The Japanese Society of Developmental Biologists
Develop. Growth Differ. (2014) 56, 92–97
doi: 10.1111/dgd.12099
Original Article
Targeted mutagenesis in sea urchin embryos using TALENs Sayaka Hosoi, Tetsushi Sakuma, Naoaki Sakamoto and Takashi Yamamoto* Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8526, Japan
Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos. Key words: sea urchin, targeted mutagenesis, transcription activator-like effector nuclease.
Introduction Recently, genome editing with engineered nucleases has been reported in animals, plants and cultured cells. Engineered nucleases comprise a customized array of DNA-binding modules that interact with a specific DNA sequence and a nuclease domain of the restriction enzyme FokI. When a pair of engineered nucleases is expressed in cells, they bind to their target sequences and induce a double-stranded break (DSB). The induced DSB is repaired by either errorprone non-homologous end-joining (NHEJ) repair or homology-directed repair (HDR). Therefore, site-specific mutations such as insertions and/or deletions can be induced during the NHEJ repair process and a precise genetic modification can be produced with an exogenous donor DNA template during HDR (Joung & Sander 2013). Two types of engineered nuclease have mainly been used for genome editing thus far. One type is zinc finger nucleases (ZFNs), which have zinc fingers as
*Author to whom all correspondence should be addressed. Email: [email protected] Received 13 September 2013; revised 1 October 2013; accepted 2 October 2013. ª 2013 The Authors Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
DNA-binding domains, and the other type is transcription activator-like effector nucleases (TALENs), which have DNA-binding modules of the TAL effector from Xanthomonas sp. Using these engineered nucleases, NHEJ-mediated targeted gene disruption has been successful in various animals, including Caenorhabiditis elegans (Wood et al. 2011), Drosophila (Bibikova et al. 2002; Liu et al. 2012), cricket (Watanabe et al. 2012), Ciona (Kawai et al. 2012), medaka (Ansai et al. 2012, 2013), zebrafish (Doyon et al. 2008; Meng et al. 2008; Hisano et al. 2013), frogs (Young et al. 2011; Suzuki et al. 2013), mice (Carbery et al. 2010; Sung et al. 2013), rats (Mashimo et al. 2010, 2013), and pigs (Hauschild et al. 2011; Carlson et al. 2012). In contrast, reports on HDR-mediated targeted gene addition are limited to a few species, such as Drosophila (Beumer et al. 2008), zebrafish (Zu et al. 2013) and mice (Cui et al. 2011). Previously, we established a construction system for ZFNs using a bacterial one-hybrid screening with ZF randomized libraries and showed that the ZFNs introduced mutations at their target sites in sea urchin embryos (Ochiai et al. 2010). Furthermore, ZFN-mediated targeted insertion of reporter genes with a donor template was feasible in the sea urchin (Ochiai et al. 2012). However, the construction of ZFNs is a laborious process, the success rate of ZFN construction is low, and the toxicity of ZFNs is relatively high, compared with TALENs.
TALEN-mediated mutagenesis in sea urchin
In this study, we examined the feasibility of TALENmediated genome editing in the sea urchin, Hemicentrotus pulcherrimus. We constructed TALENs targeting HpEts, the H. pulcherrimus homologue of Ets1, which plays a pivotal role in the specification of primary mesenchyme cells (PMCs) in sea urchin embryos (Kurokawa et al. 1999) and showed the efficacy of these TALENs by injecting them into sea urchin embryos and performing sequence analyses of the resulting mutations in the HpEts gene in the injected embryos. The results indicated that TALENs can introduce mutations into a specific genomic locus in sea urchin embryos.
Materials and methods Sea urchin culture Sea urchins (H. pulcherrimus) were harvested from Seto Inland Sea or Tateyama Bay, and their gametes were obtained by coelomic injection of 0.55 mol/L KCl. Fertilized eggs were cultured in filtered seawater at 16°C. Construction of TALENs Transcription activator-like effector repeats were cloned into pBluescript SK and assembled using the Golden Gate cloning method. The N- and C-terminal domains of TALE and the FokI nuclease domain were taken from pTALEN_v2 (Addgene) (Sanjana et al. 2012). Single strand annealing assay Single strand annealing (SSA) assays using HEK293T cells were performed according to the method reported by Sakuma et al. (2013). Briefly, the SSA-EtsS and SSA-Ets-A oligonucleotides (Table S1) were annealed and inserted between the BsaI sites of the pGL4-SSA vector to generate pGL4-SSA-Ets. HEK293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. The cells were cotransfected with 200 ng of TALEN expression vector, 100 ng of pGL4-SSA reporter plasmid, and 20 ng of pRL-CMV reference vector (Promega) using Lipofectamine LTX (Life Technologies) in 96-well plates at 50 000 cells⁄well. pSTL-ZFA36 (Ochiai et al. 2010), HPRT1 TALEN (Sakuma et al. 2013), and their SSA reporter vectors were used as positive controls. After 24 h, dual-luciferase assays were conducted using a Dual-Glo luciferase assay system (Promega) in a TriStar LB 941 plate reader (Berthold) based on the manufacturer’s instructions.
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mRNA synthesis and microinjection The plasmids containing TALEN cDNAs were linearized with SmaI. TALEN mRNAs were synthesized from the linearized plasmids using an mMESSAGE mMACHINE T7 Ultra Kit (Ambion). Microinjection was carried out as described by Rast (2000) with some modifications reported by Ochiai et al. (2008). Briefly, 2 pl of solution was injected into each fertilized egg. To observe the phenotypes, the embryos were fixed in filtered seawater containing 0.01% formaldehyde. Restriction fragment length polymorphism analysis and DNA sequencing For restriction fragment length polymorphism (RFLP) analysis, genomic DNAs were isolated from about 120 embryos at 4 h postfertilization (hpf) and about 40 embryos at 8, 12 and 24 hpf that were injected either with 2 pg of HpEtsL TALEN mRNAs or with 1 pg each of HpEts TALEN mRNAs. A 154-bp polymerase chain reaction (PCR) fragment containing the target sequence for the HpEts TALENs was amplified by PCR using the primers HsT1F and HsT1R primers (Table S1) using KOD FX Neo (Toyobo). The PCR products were then phenol/chloroform-extracted, digested with BglII (Takara) overnight, and analyzed by 3% agarose gel electrophoresis. For DNA sequencing, genomic DNAs were isolated from about 100 embryos at 24 hpf that were injected with 1 pg each of HpEts TALEN mRNAs. A 154-bp PCR fragment containing the target sequence for the HpEts TALENs was amplified by PCR with the HsT1F and HsT1R primers using LA Taq polymerase (Takara). The PCR products were cloned into the TA cloning vector pCR2.1 (Life Technologies). The nucleotide sequences of the clones were analyzed to determine the types of mutations around the HpEts TALEN target site.
Results and discussion To validate the feasibility of genome editing with TALENs in the sea urchin, we decided to make TALENs targeting the HpEts gene. Zygotic expression of HpEts is required for the specification of PMCs (Kurokawa et al. 1999). When this gene is repressed by injection of a dominant-negative form of HpEts mRNA, skeletogenesis is not observed in the embryos. In this study, we chose a target site in exon 3, which encodes the N-terminal region of the HpEts protein, to efficiently disrupt the function of the HpEts gene (Fig. 1). We constructed TALENs by Golden Gate cloning, and the activity of the TALENs was confirmed by SSA assays (Fig. S1).
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Fig. 1. HpEts gene showing the transcription activator-like effector nuclease (TALEN) target site. A schematic representation of the HpEts gene is shown. The exons are indicated by gray and black boxes, representing untranslated regions and coding regions, respectively. The bent arrow depicts the transcription start site. The TALEN-targeted sequence and a pair of TALENs used in this study are shown below at the bottom.
To examine the effects of TALENs in sea urchin embryos, HpEts TALEN mRNAs were injected into fertilized eggs (Fig. 2). When a pair of HpEts TALEN mRNAs (HpEtsL and HpEtsR: 1 pg each) was injected, loss of PMCs was observed in some embryos at the mesenchyme blastula stage. At the prism stage, 12.6% of HpEts TALEN pair-injected embryos showed impairment of skeletogenesis such as loss of skeleton or partial formation of skeleton (Fig. 2C,D; Table 1). These findings indicate that impairment of skeletogenesis was induced by introduction of the HpEts TALEN mRNA pair, suggesting that disruption of the HpEts gene occurred in the HpEts TALEN pair-injected
(A)
(B)
(C)
(D)
Fig. 2. Effects of injection of HpEts transcription activator-like effector nuclease (TALEN) mRNAs into sea urchin embryos. (A, B) Control embryo. (C, D) HpEts TALEN mRNA pair-injected embryo showing impairment of skeletogenesis. (A, C) Bright-field images of embryos. (B, D) Polarized microscopic images of embryos.
embryos. In contrast, in the embryos injected with the HpEtsL TALEN mRNA alone (2 pg), most of the embryos developed normally, although 2.9% of embryo showed impairment of skeletogenesis (Table 1). This may be because we used homodimeric TALENs harboring a wild-type FokI nuclease domain. It is considered that homodimerized HpEtsL TALEN induced disruption of the HpEts gene in the embryos showing abnormal skeletogenesis. To prevent this effect, we will need to apply the heterodimeric TALENs harboring a variant of the FokI nuclease domain in future studies. To examine the timing of the introduction of mutations by TALENs during sea urchin development, we performed genomic PCR and RFLP analysis. If there is a unique restriction site in the TALEN spacer sequence, mutated alleles can be detected as resistant fragments against the restriction enzyme. The target region of HpEts was amplified by genomic PCR using genomic DNA extracted from HpEts TALEN pairinjected embryos and HpEtsL TALEN-injected embryos as controls at several stages of development. The PCR products were digested with the restriction enzyme BglII, which has a recognition site in the target site for the HpEts TALENs (Fig. 3A). As shown in Figure 3B, the DNA fragments derived from the HpEtsL TALEN-injected embryos were completely cut by BglII at all stages. In contrast, a BglII-resistant fragment was observed weakly among the DNA fragments from the HpEts TALEN pair-injected embryos at 8 hpf (morula stage) and the amount of this fragment increased at 12 hpf (unhatched blastula stage) to reach almost a plateau at 24 hpf (mesenchyme blastula stage) (Fig. 3B). These findings suggest that TALEN-mediated mutations were introduced from the morula stage and mainly at between unhatched blastula and mesenchyme blastula stages. Previously, we reported that ZFN-mediated mutagenesis mainly occurred between the 8-cell and hatched blastula stages (Ochiai et al. 2012). Therefore, it appears that the timing of the mutagenesis induced by TALENs is later than that induced by ZFNs during sea urchin development. Next, we analyzed the mutations introduced into the HpEts target site by the TALENs. DNA fragments amplified by PCR using genomic DNA extracted from HpEts TALEN pair-injected embryos at 24 hpf were subcloned and their nucleotide sequences were determined (Fig. 3C). Among 27 clones examined, deletion (13 clones; 48.1%), insertion (one clone; 3.7%) and deletion/insertion (three clones; 11.1%) were observed. Overall, 51.9% of the HpEts genes were disrupted by frameshifts. Despite the high level of mutations, mutant phenotypes such as abnormal skeletogenesis were limited to 12.6% of the TALEN pair-injected embryos.
2013 The Authors Development, Growth & Differentiation 2013 Japanese Society of Developmental Biologists
TALEN-mediated mutagenesis in sea urchin
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Table 1. Effects of injection of HpEts transcription activator-like effector nuclease (TALEN) mRNAs TALEN mRNA Uninjected control HpEtsL TALEN mRNA HpEtsL+HpEtsR TALEN mRNA
RNA (pg/embryo)
Injected embryos
Normal (%)
Impairment of skeletogenesis (%)
Dead (%)
0 2 1 pg each
168 103 214
99.4 92.2 76.2
0.6 2.9 12.6
0 4.9 11.2
(A)
(B)
(C)
Fig. 3. Analysis of mutations induced by transcription activator-like effector nucleases (TALENs). (A) Schematic representation of the HpEts genomic region used for the polymerase chain reaction (PCR)-based analysis. The region contains a target site for the HpEts TALENs and one BglII sites. The BglII site is positioned in the middle of the HpEts TALEN target site. (B) Representative analysis of the PCR products during development. The PCR products from genomic DNA of HpEts TALEN mRNA pair-injected embryos (represented as L+R) or HpEtsL TALEN mRNA-injected embryos (represented as L) were purified, digested with BglII and analyzed by agarose gel electrophoresis. The times of the genomic DNA extractions are shown at the top. (C) Sequences observed in sea urchin embryos injected with the HpEts TALEN mRNA pair. The wild-type sequence is shown at the top with the TALEN-binding sites (boxed). Deletions are indicated by red dashes and insertions and substitutions are indicated by red letters.
This may arise because the delay in timing of the introduction of mutations contributed to a difference in the rates of the genotype and phenotype incidences. In fact, it was shown that zygotic expression of HpEts begins to occur from the morula stage and is increased at the unhatched blastula stage (Yajima et al. 2010). Therefore, it is considered that mutagenesis by HpEts TALEN pairs mainly occurs after the zygotic expression of HpEts.
In summary, we have shown that injection of a pair of HpEts TALEN mRNAs into sea urchin eggs can induce mutations into the targeted region of the HpEts gene, resulting in disruption of the gene. Although the mutant phenotypes were limited because the timing of the mutagenesis occurred after the expression of the gene, to the best of our knowledge, this is the first report on TALEN–mediated targeted mutagenesis in sea urchin embryos.
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Acknowledgments The authors wish to express their thanks to Dr Masato Kiyomoto for supplying live sea urchins. The authors also thank the Fisheries and Ocean Technology Center, Hiroshima Prefectural Technology Research Institute for supplying seawater. We also thank the Cryogenic Center of Hiroshima University for supplying liquid nitrogen. This study was supported by a Grantin-Aid for Challenging Exploratory Research (No. 24657156) to T.Y.
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Kurokawa, D., Kitajima, T., Mitsunaga-Nakatsubo, K., Amemiya, S., Shimada, H. & Akasaka, K. 1999. HpEts, an ets-related transcription factor implicated in primary mesenchyme cell differentiation in the sea urchin embryo. Mech. Dev. 80, 41– 52. Liu, J., Li, C., Yu, Z., Huang, P., Wu, H., Wei, C., Zhu, N., Shen, Y., Chen, Y., Zhang, B., Deng, W. M. & Jiao, R. 2012. Efficient and specific modifications of the Drosophila genome by means of an easy TALEN strategy. J. Genet. Genomics 39, 209–215. Mashimo, T., Takizawa, A., Voigt, B., Yoshimi, K., Hiai, H., Kuramoto, T. & Serikawa, T. 2010. Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases. PLoS ONE 5, e8870. Mashimo, T., Kaneko, T., Sakuma, T., Kobayashi, J., Kunihiro, Y., Voigt, B., Yamamoto, T. & Serikawa, T. 2013. Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes. Sci. Rep. 3, 1253. Meng, X., Noyes, M. B., Zhu, L. J., Lawson, N. D. & Wolfe, S. A. 2008. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol. 26, 695– 701. Ochiai, H., Sakamoto, N., Momiyama, A., Akasaka, K. & Yamamoto, T. 2008. Analysis of cis-regulatory elements controlling spatiotemporal expression of T-brain gene in sea urchin, Hemicentrotus pulcherrimus. Mech. Dev. 125, 2–17. Ochiai, H., Fujita, K., Suzuki, K., Nishikawa, M., Shibata, T., Sakamoto, N. & Yamamoto, T. 2010. Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases. Genes Cells 15, 875–885. Ochiai, H., Sakamoto, N., Fujita, K., Nishikawa, M., Suzuki, K., Matsuura, S., Miyamoto, T., Sakuma, T., Shibata, T. & Yamamoto, T. 2012. Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos. Proc. Natl Acad. Sci. USA 109, 10915–10920. Rast, J. P. 2000. Transgenic manipulation of the sea urchin embryo. Methods Mol. Biol. 136, 365–373. Sakuma, T., Hosoi, S., Woltjen, K., Suzuki, K., Kashiwagi, K., Wada, H., Ochiai, H., Miyamoto, T., Kawai, N., Sasakura, Y., Matsuura, S., Okada, Y., Kawahara, A., Hayashi, S. & Yamamoto, T. 2013. Efficient TALEN construction and evaluation methods for human cell and animal applications. Genes Cells 18, 315–326. Sanjana, N. E., Cong, L., Zhou, Y., Cunniff, M. M., Feng, G. & Zhang, F. 2012. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 7, 171–192. Sung, Y. H., Baek, I. J., Kim, D. H., Jeon, J., Lee, J., Lee, K., Jeong, D., Kim, J. S. & Lee, H. W. 2013. Knockout mice created by TALEN-mediated gene targeting. Nat. Biotechnol. 31, 23–24. Suzuki, K. T., Isoyama, Y., Kashiwagi, K., Sakuma, T., Ochiai, H., Sakamoto, N., Furuno, N., Kashiwagi, A. & Yamamoto, T. 2013. High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos. Biol. Open 2, 448–452. Watanabe, T., Ochiai, H., Sakuma, T., Horch, H. W., Hamaguchi, N., Nakamura, T., Bando, T., Ohuchi, H., Yamamoto, T., Noji, S. & Mito, T. 2012. Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. Nat. Commun. 3, 1017. Wood, A. J., Lo, T. W., Zeitler, B., Pickle, C. S., Ralston, E. J., Lee, A. H., Amora, R., Miller, J. C., Leung, E., Meng, X., Zhang, L., Rebar, E. J., Gregory, P. D., Urnov, F. D. &
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Meyer, B. J. 2011. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307. Yajima, M., Umeda, R., Fuchikami, T., Kataoka, M., Sakamoto, N., Yamamoto, T. & Akasaka, K. 2010. Implication of HpEts in gene regulatory networks responsible for specification of sea urchin skeletogenic primary mesenchyme cells. Zoolog. Sci. 27, 638–646. Young, J. J., Cherone, J. M., Doyon, Y., Ankoudinova, I., Faraji, F. M., Lee, A. H., Ngo, C., Guschin, D. Y., Paschon, D. E., Miller, J. C., Zhang, L., Rebar, E. J., Gregory, P. D., Urnov, F. D., Harland, R. M. & Zeitler, B. 2011. Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases. Proc. Natl Acad. Sci. USA 108, 7052–7057. Zu, Y., Tong, X., Wang, Z., Liu, D., Pan, R., Li, Z., Hu, Y., Luo, Z., Huang, P., Wu, Q., Zhu, Z., Zhang, B. & Lin, S. 2013. TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat. Methods 10, 329–331.
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Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Functional evaluation of HpEts TALENs by SSA assays. The relative activity is defined as the ratio of measured activity to the activity score of pSTLZFA36. As a positive control for the TALENs, the activity of HPRT1 TALEN was measured by the SSA assay. Table S1. Sequences of oligonucleotides used in this study.
2013 The Authors Development, Growth & Differentiation 2013 Japanese Society of Developmental Biologists
Develop. Growth Differ. (2014) 56, 92–97
doi: 10.1111/dgd.12099
Original Article
Targeted mutagenesis in sea urchin embryos using TALENs Sayaka Hosoi, Tetsushi Sakuma, Naoaki Sakamoto and Takashi Yamamoto* Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8526, Japan
Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos. Key words: sea urchin, targeted mutagenesis, transcription activator-like effector nuclease.
Introduction Recently, genome editing with engineered nucleases has been reported in animals, plants and cultured cells. Engineered nucleases comprise a customized array of DNA-binding modules that interact with a specific DNA sequence and a nuclease domain of the restriction enzyme FokI. When a pair of engineered nucleases is expressed in cells, they bind to their target sequences and induce a double-stranded break (DSB). The induced DSB is repaired by either errorprone non-homologous end-joining (NHEJ) repair or homology-directed repair (HDR). Therefore, site-specific mutations such as insertions and/or deletions can be induced during the NHEJ repair process and a precise genetic modification can be produced with an exogenous donor DNA template during HDR (Joung & Sander 2013). Two types of engineered nuclease have mainly been used for genome editing thus far. One type is zinc finger nucleases (ZFNs), which have zinc fingers as
*Author to whom all correspondence should be addressed. Email: [email protected] Received 13 September 2013; revised 1 October 2013; accepted 2 October 2013. ª 2013 The Authors Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
DNA-binding domains, and the other type is transcription activator-like effector nucleases (TALENs), which have DNA-binding modules of the TAL effector from Xanthomonas sp. Using these engineered nucleases, NHEJ-mediated targeted gene disruption has been successful in various animals, including Caenorhabiditis elegans (Wood et al. 2011), Drosophila (Bibikova et al. 2002; Liu et al. 2012), cricket (Watanabe et al. 2012), Ciona (Kawai et al. 2012), medaka (Ansai et al. 2012, 2013), zebrafish (Doyon et al. 2008; Meng et al. 2008; Hisano et al. 2013), frogs (Young et al. 2011; Suzuki et al. 2013), mice (Carbery et al. 2010; Sung et al. 2013), rats (Mashimo et al. 2010, 2013), and pigs (Hauschild et al. 2011; Carlson et al. 2012). In contrast, reports on HDR-mediated targeted gene addition are limited to a few species, such as Drosophila (Beumer et al. 2008), zebrafish (Zu et al. 2013) and mice (Cui et al. 2011). Previously, we established a construction system for ZFNs using a bacterial one-hybrid screening with ZF randomized libraries and showed that the ZFNs introduced mutations at their target sites in sea urchin embryos (Ochiai et al. 2010). Furthermore, ZFN-mediated targeted insertion of reporter genes with a donor template was feasible in the sea urchin (Ochiai et al. 2012). However, the construction of ZFNs is a laborious process, the success rate of ZFN construction is low, and the toxicity of ZFNs is relatively high, compared with TALENs.
TALEN-mediated mutagenesis in sea urchin
In this study, we examined the feasibility of TALENmediated genome editing in the sea urchin, Hemicentrotus pulcherrimus. We constructed TALENs targeting HpEts, the H. pulcherrimus homologue of Ets1, which plays a pivotal role in the specification of primary mesenchyme cells (PMCs) in sea urchin embryos (Kurokawa et al. 1999) and showed the efficacy of these TALENs by injecting them into sea urchin embryos and performing sequence analyses of the resulting mutations in the HpEts gene in the injected embryos. The results indicated that TALENs can introduce mutations into a specific genomic locus in sea urchin embryos.
Materials and methods Sea urchin culture Sea urchins (H. pulcherrimus) were harvested from Seto Inland Sea or Tateyama Bay, and their gametes were obtained by coelomic injection of 0.55 mol/L KCl. Fertilized eggs were cultured in filtered seawater at 16°C. Construction of TALENs Transcription activator-like effector repeats were cloned into pBluescript SK and assembled using the Golden Gate cloning method. The N- and C-terminal domains of TALE and the FokI nuclease domain were taken from pTALEN_v2 (Addgene) (Sanjana et al. 2012). Single strand annealing assay Single strand annealing (SSA) assays using HEK293T cells were performed according to the method reported by Sakuma et al. (2013). Briefly, the SSA-EtsS and SSA-Ets-A oligonucleotides (Table S1) were annealed and inserted between the BsaI sites of the pGL4-SSA vector to generate pGL4-SSA-Ets. HEK293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. The cells were cotransfected with 200 ng of TALEN expression vector, 100 ng of pGL4-SSA reporter plasmid, and 20 ng of pRL-CMV reference vector (Promega) using Lipofectamine LTX (Life Technologies) in 96-well plates at 50 000 cells⁄well. pSTL-ZFA36 (Ochiai et al. 2010), HPRT1 TALEN (Sakuma et al. 2013), and their SSA reporter vectors were used as positive controls. After 24 h, dual-luciferase assays were conducted using a Dual-Glo luciferase assay system (Promega) in a TriStar LB 941 plate reader (Berthold) based on the manufacturer’s instructions.
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mRNA synthesis and microinjection The plasmids containing TALEN cDNAs were linearized with SmaI. TALEN mRNAs were synthesized from the linearized plasmids using an mMESSAGE mMACHINE T7 Ultra Kit (Ambion). Microinjection was carried out as described by Rast (2000) with some modifications reported by Ochiai et al. (2008). Briefly, 2 pl of solution was injected into each fertilized egg. To observe the phenotypes, the embryos were fixed in filtered seawater containing 0.01% formaldehyde. Restriction fragment length polymorphism analysis and DNA sequencing For restriction fragment length polymorphism (RFLP) analysis, genomic DNAs were isolated from about 120 embryos at 4 h postfertilization (hpf) and about 40 embryos at 8, 12 and 24 hpf that were injected either with 2 pg of HpEtsL TALEN mRNAs or with 1 pg each of HpEts TALEN mRNAs. A 154-bp polymerase chain reaction (PCR) fragment containing the target sequence for the HpEts TALENs was amplified by PCR using the primers HsT1F and HsT1R primers (Table S1) using KOD FX Neo (Toyobo). The PCR products were then phenol/chloroform-extracted, digested with BglII (Takara) overnight, and analyzed by 3% agarose gel electrophoresis. For DNA sequencing, genomic DNAs were isolated from about 100 embryos at 24 hpf that were injected with 1 pg each of HpEts TALEN mRNAs. A 154-bp PCR fragment containing the target sequence for the HpEts TALENs was amplified by PCR with the HsT1F and HsT1R primers using LA Taq polymerase (Takara). The PCR products were cloned into the TA cloning vector pCR2.1 (Life Technologies). The nucleotide sequences of the clones were analyzed to determine the types of mutations around the HpEts TALEN target site.
Results and discussion To validate the feasibility of genome editing with TALENs in the sea urchin, we decided to make TALENs targeting the HpEts gene. Zygotic expression of HpEts is required for the specification of PMCs (Kurokawa et al. 1999). When this gene is repressed by injection of a dominant-negative form of HpEts mRNA, skeletogenesis is not observed in the embryos. In this study, we chose a target site in exon 3, which encodes the N-terminal region of the HpEts protein, to efficiently disrupt the function of the HpEts gene (Fig. 1). We constructed TALENs by Golden Gate cloning, and the activity of the TALENs was confirmed by SSA assays (Fig. S1).
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Fig. 1. HpEts gene showing the transcription activator-like effector nuclease (TALEN) target site. A schematic representation of the HpEts gene is shown. The exons are indicated by gray and black boxes, representing untranslated regions and coding regions, respectively. The bent arrow depicts the transcription start site. The TALEN-targeted sequence and a pair of TALENs used in this study are shown below at the bottom.
To examine the effects of TALENs in sea urchin embryos, HpEts TALEN mRNAs were injected into fertilized eggs (Fig. 2). When a pair of HpEts TALEN mRNAs (HpEtsL and HpEtsR: 1 pg each) was injected, loss of PMCs was observed in some embryos at the mesenchyme blastula stage. At the prism stage, 12.6% of HpEts TALEN pair-injected embryos showed impairment of skeletogenesis such as loss of skeleton or partial formation of skeleton (Fig. 2C,D; Table 1). These findings indicate that impairment of skeletogenesis was induced by introduction of the HpEts TALEN mRNA pair, suggesting that disruption of the HpEts gene occurred in the HpEts TALEN pair-injected
(A)
(B)
(C)
(D)
Fig. 2. Effects of injection of HpEts transcription activator-like effector nuclease (TALEN) mRNAs into sea urchin embryos. (A, B) Control embryo. (C, D) HpEts TALEN mRNA pair-injected embryo showing impairment of skeletogenesis. (A, C) Bright-field images of embryos. (B, D) Polarized microscopic images of embryos.
embryos. In contrast, in the embryos injected with the HpEtsL TALEN mRNA alone (2 pg), most of the embryos developed normally, although 2.9% of embryo showed impairment of skeletogenesis (Table 1). This may be because we used homodimeric TALENs harboring a wild-type FokI nuclease domain. It is considered that homodimerized HpEtsL TALEN induced disruption of the HpEts gene in the embryos showing abnormal skeletogenesis. To prevent this effect, we will need to apply the heterodimeric TALENs harboring a variant of the FokI nuclease domain in future studies. To examine the timing of the introduction of mutations by TALENs during sea urchin development, we performed genomic PCR and RFLP analysis. If there is a unique restriction site in the TALEN spacer sequence, mutated alleles can be detected as resistant fragments against the restriction enzyme. The target region of HpEts was amplified by genomic PCR using genomic DNA extracted from HpEts TALEN pairinjected embryos and HpEtsL TALEN-injected embryos as controls at several stages of development. The PCR products were digested with the restriction enzyme BglII, which has a recognition site in the target site for the HpEts TALENs (Fig. 3A). As shown in Figure 3B, the DNA fragments derived from the HpEtsL TALEN-injected embryos were completely cut by BglII at all stages. In contrast, a BglII-resistant fragment was observed weakly among the DNA fragments from the HpEts TALEN pair-injected embryos at 8 hpf (morula stage) and the amount of this fragment increased at 12 hpf (unhatched blastula stage) to reach almost a plateau at 24 hpf (mesenchyme blastula stage) (Fig. 3B). These findings suggest that TALEN-mediated mutations were introduced from the morula stage and mainly at between unhatched blastula and mesenchyme blastula stages. Previously, we reported that ZFN-mediated mutagenesis mainly occurred between the 8-cell and hatched blastula stages (Ochiai et al. 2012). Therefore, it appears that the timing of the mutagenesis induced by TALENs is later than that induced by ZFNs during sea urchin development. Next, we analyzed the mutations introduced into the HpEts target site by the TALENs. DNA fragments amplified by PCR using genomic DNA extracted from HpEts TALEN pair-injected embryos at 24 hpf were subcloned and their nucleotide sequences were determined (Fig. 3C). Among 27 clones examined, deletion (13 clones; 48.1%), insertion (one clone; 3.7%) and deletion/insertion (three clones; 11.1%) were observed. Overall, 51.9% of the HpEts genes were disrupted by frameshifts. Despite the high level of mutations, mutant phenotypes such as abnormal skeletogenesis were limited to 12.6% of the TALEN pair-injected embryos.
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Table 1. Effects of injection of HpEts transcription activator-like effector nuclease (TALEN) mRNAs TALEN mRNA Uninjected control HpEtsL TALEN mRNA HpEtsL+HpEtsR TALEN mRNA
RNA (pg/embryo)
Injected embryos
Normal (%)
Impairment of skeletogenesis (%)
Dead (%)
0 2 1 pg each
168 103 214
99.4 92.2 76.2
0.6 2.9 12.6
0 4.9 11.2
(A)
(B)
(C)
Fig. 3. Analysis of mutations induced by transcription activator-like effector nucleases (TALENs). (A) Schematic representation of the HpEts genomic region used for the polymerase chain reaction (PCR)-based analysis. The region contains a target site for the HpEts TALENs and one BglII sites. The BglII site is positioned in the middle of the HpEts TALEN target site. (B) Representative analysis of the PCR products during development. The PCR products from genomic DNA of HpEts TALEN mRNA pair-injected embryos (represented as L+R) or HpEtsL TALEN mRNA-injected embryos (represented as L) were purified, digested with BglII and analyzed by agarose gel electrophoresis. The times of the genomic DNA extractions are shown at the top. (C) Sequences observed in sea urchin embryos injected with the HpEts TALEN mRNA pair. The wild-type sequence is shown at the top with the TALEN-binding sites (boxed). Deletions are indicated by red dashes and insertions and substitutions are indicated by red letters.
This may arise because the delay in timing of the introduction of mutations contributed to a difference in the rates of the genotype and phenotype incidences. In fact, it was shown that zygotic expression of HpEts begins to occur from the morula stage and is increased at the unhatched blastula stage (Yajima et al. 2010). Therefore, it is considered that mutagenesis by HpEts TALEN pairs mainly occurs after the zygotic expression of HpEts.
In summary, we have shown that injection of a pair of HpEts TALEN mRNAs into sea urchin eggs can induce mutations into the targeted region of the HpEts gene, resulting in disruption of the gene. Although the mutant phenotypes were limited because the timing of the mutagenesis occurred after the expression of the gene, to the best of our knowledge, this is the first report on TALEN–mediated targeted mutagenesis in sea urchin embryos.
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Acknowledgments The authors wish to express their thanks to Dr Masato Kiyomoto for supplying live sea urchins. The authors also thank the Fisheries and Ocean Technology Center, Hiroshima Prefectural Technology Research Institute for supplying seawater. We also thank the Cryogenic Center of Hiroshima University for supplying liquid nitrogen. This study was supported by a Grantin-Aid for Challenging Exploratory Research (No. 24657156) to T.Y.
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Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Functional evaluation of HpEts TALENs by SSA assays. The relative activity is defined as the ratio of measured activity to the activity score of pSTLZFA36. As a positive control for the TALENs, the activity of HPRT1 TALEN was measured by the SSA assay. Table S1. Sequences of oligonucleotides used in this study.
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