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Keywords:

  • ascidian;
  • Ciona intestinalis;
  • enhanced green fluorescent protein;
  • gene targeting;
  • zinc-finger nuclease

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Zinc-finger nucleases (ZFNs) are engineered nucleases that induce DNA double-strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non-homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non-homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP-ZFN mRNAs into the embryos of an EGFP-transgenic line and observed the mutation frequency in the target site of EGFP. We also examined the effects of the EGFP-ZFNs at off-target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on-target site with less effect on the off-target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Unraveling the functions of genes is an essential subject for biology. The mutant organisms that lose gene functions are extremely helpful for obtaining deep insights into the genetic functions. Mutants can be generated by random mutagenesis with mutagens, but such forward genetic approaches are time-consuming except for a few model organisms, and mutants of the genes-of-interest can be created only by chance. The reverse genetic approaches, as exemplified by gene knockout technology, are an easier way to obtain mutagenesis since the technologies directly create mutant organisms of the genes-of-interest. Historically, the knockout technologies can be successfully conducted in a limited number of multicellular organisms in which specialized technological innovations have been achieved (Gossler et al. 1986; Ueda et al. 2008), like totipotent stem cell lines, splendid transgenic technologies or large scale mutant screening (Spradling et al. 1999; Duverger et al. 2007).

The situation has been changed recently by the application of the zinc-finger nucleases (ZFNs) for targeted mutagenesis (Bibikova et al. 2003; Porteus & Carroll 2005). ZFNs are engineered nucleases consisting of a customized array of zinc fingers (ZFs) that bind to a specific DNA sequence and a cleavage domain of the restriction enzyme FokI. One ZF recognizes three or four nucleotides, and a ZF array consisting of three ZFs recognizes nine to ten nucleotides total. When a pair of ZFNs binds to the specific sequence with an optimal spacer sequence, the nuclease domains are dimerized and introduce a double-strand break (DSB) at the spacer sequence. The DSB is correctly repaired by the homologous recombination-mediated repair system (HR) or by non-homologous end joining (NHEJ), which is independent of a template DNA for repair and frequently induces some errors (Wyman & Kanaar 2006). By the later mechanism, ZFNs introduce site-specific insertions or deletions (Doyon et al. 2008; Maeder et al. 2008; Meng et al. 2008). The ZFN-based gene knockout is superior to the previous knockout technologies since the ZFN method does not require a specialized cell line or genetic approach. ZFNs can be introduced into organisms by simple microinjection or electroporation methods, and the introduced ZFNs can generate mutations with high efficiency. In fact, ZFN-based mutagenesis has successfully been reported in multiple organisms and culture cells such as Drosophila, sea urchin, zebrafish, silk worm, plants, human culture cells and pig culture cells (Urnov et al. 2005; Beumer et al. 2008; Doyon et al. 2008; Meng et al. 2008; Foley et al. 2009; Hockemeyer et al. 2009; Shukla et al. 2009; Townsend et al. 2009; Zou et al. 2009; Ochiai et al. 2010; Takasu et al. 2010; Watanabe et al. 2010).

The chordate Ciona intestinalis is a splendid model animal for studying embryogenesis because of its mosaic development and its simple body plan consisting of countable numbers of cells. Ciona intestinalis also provides a useful experimental system for studying genetic functions. The draft genome sequence of C. intestinalis has already been determined, and large-scale expressed sequence tag (EST) information has been obtained (Dehal et al. 2002; Satou et al. 2002). The life cycle of C. intestinalis is approximately 2–3 months, and inland culture systems have been established (Joly et al. 2007). By using the culture systems, genetic approaches such as transposon-based transgenesis and ENU (N-ethyl-N-nitrosourea) mutagenesis have been introduced in this ascidian (Sasakura et al. 2005; Chiba et al. 2009). However, targeted mutagenesis of genes has not been established in C. intestinalis.

In the present study, we examined the activity, specificity and toxicity of ZFNs in C. intestinalis. As the target of ZFNs, we chose the EGFP gene inserted in the C. intestinalis genome through transposon-based transgenesis, because this locus is neutral and the usability of ZFNs can be determined without any bias. We introduced EGFP-ZFN mRNAs into embryos of an EGFP-transgenic line and then examined the mutation frequency on the target site. We also examined the effects of the EGFP-ZFNs on the off-target sites that had homology to the EGFP target sequence in the C. intestinalis genome in order to address whether ZFNs can specifically select the target sequence in the C. intestinalis genome. We showed that ZFNs can induce mutations on the target site of the C. intestinalis genome with high efficiency. The ZFNs effectively introduce mutations at an amount that does not show toxicities on the embryogenesis, and the mutations introduced by the ZFNs can be inherited to the next generation. EGFP-ZFNs introduced some mutations on the off-target sites, but the mutation frequency was far less than on the on-target site. These data suggest that target mutagenesis with ZFNs will be a powerful technique for studying gene functions in C. intestinalis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Animals

Wild-type Ciona intestinalis was collected from or cultivated at Maizuru (Kyoto), Mukaishima (Hiroshima) and Usa (Kochi). The EGFP-transgenic line Tg[MiCiTnIG]2 (Joly et al. 2007) was used as the donor of EGFP. This line has a single EGFP insertion in the genome, as described previously (Hozumi et al. 2010). Transgenic animals were cultured by an inland system described previously (Joly et al. 2007).

Constructs

ZF arrays that target the specific position (5′-GGCATCGACnnnnnnGAGGACGGC-3′) of the EGFP open reading frame were screened with ZF randomized libraries and a single-strand annealing assay as described previously (Ochiai et al. 2010). The ZF arrays were subcloned into the XbaI and SacI sites of the pNSe2-DS/RR vector containing the cleavage domain of the FokI Sharkey variant to create pNSe2-EGFPZFNL-DS and pNSe2-EGFPZFNR-RR (Guo et al. 2010). The ZFN cDNAs were subcloned into the BamHI and PstI sites of the pBS-HTB vector (Akanuma et al. 2002) by polymerase chain reaction (PCR) using the following primers (5′ -GAGGATCCCGGGATCTAGCACCAT-3′and 5′-GGGCTGCAGAAAGATCCCAAGCAAG-3′) to create pHTB-EGFPZFN-L and pHTB-EGFPZFN-R.

mRNA synthesis and microinjection

pHTB-EGFPZFN-L and pHTB-EGFPZFN-R were linearized with the restriction enzyme KpnI, and then used as templates for the in vitro synthesis of mRNA with the mMESSAGE mMACHINE T3 kit (Ambion). Unfertilized eggs were microinjected with synthetic 5′-capped mRNAs in approximately 30 pL of solution as described previously (Satou et al. 2001). The eggs were then inseminated with sperm of the wild-type or the Tg[MiCiTnIG]2 transgenic line. After insemination, the embryos were reared at about 16°C.

Analyses of genomes

Genomic DNAs were isolated from approximately 70–100 late tailbud stage embryos injected with 30, 150, 300 or 750 fg each of EGFP-ZFN mRNAs using a Wizard genomic DNA purification kit (Promega). The genomic regions containing the on-target or off-target sites were amplified by PCR with the primer sets described in Table S1. The PCR products were purified using a QIAquick Gel Extraction kit (QIAGEN) and then subcloned into the pGEMT vector (Promega). After transformation of E. coli with the ligated vectors, the E. coli colonies were subjected to PCR with the primers 5′-GTAAAACGACGGCCAGTG-3′ and 5′-GAAACAGCTATGACCATGATT-3′ to amplify the subcloned fragments. The PCR products were purified using a QIAquick PCR Purification kit (QIAGEN) or a Nucleospin 96 PCR Clean-up kit (MACHEREY-NAGEL), and their sequences were determined.

One embryo injected with 300 fg each of EGFP-ZFN mRNAs was reared until the reproductive stage. Sperm was collected from the animal, and genomic DNA was isolated from the sperm to examine mutations in the genomes of the germ cells. A part of the sperm of this animal was used to fertilize eggs from wild-type animals to obtain the progeny. The progeny were reared until the juvenile stage, and their genomes were analyzed to investigate inheritance of the mutations introduced by ZFNs. To extract genomic DNA from individual juveniles, each of them was digested in 50 μL of 1× TE buffer containing 0.2 mg/mL Proteinase K for 3 h at 50°C, followed by incubation for 15 min at 95°C to inactivate Proteinase K. One microlitre of the solution was used for PCR analyses with primers 5′-GACCATGTGATCGCGCTTCTCGT-3′ and 5′-GACCATGTGATCGCGCTTCTCGT-3′. After the first round of PCR, the PCR products were subjected to a second round of PCR with primers 5′-CAACATCCTGGGGGG-3′ and 5′-ATGTTGTGGCGGATCTTGAAG-3′. The PCR fragments were subjected to the sequence analysis as described above. As the positive control for genomic PCR, the DNA fragment of the snail gene was amplified using primers (5′-TGATGTCGCCACCACAAC-3′ and 5′-GAAGTGCTCCAAGAGAACTG-3′).

SURVEYOR nuclease assay

Genomic DNAs were isolated from 100 embryos at 4 hpf (hours post-fertilization) and 30 embryos at 5.5 hpf injected with 150 or 750 fg each of the EGFP-ZFN mRNAs using a Wizard genomic DNA purification kit (Promega). The genomic region containing the target site was amplified by PCR with primers 5′-CATTTTCATCTTCAGCAG-3′ and 5′-ATATGGTTGATGTCATGTAGCC-3′. After the first round of PCR, the PCR products were subjected to a second round of PCR with primers 5′-CAAGCTGACCCTGAAG-3′ and 5′-ATGTTGTGGCGGATCTTGAAG-3′. The PCR fragments were purified using a QIAquick Gel Extraction Kit (QIAGEN). The purified DNA fragments were denatured by heat, followed by annealing in accordance with the manufacturer's protocol in the hybridization buffer (10 mmol/L Tris–HCl (pH 8.5), 75 mmol/L KCl and 1.5 mmol/L MgCl2). The annealed DNAs were then digested by Surveyor nuclease (Transgenomic) and subjected to electrophoresis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

ZFNs targeting EGFP gene disrupt expression of this gene in the C. intestinalis embryos

To determine whether ZFNs function in the C. intestinalis embryos, we attempted to knockout the EGFP gene inserted in the C. intestinalis genome by using a pair of ZFNs designed to target EGFP (EGFP-ZFNs). The nuclease domain of these ZFNs is the modified FokI cleavage domain with higher activity (Sharkey variant; Guo et al. 2010). We selected a previously established transgenic line Tg[MiCiTnIG]2 as the source of the EGFP gene (Joly et al. 2007). Tg[MiCiTnIG]2 has a single EGFP gene in the genome (Hozumi et al. 2010), and this line expresses EGFP in the tail muscle cells at the tailbud stage in a non-mosaic fashion (Fig. 1A–D). We injected solutions of mRNAs encoding EGFP-ZFNs into unfertilized C. intestinalis eggs and then inseminated the eggs with sperm of a heterozygous animal of Tg[MiCiTnIG]2. After the experimental embryos reached the late tailbud stage, we observed their EGFP fluorescence. Normally, the frequency of embryos expressing EGFP is about 50% according to Mendel's law when embryos are obtained by crossing a heterozygous Tg[MiCiTnIG]2 animal and a wild type animal. However, the population of embryos injected with 750 fg each of EGFP-ZFN mRNAs showed a lower frequency of EGFP-positive animals (Fig. 1I). Almost none of the injected embryos had EGFP fluorescence. When the amount of EGFP-ZFN mRNAs was reduced, the frequency of embryos expressing EGFP was increased (Fig. 1I). However, most of the embryos showed EGFP fluorescence in only a few muscle cells (Fig.1E–H), suggesting that the EGFP genes in most muscle cells were impaired. These results suggest that EGFP-ZFNs can disrupt the EGFP gene in C. intestinalis embryos.

image

Figure 1. Enhanced green fluorescent protein-zinc-finger nucleases (EGFP-ZFNs) disrupt expression of EGFP in Ciona intestinalis. (A–H) A larva of the transgenic line Tg[MiCiTnIG]2 expressing EGFP in the tail muscle cells at the late tailbud stage. (A–D) Uninjected embryos. (A, B) An EGFP-positive embryo. (C, D) An EGFP-negative embryo. (E–H) Two embryos injected with 300 fg each of ZFN mRNAs. They showed EGFP signals in only a few tail muscle cells (arrowheads). (A, C, E, G) are the fluorescent images and (B, D, F, H) are the merged images of the fluorescent and bright field images. Left is toward anterior. Bar, 100 μm. (I) Graph displaying the ratio of EGFP-positive embryos injected with different amounts of EGFP-ZFN mRNAs. Results of the two independent experiments are shown. EGFP-positive late tailbud embryos showing expression of EGFP in the tail muscle cells were scored irrespective of the mosaicism of the expression. N indicates the number of counted embryos.

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EGFP-ZFNs can introduce mutations into the EGFPgene in the C. intestinalis genome

The loss of EGFP expression in embryos injected with EGFP-ZFNs suggests that EGFP genes were mutated by the ZFNs. To examine whether mutations were introduced by ZFNs on the target site of EGFP, we determined the nucleotide sequences of the EGFP genes in the genome of the Tg[MiCiTnIG]2 late tailbud embryos injected with EGFP-ZFN mRNAs. As a result, mutations were introduced in the target site of EGFP genes (Fig. S1). In the embryos injected with 150, 300 or 750 fg each of EGFP-ZFN mRNAs solutions, the mutation frequency reached about 100% (Table 1). In the embryos injected with 30 fg each of EGFP-ZFN mRNAs, the mutation frequency was reduced to about 27%. The EGFP genes were mutated by insertion of additional nucleotides ranging from 1 to 43 bp, deletion of a part of their sequence ranging from 1 to 17 bp or complex types containing both deletions and insertions. Deletion mutations tended to occur more frequently (76% in total) than others (insertions were 4% and complex types were 20% in total, respectively). These results indicate that EGFP-ZFNs can introduce insertions, deletions and complex types into the EGFP gene in the genome of C. intestinalis embryos.

Table 1. Mutation frequency of the target site
Amount of EGFP-ZFN mRNAs in the injection mediumExperimentNumber of sequenced clonesNormalDeletionInsertion§ComplexMutation frequency (%)
  1. Clones whose sequence was the same as the normal EGFP. Clones with deletional mutations of the EGFP gene. §Clones with insertional mutations of the EGFP gene. Clones with deletional and insertional mutations of the EGFP gene.

Uninjected111110000
214140000
30 fg each1352271527
2321783427
150 fg each13302814100
2371281797
300 fg each13402626100
23102515100
750 fg each14513201297
25404518100

EGFP-ZFNs introduce mutations on the target site from early embryonic stage

To determine when EGFP-ZFNs start to introduce mutations on the target site in the C. intestinalis embryos, we carried out a Surveyor nuclease assay of the genomic DNAs of embryos injected with EGFP-ZFN mRNAs (Fig. 2). We performed this assay with 32-cell stage (approximately 4.0 hpf) and 110-cell stage (approximately 5.5 hpf) embryos. While DNA fragments derived from the uninjected embryos were not cut by the Surveyor nuclease (Fig. 2), digested fragments were observed when the genomic DNAs were isolated from embryos injected with 750 or 150 fg of EGFP-ZFN mRNAs (Fig. 2). The mutations were introduced as early as the 32-cell or 110-cell stage when 750 or 150 fg of EGFP-ZFN mRNA solutions were injected, respectively (Fig. 2). These results suggest that the ZFNs start introducing mutations by the 32-cell stage, when a 750 fg or higher amount of EGFP-ZFN mRNA solutions are introduced into C. intestinalis embryos.

image

Figure 2. Enhanced green fluorescent protein-zinc-finger nucleases (EGFP-ZFNs) introduce mutations in the early embryonic stages. Surveyor assay was performed using genomic DNAs isolated from a 32-cell or 110-cell stage embryo. A schematic representation of the target site of the EGFP gene, the primer sites used for this assay and the expected fragment sizes are shown on the bottom diagram.

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Effect of EGFP-ZFNs on the off-target sites

Although ZFNs can cleave and induce mutations at specific sites within animal genomes, the possibility of mutations being introduced to non-specific sites cannot be ruled out completely (Gupta et al. 2011). To address this issue, we determined sequences of the off-target sites in the genome of embryos injected with EGFP-ZFNs. In the genome of C. intestinalis, we found 15 potential off-target sites of EGFP-ZFNs which contained three mismatched nucleotides compared with the on-target site of EGFP-ZFNs used in the in silico search. Seven out of 15 potential off-target sites were selected for analyses because these sites could be amplified by PCR and did not have a detectable polymorphism (Table 2). Among the seven potential off-target sites, we identified two off-target sites that were mutated with lower frequency than the on-target site in the embryos injected with 750 fg each of EGFP-ZFN mRNAs (Table 3). The other sites did not have a mutation. To confirm this, we further sequenced more than 100 clones in two of the non-mutated sites (OffT11-1 and OffT38 in Table 3), and did not detect any mutation (Table 3).

Table 2. Potential enhanced green fluorescent protein-zinc-finger nucleases (EGFP-ZFNs)' off-target sites searched in the Ciona intestinalis genome in silico
NameNumber of mismatchSequence in the genome browseraPosition in the genomePosition in the geneComment
  1. a

    Potential ZFNs-binding sequences are shown in capital letters. Underline indicates mismatched nucleotide compared to the on-target site.

EGFP0agGGCATCGACttcaagGAGGACGGCaaExonOn-target
OffT11-13ttCCCGTCATCtattggTTCGATGCCaaKhc11 2603458-2603481Exon 
OffT11-33aaTGCCTCGACatcgaaGAGGACGCCgcKhc11 4870131-4870154Exon 
OffT383atGTCATCGACgatgcaGACGACAGCggKhl38 176235-176258Exon 
OffT61-23acGACGACCTCattctcGTCGAAGCCgtKhl61 105831-105854Exon 
OffT99-13caGGCATTGACactttgGACGACGTCggKhl99 1093-1116Exon 
OffT1683aaGAGATCGACtatgacGGGGACGGCgtKhl168 38180-38203Exon 
OffT5343caGGCATTGACactttgGACGACGTCggKhs534 2265-2288Exon 
OffT103gcGACAACGACgtttgtGAGAACGGCgaKhc10 4035251-4035274ExonNot amplified
OffT11-23taGGCATCGGCagtgttGCGGACAGCggKhc11 4082729-4082752NoPolymorphism
OffT123ttGCCGTCCTGgtctgtGTCGAAGACttKhc12 4445747-4445764ExonPolymorphism
OffT61-13aaGGCATCGAAcgtaacGAGTACGGGttKhl61 79662-79685IntronSubstitution of one base
OffT633ccGCCGTCGTCgtcgtcGTCGCCGCCgcKhl63 44918-44941ExonPolymorphism
OffT99-23caGGCATCGAGgtcgttGATGACGTCatKhl99 12977-13000ExonNot amplified
OffT1323tcGCCGTCAACttgctgGTCGACGCCgtKhl132 10157-10180ExonInsertion of one base
OffT6753gcTCCGTCCTCttcttcGTTGTTGCCtcKhl675 2375-2398ExonIncluding gap
Table 3. Mutation frequency of the off-target sites in embryos injected with 750 fg each of enhanced green fluorescent protein-zinc-finger nucleases (EGFP-ZFNs) mRNA
NameExperimentNumber of sequenced clonesNormalDeletionInsertionMutation frequency (%)
OffT11-116363000
25555000
OffT11-3115104133.3
21565460
OffT3816262000
26161000
OffT61-211515000
21616000
OffT99-111616000
21616000
OffT16811919000
216142012.5
OffT53411616000
21616000

About the two off-target sites, we examined whether the frequency of their mutations is dependent on the amount of EGFP-ZFN mRNAs introduced into embryos. We analyzed genomic DNAs from embryos injected with different amounts of EGFP-ZFN mRNAs. As a result, mutations on the two off-target sites were induced in a concentration-dependent manner (Tables 4 and 5), supporting that the mutations were introduced by EGFP-ZFNs. We did not detect mutations in either off-target site when EGFP-ZFNs <150 fg were introduced. These results indicate that off-target sites with three-nucleotide mismatches can be mutated by ZFNs with lower frequency than on-target site. However, not all of the off-target sites with three-nucleotide mismatches were targeted by the ZFNs. The chance of the off-target mutations occurring can be reduced by adjusting the amount of ZFNs introduced into the embryos.

Table 4. Mutation frequency of OffT11-3 induced by different amounts of the enhanced green fluorescent protein-zinc-finger nucleases (EGFP-ZFNs)
Amount of EGFP-ZFN mRNAs in the injection mediumExperimentNumber of sequenced clonesNormalDeletionInsertionMutation frequency (%)
Uninjected11616000
21616000
30 fg each11616000
21616000
150 fg each11515000
21414000
300 fg each115122120
214121114.3
750 fg each115104133.3
21565460
Table 5. Mutation frequency of OffT168 induced by different amounts of the enhanced green fluorescent protein-zinc-finger nucleases (EGFP-ZFNs)
Amount of EGFP-ZFN mRNAs in the injection mediumExperimentNumber of sequenced clonesNormalDeletionInsertionMutation frequency (%)
Uninjected11616000
21616000
30 fg each11616000
21616000
150 fg each11515000
21616000
300 fg each116140212.5
216142012.5
750 fg each11919000
216142012.5

Toxicity of EGFP-ZFNs on the embryogenesis

In order to learn the effects of ZFNs on the normal embryogenesis of C. intestinalis, we counted the number of normally or abnormally developed embryos that were injected with different amounts of EGFP-ZFN mRNAs. In this experiment, we used wild type animals that did not have the EGFP gene in their genomes because we wanted to demonstrate the toxicities of EGFP-ZFNs on embryogenesis without the damage from the double-strand break of the genome. The percentage of normally developed embryos was normalized using the score of uninjected control embryos (Fig. 3). When 750 or 1500 fg each of EGFP-ZFN mRNAs was injected, the ZFNs did not have an adverse effect on embryogenesis. A larger amount of ZFNs led to defects in embryogenesis. We concluded that 150 fg each of EGFP-ZFN mRNAs is sufficient for mutagenesis because it showed no toxicity on the embryogenesis and effectively induced mutation on the on-target site with less mutation frequency on the off-target sites (Fig. 3, Tables 1, 4, 5).

image

Figure 3. Toxicity of enhanced green fluorescent protein-zinc-finger nucleases (EGFP-ZFNs) to embryogenesis. The graph displays the rate of normally developed late tailbud embryos injected with different amounts of EGFP-ZFN mRNAs. “Relative rate of normal embryogenesis” means that the percentage of normally developed embryos is normalized with the score of uninjected embryos. N indicates the number of counted embryos.

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The mutation of the EGFP gene induced by EGFP-ZFN is inheritable to the next generation

An important issue for creating mutants with ZFNs is whether ZFNs can mutate genes in the germ line cells and the mutations are inheritable to the next generation. To test this, we cultured animals of the Tg[MiCiTnIG]2 line, into which 300 fg each of EGFP-ZFN mRNAs was injected. We obtained one animal, which grew up to the reproductive stage. We examined whether its germ cells contained the mutated EGFP gene. Genomic DNA isolated from this animal's sperm was used to amplify the target site by PCR. We determined the sequences of 69 clones, all of which had the same insertional mutation in the target site of the EGFP gene (Fig. 4A), suggesting that the sperm contained only one mutated EGFP allele. Next, the sperm of this animal was used to fertilize eggs from the wild-type animals to obtain progeny. None of the resultant larva showed expression of EGFP (data not shown). Because their paternal parent had the EGFP gene in their genome, it is thought that the progeny either inherited mutated EGFP or somehow did not inherit EGFP. To determine which, we performed PCR analysis of the genomes of the progeny. This experiment showed that three out of five progeny had the same mutated EGFP gene as the sperm of their paternal parent (Fig. 4B). These results indicate that ZFNs can introduce mutations to the genes of the germ cells as well as the somatic cells, and the mutations can be inherited to the descendants.

image

Figure 4. The mutation induced by enhanced green fluorescent protein-zinc-finger nucleases (EGFP-ZFNs) was inherited to the next generation. (A) The nucleotide and amino-acid sequences of the normal EGFP gene around the target site of the EGFP-ZFNs (top), and the nucleotide and amino-acid sequences of EGFP identified from the sperm of the animal injected with EGFP-ZFNs (bottom). The capital letters in the nucleotide sequences indicate the binding sites of EGFP-ZFNs. The underline indicates the inserted nucleotides. The arrow indicates the primer site for the polymerase chain reaction (PCR) analysis in (B). (B) Inheritance of the mutated EGFP to the progeny. The upper panel shows that the mutated EGFP locus shown in (A) was present in the genome of three progeny (J3, J4 and J5). Lower panel shows the PCR products of an endogenous locus of the snail gene as the positive control for the genomic PCR analyses. P.C. indicates the positive control using the sperm genome of the paternal parent. N.C. indicates the negative control using the sperm genome of the non-mutated Tg[MiCiTnIG]2 transgenic line.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

We have shown that ZFNs can introduce inheritable mutations to the C. intestinalis genome with high efficiency. This indicates that ZFNs will be a powerful tool for knocking out genes in this ascidian. The mutation frequency of EGFP-ZFNs in C. intestinalis reached over 90% when 150 fg each of EGFP-ZFN mRNAs was introduced into embryos. This mutation frequency is higher compared with previous studies in other animals or culture cells. For example, the mutation frequency was about 10% in zebrafish embryos (Meng et al. 2008), and 62% in sea urchin embryos (Ochiai et al. 2010). In C. intestinalis, a mutation frequency of about 100% can be achieved without disrupting embryogenesis. There are several possible reasons why EGFP-ZFNs can mutate the target gene with high efficiency in C. intestinalis. First, we used ZFNs containing the hyperactive variant of the FokI cleavage domain (Guo et al. 2010). This variant is 15 times more active than wild type FokI. Second, more errors might occur in NHEJ in C. intestinalis than in other organisms. Further study of the NHEJ system in C. intestinalis is needed to confirm this. We used a heterozygous EGFP gene locus as the target of ZFNs in this study, which cannot be repaired through the homologous recombination (HR) system. The mutation frequency of the endogenous and homozygous genes would be lower than that of EGFP in this study because the endogenous gene is repaired by both the NHEJ and HR systems. However, we do not think that the repair of the DSB by the HR system is problematic over mutagenesis. DSB repair with the HR system is accurate, and thus the repaired DNA will again be the target of ZFNs until mutations are introduced. In fact, EGFP-ZFNs introduced mutations at some homozygous off-target sites, suggesting that ZFNs can mutate homozygous endogenous genetic loci in C. intestinalis. The optimal expression system of ZFNs, in which EGFP-ZFN mRNAs may be translated from the early embryonic stage, may be another factor affecting the efficiency of mutagenesis with EGFP-ZFN. In this study, we used the pBS-HTB vector as the backbone of EGFP-ZFNs open reading frames (ORFs). The pBS-HTB vector contains 5′- and 3′-UTRs of the βtubulin gene of another ascidian Halocynthia roretzi (Akanuma et al. 2002), and the mRNAs synthesized based on this vector have high translational efficiency in C. intestinalis (Sasakura et al. 2010). The efficient translation of EGFP-ZFNs may help to increase the mutation frequency by the ZFNs. Surveyor nuclease assay showed EGFP-ZFN mRNAs introduced the mutations as early as the 32-cell stage (approximately 4 hpf; Fig. 2), supporting that introduction of the mutations starts at the earlier embryonic stage.

An inferiority of ZFN in C. intestinalis is the introduction of mutations in the off-target sites even though they have three mismatch nucleotides compared to the on-target site. In zebrafish, ZFNs mutate four among 17 off-target sites with lower frequency (approximately 1%) (Meng et al. 2008) than in C. intestinalis (approximately 60%). However, the mutation frequency on the on-target site was lower (10–19%) in zebrafish than in C. intestinalis (approximately 100%). Thus, the frequent off-target mutations in C. intestinalis may be caused by too strong activity of ZFNs in C. intestinalis embryos. There are three ways of decreasing off-target mutations by ZFNs. The first is to design ZFNs with a target sequence that does not have off-target sites with three or less mismatched nucleotides. The second is to find the optimal amount of ZFNs introduced into embryos for mutagenesis. The non-specific activity of ZFNs can be decreased by reducing the amount of EGFP-ZFNs introduced into embryos without strongly undermining ZFNs' efficacy on the target site (Tables 1, 4, 5). In fact, we found that the off-target sites were mutated with about one-eighth to three-fifths lower frequency than the on-target site. Therefore, we can generate on-target mutations while reducing the effect on the off-target sites by using an optimal amount of ZFN mRNAs introduced in C. intestinalis embryos. The present study suggests that 150 fg each of ZFN mRNAs will be sufficient for on-target mutagenesis without a strong effect on the off-target sites. The third way of decreasing off-target mutations by ZFN is conducting another experiment that shows the specificity of phenotypes obtained by the mutations with ZFNs. It has been reported that the genomic regions that do not show sequence similarity to the on-target sites could be potential off-target sites (Gabriel et al. 2011), suggesting that it is difficult to completely rule out ZFNs effects against off-target sites. Therefore, it will be necessary to conduct an experiment to determine whether the mutant phenotypes obtained with ZFNs surely reflect the functions of genes. The adverse effects of off-target mutations will be minimized by these experimental attempts.

We obtained one founder animal that inherited the EGFP locus mutated by ZFNs. This indicates that ZFNs are active in the germ line cells as well as the somatic cells. The mutated allele was the only EGFP allele identified from the sperm of this animal, suggesting that the EGFP allele is possessed by most of the sperm of this animal. There is a possible explanation for the non-mosaicism of the sperm of this animal. The possibility is that although many germ cells were mutated independently by EGFP-ZFNs, most of the mutated germ cells were dead by the damage from the DSB, and only one cell that somehow escaped death survived to produce sperm. Our result suggests that ZFNs have the potential to mutate germ cells with less mosaicism, which is advantageous for screenings of mutants.

The present study has demonstrated that an optimal amount of ZFNs can induce inheritable mutations in the gene of interest with high efficiency and low toxicity in C. intestinalis. The data collected in this study will be used for setting the conditions for mutagenesis of endogenous genes in future studies. Recent studies of other organisms have achieved some improvements of the artificial nuclease-based targeted mutagenesis, like exchange of the DNA-binding site derived from the TAL effector (Boch et al. 2009; Moscou & Bogdanove 2009). Our present data can be applied to the TAL effector-based nucleases (TALENs) as well as the ZFNs, because the same nuclease domain of FokI is used in both ZFNs and TALENs (Christian et al. 2010). The targeted mutagenesis of genes with artificial nucleases will bring about technological innovation in the study of gene functions in C. intestinalis.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

We would like to thank the members of the Shimoda Marine Research Center at the University of Tsukuba for their cooperation with this study. We would also like to thank Drs Shigeki Fujiwara, Nobuo Yamaguchi, and all members of the Department of Zoology, Kyoto University, and the Maizuru Fishery Research Station of Kyoto University for collecting the Ciona adults. We are grateful to Dr Sumihare Noji for allowing generous use of the EGFP-ZFN. This study was supported by Grants-in-Aid for Scientific Research from JSPS, MEXT and Innovative Research Support Programs (Pilot Models) from University of Tsukuba to YS. Further support was provided by grants from the National Bioresource Project.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
dgd1355-sup-0001-TableS1.docxWord document12KFig. S1. Sequences observed in Ciona intestinalis embryos injected with 750 fg each of EGFP-ZFN mRNAs in experiment 1 of Table 1.
dgd1355-sup-0002-FigS1.docxWord document12KTable S1. Primers for PCR amplification of the on-target or off-target sites.

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