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0Please cite this article in press as: Sato et al., Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9, Stem Cell Reports (2015), http://dx.doi.org/10.1016/j.stemcr.2015.05.011
Stem Cell Reports
Article
-OPEN ACCESS
Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9
Takuya Sato,1'* Tetsushi Sakuma,2 Tetsuhiro Yokonishi,3 Kumiko Katagiri,1 Satoshi Kamimura,4 Narumi Ogonuki,4 Atsuo Ogura,4 Takashi Yamamoto,2 and Takehiko Ogawa1,3,*
laboratory of Proteomics, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama 236-0004, Japan
2Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan 3Department of Urology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan 4RIKEN, Bioresource Center, Ibaraki 305-0074, Japan
Correspondence: tsato@yokohama-cu.ac.jp (T.S.), ogawa@yokohama-cu.ac.jp (T.O.) http://dx.doi.org/10.10167j.stemcr.2015.05.011
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
SUMMARY
Mouse spermatogonial stem cells (SSCs) can be cultured for multiplication and maintained for long periods while preserving their spermatogenic ability. Although the cultured SSCs, named germline stem (GS) cells, are targets of genome modification, this process remains technically difficult. In the present study, we tested TALEN and double-nicking CRISPR/Cas9 on GS cells, targeting Rosa26 and Stra8 loci as representative genes dispensable and indispensable in spermatogenesis, respectively. Harvested GS cell colonies showed a high targeting efficiency with both TALEN and CRISPR/Cas9. The Rosa26-targeted GS cells differentiated into fertility-competent sperm following transplantation. On the other hand, Stra8-targeted GS cells showed defective spermatogenesis following transplantation, confirming its prime role in the initiation of meiosis. TALEN and CRISPR/Cas9, when applied in GS cells, will be valuable tools in the study of spermatogenesis and for revealing the genetic mechanism of spermatogenic failure.
INTRODUCTION
Spermatogonial stem cells (SSCs) are pivotal for maintaining the lifelong sperm production of mammalian males. They maintain a delicate balance between self-renewal and commitment to differentiate for sperm production. Mouse SSCs can be maintained under culture conditions, continuing to proliferate for up to 2 years without losing their spermatogenic ability as well as maintaining genetic, epigenetic, and karyotypic stability. The cultured SSCs, named germline stem (GS) cells, are now useful in research on various aspects of spermatogenesis. (Kanatsu-Shinohara et al., 2003;Kubota et al., 2004). In order to make GS cells more widely applicable for the study of spermatogenesis at the genetic and molecular levels, it is desirable to be able to modify their genome without disturbing their spermato-genic ability. In fact, this was shown to be possible by the transfection of genes into GS cells (Kanatsu-Shinohara et al., 2005) or by targeting particular genes in GS cells by homologous recombination (Kanatsu-Shinohara et al., 2006a). Successfully treated GS cells were selected during the subsequent cultivation and transplanted into the seminiferous tubules of host mice to produce genome-modified sperm. These results proved that GS cells, like embryonic stem cells (ESCs), can be used as genetically modified cells for the production of animals with modified genomes. Thus, GS cells appear to be a useful tool not only for the study of spermatogenesis but also for many other areas of biological research. However, it is rather difficult to manip-
ulate the genome of GS cells compared to ESCs because of their low transfection efficiency, slow cycling, and difficulty in cloning (Kanatsu-Shinohara et al., 2005, 2006b; Tamm etal., 2013). Thus, targeted genome-modifying experiments using GS cells are not common, and, to the best of our knowledge, only three reports have been published (Iwamori et al., 2012; Kanatsu-Shinohara et al., 2006a, 2008a).
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 (Fanslowetal., 2014;Wuetal., 2015, Chapman et al., 2015).
In the present study, we adopted the double-nicking system of CRISPR/Cas9 and tested it, along with TALEN, on GS cells. Our results demonstrated that these artificial endonu-cleases are powerful tools for genetic-modification experiments in GS cells, providing an effective way to study the genetic mechanism of spermatogenic failure in particular.
RESULTS
Preparation of TALEN and CRISPR/Cas9 Plasmids Targeting the Rosa26 Locus
We designed TALEN and CRISPR/Cas9 constructs targeting a site in the first intron of the Rosa26 locus that was
Please cite this article in press as: Sato et al., Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9, Stem Cell Reports (2015), http://dx.doi.org/10.1016/j.stemcr.2015.05.011
Mouse Rosa26 allele
Pl 2,552 bp P2
a ^■■■■■r-p ^ [T
M W 1 2 3 4 5 6 7 8 9 10 11
Targeting vector
m~EGFP il iresî Puro i
-.-''—r
800 bP 2,832 bp
Targeted --—-^^-^^-
allele |_l| |sa| EGFP |||RES|| Puro
P1 1,678 bp P3
■Xbal
5,384 bp
W 1 2 3 4 5 6 7 8 9 10 11
CRISPR/Cas9
1 2 3 4 5 6 7 8
10K_ 7K-5K ■
10K-7K 5K"
Figure 1. Gene Targeting of the Rosa26 Locus in GS Cells
(A) Schematic overview of the targeting strategy for the Rosa26 Locus. Arrows (P1, P2, and P3) are primer sites for PCR genotyping. P1 and P2 primers are Located at outside the region of homology arms. The P3 primer is Located in the transgene. Expected sizes of PCR products are 2,552 bp in wild-type and 1,678 bp in targeted alleles. The probe used for Southern blot analysis is shown as a green bar. SA, splicing acceptor;IRES, internal ribosomaL entry site;Puro, puromycin-resistance gene.
(B) Targeted GS ceLLs using TALEN express GFP, demonstrated by a merged image of GFP (green) and differential interface contrast (DIC).
(C) Genotyping PCR of GS ceLLs targeted using TALEN or CRISPR/Cas9, seLected by puromycin treatment, and picked up without confirming GFP expression. The M and W Lanes show a 200-bp Ladder marker and wiLd-type controLs, respectiveLy. Asterisks indicate homozygousLy targeted cLones. WT indicates the position of the band derived from the originaL wiLd-type Rosa26 Locus, whiLe TA indicates that from the targeted aLLeLe.
(D) Southern bLot anaLysis of GS ceLLs targeted with TALEN or CRISPR/Cas9. XboJ-digested genomic DNA was hybridized with a GFP probe. Expected fragment size is 7 kb.
ScaLe bar, 100 mm (B).
previously targeted successfully with the ZFN system (Perez-Pinera et al., 2012) (Figure S1A). The Rosa26 locus was chosen because inserted genes will be expressed ubiquitously without any additional effects on the characteristics of target cells (Zambrowicz et al., 1997). Thus, the targeted GS cells were expected to maintain spermato-genic competency. We used the Platinum TALEN plasmid, which has enhanced cleavage activity compared to conventional TALEN (Sakuma et al., 2013). As for CRISPR/ Cas9, we chose the pX335 CRISPR/Cas9 expression vector, which uses a double-nicking strategy, by which the occurrence of a double-strand break (DSB) becomes more specific and accurate (Mali et al., 2013;Ran et al., 2013). To confirm the target-site-specific cutting efficiency of the TALEN and CRISPR/Cas9-expressing vectors, we performed a Surveyor nuclease assay with the cell line
15P-1, which confirmed their accurate and sufficient cutting efficiency (Figure S1B).
Gene Targeting of the Rosa26 Locus Using TALEN and CRISPR/Cas9
The construct of the targeting vector was composed of a splice acceptor, an EGFP, internal ribosome entry sites, and puromycin-resistant gene sequences, sequentially connected in this order and flanked with short homologous sequences on both the 5' and 3' sides (Figure 1A). When gene targeting is successful, the targeted GS cells express GFP and become resistant to puromycin.
We performed transfection with the targeting vector together with the TALEN or CRISPR/Cas9-expressing vector by electroporation of GS cells derived from the wildtype mouse. Approximately 2 weeks after electroporation,
Please cite this article in press as: Sato et al., Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9, Stem Cell Reports (2015), http://dx.doi.org/10.1016/j.stemcr.2015.05.011
Table 1. Summary of Targeting Experiments Using TALEN and CRISPR/Cas9
Targeting Vector No. of Clones Analyzed No. of Non-targeted Clones No. of Targeted Clones No. of Random Integration Homozygous Insertion
TALEN Rosa-SA-GFPiPuro 12 1 11 0 8/11
CRISPR Rosa-SA-GFPiPuro 8 0 8 0 5/8
CRISPR Stra8-tdTomato-PGK-Puro 18 0 18 0 3/9 6/9a
aDouble doses of CRISPR/Cas9 and targeting vectors were used for transfection.
the selection of GS cells with puromycin was initiated. Pu-romycin-resistant colonies became apparent in week, and some of them were picked up for cloning without checking GFP expression. We obtained 12 and 8 clones of transfected GS cells with TALEN and CRISPR/Cas9, respectively. Excluding one clone produced by TALEN, every GS cell clone expressed GFP (Figure 1B). We used PCR genotyp-ing to examine whether the Rosa26-targeting construct was integrated into the target site. All of the 11 and 8 GFP-expressing clones induced by TALEN and CRISPR/Cas9, respectively, showed the correct insertion of the target sequence into the Rosa26 locus (Figure 1C). Among them, 8 out of the 11 clones and 5 out of the 8 clones showed homozygous insertion (Figure 1C). Southern blotting analysis demonstrated that there was no integration of the donor construct other than at the target site (Figure 1D). Collectively, targeting efficiencies mediated by TALEN and CRISPR/Cas9 were quite high: 11 clones out of 12 and all 8 clones, respectively (Table 1). Thus, these results indicate that genome editing mediated by the TALEN or CRISPR/ Cas9 double-nicking system was highly effective in introducing a transgene at target site in the genome of GS cells.
Spermatogenic Ability of Targeted GS Cells
We tested whether those GS cells with a target construct at the Rosa26 site (Roas26-GS cell) remain as functional SSCs and differentiate into competent sperm. We transplanted lines of Rosa26-GS cells into the testes of W/Wv (W) mice whose testes contain very few germ cells because of genetic defects in the c-kit gene, making them an ideal host testis. The transplanted Rosa26-GS cells colonized the seminiferous tubules and formed stretches of colonies that expressed GFP (Figure 2A). By histological observation, we confirmed that those colonies supported spermatogenesis up to sperm formation. The immunohistologic features, as well as regular histological findings on periodic acid-Schiff (PAS) stain, demonstrated normal spermatogenesis (Figures 2B, 2D, and 2E). When testis tissue was dissociated, sperm were obtained (Figure 2C). In summary, we transplanted 11 and 4 clones of Rosa26-GS cells of TALEN and CRISPR/Cas9, respectively, resulting in 9 and 4 clones giving rise to sper-
matogenesis, respectively. The two clones of Rosa26-GS cells with the TALEN procedure showed neither colonization nor spermatogenesis in the W mouse testes, for unknown reasons.
Next, we tested the fertility of those sperm by intracy-toplasmic sperm injection (ICSI). We chose the clones of TALENs #8 and #9 (Figures 1C and 1D). All (n = 113) oocytes fertilized by ICSI developed into the two-cell stage on the next day (Table S1). Following embryo transfer, 8 and 23 offspring were delivered from clones #8 and #9, respectively, and all grew healthily (Figure 2F;Table S1). As both clones were targeted homozygously (Figure 1C), all offspring carried the inserted GFP heterozygously (Figure S2A) and expressed GFP throughout the body (Figure 2F). When their reproductive potential was examined by brother-sister mating, they gave birth to offspring, proving its reproductive competency. The second-generation progeny also grew normal and were healthy (Figures S2B-S2D). These results demonstrate that gene modification of GS cells using a TALEN and CRISPR/Cas9 double-nicking system did not affect the characteristics of GS cells, both as stem and spermatogenic cells.
Gene Targeting of Stra8 Gene Using CRISPR/Cas9
The genetic modification techniques when applied to GS cells could be a useful means to examine the function of particular genes during spermatogenesis. Using the double-nicking CRISPR/Cas9, we tried to disrupt the gene of Stimulated by retinoic acid gene 8 (Stra8) by inserting the targeting vector sequence. Stra8 expression is induced by retinoic acid (RA) in spermatogonia and is responsible for entry into meiosis (Baltus et al., 2006;Zhou et al., 2008). We constructed a CRISPR/Cas9-expressing vector, which includes sequence of guide RNA specific to a site in the fourth exon of the Stra8 gene (Table S2), and a targeting vector, which contained tdTomato fluorescence, PGK promoter, and Puro sequences (Figure 3A). We used these three vectors to transfect GFP-GS cells that express GFP constitu-tively. Amounts of vector DNA transfected were 2 and 6 mg of CRISPR/Cas9 and targeting vectors, respectively, or double doses of them. Nine colonies resistant to puromycin
Please cite this article in press as: Sato et al., Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9, Stem Cell Reports (2015), http://dx.doi.org/10.1016/j.stemcr.2015.05.011
Day 159
Figure 2. Spermatogenic Ability of Targeted GS Cells
(A) Recipient testes transpLanted with GS ceLLs targeted by TALEN and CRISPR/Cas9 contained seminiferous tubuLes expressing GFP, which indicates GS ceLL coLonization. A non-transpLanted recipient testis (W mouse) did not show GFP expression.
(B) Immunostaining of recipient testis tissue cryosections, transpLanted with Rosa26-GS ceLLs targeted by TALEN. Peanut aggLutinin (PNA), which reacts with the acrosome, appears as red. Anti-GFP (green) and counterstain with Hoechst dye (bLue) were merged.
(C) FLageLLated sperm derived from mechanicaLLy dissociated host testis transpLanted with Rosa26-GS ceLLs targeted with TALEN.
(D) Immunostaining of recipient testis tissue transpLanted with Rosa26-GS ceLLs targeted by CRISPR. PNA (red), anti-GFP (green), and counterstain with Hoechst dye (bLue) were merged.
(E) A recipient testis transpLanted with GS ceLLs targeted by CRISPR/Cas9 shows extensive coLonization on day 159 after transpLantation (Left). This testis processed for histoLogicaL examination showed normaL spermatogenesis with PAS stain (right).
(F) Offspring obtained using the ICSI procedure with sperm derived from two Rosa26-GS ceLL cLones (#8 and #9) targeted with TALEN. GFP expression throughout the bodies of the offspring was confirmed under UV Light.
ScaLe bars represent 1 mm (A and E, Left), 200 mm (E, right), 100 mm (B), 50 mm (D), or 20 mm (C).
were picked up from each plasmid dosage group, resulting in 18 clones in total (termed Stra8-GS cells). Each clone was examined by PCR genotyping and Southern blotting to show that the Stra8-targeting construct had been integrated into the target site correctly, without random integration (Figures 3B and 3C). The homologous insertion was noted in three out of nine clones and six out of nine clones, respectively, for each dose set of vectors (Figure 3B).
When Stra8-GS cells were cultured in a medium with RA added for 2 days, almost all Stra8-GS cells expressed tdTo-
mato, confirming that the expression of the inserted construct was induced by RA (Figure 3D). We then chose a line of homozygously knocked in Stra8-GS cells and transplanted them into the testes of W mice. Eighty-four days after, on analysis, the recipient testis showed extensive colonization by GS cells, appearing as tubules colored with GFP (Figure 3E). The colonized cells, however, appeared to be restricted to the periphery of the seminiferous tubules, indicating that they were located on the basement membrane without differentiation to the inside of the
Please cite this article in press as: Sato et al., Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9, Stem Cell Reports (2015), http://dx.doi.org/10.1016/j.stemcr.2015.05.011
Stra8 allele | 3 | Targeting vector
^ .....1,687 bp
B CRISPR 2 pg / TV 6pg CRISPR 4 pg / TV 12 pg
* 2 3 4 5 6 7 8 9 *0 11 12 13 1415 16 1* 18 W
640 bp
700 bp
Targeted allele
6.6 kb 3,678 bP
......■■",■.........Spel
Probe f
41 Tomato | IpgkII Purol 0 H 7K-
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
.............<-
P1 1,542 bp P3
Figure 3. Targeting of the Stra8 Gene in GS Cells Using CRISPR/Cas9
(A) Schematic overview of the targeting strategy for the Stra8 gene. Arrows (P1, P2, and P3) show primer sites for the genotyping PCR. P1 and P2 primers are Located outside the region of the homology arm. The P3 primer is Located in the transgene. Expected sizes of the PCR products are 1,687 bp in wiLd-type and 1,542 bp in targeted alleles. The probe used for Southern bLot analysis is shown as a green bar.
(B) PCR genotyping of StroS-GS ceLLs targeted with CRISPR/Cas9. The Leftmost Lane shows a 200-bp Ladder marker. The W Lane shows wiLdtype controls. Asterisks indicate homozygousLy targeted cLones.
(C) Southern bLot anaLysis of StroS-GS ceLLs. Spel-digested genomic DNA was hybridized with the tdTomoto probe. The expected fragment size is 6.6 kb.
(D) Expression of the tdTomato was induced in StroS-GS ceLLs by RA addition to the medium in 2 days. A merged image of tdTomato (red), GFP (green), and DIC is shown.
(E) A recipient testis transpLanted with StroS-GS ceLLs shows extensive coLonization of the GS ceLLs. The inset shows a magnified view of the same testis.
(F) H&E staining of a cryosection of the recipient testis, showing no meiotic differentiation.
(legend continued on next page)
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tubules. Histological analysis showed that there were tubules containing spermatogonia lining the basement membrane with no progression of spermatogenesis (Figure 3F), which was exactly the same as the phenotype of StraS-deficient mice (Baltus et al., 2006). Furthermore, we performed an immunohistological examination with antibodies to tdTomato and GFP and found that the expression of tdTomato in spermatogonia was different from tubule to tubule (Figure 3G). This observation is consistent with a previous report that the expression of StraS was periodic in spermatogonia and spermatocytes, depending on the wave of the seminiferous epithelium (Zhou et al., 2008). Together, these results demonstrate that CRISPR/Cas9-mediated gene disruption is effective in GS cells, the effects of which could appear as a particular phenotype in sper-matogenesis following transplantation into the host testis.
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 cells without genome-editing technology was as low as 1.7%, as 2 out of 120 clones were selected (Kanatsu-Shino-hara 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.
As was shown in this study, along with many other previous studies, TALEN and double-nicking CRISPR/ Cas9 genome-editing techniques are significantly accurate, with only a negligible risk of random gene disruption, in the drug-selected clones. This result indicates that disrupting a particular gene without disturbing any other genes in GS cells could replace gene-manipulating
(G) Immunostaining of recipient testis tissue transplanted with Stra8-GS ceLLs, confirming colonization of the GS cells, some of which express tdTomato. tdTomato (red), anti-GFP (green), and counterstain with Hoechst dye (blue) were merged. The red and white dotted Lines delineate seminiferous tubules containing tdTomato-positive and tdTomato-negative spermatogonia, respectively. Scale bars represent 1 mm (E), 100 mm (D), or 50 mm (F and G).
Please cite this article in press as: Sato et al., Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9, Stem Cell Reports (2015), http://dx.doi.org/10.1016/j.stemcr.2015.05.011
technologies with ESCs in the study of spermatogenesis. When GS cells of species other than mice become available for gene targeting as well, it may become possible to study the role of particular genes in their spermatogenesis, which would be rather difficult or practically impossible with ESC technology. In fact, culturing of the SSCs of the rat, hamster, and rabbit was reported to be feasible (Hamra et al., 2005;Kanatsu-Shinohara et al., 2008b;Ku-bota et al., 2011;Ryu et al., 2005), making this research strategy realistic. In addition, whole-genome sequencing is now available, owing to the many technological innovations in that field. Based on such a huge volume of genome information along with sophisticated analysis methods, genetic analysis of infertile patients (azoo-spermia in particular) could reveal genes responsible for spermatogenic failure. The genome-modifying technologies shown in this study will be extremely useful to examine such candidate genes responsible for spermato-genic defects in the future.
EXPERIMENTAL PROCEDURES
Animals and Culture of GS Cells
To establish wild-type GS and GFP-GS cells, male C57BL/6 (CLEA Japan) and C57BL/6-Tg (CAG/Acr-GFP) (Okabe et al., 1997) trans-genic mice, respectively, were mated with wild-type ICR females (CLEA Japan) to produce F1 pups. The testes of F1 pups were dissected out and used as a source of GS cells. The culture medium used for GS cells was StemPro-34 based (Kanatsu-Shinohara et al., 2003), with minor modifications (Sato et al., 2013). Wild-type GS and GFP-GS cells were used for targeting experiments of Rosa26 and Stra8, respectively. WBB6F1-W/Wv (Japan SLC), at 6-15 weeks old, were used as recipients for spermatogonial transplantation. W/Wv allows colonization of GS cells of ICR/B6 background without apparent rejection (Araki et al., 2010). The spermatogenic ability of GS cell lines was checked by transplantation into the testes of W/Wv before using for targeting experiments (Figures S3A and S3B). All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation (Animal Research Center of Yokohama City University, Yokohama, Japan).
Gene Targeting of GS Cells
GS cells used for targeting to Rosa26 and Stra8 were at passages 6 and 8, respectively. They were harvested using 0.25% trypsin, and 1.5-4 x 106 cells resuspended in T solution of Cell Line Nucleofector Kit were electroporated with 6 mg of targeting vector plasmids and 2 mg of each TALEN or CRISPR/Cas9-expressing plasmid using Nucleofector 2b with program A-023. Selection with puromycin (0.15 mg/ml) was performed 10-14 days after electroporation. When puromycin-resistant colonies expanded, usually at ~4 weeks after electroporation, we picked up individual colonies using a micropipette (P200 pipetman) under an inverted microscope for successive culturing.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, three figures, and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr.2015.05. 011.
ACKNOWLEDGMENTS
We thank Ms. Y. Koushige for technical assistance. This study was funded by a Grant-in-Aid for Scientific Research on Innovative Areas, ''Mechanisms regulating gamete formation in animals'' 25114007 (to T.O.); a Grant-in-Aid for Scientific Research (B) 24390371 (to T.O.); Strategic Research Promotion of Yokohama City University, No. IR2502 (to T.O.); and a Grant-in-Aid for Young Scientists (A) 26713012 (to T. Sato).
Received: February 25, 2015 Revised: May 15, 2015 Accepted: May 18, 2015 Published: June 18, 2015
REFERENCES
Araki, Y., Sato, T., Katagiri, K., Kubota, Y., Araki, Y., and Ogawa, T. (2010). Proliferation of mouse spermatogonial stem cells in microdrop culture. Biol. Reprod. 83, 951-957.
Baltus, A.E., Menke, D.B., Hu, Y.-C., Goodheart, M.L., Carpenter, A.E., de Rooij, D.G., and Page, D.C. (2006). In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premei-otic DNA replication. Nat. Genet. 38, 1430-1434. Chapman, K.M., Medrano, G.A., Jaichander, P., Chaudhary, J., Waits, A.E., Nobrega, M.A., Hotaling, J.M., Ober, C., and Hamra, F.K. (2015). Targeted Germline Modifications in Rats Using CRISPR/Cas9 and Spermatogonial Stem Cells. Cell Rep. 10, 1828-1835.
Fanslow, D.A., Wirt, S.E., Barker, J.C., Connelly, J.P., Porteus, M.H., and Dann, C.T. (2014). Genome editing in mouse spermatogonial stem/progenitor cells using engineered nucleases. PLoS ONE 9, e112652.
Gaj, T., Gersbach, C.A., and Barbas, C.F., 3rd. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397-405.
Hamra, F.K., Chapman, K.M., Nguyen, D.M., Williams-Stephens, A.A., Hammer, R.E., and Garbers, D.L. (2005). Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture. Proc. Natl. Acad. Sci. USA 102, 17430-17435. Hockemeyer, D., Wang, H., Kiani, S., Lai, C.S., Gao, Q., Cassady, J.P., Cost, G.J., Zhang, L., Santiago, Y., Miller, J.C., et al. (2011). Genetic engineering of human pluripotent cells using TALE nucle-ases. Nat. Biotechnol. 29, 731-734.
Iwamori, N., Iwamori, T., and Matzuk, M.M. (2012). Characterization of spermatogonial stem cells lacking intercellular bridges and genetic replacement of a mutation in spermatogonial stem cells. PLoS ONE 7, e38914.
Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., Miki, H., Ogura, A., Toyokuni, S., and Shinohara, T. (2003). Long-term proliferation
Please cite this article in press as: Sato et al., Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9, Stem Cell Reports (2015), http://dx.doi.org/10.1016/j.stemcr.2015.05.011
in culture and germline transmission of mouse male germline stem cells. Biol. Reprod. 69, 612-616.
Kanatsu-Shinohara, M., Inoue, K., Lee, J., Yoshimoto, M., Ogonuki, N., Miki, H., Baba, S., Kato, T., Kazuki, Y., Toyokuni, S., et al. (2004). Generation of pluripotent stem cells from neonatal mouse testis. Cell 119, 1001-1012.
Kanatsu-Shinohara, M., Toyokuni, S., and Shinohara, T. (2005). Genetic selection of mouse male germline stem cells in vitro: offspring from single stem cells. Biol. Reprod. 72, 236-240.
Kanatsu-Shinohara, M., Ikawa, M., Takehashi, M., Ogonuki, N., Miki, H., Inoue, K., Kazuki, Y., Lee, J., Toyokuni, S., Oshimura, M., et al. (2006a). Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 103, 8018-8023.
Kanatsu-Shinohara, M., Inoue, K., Lee, J., Miki, H., Ogonuki, N., Toyokuni, S., Ogura, A., and Shinohara, T. (2006b). Anchorage-independent growth of mouse male germline stem cells in vitro. Biol. Reprod. 74, 522-529.
Kanatsu-Shinohara, M., Lee, J., Inoue, K., Ogonuki, N., Miki, H., Toyokuni, S., Ikawa, M., Nakamura, T., Ogura, A., and Shinohara, T. (2008a). Pluripotency of a single spermatogonial stem cell in mice. Biol. Reprod. 78, 681-687.
Kanatsu-Shinohara, M., Muneto, T., Lee, J., Takenaka, M., Chuma, S., Nakatsuji, N., Horiuchi, T., and Shinohara, T. (2008b). Long-term culture of male germline stem cells from hamster testes. Biol. Reprod. 78, 611-617.
Kubota, H., Avarbock, M.R., and Brinster, R.L. (2004). Growth factors essential for self-renewal and expansion of mouse spermatogo-nial stem cells. Proc. Natl. Acad. Sci. USA 101, 16489-16494.
Kubota, H., Wu, X., Goodyear, S.M., Avarbock, M.R., and Brinster, R.L. (2011). Glial cell line-derived neurotrophic factor and endo-thelial cells promote self-renewal of rabbit germ cells with sper-matogonial stem cell properties. FASEB J. 25, 2604-2614.
Mali, P., Aach, J., Stranges, P.B., Esvelt, K.M., Moosburner, M., Ko-suri, S., Yang, L., and Church, G.M. (2013). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833-838.
Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., and Nishi-mune, Y. (1997). 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 407, 313-319.
Perez-Pinera, P., Ousterout, D.G., Brown, M.T., and Gersbach, C.A.
(2012). Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases. Nucleic Acids Res. 40, 3741-3752.
Ran, F.A., Hsu, P.D., Lin, C.Y., Gootenberg, J.S., Konermann, S., Tre-vino, A.E., Scott, D.A., Inoue, A., Matoba, S., Zhang, Y., and Zhang, F. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380-1389. Ryu, B.Y., Kubota, H., Avarbock, M.R., and Brinster, R.L. (2005). Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. Proc. Natl. Acad. Sci. USA 102, 1430214307.
Sakuma, T., Ochiai, H., Kaneko, T., Mashimo, T., Tokumasu, D., Sa-kane, Y., Suzuki, K., Miyamoto, T., Sakamoto, N., Matsuura, S., et al.
(2013). Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Scientific reports 3, 3379. Sato, T., Katagiri, K., Kubota, Y., and Ogawa, T. (2013). In vitro sperm production from mouse spermatogonial stem cell lines using an organ culture method. Nat. Protoc. 8, 2098-2104. Tamm, C., Pijuan Galito, S., and Anneren, C. (2013). A comparative study of protocols for mouse embryonic stem cell culturing. PLoS ONE 8, e81156.
Wu, Y., Zhou, H., Fan, X., Zhang, Y., Zhang, M., Wang, Y., Xie, Z., Bai, M., Yin, Q., Liang, D., et al. (2015). Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogo-nial stem cells. Cell Res. 25, 67-79.
Zambrowicz, B.P., Imamoto, A., Fiering, S., Herzenberg, L.A., Kerr, W.G., andSoriano, P. (1997). Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc. Natl. Acad. Sci. USA 94, 3789-3794. Zhou, Q., Nie, R., Li, Y., Friel, P., Mitchell, D., Hess, R.A., Small, C., and Griswold, M.D. (2008). Expression of stimulated by retinoic acid gene 8 (Stra8) in spermatogenic cells induced by retinoic acid: an in vivo study in vitamin A-sufficient postnatal murine testes. Biol. Reprod. 79, 35-42.
