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31. =+Targeted gene editing in the sea urchin embryo using zinc-finger nucleases(

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2. ;=+
Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases.
Ochiai, H., Fujita, K., Suzuki, K., Nishikawa, M., Shibata, T., Sakamoto, N. &Yamamoto, T.
Genes to Cells, 15, 875-885, 2010.
3. 8=+(1) Analysis of cis-regulatory elements controlling spatio-temporal expression of
T-brain gene in sea urchin, Hemicentrotus pulcherrimus.
Ochiai, H., Sakamoto, N., Momiyama, A., Akasaka, K. & Yamamoto, T.
Mechanisms of Development, 125, 2– 17, 2008.
(2) The Ars insulator facilitates I-SceI meganuclease-mediated transgenesis in the sea
urchin embryo.
Ochiai, H., Sakamoto, N., Suzuki, K., Akasaka, K. & Yamamoto, T.
Developmental Dynamics, 237, 2475–2482, 2008.
(3) Suppressor of Hairless (Su(H)) is required for foregut development in the sea
urchin embryo.
Karasawa, K., Sakamoto, N., Fujita, K., Ochiai, H., Fujii, T., Akasaka, K. &Yamamoto, T.
Zoological Science, 26, 686-690, 2009.
(4) Role of the nanos homolog during sea urchin development.
Fujii, T., Sakamoto, N., Ochiai, H., Fujita, K., Okamitsu, Y., Sumiyoshi, N.,
Minokawa, T. & Yamamoto, T.
Developmental Dynamics, 238, 2511–21, 2009.
(5) Dicer is required for the normal development of sea urchin, Hemicentrotus
pulcherrimus.
Okamitsu, Y., Yamamoto, T., Fujii, T., Ochiai, H. & Sakamoto, N.
Zoological Science, 27, 477-486, 2010.



=+

Targeted gene editing in the sea urchin embryousing zinc-finger nucleases

Hiroshi Ochiai
2011

Contents
Introduction ................................................................................................................................. 1
Experimental Procedures ........................................................................................................... 6
Determination of the cDNA sequence of HpHesC and HpLig4.......................................................... 6
Confirmation of genetic variations in the target gene locus .............................................................. 6
Construction of randomized ZF libraries and B1H selection ............................................................ 6
SSA assay using HEK293T cells ........................................................................................................... 9Construction of a targeting donor construct ..................................................................................... 11
Sea urchin culture ................................................................................................................................ 12mRNA synthesis and microinjection .................................................................................................. 12
Quantitative PCR ................................................................................................................................. 13
Whole mount in situ hybridization ..................................................................................................... 13
Sequence analysis of mutations........................................................................................................... 13
Genomic PCR analysis ........................................................................................................................ 14
Reverse transcription PCR analysis ................................................................................................... 14
Live imaging of sea urchin embryos................................................................................................... 15
Statistical analysis ................................................................................................................................ 16

Results......................................................................................................................................... 17
Generation of functional ZFNs targeting the HpHesC gene ............................................................ 17
Effects of injecting mRNAs for the ZFNs targeting the HpHesC gene into sea urchin embryos . 20
Necessity of the SSA assay for selection of functional ZFNs ............................................................ 23
Targeted gene addition into the HpEts locus using ZFNs ................................................................ 23

Discussion ................................................................................................................................... 27
Generation of functional ZFNs by the combined use of B1H and SSA screenings........................ 27
Targeted mutagenesis in sea urchin embryos using ZFNs ............................................................... 29
Targeted gene addition in sea urchin embryos using ZFNs ............................................................. 30

Figures ........................................................................................................................................ 33
References .................................................................................................................................. 46
Acknowledgements .................................................................................................................... 53

Introduction
Targeted gene editing, such as targeted mutagenesis and targeted gene addition, is a
powerful approach to examine the function of genes and the regulatory mechanism fortheir expression during animal development. However, this approach is available only
in limited animal models, such as the mouse, in which embryonic stem (ES) cells
required for gene targeting have been established. In other animal models in which agene editing approach is not available, to analyze the function of a gene of interest,
loss-of-function approaches, such as the introduction of small interfering RNA and/or
morpholino antisense oligonucleotide (MO) that reduce the production of the encoded
protein, are employed as alternatives to targeted mutagenesis (Nasevicius et al., 2000;
Harborth et al., 2001). However, it has been reported that, in many cases, the complete
inactivation of a gene of interest is not achieved by these loss-of-function approaches(so-called gene knockdown) (Cost et al., 2010). In animal models in which a gene
editing approach is available, targeted gene addition enables in vivo monitoring of theexpression of the endogenous gene of interest and aids in examining the regulatory
mechanism of its expression. On the other hand, in animal models in which a gene
editing approach is not available, the random introduction of a reporter construct that
contains cis-regulatory elements of the gene of interest is used as an alternative totargeted gene addition to gain insights into the regulatory mechanism of gene
expression. However, it has been reported that the expression profile of the randomly
integrated reporter gene might be affected by the chromosomal environment (Levis et1

al., 1985). Accordingly, for the quantitative analysis of gene expression, insertion of areporter gene into the specific genomic locus of interest is used in model organisms inwhich a gene editing approach is available (Elowitz et al., 2002; Yoo et al., 2004; Raser
et al., 2004). Therefore, the development of novel strategies to further targetedmutagenesis and targeted gene addition in different model organisms is required.
Recently, a new method for targeted mutagenesis using engineered zinc-finger
nucleases (ZFNs) has been used in Drosophila (Beumer et al., 2008), zebrafish (Doyonet al., 2008; Meng et al., 2008; Foley et al., 2009), plants (Shukla et al., 2009;
Townsend et al., 2009) and human cultured cells (Urnov et al., 2005; Hockemeyer et al.,2009; Zou et al., 2009). ZFNs consist of a customized array of zinc fingers (ZFs) thatbind to a specific DNA sequence and the nuclease domain of the restriction enzyme
FokI. When two ZFNs bind to their associated target sequences in the appropriate
direction, the nuclease domains dimerize and a double-stranded break (DSB) is
introduced. The ZFN-induced DSB can then be repaired with high efficiency either byhomology-directed repair (HDR) or by error-prone nonhomologous end joining (NHEJ)
repair independent of a DNA template for repair. ZFNs, therefore, induce a site-specificinsertion or deletion at the DSB site after NHEJ repair (Doyon et al., 2008; Maeder etal., 2008). In the zebrafish, co-injection of mRNAs for ZFNs whose target site is
positioned in the kinase insert domain receptor, kdr gene, into one-cell-stage zebrafish
embryos led to mutagenic lesions at the target site and the mutations were transmitted
through the germ line with high frequency (Meng et al., 2008). Alternatively, ZFNs can
produce defined genetic modifications that include insertion of a reporter gene near the2site of the DSB by HDR using an exogenous targeting donor construct (Moehle et al.,2007; Beumer et al., 2008; Shukla et al., 2009). In the human cultured cells, it has beenreported that the X-linked severe combined immune deficiency mutation in theinterleukin-2 receptor- (IL2RG) gene was efficiently modified by ZFN-mediatedgene correction (Urnov et al., 2005). It has been noted that, in animals, ZFN-induced
DSBs are mainly repaired by NHEJ, so ZFN-mediated targeted gene correction andaddition are considered to be challenging (Beumer et al., 2008; Meyer et al., 2010). In
addition, because of the complexity of DNA sequence recognition by a ZF array,screening for ZFNs that introduce DSB at a specific genomic site is laborious.
Consequently the application of targeted gene editing using ZFNs is not widespread.
The sea urchin embryo, which is transparent, simple and readily accessible toexperimental perturbations using MO, offers a unique opportunity to study theregulation of morphogenesis during early development (McClay et al., 1992; Hardin,
1996). However, since the concentration of injected MO might be reduced throughsuccessive cell divisions, it was suggested that inactivation of the gene of interest could
become insufficient at later stages of development. Therefore, alternatives to theknockdown approach using MO are required for the functional analyses of genes thatare expressed at later stages in sea urchin development. In sea urchin embryos, some of
the regulatory mechanisms underlying the expression of the genes involved inendomesoderm specification have been analyzed by means of the microinjection ofreporter gene constructs that were randomly integrated into the genomic DNA (Makabe
et al., 1995; Revilla-i-Domingo et al., 2004; Ransick & Davidson, 2006; Ochiai et al.,3

2008a). Additionally, because of its transparency and small size, in vivo quantitative
imaging methodology at cellular level has been established in this organism (Damle etal., 2006). However, because of the unavailability of targeted gene addition, quantitative
imaging analysis has been limited to examine the expression of exogenous reporter
genes that, as mentioned above, might be quantitatively different from the expression ofendogenous genes.
In this thesis, to establish a system of targeted mutagenesis and targeted gene
addition in the sea urchin, I applied ZFN technology to the sea urchin, Hemicentrotus
pulcherrimus. To generate functional ZFNs, I adopted a bacterial one-hybrid (B1H)
system using ZF randomized libraries (Meng et al., 2008) and a single-strand annealing(SSA) assay using cultured cells. Using these screenings, I selected a pair of ZFNs
targeting HpHesC, the H. pulcherrimus homologue of HesC, which was reported torepress several transcription factor genes responsible for the differentiation of primarymesenchyme cells (PMCs) in the sea urchin embryo (Revilla-i-Domingo et al., 2007;
Yamazaki et al., 2009). I have shown the efficacy of the ZFNs by injecting them into
sea urchin embryos and performing sequence analysis to identify mutations in the
HpHesC gene in the injected embryos. Moreover, I have demonstrated that the insertionof a reporter cassette into a specific genomic locus was successfully achieved byinjecting another pair of ZFNs for HpEts gene, which encodes an Ets transcription
factor, along with a targeting donor construct. I have also shown that the introduction ofmRNA for a dominant negative form of HpLig4, a gene that encodes the H.
pulcherrimus homologue of the DNA ligase IV required for NHEJ, increased the4efficiency of ZFN-mediated targeted gene addition. These results suggest that targeted
gene editing using ZFNs is feasible in sea urchin.

5Experimental Procedures

Determination of the cDNA sequence of HpHesC and HpLig4
The cDNA of HpHesC and of the region of the Hemicentrotus pulcherrimus homologue
of DNA ligase IV (HpLig4) that encodes the carboxy-terminal tandem BRCT repeat(DN-lig4) were amplified by PCR from a cDNA library using primers for HpHesC(HpHesCF and HpHesCR) (Table 1) and HpLig4 (CTlig4F and CTlig4R) (Table 1),
respectively. The amplified cDNA was subcloned into pBluescriptII SK(+) (Stratagene),and the nucleotide sequence was determined using a CEQ 8000 Genetic Analysis
System (Beckman Coulter). The nucleotide sequence of HpHesC cDNA was depositedin GenBank (Accession No. GU390551).

Confirmation of genetic variations in the target gene locus
Genomic DNA was extracted from the tube feet of adult H. pulcherrimus. Regions of
153 bp in the third exon of the HpHesC gene was amplified from the genomic DNA
using a primer sets for HpHesC target site (HT1F and HT1R). The amplified genomic
DNA fragments were sequenced directly.

Construction of randomized ZF libraries and B1H selection
Construction of the zinc-finger randomized libraries and B1H selections were carriedout as described by Meng & Wolfe (2006) with some modifications. In these6experiments, ZF randomized libraries, which express fusion proteins consisting of
three-finger ZF arrays and the omega subunit of RNA polymerase, and HIS3 reporter
vectors, which contain the target sequences for the ZF arrays, were cotransfected into
the US0!hisB!pyrF!rpoZ bacterial strain and plated on histidine-deficient plates with
the HIS3 inhibitor 3-amino-1,2,4-triazole (3-AT) (Fig. 1A).
To construct the randomized ZF libraries, randomized sequence-receptive vectors
were constructed by modification of pc3XB-ZF70, a ZF module encoding a vector for
restriction digestion-based modular assembly (Wright et al., 2006; ZF Consortium
Modular Assembly ZF Set (Addgene)). Briefly, two BbsI sites were introduced into
pc3XB-ZF70 by inverse PCR to insert a randomized ZF cassette for a recognition helix
and ZF-specific mutations to enable PCR-based modular assembly. The first, second
and third modules from the N-terminus were designated ZF1-BbsI, ZF2-BbsI and
ZF3-BbsI, respectively (Fig. 1B). ZF-specific mutations were also introduced into
pc3XB-ZF64 and pc3XB-ZF63 (ZF module-encoding vectors; ZF Consortium Vector
Kit v1.0) by inverse PCR (Fig. 1B). pc3XB-ZFA36, which encodes a three-finger ZF
protein comprised of ZF60, ZF64 and ZF63 from the N-terminus (Ramirez et al., 2008;
Table 2), was constructed by restriction digestion-based modular assembly as described
previously (Wright et al., 2006). Furthermore, one of ZF60, ZF64 or ZF63 within
ZFA36 was substituted to the ZF1-BbsI, ZF2-BbsI and ZF3-BbsI modules by restriction
digestion-based modular assembly to construct pc3XB-Z1B, pc3XB-Z2B and
pc3XB-Z3B, respectively (Table 2). DNA fragments encoding three ZFs within
pc3XB-ZFA36, pc3XB-Z1B, pc3XB-Z2B and pc3XB-Z3B were amplified using the7

oligonucleotide primers 3Z-F and 3Z-R (Table 1). The amplified fragments weredigested with NotI and SpeI and inserted into the NotI/XbaI site of pB1H2!2-zif268,
which expresses a fusion protein of the DNA-binding domain of zif268 and the omega
subunit of RNA polymerase in bacteria (Meng et al., 2008) to generate
pB1H2!2-ZFA36, pB1H2!2-Z1B, pB1H2!2-Z2B and pB1H2!2-Z3B. A randomized
sequence flanked by BbsI sites (Random+BbsI-L) was synthesized and converted to
double-stranded DNA by primer extension with the Random+BbsI-S primer (Table 1)
and KOD-plus- (TOYOBO). The randomized sequence was digested with BbsI and
inserted into the BbsI sites of pB1H2!2-Z1B, pB1H2!2-Z2B and pB1H2!2-Z3B togenerate randomized libraries containing more than 2 " 109 types.
To construct a series of pH3U3-B1H derivatives as HIS3 reporter vectors for B1H
selections, several sets of two complementary oligonucleotides corresponding to partsof the HpHesC and HpEts targets sequence (Table 3) were synthesized chemically,annealed and inserted into the EcoRI/XmaI site of pH3U3-mcs (Meng et al., 2008). To
select the ZF recognizing each subsite of the HpHesC and HpEts target sequences, afirst-stage library screening in the B1H system was carried out as described by Meng &Wolfe (2006). After this selection, pools of ZFs of positive clones were amplified with
KOD-plus- from the pB1H2!2-Z1B-derived library using ZF1-forward and
ZF1-reverse primers, from the pB1H2!2-Z2B-derived library using ZF2-forward and
ZF2-reverse primers and from the pB1H2!2-Z3B-derived library using ZF3-forward
and ZF3-reverse primers (Table 1). The PCR products were analyzed by 1.5% agarose
gel electrophoresis in Tris-borate-EDTA (TBE) buffer, and the appropriate bands were8

excised and purified from the gel using a Gel Extraction Kit (Qiagen).
To construct a vector for the second stage of B1H selection, pB1H2!2-ZFA36
was subjected to inverse PCR with pB1H2!2-BbsI-F and pB1H2!2-BbsI-R primers(Table1),

followedbyinsertion

of

a

DNA

fragment(5#–

GCCTGAGTCTTC-(55bp)-GAAGACATAGAG–3#), which includes two BbsI sites atboth ends. A library of three-finger ZF arrays was assembled from three pools of
individual PCR-amplified ZFs by overlapping PCR (Fig. 1A) (Meng et al., 2008). Next,
the purified PCR products were analyzed by 1.5% agarose gel electrophoresis in TBE
buffer, and the band with appropriate sizes was excised and purified using theabove-mentioned Gel Extraction Kit. The PCR-amplified three-finger ZF arrays weredigested with BbsI and inserted into the BbsI sites of the vector to construct the ZF
array libraries for the second-stage B1H selection. The second stage of selection was
carried out using the ZF array libraries and pH3U3-B1Hs containing either an HpHesC1
site (5#–GGGCGAGTCT–3#), HpHesC2 site (5#–GGGGTCTGGA–3#), HpEts1 site (5–GGGGTTGACG–3#)

or

HpEts2site(5–GATGATGACT–3#)

(Fig.

1A).

ZF

array-positive clones were isolated from 20 mM 3-AT stringency plates, and thenucleotide sequences of the inserted cDNAs encoding the ZF arrays were analyzed.

SSA assay using HEK293T cells
To construct the pSTL vector as a ZFN expression vector for the SSA assay, the BsgIsite of pST1374 (ZFN expression vector; ZF Consortium Vector Kit v1.0) was
eliminated by inverse PCR with delta-BsgI-L and delta-BsgI-R primers (Table 1) and a
9

DNA region of pST1374 between the XbaI and EcoRI sites was substituted with a DNA
fragment, pST (Fig. 1C). Next, the cDNAs of the ZF arrays selected by the B1Hprocedure were excised by digestion with XbaI and BsgI and inserted between the XbaIand BsgI sites to generate the pSTL-ZFN expression vector. This vector includes a6-amino acid linker (TGAAAR) between the ZF DNA-binding domain and the FokI
nuclease domain that restricts the nuclease activity to sites containing a 6-bp spacer(Shimizu et al., 2009).
pGL4-SSA reporter constructs containing a ZFN target sequence between theinactive fragments of the luciferase gene were generated using a pGL4 vector(Promega). PCR was conducted to amplify 5# and 3# fragments of the luciferase gene,including a 700-bp region of homologous overlap, using the following oligonucleotides
as primers: SSA-luc2-1 and SSA-luc2-2 for the 5# fragment and SSA-luc2-3 andSSA-luc2-4 for the 3# fragment (Table 1). The luciferase gene of pGL4 was substituted
into the amplified fragments that were joined to each other by introduced XmaI sites.
The cytomegalovirus (CMV) immediate-early enhancer/promoter region was inserted
into the upstream of the inactive luciferase gene. The following oligonucleotides wereannealed and inserted between the BsaI sites of pGL4-SSA vector: SSA-GL4-C1C1-S
and SSA-GL4-C1C1-A were used to generate pGL4-SSA-C1C1, SSA-GL4-C2C2-S
and SSA-GL4-C2C2-A were used to generate pGL4-SSA-C2C2, SSA-GL4-C1C2-S
and SSA-GL4-C1C2-A were used to generate pGL4-SSA-C1C2, SSA-GL4-Ets1-S and
SSA-GL4-Ets1-A were used to generate pGL4-SSA-Ets1 and SSA-GL4-Ets2-S and
SSA-GL4-Ets2-A were used to generate pGL4-SSA-Ets2 (Table 1).
10HEK293T cells were grown in DMEM supplemented with 10% FCS. The cells
were cotransfected with 200 ng of the pSTL-ZFN expression vector, 100 ng of thepGL4-SSA reporter plasmid and 20 ng of the pRL-CMV reference vector (Promega)using Lipofectamine 2000 (Invitrogen) in 96-well plates at 50,000 cells/well. After 24 h,dual-luciferase assays were conducted using the Dual-Glo luciferase assay system(Promega) in a TriStar LB 941 plate reader (Berthold) based on the manufacturer’s
instructions.

Construction of a targeting donor construct
A genomic region spanning 2410 bp (-1137 to +1273 from the stop codon of the HpEts
gene) from sea urchin genomic DNA was amplified by PCR using Ets-HRD-F and
Ets-HRD-R primers (Table 1) and subcloned into pBluescriptII SK(+) to construct a
pBSK-Ets-HRD plasmid. Two oligonucleotides (2A-peptide-S and 2A-peptide-A)(Table 1) were synthesized and converted to double-stranded DNA by primer extension
with KOD-plus-. The 2A-peptide coding DNA fragment was subcloned into the EcoRV
site of pBluescriptII SK(+) and an H2B-GFP cassette (Fujii et al., 2009) was cloned
downstream of the 2A-peptide coding sequence to generate pBSK-2A-H2B-GFP vector.
A 2A-H2B-GFP cassette was amplified by PCR with KOD-plus using 2A peptide-2F
and T3 promoter primers (Table 1). To generate a targeting donor construct,
pBSK-Ets-HRD was subjected to inverse PCR with Ets HRD-inv-F and Ets HRD-inv-R
primers (Table 1), followed by insertion of the amplified 2A-H2B-GFP cassette.

11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 M KCl. Fertilized eggs
were cultured in filtered seawater at 16 °C.

mRNA synthesis and microinjection
cDNAs encoding ZFNs and nuclear localization signal (NLS) - the carboxy-terminal
tandem BRCT repeat of HpLig4 (designated DN-lig4) were subcloned into atranscription vector containing a T7 promoter and the $-globin leader sequence. The
plasmids containing ZFN or DN-lig4 cDNAs were linearized with restriction enzymes.
HesC ZFN and Control ZFN mRNAs capped with m7G(5')ppp(5')G were synthesizedusing a mMESSAGE mMACHINE T7 Kit (Ambion). The polyadenylated HpEts ZFN
and DN-lig4 mRNAs capped with anti-reverse cap analog were synthesized using a
mMESSAGEmMACHINE T7 ultra kit (Ambion). An MO complementary to thesequence

containing

thetranslation

start

site

of

HpHesCmRNA(5#–

ATGCTTACTTCATCTGGATACCAAC–3#) and a standard control MO (5#–CCTCTTACCTCAGTTACAATTTATA–3#) were obtained from Gene Tools. The MOs
and mRNAs were dissolved in 10% glycerol. Microinjection was carried out asdescribed by Rast (2000) with some modifications by Ochiai et al. (2008a). Two or four
picoliters of the solution was injected into each fertilized egg. To observe thephenotypes, embryos were fixed in filtered seawater containing 0.01% formaldehyde.

12Quantitative PCR
Quantitative PCR was conducted as described previously (Ochiai et al., 2008b) using
qHpDelta-F and qHpDelta-R primers (Table 1).

Whole mount in situ hybridization
Whole mount in situ hybridization was carried out following the method in Minokawa
et al. (2004). Digoxigenin-labeled RNA probes for HpDelta were prepared using T3 or
T7 RNA Polymerases (Roche) with a DIG RNA Labeling Mix (Roche).

Sequence analysis of mutations
Genomic DNAs were isolated from approximately 300 control noninjected embryos andapproximately 300 embryos injected with 10 pg each of HpHesC ZFN mRNAs at
different developmental stages. A 153-bp PCR fragment containing the target sequence
for the HpHesC ZFNs was amplified by PCR using the HT1F and HT1R as primers.
The PCR products derived from genomic DNA from HpHesC ZFN mRNA-injected
embryos at 8 h postfertilization (hpf) were purified using QIAquick PCR Purification
Kit (Qiagen) and subcloned into pBluescript II SK+ (Stratagene). The nucleotide
sequences of the clones were analyzed to determine the types of mutations around the
HpHesC ZFN target site. Aliquots (200 ng) of the PCR products were digested with
BslI (New England Biolabs) overnight, purified and analyzed by 3% agarose gelelectrophoresis.

13

Genomic PCR analysis
Forty fg of the targeting donor construct alone or in combination with 1 pg of each
HpEts ZFN mRNA were injected into fertilized sea urchin eggs. Genomic DNAs wereisolated from 300 control noninjected embryos, 300 donor-injected embryos and 300
co-injected embryos at 24 hpf using DNeasy Blood & Tissue Kit (Qiagen). PCR was
performed with KOD-plus- using extracted genomic DNA and with 0.3 µM of gene
specific primers for HpArs (Ochiai et al., 2008a) for 25 cycles (94°C 15 sec, 60°C 15
sec, 68°C 1 min) or for confirmation of targeted gene addition (primer1 and 2) (Table 1)
for 37 cycles (94°C 30 sec, 65°C 30 sec, 68°C 4 min). PCR products for HpArs andconfirmation of targeted gene addition were separated on 2% and 1% agarose gels,
respectively.

Reverse transcription PCR analysis
Total RNA was extracted from approximately 300 control noninjected embryos,
approximately 300 embryos injected with 40 fg of the targeting donor construct,approximately 300 embryos injected with 40 fg of the targeting donor construct and 1pg
of HpEts ZFN mRNA and approximately 300 embryos injected with 40 fg of thetargeting donor construct and 1pg of HpEts ZFN mRNA in combination with 5 pg of
DN-lig4 mRNA at 24 hpf using ISOGEN (NIPPONGENE, Japan) as described in the
manufacturer’s manual. The cDNAs were prepared by using a ThermoScript RT-PCR
System (Invitrogen) with random hexamers following the manufacturer’s instructions.
Targeted gene additions were detected by the following PCR with KOD-plus- using 0.3
14µM of primers (primer3 and primer4) (Table 1) for 35 cycles (94°C 30 sec, 60°C 30 sec,
68°C 4 min). As a control, mitochondrial cytochrome oxidase subunit I (HpMitCOI)
mRNA, which is expressed constitutively at the same level during sea urchindevelopment (Okabayashi & Nakano, 1983; Yamaguchi & Ohba, 1994; Fujiwara &Yasumasu, 1997) was detected by PCR using reaction mixtures containing 0.3 µM of
HpMitCOI-specific primers (Ochiai et al., 2008a) for 23 cycles (94°C 15 sec, 60°C 15
sec, 68°C 1 min). The PCR products for the targeted gene insertion and for HpMitCOI
were separated on 1% and 2% agarose gels, respectively.

Live imaging of sea urchin embryos
Injected embryos were reared in 35-mm tissue culture dishes and allowed to develop to
the late gastrula stage (30 hpf). Embryos were then mounted in a microchamberassembled as follows: (i) High vacuum grease (DOW CORNING) was applied at thecorners of a square 15 mm wide on a 35-mm glass based dish (Iwaki): (ii)
Approximately 100 embryos were mouth-pipetted onto the center of the glass surface:(iii) The microchamber containing the embryos was closed with a 18 x 18-mm #1
coverslip (Matsunami): (iv) The corners of the coverslip were gently pressed to squeeze
and the immobilize swimming embryos: and (v) The microchamber was filled withfiltered sea water and sealed with mineral oil (Sigma-Aldrich). The embryos werevisualized on IX-81 fluorescence microscope (Olympus) equipped with MetaMorphsoftware (Universal Imaging).

15

Statistical analysis
Data are presented as means ± SD. Differences were evaluated for significance byStudent’s t-test, with significance defined as values of P < 0.01 using a two-tailed
unpaired t-test.

16

Results

Generation of functional ZFNs targeting the HpHesC geneTo evaluate the functionality of the engineered ZFNs in the sea urchin, I generated
ZFNs targeting the HpHesC gene. The sea urchin HesC gene, which is expressed in thewhole embryo except for the micromere lineage during early embryogenesis, represses
several transcription factor genes that are responsible for PMC differentiation(Revilla-i-Domingo et al., 2007; Yamazaki et al., 2009). When this gene is repressed at
the translational level by an MO, the number of PMCs increases (Revilla-i-Domingo etal., 2007). First, I cloned a cDNA for HpHesC and determined its nucleotide sequence.
The HpHesC protein was predicted to have a basic helix-loop-helix DNA-binding
domain and a Hairy Orange domain. Therefore, I designed ZFNs targeting a site
between these two conserved domains. If an NHEJ-mediated mutation was introduced
at the target site by ZFNs, I expected that the function of the HpHesC gene would be
disrupted (Fig. 2). In the genomic sequence of Strongylocentrotus purpuratus, which is
closely related to H. pulcherrimus, a nucleotide sequence identical to the target site (5’–TCCAGACCCCNNNNNNGGGCGAGTCT–3’) was found only in the SpHesC gene.
However, single nucleotide polymorphisms (SNPs) can be detected in the coding region
as well as the intron in the H. pulcherrimus gene (Yamamoto et al., 2007). Therefore, toconfirm that no SNPs were present in the target site, I examined the genetic variationsin the target sequence of the HpHesC gene. No polymorphisms were found for H.

17

pulcherrimus in the target site (data not shown).
To generate three-finger ZFNs targeting the HpHesC gene, I adopted the directeddomain shuffling and B1H system described by Meng et al. (2008) with somemodifications (Fig. 1A). The Cys2His2 ZF domain, which consists of approximately 30
amino acid residues, recognizes a target subsite that consists of 3 or 4 bp within its
recognition helix (Wolfe et al., 2000). In the engineering strategy, highly specificthree-finger ZF arrays recognizing 9 or 10 bp were selected from the libraries, in which
each recognition helix had been randomized (codon encoded VNS in which V
represents A, C or G, N represents A, C, G or T and S represents G or C, seeExperimental Procedures for details). The framework involving sequences outside therecognition helix in the ZF domain was reported to be involved in structural stability(Shi & Berg 1995). Therefore, to construct ZF randomized libraries, I adopted the Sp1C
consensus framework, which has been shown to have enhanced stability towardchelating agents (Shi & Berg 1995; Mandell & Barbas 2006). In addition, many species
of designed ZFs that recognize DNA triplets have been generated using this framework(Segal et al. 1999; Dreier et al., 2000, 2001, 2005).
After two rounds of screening with the randomized libraries (Fig. 1A, seeExperimental Procedures for details), I obtained 10 cDNA clones for three-finger ZF
arrays targeting the HpHesC1 site (5’–GGGGTCTGGA–3’) and 30 cDNA clones forthree-finger ZF arrays targeting the HpHesC2 site (5’–GGGCGAGTCT–3’). I
determined the nucleotide sequences of 10 cDNA clones of each type. Alignment of thepredicted amino acid sequences of the recognition helices in the ZF arrays showed that18

some residues in the recognition helix occurred frequently (Fig. 3). For example, the +6
residues in the second recognition helix of the HpHesC1-binding ZF array clones were
all arginine residues.
Next, to screen for functional ZFNs, ZFN expression constructs were generated
by fusion of the cDNAs for the selected ZF arrays and a nuclease domain of FokI, andan SSA assay was carried out (Szczepek et al., 2007). In the SSA assay, a ZFN
construct and a reporter construct, in which the luciferase gene is split into two inactive
fragments separated by both insertion of a stop codon and a pair of target sites for either
HpHesC1 or HpHesC2, were cotransfected into cultured cells. When a DSB at the target
site is induced by the ZFN homodimers, SSA occurs between the homologous regionsof the inactive fragments, producing an active luciferase gene (Fig. 4A). As shown in
Fig. 4B, the individual ZFNs for HpHesC1 or HpHesC2 showed different levels ofreporter activity as homodimers. However, some ZFNs had low levels of activity in the
SSA assay (Fig. 4B), although all the selected ZF arrays possessed the potential to bindto HpHesC sites. In the assayed samples, clone C1-4 for the HpHesC1 site and clone
C2-2 for the HpHesC2 site showed the highest activities. To examine the activity of
ZFNs as heterodimers, an SSA reporter vector containing both HpHesC1 and HpHesC2
sites was prepared and the SSA assays were carried out using the C1-4 and C1-8 clones
for the HpHesC1 site, which showed the highest and lowest activities as homodimers,
respectively (Fig. 4B), and the C2 clones for the HpHesC2 site (Fig. 4C). Consistent
with the activity of the ZFNs as homodimers, the combination of clone C1-4 for the
HpHesC1 site and clone C2-2 for the HpHesC2 site showed the highest activity among19combinations examined. I referred to these ZFNs as HpHesC1 ZFN and HpHesC2 ZFN
and used them for use in further experiments.

Effects of injecting mRNAs for the ZFNs targeting the HpHesC gene into sea urchin
embryos
It has been reported that the FokI nuclease domain variants RR and DD have a reduced
their propensity for homodimerization, thereby decreasing the frequency of off-target
cleavage events through homodimerization of an individual ZFN (Szczepek et al., 2007).
Therefore, the nuclease domains of HpHesC1 ZFN and HpHesC2 ZFN were mutated to
DD and RR versions, respectively, generating HpHesC1DD and HpHesC2RR ZFNs.
To validate the functionality of ZFNs in sea urchin embryos, HpHesC ZFNmRNAs or HpHesC MO, which target the translation start site of HpHesC mRNA, were
injected (Fig. 5). In the embryos injected with HpHesC MO, an increase in the numberof PMCs in the blastocoel was observed at 24 hpf (Fig. 5C) consistent with a previous
report for S. purpuratus (Revilla-i-Domingo et al., 2007). In this thesis, this phenotype
is called the HpHesC morphant phenotype. In the embryos injected with 6.5 pg each of
the HpHesC1DD and HpHesC2RR ZFN mRNAs, most developed normally to the earlygastrula stage, although some embryos showed the HpHesC morphant phenotype (Fig.
5B). When 13 pg each of the HpHesC ZFN mRNAs were injected, the HpHesC
morphant phenotype was observed in 9.5% of the ZFN-injected embryos, but severely
malformed embryos were also observed (Table 4). To verify whether the observed
effects were HpHesC ZFN mRNA-specific, I prepared a control set of three-finger
20

ZFNs targeting 5’–GCGGCCAGGG–3’ and 5’–GGTGTCCATG–3’. The target
sequence for these control ZFNs, 5’–CCCTGGCCGCNNNNNNGGTGTCCATG–3’,
was not found in the genomic sequence of S. purpuratus. When 13 pg of each of thecontrol ZFN mRNAs were introduced into sea urchin eggs, most embryos developed
normally and no embryos with the HpHesC morphant phenotype were observed (Fig.
5A, Table 4). These findings indicate that the HpHesC morphant phenotype was
induced by the introduction of the HpHesC ZFN mRNAs.
To confirm that the HpHesC morphant phenotype found in the embryos injectedwith HpHesC ZFN mRNAs was caused by disruption of the HpHesC gene, theexpression of the Delta gene, which was reported to be down-regulated by HesC in S.
purpuratus (Revilla-i-Domingo et al., 2007), was analyzed at 15 hpf by quantitative
PCR. In embryos injected with both the HpHesC1DD and HpHesC2RR ZFN mRNAs, theamount of HpDelta mRNA was significantly increased compared with that in embryos
injected with the HpHesC1DD or HpHesC2RR ZFN mRNA alone (Fig. 5D). The
expression of HpDelta mRNA was also examined at 12 hpf by whole mount in situ
hybridization. In embryos injected with control ZFN mRNAs, HpDelta mRNA
expression was observed in the presumptive PMCs at the vegetal pole (Fig. 5E). In
embryos injected with HpHesC ZFN mRNAs, although most embryos showed the
HpDelta mRNA expression in presumptive PMCs as observed in control ZFN
mRNA-injected embryos, in some embryos an expansion of mRNA expression at thevegetal pole and ectopic expression in ectoderm was observed (Fig. 5F).
Next, I analyzed the mutations introduced into the HpHesC target site by the21

ZFNs (Fig. 6). DNA fragments amplified by PCR using genomic DNA extracted from
HpHesC ZFN mRNA-injected embryos at 8 hpf were subcloned and their nucleotide
sequences were determined (Fig. 6A). Among the 34 clones examined, deletions (17clones, 50%) and insertions (four clones, 12%) were observed. In some of the clones
that had deletions, substitutions were also observed. Overall, 44% of the HpHesC genes
were disrupted by frameshifts. These results indicated that the endogenous HpHesC
gene was disrupted by ZFN-induced site-specific mutagenesis in these sea urchin
embryos.
To examine when the introduction of mutations by ZFNs occurred during the sea
urchin development, DNA fragments amplified by PCR using genomic DNA extracted
from HpHesC ZFN mRNA-injected embryos and control embryos at several stages ofdevelopment were digested with the restriction enzyme BslI, which has two recognition
sites in the target site for the HpHesC ZFNs (Fig. 6B). The DNA fragments derived
from the control embryos were cut completely by BslI at all developmental stages (Fig.
6B). In contrast, a BslI-resistant fragment was faintly observed among the DNA
fragments from the HpHesC ZFN mRNA-injected embryos at 4 hpf (eight-cell stage)
and the amount of this fragment increased from 8 hpf (morula stage) to reach a plateau
at 12 hpf (unhatched blastula stage) (Fig. 6B). These findings suggest that the
ZFN-mediated mutations were introduced mainly between the eight-cell and unhatchedblastula stages.

22

Necessity of the SSA assay for selection of functional ZFNs
Although B1H-selected ZF arrays have the potential to bind to their target sites, some
ZFNs showed low activities in the SSA assays (Fig. 4B). To investigate if thesedifferences in activity in the SSA assay originate from the functional differences of
ZFNs, several mRNAs for ZFNs that show higher (C1-4, C2-1 and C2-2) or lower
activities (C1-8 and C2-7) in the SSA assay were injected in different combinations into
sea urchin eggs. PCR-based analysis was then performed using genomic DNA extracted
from the injected embryos at 14 hpf (Fig. 6C). In agreement with the ZFN activities
observed in the SSA assay, mutations at the HpHesC target site were more efficiently
introduced in the sea urchin embryos by the injection of clone C1-4 for the HpHesC1
site in combination with clone C2-2 for the HpHesC2 site than by the injection of clone
C2-1 (Figs 4C and 6C). This suggests that the activities of the ZFNs in the SSA assay
are consistent with their activity in the sea urchin embryos and supports the proposalthat functional ZFN clones can be selected by the SSA assay. Taken together, the results
suggest that, subsequent to B1H selection, the SSA assay can be used to efficiently
generate functional ZFNs.

Targeted gene addition into the HpEts locus using ZFNs
Recently, in mouse zygotes, targeted gene addition was achieved by the introduction of
ZFN mRNAs with a targeting donor construct (Meyer et al., 2010). This prompted me
to explore the possibility of inserting a reporter cassette into a genomic site of interest in
the sea urchin embryos. To explore this further, HpEts ZFNs, whose target site (5’–23

CGTCAACCCCNNNNNNGATGATGACT–3’) is located immediately upstream of thestop codon of the HpEts gene, were selected by B1H and SSA screening (Fig. 7A). The
Ets transcription factor, coded for by the HpEts gene, is expressed in PMCs andsecondary mesenchyme cells (SMCs) at late gastrula stage (Kurokawa et al., 1999;
Rizzo et al., 2006). These ZFNs are subsequently referred to as HpEts1 and HpEts2
ZFNs. The FokI nuclease domains of the HpEts1 and HpEts2 ZFNs were mutated tosharkey RR and sharkey DS variants, respectively, because they were recently reported
to have enhanced cleavage activities (Guo et al., 2010). A targeting donor constructwith approximately 1-kb homology arms containing a 2A-histone H2B-GFP cassette(2A is a self cleaving peptide sequence, Szymczak et al., 2004), was also generated (Fig.
7A). Therefore, insertion of the reporter cassette into the HpEts locus is expected toresult in the expression of two proteins: a fusion protein comprising the full length of
HpEts fused to the 17 amino acid sequence of the 2A peptide; and H2B-GFP, which
localizes to cell nuclei where its fluorescence can be more accurately quantified(Sprinzak et al., 2010). Both proteins are under the control of the endogenous HpEts
promoter (Fig. 7A).
To validate the utility of ZFN-mediated targeted gene addition in the sea urchin,injections of the targeting donor construct with or without the HpEts ZFN mRNAs werecarried out and genomic PCR analysis was performed using genomic DNA extracted
from the embryos at 24 hpf (Fig. 7B). While, as expected, no PCR product was detectedin the noninjected and donor-injected samples, a PCR product of the expected size wasobserved in the co-injected sample, suggesting that targeted gene addition was induced
24

by the introduction of ZFNs.
Recently, in Drosophila, although it was reported that HDR with a targeting
donor construct is a minor event compared with error-prone NHEJ repair in wild-type
flies, the efficiency of HDR with an exogenous donor construct was greatly enhanced inrecipients deficient in the NHEJ component DNA ligase IV (Beumer et al., 2008),
suggesting that inactivation of DNA ligase IV increases the efficiency of ZFN-mediatedtargeted gene addition. However, based on a microarray analysis, mRNA for SpDnl4, agene that encodes the S. purpuratus homologue of DNA ligase IV, exists in sea urchin
embryoimmediately

after

fertilization(Sp-Dnl4,

SPU_018243,

http://urchin.nidcr.nih.gov/blast/exp.html), suggesting that the DNA ligase IV proteins
may be maternally deposited in the egg and that inhibition of their function using MO
may not work in the sea urchin embryos. Therefore, I cloned cDNA for the
carboxy-terminal tandem BRCT repeat of DNA ligase IV (DN-lig4), the overexpressionof which was reported to achieve a dominant-negative effect in human cultured cells(Wu et al., 2009), and examined the effect of DN-lig4 overexpression on the efficiency
of ZFN-mediated targeted gene addition by reverse transcription (RT)-PCR analysis
using RNAs that were extracted from injected embryos at 24 hpf (Fig. 7C). The
expression of the HpEts-2A-H2B-GFP fusion gene was detected in the embryos into
which HpEts ZFN mRNAs and the targeting donor construct were injected without
DN-lig4 mRNA and the expression was significantly increased in the embryos into
which HpEts ZFN mRNA and the targeting donor construct were coinjected in
combination with DN-lig4 mRNA. This result suggests that introduction of DN-lig4
25

mRNA enhances the efficiency of ZFN-mediated targeted gene addition. Next, I
examined H2B-GFP expression in the embryos into which HpEts ZFN mRNAs and thetargeting donor construct were injected in combination with DN-lig4 mRNA using
fluorescence microscopy at 30 hpf (late gastrula stage) (Fig. 7D–I). About 10 % of theinjected embryos showed GFP expression in the nuclei of some PMCs and SMCs, inwhich Ets gene was reported to be expressed (Kurokawa et al., 1999; Rizzo et al.,2006).

26

Discussion
Generation of functional ZFNs by the combined use of B1H and SSA screenings
Recently, genetic manipulation using ZFN technology has been reported in several
model organisms (Beumer et al., 2008; Doyon et al., 2008; Shukla et al., 2009; Zou etal., 2009). Despite its significance and utility, ZFN technology has not been widely usedbecause the methods for generating functional ZFNs are considered to be very
complicated. In this thesis, I successfully selected functional ZFNs by the combined useof a B1H system involving ZF randomized libraries and an SSA assay using cultured
cells. In addition, I showed that targeted gene editing in sea urchin embryos wasachieved using the selected ZFNs.
In this work, I selected several ZF arrays that bind to HpHesC1, HpHesC2,
HpEts1 and HpEts2 target sites using B1H selection with ZF randomized libraries. It
was reported earlier that individual ZF domains in a ZF array recognize their target
subsites through the recognition helix (Wolfe et al., 2000). Among the selected ZF
arrays, specific residues in the recognition helices were found to be correlated withspecific bases in the target subsites. For example, when guanine was the first andsecond nucleotide from the 5’ end of the subsite, arginine and histidine residues
occurred most frequently at positions six and three, respectively of the recognition
helices of the B1H-selected ZF arrays. The relationship between a specific base in thetarget subsite and amino acid residue in the recognition helix has been observed innatural ZF proteins and ZF arrays selected by phage display (Wolfe et al., 2000),27

suggesting that I successfully selected appropriate ZF arrays by using B1H selection
with randomized ZF libraries. However, there were several exceptions to these
relationships among the frequent residues in the recognition helices of the B1H-selected
ZF arrays. For example, when cytosine was the third nucleotide from the 5’ end of thesubsites, aspartic acid occurred most frequently at the -1 position in the second
recognition helix from the N-terminal end of the B1H-selected ZF arrays that bound to
the HpHesC1 target site, whereas threonine was found most frequently at position -1 ofthe first recognition helix from the N-terminal end of the B1H-selected ZF arrays thatbound to the HpHesC2 target site. The mechanisms for DNA sequence recognition by
ZF arrays are thought to be complex, because their specificities are affected by several
factors, including sequence-dependent conformational flexibility of the DNA, and side
chain-side chain interactions in the ZF array (Wolfe et al., 2000). Therefore, theinteraction residues in the recognition helices were assumed to be determined by several
factors, rather than a simple one-to-one interactions between specific positions on thehelix and specific base-pairs in the ZF recognition site.
Although the B1H-selected ZF arrays possibly have the potential to bind to theirtarget sequences, some ZFNs, which consist of both a B1H-selected ZF array and anuclease domain of FokI, showed low activities in the SSA assay and the efficiency ofmutagenesis using these ZFNs was also found to be low in the sea urchin embryos.
Because DNA sequence recognition by ZF arrays is thought to be affected by several
factors (Wolfe et al., 2000), it is also possible that the interaction between a ZF array
and its fusion partner could affect the DNA-binding activity of the ZF array. Indeed, the28

fusion partner of a ZF array in B1H selection is different from that in an SSA assay
because, in the B1H selection, the omega subunit of RNA polymerase is used as thefusion partner of the ZF array while in the SSA assay, a nuclease domain of FokI isused. Thus, the fusion of a ZF array with the nuclease domain of FokI is likely tochange the DNA-binding activity of the ZF array. Alternatively, the nuclease activity ofa ZFN could be considered to decrease when the ZF array and the nuclease domain are
fused. Thus, for efficient generation of highly functional ZFNs, the assessment of boththe DNA-binding activity of ZF arrays by bacterial-based selection and the evaluationof ZFNs by the SSA assays are necessary.

Targeted mutagenesis in sea urchin embryos using ZFNs
The use of the ZFNs evaluated by the SSA assay was successful in introducing
several types of mutations into a specific genomic site in sea urchin embryos. Most ofthese mutations were frameshift mutations that disrupted the HpHesC gene. However,
because the target sites of the HpHesC ZFNs were designed to lie between the
DNA-binding domain and the Hairy Orange domain, it is possible that the expression of
the DNA-binding domain of HpHesC induces dominant-negative effects on theregulation of genes that are regulated by HesC. To examine this possibility, I injected anmRNA for the N-terminal DNA-binding domain of HpHesC into fertilized eggs andobserved that these embryos did not show the HpHesC morphant phenotype anddeveloped normally into pluteus larvae, suggesting that the N-terminal DNA-binding
domain of HpHesC does not act in a dominant-negative manner (data not shown).
29

Regardless of the mutagenic frequency (44%), only 9.5% of the HpHesC ZFN
mRNA-injected embryos showed the HpHesC morphant phenotype. Considering thefinding that the introduction of ZFN-induced mutations mainly occurred between theeight-cell and hatched blastula stages in HpHesC ZFN mRNA-injected embryos, it ispossible that this delay in the timing of the introduction of mutations may contribute to
the difference in the rates of the genotype and phenotype incidences. In the sea urchin S.
purpuratus, the expression of HesC begins at the morula stage (8 hpf) repressing theseveral of the transcription factor genes responsible for the differentiation of PMCs
(Revilla-i-Domingo et al., 2007). Therefore, it is possible that the disruptions of
HpHesC may occur after the stage when HesC exerts its functions. Another possibility
is that only cell descendants in which both alleles of the HpHesC gene were disrupted
exhibited the HpHesC morphant phenotype and cells in which only one allele of the
HpHesC gene was disrupted in the ZFN mRNA-injected embryos did not show thephenotype.

Targeted gene addition in sea urchin embryos using ZFNs
Insertion of a reporter gene into a target site was detected by PCR-based analysis
in embryos coinjected with the targeting donor construct and ZFN mRNAs. In embryos
injected with the targeting donor alone, targeted gene addition was not observed. These
results indicate that spontaneous homologous recombination is a rare event in the sea
urchin and that ZFN-induced DSB at the target site stimulate HDR, resulting in an
induction of targeted gene addition at a detectable level in agreement with earlier work30

using different models (Beumer et al., 2008; Meyer et al., 2010). Moreover,introduction of DN-lig4 mRNA further increased the efficiency of ZFN-mediatedtargeted gene addition. It has been reported that DNA Ligase IV, a major component of
the NHEJ pathway, forms a complex with XRCC4 and seals DNA ends (Wu et al.,2009) and that DSB repair is biased toward HDR by a disruption of the function of
DNA ligase IV resulting in an increase in the efficiency of targeted gene modification(Beumer et al., 2008). Therefore, in the sea urchin, it is proposed that an inhibition of
DNA ligase IV by introduction of DN-lig4 increases the propensity to repair
ZFN-induced DSB through HDR, resulting in an enhancement in the efficiency of
ZFN-mediated targeted gene addition. Although, in the Lig4 mutant of Drosophila,
DSB repair was reported to be almost completely biased toward HDR, the enhancementin the efficiency of ZFN-mediated targeted gene addition using DN-lig4 was modest in
the sea urchin (Beumer et al., 2008). This may be because, when DSBs were introduced
by ZFNs, DN-lig4 proteins are not translated at a rate high enough to completely inhibit
the function of endogenous DNA ligase IV proteins. Therefore, the introduction ofrecombinant DN-lig4 and ZFN proteins into fertilized sea urchin eggs might
considerably enhance the efficiency of ZFN-mediated targeted gene addition. The
inhibition of NHEJ activity and the simultaneous enhancement of the activity of HDR
may efficiently induce ZFN-mediated targeted gene addition in sea urchin embryos. It
has been reported that overexpression of Rad51, a protein with homologous DNA
pairing and strand exchange activities, increases the efficiency of gene targeting inhuman cells (Yáñez & Porter, 1999).
31

In sea urchin embryos, the injection of DN-lig4 mRNA did not induce any
developmental abnormality until at least the 72 hpf pluteus stage (data not shown),suggesting that DNA ligase IV-dependent NHEJ is not necessary in the early
development of the sea urchin. In mice, inactivation of DNA Ligase IV results inembryonic lethality as a consequence of massive apoptosis in the central nervous
system (Frank et al., 2000), while Drosophila deficient for Lig4 are viable and fertileand show no obvious signs of defects or other abnormalities (Gorski et al., 2003). Thesesuggest that an enhancement in the efficiency of ZFN-mediated targeted gene addition
through inactivation of DNA ligase IV may work only in a limited number of modelorganisms.

In conclusion, I have demonstrated here that functional ZFNs can be selected by
the combined use of B1H and SSA screenings, and that using the selected ZFNs,
targeted mutagenesis and targeted gene addition were achieved in sea urchin embryos.
Moreover, I have shown that endogenous gene expression can be visualized by theinsertion of a 2A-H2B-GFP cassette into the HpEts locus in living sea urchin. These
techniques provide useful tools for the analysis of the expression dynamics ofendogenous genes during the development of sea urchin embryos that could be used infuture work.

32

Figures

33

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3B"/%#'(%C6%./%'D==7BE:=<&'%!"!#$#$!$"#$#!$!"$##"!$"#!"#"#"$$$!$"!$"$$"##$#$"!"!#$!"#### )*$!$##"!$$#$$#$##$###!"!$##"!!"$!"#!###!!$###!#!$!$""!"#!$##! +,*+)#!#!!$##!!##!!$###!!$""#$###!!""#"!"#$$#!#$##!!"% ('Figure 1. B1H screening and the construction of randomized ZF libraries. (A) Bacterial
one-hybrid (B1H) screening. Schematic representations of the first selection stage of
B1H screening to identify single zinc fingers (ZFs) with optimized binding to each 3-bp
subsite within a recognition element is shown in the left panel. Schematic
representations of in vitro recombination of individual zinc fingers and subsequent
second selection stage against full ZF array recognition elements are shown on the rightside of the panel. (B) Nucleotide sequences of randomized sequence-receptible modules,
ZF1-BbsI, ZF2-BbsI and ZF3-BbsI. (C) Nucleotide sequence of DNA fragment pST.

34

Figure 2. The HpHesC gene showing the ZFN target site. A schematic representationof the Hemicentrotus pulcherrimus homologue of the HesC gene (HpHesC) is shown.
Exons are indicated by boxes. Gray and black boxes represent untranslated regions andcoding regions, respectively. The bent arrow depicts the transcription start site. The
ZFN-targeted sequence and the interaction site of a pair of the ZFNs used in this thesis
are shown.

35

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derived from selected clones for the HpHesC1 (5’GGGGTCTGGA3’) and HpHesC2(5’GGGCGAGTCT3’) target sites, respectively. The numbers of residues in therecognition helix (No. in RH) and the target sequences are shown above and below eachpanel, respectively. The phylogenetic trees are shown on the right side of each panel.

36

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Figure 4. Evaluation of the functionality of engineered ZFNs using the SSA assays.
For the dual-luciferase assay, HEK293T cells were transfected with three types ofplasmids: a ZFN-expressing plasmid, a reporter plasmid and a reference plasmid. Aftertransfection, the cells were incubated for 24 h, lysed and analyzed for their luciferase
activity. (A) Schematic diagram of the SSA assay. The reporter plasmid encodes two
split inactive parts of the luciferase gene with overlapping repeated sequences.
Following a DSB caused by the ZFNs, a functional luciferase gene is generated by an
SSA reaction. The hatched box represents a stop codon. (B) Cellular activities of theengineered ZFNs as homodimers. The fold activation of recombination by each ZFN
represents the luciferase expression level in cells cotransfected with the ZFN vector,
SSA reporter vector (either pGL4-SSA-C1C1 or pGL4-SSA-C2C2, see Experimental
Procedures for details) and pRL-CMV reference vector compared with that in cells
transfected with a control empty vector, SSA reporter vector and reference vector. Data
are expressed as means ± SEM (n = 3). (C) Cellular activities of the engineered ZFNs asheterodimers. The expression level in cells cotransfected with SSA reporter vector(pGL4-SSA-C1C2, see Experimental Procedures for details), reference vector andindicated ZFN vector set was analyzed. The data were normalized to reference Renillaluciferase activity.
37

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Figure 5. The effect of injection of HpHesC ZFN mRNAs into sea urchin embryos.
(A–C) Bright-field images of embryos at 24 h postfertilization (hpf). (A) Control ZFN
mRNA-injected embryo. (B) HpHesC ZFN mRNA-injected embryo. (C) HpHesC
MO-injected embryo. Among the HpHesC ZFN mRNA-injected embryos, mostembryos developed normally, although some embryos showed the HpHesC morphantphenotype (B). (D) Relative amounts of HpDelta mRNA in the sea urchin embryos
injected with HpHesC ZFN mRNAs. The indicated amounts of ZFN mRNAs were
injected into fertilized eggs. Total RNA was isolated from the embryos at 15 hpf, and
HpDelta mRNA was analyzed by quantitative PCR. The expression levels in the ZFN
mRNA-injected samples were normalized by those in the HpHesC1DD ZFNmRNA-injected samples. *P < 0.01, by Student’s t-test. (E–F) Whole mount in situ
hybridization showing the spatial expression of HpDelta mRNA at 12 hpf. (E) Control
ZFN mRNA-injected embryo. (F) HpHesC ZFN mRNA-injected embryo. Arrow
indicates HpDelta mRNA expression in ectoderm. Asterisk indicates the vegetal pole.

38

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Figure 6. Analysis of the mutations induced by ZFNs. (A) Sequences from in sea
urchin embryos injected with HpHesC ZFN mRNAs. The wild-type sequence is shownat the top with the ZFN-binding sites (boxed). Deletions are indicated by red dashes andinsertions and substitutions are indicated by red letters. (B) A schematic representationof the HpHesC genomic region used for the PCR-based analysis is shown in the left
panel. This region contains a target site for HpHesC ZFNs and three BslI sites. Two of
the BslI sites are positioned in the middle of the HpHesC ZFN target site. In the rightpanel, a representative analysis of the PCR products during development is shown. The
PCR products from genomic DNA of sea urchin embryos injected with HpHesC ZFNmRNAs (represented as Z) or control embryos (represented as C) were purified,digested with BslI and analyzed by agarose gel electrophoresis. The times of the
genomic DNA extractions are shown at the top of the image. (C) The in vivo activity ofseveral ZFN clones. The activity of the indicated combinations of C1 and C2 ZFNs wasanalyzed by the PCR-based analysis as described in (B). The genomic DNA wasextracted at 14 h postfertilization.
39

Figure 7. ZFN-mediated targeted gene addition. (A) The targeting donor construct for
the insertion of 2A-histone H2B-green fluorescent protein (GFP) cassette into the
HpEts locus. The ZFN-targeted sequence and a pair of ZFNs for HpEts target site areshown. The structure of HpEts locus (part of intron 7 and exon 8) and the targeted
HpEts allele is shown below. Gray and black boxes represent coding and non-codingexons, respectively. Schematic representation of the transcript and proteins, which areseparated into the full length HpEts protein and H2B-GFP during translation in order to
2A self cleavage peptide, derived from the targeted HpEts allele are also shown. Primer
sites for genomic PCR and reverse transcription-PCR (RT-PCR) analysis are indicated.
(B) Representative results of PCR-based genotyping analysis of HpEts locus. PCR was
performed using genomic DNA extracted from embryos, into which indicated samples
40

were injected immediately after fertilization, at 24 h postfertilization (hpf) and primers
either for confirmation of targeted gene addition (primer1 and primer2) (A) or for
HpArs. The PCR products were separated by gel electrophoresis. (C) RT-PCR approach
using HpMtCOI as an internal standard. Total RNAs were extracted from 24-hpf
embryos, into which indicated samples were injected, and reverse transcribed. These
cDNAs were subjected to RT-PCR using primer3 and primer4 (A). (D-I) GFP
expressing embryos at 30 hpf injected with HpEts ZFN mRNAs, the targeting donor
construct and DN-lig4 mRNA. (D-F) A representative embryo showed GFP expression
in primary mesenchyme cells (PMCs) viewed from vegetal pole (vv). (G-I) Anotherrepresentative embryo showed GFP expression in a secondary mesenchyme cell (SMC)
viewed from animal pole (av). D and G are brightfield images. E and H are fluorescentimages. F and I are merged images of D-E and G-H, respectively. Arrowheads andarrows indicate GFP fluorescence in the nuclei of PMCs and a SMC, respectively.
Background autofluorescences were denoted by asterisks.

41

Table 1. Nucleotide sequences of oligonucleotides used in this thesis
Table 1. Nucleotide sequences of oligonucleotides used in this study
Oligonucleotides

Nucleotide sequences

HpHesCF

5!–ATCTCTGCCTCTAAGGAATC–3!

HpHesCR

5!–GACAATCAACGCAATTCATC–3!

CTlig4F

5!–CTGAATTCCTGGAGGGACGAGAGCTCTGTATCATG–3!

CTlig4R

5!–CTCTCGAGTCAAGACATTGCTGCTTC * RTA–3!

HT1F

5!–AAAGCTGACATCTTGGAGATG–3!

HT1R

5!–GATGCTCTCGCAATTCGACAT–3!

ET1F

5!– ACCCAAGATGAACTACGAGAAGC –3!

ET1R

5!– GGAAAAAGCGACACTCCTCC –3!

3Z-F

5!–ACAATTGGGTAGTACGATGA–3!

3Z-R

5!–GCATCATCTCACTAGTGTGCAGAGGATCCACGCAG–3!

Random+BbsI-S

5!–GCGATCATGATCATGGAAGAC–3!

Random+BbsI-L

5!–TGGTGCAGCGTACTAGAAGACGTGTGTACGCTGGTGSNBSNBAAGSNBSNBSNBSNBGCTAAAACTCTTTCCACCAGTCTTCCATGATCATGATCGC–3!

ZF1-forward

5!–AAGGTCGTGCGGCCGGAAGACAAGCCTTACAAATGCCCAGAA–3!

ZF1-reverse

5!–CAGGACACTTATAGGGTTTTTCCCCGGTATGTGTA–3!

ZF2-forward

5!–GAAAAACCCTATAAGTGTCCT–3!

ZF2-reverse

5!–CCGGGCACTTGTATGGCTTCTCCCCGGTATGTGTA–3!

ZF3-forward

5!–GAGAAGCCATACAAGTGCCCG–3!

ZF3-reverse

5!–GACTTGTCGGCCTTGAAGACGTCTCTAGTGTGCAGAGGATCCACGCAGG–3!

pB1H2!2-BbsI-F

5!–AGAGACTAGAAAAAGGCCGACA–3!

pB1H2!2-BbsI-R

5!–AGGCTTCTCCCCGGGTCTAGAT–3!

delta-BsgI-L

5!–TGACACCAGTGAAGATGCGG–3!

delta-BsgI-R

5!–ACTGCTGCTGCTGCGGCAGC–3!

SSA-luc2-1

5!–ATGCGCGGTACCTAGTTATTAATAGTAATCAA–3!

SSA-luc2-2

5!–ACCGGTCCCGGGCACGACAGAGACCCTAGTCCAAGTCCACCACCTTAGC–3!

SSA-luc2-3

5!–CCCGGGTACTGATGTACCGTGAGACCTAGGAGCGCGAGCTGCTGAACAG–3!

SSA-luc2-4

5!–ATGATCTAGACTGCAGTTAAGAATTC–3!

SSA-GL4-C1C1-S

5!–GTCGGATCCAGACCCCATGACTGGGGTCTGGAGGT–3!

SSA-GL4-C1C1-A

5!–CGGTACCTCCAGACCCCAGTCATGGGGTCTGGATC–3!

SSA-GL4-C2C2-S

5!–GTCGGAAGACTCGCCCTTGACAGGGCGAGTCTGGT–3!

SSA-GL4-C2C2-A

5!–CGGTACCAGACTCGCCCAGTCAAGGGCGAGTCTTC–3!

SSA-GL4-C1C2-S

5!–GTCGGATCCAGACCCCAGAGCAGGGCGAGTCTGGT–3!

SSA-GL4-C1C2-A

5!–CGGTACCAGACTCGCCCTGCTCTGGGGTCTGGATC–3!

SSA-GL4-Ets1-S

5!–GTCGGACGTCAACCCCGTGACCGGGGTTGACGGGT–3!

SSA-GL4-Ets1-A

5!–CGGTACCCGTCAACCCCGGTCACGGGGTTGACGTC–3!

SSA-GL4-Ets2-S

5!–GTCGGAAGTCATCATCGGTGTCGATGATGACTGGT–3!

SSA-GL4-Ets2-A

5!–CGGTACCAGTCATCATCGACACCGATGATGACTTC–3!

qHpDelta-F

5!–GAGACAGCCGAGACTTGTCC–3!

qHpDelta-R

5!–CATGTAGCTCCGTTGAAGCA–3!

Ets-HRD-F

5!–ATTACTGGCAGTGGTAACAG–3!

Ets-HRD-R

5!–GACATTCTGCATGAAACCTAG–3!

2A peptide-S

5!–GAAGATCTTGCTGCCTCGAGGAGGGAAGAGGTAGCCTGCTAACTTGCGG–3!

2A peptide-R

5!–GCTCTAGAGGGACCAGGATTTTCCTCTACGTCCCCGCAAGTTAGCAGGC–3!

2A peptide-2F

5!–GAGGGAAGAGGTAGCCTGCT–3!

T3 promoter primer

5!–ATTAACCCTCACTAAAGGGA–3!

Ets HRD-inv-F

5!–TGAGAGTCCTGCAACGTGCC–3!

Ets HRD-inv-R

5!–ATCGTCGTCGCGTGCTGGAT–3!

primer1

5!–GCATCAAGGTCAACTTCAAGATCAGA–3!

primer2

5!–TGGCAAGTTTGGACAAAATTACATTC–3!

primer3

5!–GAAGTGGTCTCGAACCCTGTTATCC–3!

primer4

5!–CTTGTGGCCGAGAATGTTTC–3!

*R represents A or G

42

Table 2. Composition of ZFs in pc3XB vectors
Vectors
pc3XB-ZFA36
pc3XB-Z1B
pc3XB-Z2B
pc3XB-Z3B

Position of ZFs
ZF1
ZF2
ZF3
60
64
63
+BbsI
64
63
60
+BbsI
63
60
64
+BbsI

60, 64 and 63 represent ZF60, ZF64 and ZF63 zinc-finger
domain of Zinc Finger Consortium Vector Kit v1.0,

43

Table 3. Nucleotide sequences of oligonucleotides used for preparation of
pH3U3-B1H-reporter vectors
ZFN target site HpHesC target subsite
subsite for ZF1, ZF2 and ZF3subsite for ZF3
HpHesC1

subsite for ZF2
subsite for ZF1
subsite for ZF1, ZF2 and ZF3subsite for ZF3

HpHesC2

subsite for ZF2
subsite for ZF1
subsite for ZF1, ZF2 and ZF3subsite for ZF3

HpEts1

subsite for ZF2
subsite for ZF1
subsite for ZF1, ZF2 and ZF3subsite for ZF3

HpEts 2subsite for ZF2
subsite for ZF1

Oligonucleotides
B1HT-HesC1S
B1HT-HesC1A
B1HT-HesC1-3S
B1HT-HesC1-3A
B1HT-HesC1-2S
B1HT-HesC1-2A
B1HT-HesC1-1S
B1HT-HesC1-1A
B1HT-HesC2S
B1HT-HesC2A
B1HT-HesC2-3S
B1HT-HesC2-3A
B1HT-HesC2-2S
B1HT-HesC2-2A
B1HT-HesC2-1S
B1HT-HesC2-1A
B1HT- Ets1S
B1HT-Ets1A
B1HT-Ets1-3S
B1HT-Ets1-3A
B1HT-Ets1-2S
B1HT-Ets1-2A
B1HT-Ets1-1S
B1HT-Ets1-1A
B1HT-Ets2S
B1HT-Ets2A
B1HT-Ets2-3S
B1HT-Ets2-3A
B1HT-Ets2-2S
B1HT-Ets2-2A
B1HT-Ets2-1S
B1HT-Ets2-1A

44

sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense

Nucleotide sequence
5!–CCGGTGGGGTCTGGATGTC–3!
5!–AATTGACATCCAGACCCCA–3!
5!–CCGGAGGGGATGGTCTGTC–3!
5!–AATTGACAGACCATCCCCT–3!
5!–CCGGGGAAGTCGGTCTGTC–3!
5!–AATTGACAGACCGACTTCC–3!
5!–CCGGGGAAGATTGGATGTC–3!
5!–AATTGACATCCAATCTTCC–3!
5!–CCGGAGGGCGAGTCTTGTC–3!
5!–AATTGACAAGACTCGCCCT–3!
5!–CCGGAGGGGATGGTCTGTC–3!
5!–AATTGACAGACCATCCCCT–3!
5!–CCGGGGAACGAGGTCTGTC–3!
5!–AATTGACAGACCTCGTTCC–3!
5!–CCGGGGAAGATGTCTTGTC–3!
5!–AATTGACAAGACATCTTCC–3!
5!–CCGGCGGGGTTGACGTGTC–3!
5!–AATTGACACGTCAACCCCG–3!
5!–CCGGCGGGGATGGTGTGTC–3!
5!–AATTGACACACCATCCCCG–3!
5!–CCGGCGAAGTTGGTGTGTC–3!
5!–AATTGACACACCAACTTCG–3!
5!–CCGGCGAAGATGACGTGTC–3!
5!–AATTGACACGTCATCTTCG–3!
5!–CCGGCGATGATGACTTGTC–3!
5!–AATTGACAAGTCATCATCG–3!
5!–CCGGCGATGATGGTTTGTC–3!
5!–AATTGACAAACCATCATCG–3!
5!–CCGGCGAAGATGGTTTGTC–3!
5!–AATTGACAAACCATCTTCG–3!
5!–CCGGCGAAGATGACTTGTC–3!
5!–AATTGACAAGTCATCTTCG–3!

Table 4.

Effects of injection of HpHesC ZFN mRNAs

ZFN mRNA
HpHesC1 DD + HpHesC2 RR
ZFN mRNAs
Control-1DD + control-2 RR
ZFN mRNAs

RNA (pg/embryo)
6.5 each
13 each
6.5 each
13 eachInjected embryos
134
200
104
235

ZFN, zinc-finger nucleases.

45

Normal (%)
99.3
87
99
99.6

HpHesC morphant phenotype (%)
0.7
9.5
0
0

Dead (%)
0
3.5
1
0.4

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Acknowledgements
The author would like to express his deepest appreciation for his major professor, Dr.
Takashi Yamamoto, for providing an understanding environment to complete his thesis
studies. The author would also like to thank Dr. Naoaki Sakamoto and Dr. Tatsuo
Shibata for the helpful suggestions and their timely advice. Sincere appreciation is also
extended to Dr. Ken-ichi Suzuki, Dr. Masatoshi Nishikawa, Dr. Sumihare Noji, Dr.
Shinya Matsuura, Dr. Tatsuo Miyamoto and Dr. Koji Akasaka for fruitful discussions. I
am grateful to members of Dr. Takashi Yamamoto laboratory for discussions. The
author also wish to express their thanks to Dr. Keith Joung for providing the pST1374
expression vector (Addgene plasmid 13426), Dr. Daniel Voytas for supplying the
pc3XB-ZF60, pc3XB-ZF63, pc3XB-ZF64 and pc3XB-ZF70 vectors (Addgene plasmid13196, 13199, 13200 and 13193, respectively), Dr. Scot Wolfe for providing thepH3U3-mcs reporter vector, pB1H2x2-zif268 plasmid and US0!hisB!pyrF!rpoZ
bacterial strain (Addgene plasmids 12609, 184045 and 18049, respectively) and Dr.
Masato Kiyomoto for supplying live sea urchins. The author also thank the Fisheries
and Ocean Technology Center, Hiroshima Prefectural Technology Research Institutefor supplying seawater. I also thank to the Cryogenic Center of Hiroshima University
for supplying liquid nitrogen. This study was supported by a Grant-in-Aid for Scientific
Research on Innovative Areas (No. 2020006) to T.Y. and a Grant-in-Aid for JSPS
Fellows (No. 09J01990) to H.O.

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