endonuclease that contains Xanthomonas transcription activator-like effector (TALE) and a nuclease domain of FokI restriction endonuclease 6 . DNA binding domain of TALE consists of a tandem repeat of 33-35 amino acid motifs in which there are two critical adjacent amino acid pairs called a repeat variable diresidue (RVD) that determines the binding specificity for single nucleotide. There is a one-toone relationship between the RVD and its recognition nucleotide 7,8 . Using this code, a TALEN can...
Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.
Genome editing, which introduces mutations in genes of interest using artificial DNA nucleases such as the ZFN, TALEN, and CRISPR/Cas9 systems in living cells, is a useful tool for generating mutant animals. Although CRISPR/Cas9 provides advantages over the two other systems, such as an easier vector construction and high efficiency of genome editing, it raises concerns of off-target effects when single guide RNA (gRNA) is used. Recently, FokI-dCas9 (fCas9), a fusion protein comprised of the inactivated mutant form of Cas9 and the DNA nuclease domain of FokI, has been developed. It enables genome editing with reduced risks of off-target effects in mammalian cultured cell lines, as fCas9 requires gRNAs to bind opposite strands with an appropriate distance between them. Here, we demonstrated that fCas9 efficiently generates living mutant mice through microinjection of its mRNA and gRNAs into zygotes. A comparison of the relative efficiencies of genome editing using fCas9 and other modified Cas9s showed that these mutagenesis efficiencies are similar when the targets of two gRNAs are separated by an appropriate distance, suggesting that in addition to the ease of vector construction, fCas9 exhibit high efficiency in producing mutant mice and in reducing risks of off-target effects.
Genomic imprinting is a phenomenon that causes parent-origin-specific monoallelic expression of a small subset of genes, known as imprinted genes, by parentally inherited epigenetic marks. Imprinted genes at the delta-like homolog 1 gene (Dlk1)-type III iodothyronine deiodinase gene (Dio3) imprinted domain, regulated by intergenic differentially methylated region (IG-DMR), are essential for normal development of late embryonic stages. Although the functions of IG-DMR have been reported by generating knockout mice, molecular details of the regulatory mechanisms are not fully understood as the specific sequence(s) of IG-DMR have not been identified. Here, we generated mutant mice by deleting a 216 bp tandem repeated sequence in IG-DMR, which comprised seven repeats of 24 bp motifs, by genome editing technologies. The mutant mice showed that paternal transmission of the deletion allele, but not maternal transmission, induces severe growth retardation and perinatal lethality, possibly due to placental defects. Embryos with a paternally transmitted deletion allele showed biallelic expression of maternally expressed genes and repression of paternally expressed genes. DNA methylation status also showed loss of methylation at IG-DMR and Gtl2-DMR, indicating that the tandem repeat sequence of IG-DMR is one of the functional sequences of IG-DMR, which is required for maintaining DNA methylation imprints of paternal allele at IG-DMR.
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