Yoshitaka Fujihara

Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.

Comparison of gene expression in male and female mouse blastocysts revealed imprinting of the X-linked gene, Rhox5/Pem, at preimplantation stages.

Mammalian male preimplantation embryos develop more quickly than females . Using enhanced green fluorescent protein (EGFP)-tagged X chromosomes to identify the sex of the embryos, we compared gene expression patterns between male and female mouse blastocysts by DNA microarray. We detected nearly 600 genes with statistically significant sex-linked expression; most differed by 2-fold or less. Of 11 genes showing greater than 2.5-fold differences, four were expressed exclusively or nearly exclusively sex dependently. Two genes (Dby and Eif2s3y) were mapped to the Y chromosome and were expressed in male blastocysts. The remaining two (Rhox5/Pem and Xist) were mapped to the X chromosome and were predominantly expressed in female blastocysts. Moreover, Rhox5/Pem was expressed predominantly from the paternally inherited X chromosome, indicating sex differences in early epigenetic gene regulation.

Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa

Formation of spermatozoa of normal shape, number, and motility is insufficient for the male siring of pups. The spermatozoa must be accompanied by sound fertilizing ability. We found that males with disrupted testis-expressed gene 101 (Tex101) produce normal-looking but fertilization-incompetent spermatozoa, which were accompanied by a deficiency of a disintegrin and metallopeptidase domain 3 (ADAM3) on sperm plasma membrane. It was also found that the existence of TEX101 on spermatozoa was regulated by angiotensin-converting enzyme (ACE). The removal of GPI-anchored protein TEX101 by ACE was essential to produce fertile spermatozoa, and the function of ACE was not depending on its well-known peptidase activity. The finding of TEX101 as a unique specific substrate for ACE may provide a potential target for the production of an awaited contraceptive medicine for men.

SPACA1-deficient male mice are infertile with abnormally shaped sperm heads reminiscent of globozoospermia

SPACA1 is a membrane protein that localizes in the equatorial segment of spermatozoa in mammals and is reported to function in sperm-egg fusion. We produced a Spaca1 gene-disrupted mouse line and found that the male mice were infertile. The cause of this sterility was abnormal shaping of the sperm head reminiscent of globozoospermia in humans. Disruption of Spaca1 led to the disappearance of the nuclear plate, a dense lining of the nuclear envelope facing the inner acrosomal membrane. This coincided with the failure of acrosomal expansion during spermiogenesis and resulted in the degeneration and disappearance of the acrosome in mature spermatozoa. Thus, these findings clarify part of the cascade leading to globozoospermia.

Sperm equatorial segment protein 1, SPESP1, is required for fully fertile sperm in mouse

Mammalian fertilization is a multistep process that culminates in the fusion of the sperm and egg plasma membrane. It is widely accepted that the equatorial segment of the acrosome-reacted sperm is important in initiating fusion with the egg plasma membrane during fertilization. There are various proteins known to be distributed only in the equatorial segment of sperm. The role of these proteins must be clarified to understand the membrane fusion process. We produced a mouse line that lacked SPESP1 (sperm equatorial segment protein 1) and analyzed the fertilizing ability of the sperm. The average number of pups that were fathered by Spesp1+/− and Spesp1−/− males was significantly lower than that of wild-type fathers. In these mouse lines, fewer sperm were found to migrate into oviducts and fewer eggs were fertilized. The Spesp1+/− and Spesp1−/− sperm showed a lower fusing ability compared with the wild-type sperm. The disruption of Spesp1 was shown to cause an aberrant distribution of various sperm proteins. Moreover, scanning electron microscopy revealed that the membrane in the equatorial segment area, which usually forms an acrosomal sheath, disappears after acrosome reaction in Spesp1-deficient mice. It was demonstrated that SPESP1 is necessary to produce the fully ‘fusion competent’ sperm.

Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes

The recombinant clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas system has opened a new era for mammalian genome editing. Here, we constructed pX330 plasmids expressing humanized Cas9 (hCas9) and single guide RNAs (sgRNAs) against mouse genes and validated them both in vitro and in vivo. When we randomly chose 291 target sequences within protein coding regions of 73 genes, an average number of off‐target candidates (exact match 13 nucleotides from 3′ target and NGG) found by Bowtie software was 9.2 ± 21.0 (~1.8 times more than the estimated value, 5.2). We next validated their activity by observing green fluorescence reconstituted by homology dependent repair (HDR) of an EGFP expression cassette in HEK293T cells. Of the pX330 plasmids tested, 81.8% (238/291) were found to be functional in vitro. We finally injected the validated pX330 plasmids into mouse zygotes in its circular form against 32 genes (including two genes previously tested) and obtained mutant mice at a 52.9 ± 22.3% (100/196) mutation frequency. Among the pups carrying mutations on the autosomes, 43.6% (47/96) carried the mutations in both alleles. When off‐target candidate sites were examined in 63 mutant mice, 0.8% (3/382) were mutated. We conclude that our method provides a simple, efficient, and cost‐effective way for mammalian gene editing that is applicable for large scale mutagenesis in mammals.

Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive

Mouse work may lead to male contraceptive Unintended pregnancies are a major health issue worldwide. Although oral contraceptives were developed decades ago for use in women, there are no male oral contraceptives. Miyata et al. show that genetic deletion or drug inhibition of sperm-specific calcineurin enzymes in mice cause male sterility (see the Perspective by Castaneda and Matzuk). Although calcineurin inhibitors resulted in male infertility within 2 weeks, fertility recovered 1 week after halting drug administration. Because the sperm-specific calcineuin complex is also found in humans, its inhibition may be a strategy for developing reversible male contraceptives. Science, this issue p. 442, see also p. 385 Inhibiting the function of a protein involved in sperm maturation may help in the development of future male contraceptives. [Also see Perspective by Castaneda and Matzuk] Calcineurin inhibitors, such as cyclosporine A and FK506, are used as immunosuppressant drugs, but their adverse effects on male reproductive function remain unclear. The testis expresses somatic calcineurin and a sperm-specific isoform that contains a catalytic subunit (PPP3CC) and a regulatory subunit (PPP3R2). We demonstrate herein that male mice lacking Ppp3cc or Ppp3r2 genes (knockout mice) are infertile, with reduced sperm motility owing to an inflexible midpiece. Treatment of mice with cyclosporine A or FK506 creates phenocopies of the sperm motility and morphological defects. These defects appear within 4 to 5 days of treatment, which indicates that sperm-specific calcineurin confers midpiece flexibility during epididymal transit. Male mouse fertility recovered a week after we discontinued treatment. Because human spermatozoa contain PPP3CC and PPP3R2 as a form of calcineurin, inhibition of this sperm-specific calcineurin may lead to the development of a reversible male contraceptive that would target spermatozoa in the epididymis.

Production of mouse pups from germline transmission-failed knockout chimeras.

Occasionally, chimeras do not transmit the gene of interest to pups in gene disruption experiments. However, the risk of failure could be reduced if we could identify embryonic stem (ES)-derived germ cells in the testis. Here, we report the production of pups from three lines of infertile chimeric male mice and the establishment of knockout lines by combining green fluorescent protein-tagged ES cells with intracytoplasmic sperm injection.

GPI-Anchored Protein Complex, LY6K/TEX101, Is Required for Sperm Migration into the Oviduct and Male Fertility in Mice

ABSTRACT A disintegrin and metallopeptidase domain 3 (ADAM3) is a sperm membrane protein reported to be critical for both sperm migration from the uterus into the oviduct in vivo and sperm binding to the zona pellucida in vitro. In order for ADAM3 to be expressed on the sperm surface, the interaction with testis-expressed gene 101 (TEX101), a glycosylphosphatidylinositol (GPI)-anchored protein, is essential. Without TEX101, ADAM3 is degraded during sperm transition through the epididymis. However, it is also known that TEX101 has to be shed and to disappear from testicular germ cells (TGCs) by the GPI-anchored protein-releasing activity of angiotensin-converting enzyme (ACE) for the correct localization of ADAM3 on the mature sperm surface to take place. Here, we found that in a mouse line with a disruption for another testis-specific GPI-anchored protein, lymphocyte antigen 6 complex, locus K (LY6K), the male mice became infertile and demonstrated a phenotype similar to that found in Adam3–/–, Tex101–/–, and Ace–/– mice. LY6K interacted with TEX101 and ADAM3 in the TGCs but disappeared from mature spermatozoa. Differing from more than 10 previously known gene knockout mouse lines that showed male infertility by impaired sperm migration into the oviduct, spermatozoa from Ly6K–/– mice had no aberrance in ADAM3. Thus, LY6K is a newly identified factor involved in sperm fertilizing ability. The lack of effect on ADAM3 in Ly6K–/– mice is indicative of an as yet undefined pathway in the mouse.

CRISPR/Cas9-Based Genome Editing in Mice by Single Plasmid Injection

CRISPR/Cas-mediated genome modification has opened a new era for elucidating gene function. Gene knockout mice can be generated by injecting humanized Cas9 (hCas9) mRNA and guide RNA (sgRNA) into fertilized eggs. However, delivery of RNA instead of DNA to the fertilized oocyte requires extra preparation and extra care with storage. To simplify the method of delivery, we injected the circular pX330 plasmids expressing both hCas9 and sgRNA and found that mutant mice were generated as efficiently as with RNA injection. Different from the linearized plasmid, the circular plasmid decreased the chance of integration into the host genome. We also developed the pCAG-EGxxFP reporter plasmid for evaluating the sgRNA activity by observing EGFP fluorescence in HEK293T cells. The combination of these techniques allowed us to develop a rapid, easy, and reproducible strategy for targeted mutagenesis in living mice. This chapter provides an experimental protocol for the design of sgRNAs, the construction of pX330-sgRNA and pCAG-EGxxFP-target plasmids, the validation of cleavage efficiency in vitro, and the generation of targeted gene mutant mice. These mice can be generated within a month.