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    • Recently new genome modifying methods using sequence

    2018-10-20

    Recently, new genome-modifying methods using sequence-specific endonucleases, such as zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR/Cas9) systems, were developed one after another (Gaj et al., 2013). In addition, gene-targeting experiments with these nuclease systems were very recently reported to be successful in SSC lines (Fanslow et al., 2014; Wu et al., 2015, Chapman et al., 2015).
    Results
    Discussion In the present study, we succeeded in genome modification of the Rosa26 locus and Stra8 gene using TALEN or CRISPR/Cas9 systems, with extremely high-level efficiency and accuracy. The accuracy of CRISPR/Cas9 was comparable to that of TALEN and was actually perfect, probably owing to the double-nicking system we used. In a previous report, the gene-targeting efficiency in GS glp-2 without genome-editing technology was as low as 1.7%, as 2 out of 120 clones were selected (Kanatsu-Shinohara et al., 2006a). Using TALEN and CRISPR/Cas9 in the present study, the targeting efficiency appeared surprisingly high, because most of the picked-up colonies showed successful gene targeting. This efficiency is comparable to or even higher than that in other reports using TALEN with human ESCs, which showed 42% to 100% (Hockemeyer et al., 2011). These results confirm that DSB induction can promote homologous recombination significantly in GS cells as well. Very recently, two groups reported gene-targeting experiments with GS cells using ZFN and CRISPR/Cas9 systems, respectively (Fanslow et al., 2014; Wu et al., 2015). Fanslow et al.’s group reported to have succeeded in genome editing in GS cells with the ZFN system. The targeted GS cells, however, appeared to have lost their spermatogenic ability, being unable to differentiate into sperm following transplantation into the host testis. Wu et al.’s group treated a genetic defect of a single-nucleotide deletion in a mutant mouse, which causes cataract, using CRISPR/Cas9 in GS cells. The sequence covering the deletion site was replaced by the 89 bp of single-stranded oligodeoxynucleotides, which resulted in correction of the mutation. The treated GS cells, after transplantation into the host mouse testis, differentiated into haploid cells, which were used for the production of progeny not showing cataract. These two reports demonstrated that GS cells, as well as many other somatic cells or cell lines, can be genomically manipulated with those sequence-specific endonuclease systems. The latter one, in particular, showed that the correction of a mutated sequence was possible in GS cells, whereby genetic diseases caused by such mutations can be eliminated from subsequent generations. In the present study, we showed that both TALEN and double-nicking CRISPR/Cas9 were effective for genome editing in GS cells. In fact, we demonstrated that 2.8–4.7 kbp of transgenes could be successfully introduced accurately into the target site in the genome of GS cells. This result suggests that genome-editing technology in GS cells allows us to repair more extensive mutations than those involving single nucleotides. More importantly, our study demonstrated that the Rosa26-GS cells retained full capacity for complete spermatogenesis up to the formation of competent sperm, which was not shown in either of the two previous studies. It is noteworthy that GS cells are prone to lose spermatogenic potential, possibly after being cultured under stressful conditions such as overgrowth, repeated freezing and thawing, or high passage numbers. In an extreme case, GS cells not only lose spermatogenic ability but also gain multipotency, turning into ESC-like cells (Kanatsu-Shinohara et al., 2004). In order to make GS cells useful for the study of spermatogenesis, this characteristic of GS cells must be kept in mind. In the present study, we successfully showed that the spermatogenic ability of GS cells was disturbed when the Stra8 gene was disrupted. This result demonstrated that GS cells can be used to test whether or not a particular gene or genes are functioning in spermatogenesis when cultured under the appropriate conditions.