JGG

Available online at www.sciencedirect.com

Journal of Genetics and Genomics 39 (2012) 421e433

REVIEW

Reverse Genetic Approaches in Zebrafish

Peng Huang a, Zuoyan Zhu a, Shuo Lin b,*, Bo Zhang a,*
a
Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, China
b
Department of Molecular, Cell & Developmental Biology, University of California, Los Angeles, CA 90095, USA

Received 10 June 2012; revised 3 July 2012; accepted 7 July 2012

Available online 11 August 2012

ABSTRACT

Zebrafish (Danio rerio) is a well-established vertebrate animal model. A comprehensive collection of reverse genetics tools has been
developed for studying gene function in this useful organism. Morpholino is the most widely used reagent to knock down target gene
expression post-transcriptionally. For a long time, targeted genome modification has been heavily relied on large-scale traditional forward
genetic screens, such as ENU (N-ethyl-N-nitrosourea) mutagenesis derived TILLING (Targeting Induced Local Lesions IN Genomes)
strategy and pseudo-typed retrovirus mediated insertional mutagenesis. Recently, engineered endonucleases, including ZFNs (zinc finger
nucleases) and TALENs (transcription activator-like effector nucleases), provide new and efficient strategies to directly generate site-
specific indel mutations by inducing double strand breaks in target genes. Here we summarize the major reverse genetic approaches
for loss-of-function studies used and emerging in zebrafish, including strategies based on genome-wide mutagenesis and methods for site-
specific gene targeting. Future directions and expectations will also be discussed.

KEYWORDS: Zebrafish; Reverse genetics; Morpholino; TILLING; Retrovirus; ZFN; TALEN; Gene targeting

1. INTRODUCTION identify genes and pathways involved in different develop-

Zebrafish (Danio rerio) has become a powerful model
organism not only for the study of vertebrate development, but
also for human disease modeling as well as for drug discovery
through small molecule screening at individual level. It is also
well established that genetic studies in zebrafish can provide
insights into the function of vertebrate genes and lead to better
understanding of human diseases. During early period in
zebrafish research, mutagenesis methods relied primarily on
forward genetics tools, which is an unbiased approach to
Abbreviations: DSB, double strand break; ENU, N-ethyl-N-nitrosourea;
HR, homologous recombination; MLV, murine leukemia virus; MO, mor-
pholino; TALE, transcription activator-like effector; TALEN, TALE nuclease;
TILLING, Targeting Induced Local Lesions IN Genomes; ZFA, zinc finger
array; ZFN, zinc finger nuclease.
* Corresponding authors. Tel/fax: þ86 10 6275 9072 (B. Zhang); Tel: þ1
310 267 4970, fax: þ1 310 267 4971 (S. Lin).
E-mail addresses: shuolin@ucla.edu (S. Lin); bzhang@pku.edu.cn (B. Zhang).
1673-8527/$ - see front matter Copyright Ó 2012, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China.
Published by Elsevier Limited and Science Press. All rights reserved.
http://dx.doi.org/10.1016/j.jgg.2012.07.004
mental processes. These forward genetic studies have been
conducted using irradiation, murine leukemia virus (MLV) and
chemical mutagens such as the DNA alkylating agent N-ethyl-
N-nitrosourea (ENU) (Fig. 1) (Chakrabarti et al., 1983; Walker
and Streisinger, 1983; Lin et al., 1994; Mullins et al., 1994;
Solnica-Krezel et al., 1994; Driever et al., 1996; Haffter et al.,
1996). Several forward genetic screens have been carried out,
through which large numbers of mutants have been isolated
(Driever et al., 1996; Haffter et al., 1996; Amsterdam et al.,
2004). The mutated genes are then identified by positional
cloning. Applications of forward genetic tools in zebrafish
have been summarized in several excellent reviews (Patton
and Zon, 2001; Lieschke and Currie, 2007; Lawson and
Wolfe, 2011). However, forward genetic screens are intrinsi-
cally limited in their effectiveness to isolate mutations of every
single gene due to functional redundancy between different
genes and the need to have measurable phenotypes. This is
especially noteworthy for zebrafish since it has undergone an
422 P. Huang et al. / Journal of Genetics and Genomics 39 (2012) 421e433

additional genome duplication event during teleost evolution.
In addition, it is also quite time-consuming but without
guarantees to identify the responsible mutated gene. It is
therefore also desirable to first identify genes of interest and
subsequently obtain their mutations followed by the evaluation
of the phenotypic effects of these mutations (i.e., “reverse”
genetics).
Genome sequencing projects of model organisms such as
zebrafish could document each transcript with protein-coding
and non-coding genes within genomes. With zebrafish
genome sequencing project nearly complete, it is now theo-
retically possible to generate mutations in all zebrafish genes
to systematically evaluate their functions, which requires
powerful reverse genetics tools. Unlike mice and Drosophila,
targeted gene knock-out through homologous recombination
(HR) is still unavailable in zebrafish. The existence of
embryonic stem (ES) cells in zebrafish is still an open ques-
tion. Instead, several alternative reverse genetics strategies for
loss-of-function studies have been developed, some of which
were derived from forward genetics approaches, such as
mutagenesis screens based on retrovirus or transposons, Tar-
geting Induced Local Lesions IN Genomes (TILLING) (Lin
et al., 1994; Kawakami et al., 2000; Wienholds et al., 2002).
In addition, antisense morpholino (MO) oligonucleotide
analogs can be used to disrupt target gene expression without
changing its genetic information (Nasevicius and Ekker,
2000). Recently, engineered endonucleases (EENs),
including zinc finger nuclease (ZFN) and transcription
activator-like effector nuclease (TALEN), are emerging as
a promising and efficient new method to achieve directly site-
specific genome modification (Fig. 1) (Doyon et al., 2008;
Meng et al., 2008; Huang et al., 2011; Sander et al., 2011a). In
this review, we summarize the major reverse genetics tools
applicable for loss-of-function studies in zebrafish and
particularly the recent developments and improvements of
each strategy (Fig. 2). We also anticipate the future directions
and open questions towards reverse genetic approaches, which
are still unavailable but crucial for zebrafish research.
2. HUNTING FOR TARGET GENES
The first step towards reverse genetic study is to find
a target gene or a group of target genes for mutagenesis. There
are several ways to achieve this purpose, including searching
for novel and functional unknown genes through bio-
informatics analysis and data mining from available cDNA
and genomic information, picking up potentially important
genes whose homologs have only been studied in lower
organisms such as Drosophila and Caenorhabditis elegans, or
screening for tissue-specifically expressed genes by in situ
hybridization or various genome-wide insertional traps medi-
ated by transposons or retroviruses, such as enhancer trap,
gene trap, poly A trap and promoter trap, etc (Fig. 2). Here we
only briefly summarize the gene hunting strategies based on
Tol2 transposon as examples.

Fig. 1. A historical view of the development of some mutagenesis tools in zebrafish.

Note that the whole issue for Volume 123 of Development (1996) was dedicated to reporting the results from two major large-scale chemical mutagenesis screens
based on ENU, therefore, no author names were mentioned in the corresponding box in this figure. EMS, ethyl methanesulfonate; ENU, N-ethyl-N-nitrosourea;
TALEN, transcription activator-like effector nuclease; TILLING, Targeting Induced Local Lesions IN Genomes; ZFN, zinc finger nuclease.
 P. Huang et al. / Journal of Genetics and Genomics 39 (2012) 421e433 423

Fig. 2. Strategies for loss-of-function studies by gene targeting in zebrafish.

Target genes (i.e., genes of interest) could be identified through bioinformatics analysis, searching for homologous genes from other species, screening for genes
with tissue specific expression patterns by in situ hybridization or various genome-wide insertional traps including enhancer trap, gene trap, etc. For functional
study of target genes, several mutagens could be used to create loss-of-function mutations in zebrafish, including retrovirus, transposons, ENU and EENs (ZFNs
and TALENs), while morpholino could be used as phenocopy agents to knock down gene expression post-transcriptionally. Researchers can screen for point
mutations using TILLING in a defined gene from the mutant archives generated through ENU mutagenesis. One can also look up insertional mutations for
a specific gene in the mutant archives generated through retrovirus insertional mutagenesis. Conversely, one can also search for their genes of interest from genetic
screens mediated by retrovirus or transposons. Loss-of-function indel mutations of target genes can be achieved with ZFNs, or more efficiently, TALENs. Large
genomic deletions might be achieved through imprecise excision of Tol2 transposon or cleavage of two pairs of EENs. Another potential application of transposons
and EENs is to stimulate HR by inducing DSBs. The solid line arrows indicate that the corresponding methods are already in practice, while the dotted arrows
indicate that the corresponding methods are only hypothetical and have not been established at present. ENU, N-ethyl-N-nitrosourea; HR, homologous recom-
bination; KI, knock-in; KO, knock-out; NHEJ, non-homologous end joining; TALEN, transcription activator-like effector nuclease; TILLING, Targeting Induced
Local Lesions IN Genomes; ZFN, zinc finger nuclease.

Transposons could integrate into genome sequence
randomly and efficiently, and thus are widely used to generate
transgenic fish lines. In addition to transgenesis, transposons
containing various sequence cassettes have also been used for
genetic screens of different purposes. Several transposons have
been shown to be functional in zebrafish, including Tol1, Tol2,
Sleeping Beauty and Ac/Ds elements, while Tol2 transposon is
more frequently used due to its high efficiency and ease to
manipulate (Kawakami et al., 2000; Davidson et al., 2003;
Emelyanov et al., 2006; Koga et al., 2008). Several large-scale
genetic screens based on Tol2 transposon, which are mainly
enhancer trap and gene trap, have been carried out and
a collection of transgenic fish lines with known insertion sites
are currently available (Kawakami et al., 2004; Parinov et al.,
2004; Zhao et al., 2008; Kondrychyn et al., 2009; Tian et al.,
2009; Xue et al., 2010). One can identify their genes of interest
through the analysis of the expression pattern of the reporter
gene and genomic sequences flanking the insertion sites.
Enhancer trap lines usually do not disrupt endogenous genes
while the genes identified from gene trap are generally already
mutation alleles. Strictly speaking, since gene trap could
reveal genes and their mutations at the same time, it is more
accurate to be considered as a mixed strategy of forward
genetics and reverse genetics approaches. Curiously, our and
others’ results suggest that Tol2 transposon tends to insert into
transcribed regions in the genome revealed by gene trap as
well as enhancer trap screens, which is similar to the pseudo-
typed retrovirus used for creating insertional mutations in
zebrafish (Kondrychyn et al., 2009).
Recently, three groups reported strategies to achieve
conditional gene inactivation with various gene-trap cassettes
using transposons, where one can simultaneously obtain
a conditional allele while identifying the genes of interest
(Clark et al., 2011; Maddison et al., 2011; Trinh et al., 2011).
Clark et al. (2011) applied a gene-breaking mutagenic cassette
with a terminator that can disrupt the trapped protein. Since
the mutagenic cassette contains splicing acceptor elements
flanked by two loxP sites, the trapped genes can be rescued by
Cre recombinase or MOs targeting the splicing sites.
Maddison et al. (2011) used a Tol2 transposon based Flip and
Excision (FlEx) module which contains a mCherry-EGFP dual
reporter and loxP and FRT elements. By combination with
tissue and temporal specific expressed Cre or Flp transgenic
fish lines, FLEx module could invert and conditionally inac-
tivate target gene expression (Maddison et al., 2011). Similar
to protein-trap strategy used in Drosophila, Trinh et al. (2011)
performed another screening using Tol2 transposon and Ac/Ds
elements with a “FlipTrap” cassette, which contains a Citrine
reporter flanked by a splicing acceptor and donor. In combi-
nation with transgenic fish lines carrying Cre recombinase, the
Citrine reporter and splicing donor elements can be flipped-out
in a tissue and temporal manner, which could generate
424 P. Huang et al. / Journal of Genetics and Genomics 39 (2012) 421e433
a truncated protein of the trapped gene (Trinh et al., 2011).
Although alleles generated with these strategies are not
complete knock-outs and genes of interest cannot be directly
targeted, they provide useful resources to investigate gene
function in a conditional manner, especially when HR is
currently unavailable in zebrafish.
3. GENE KNOCK-DOWN VIA PHENOCOPY
Zebrafish is originally discovered as an ideal embryological
model to study embryogenesis due to its transparency from
very beginning, and soon also considered to be a good
organism for genetic screens due to its capability of producing
large quantity of offspring. However, it had been a big chal-
lenge to explore reverse genetic study in zebrafish, and it is
only until recently that bona fide direct site-specific gene
targeting methods are gradually established. Before that, most
loss-of-function studies have heavily relied on knocking-down
target gene expression via MOs. MOs are a kind of artificially
synthesized oligonucleotide analogs which use morpholine
rings to replace the ribose backbone of nucleic acids
(Summerton and Weller, 1997). The replacement with analo-
gous structures enables MOs to be resistant to nuclease
digestion in addition to its enhanced binding activity to their
complementary RNA sequences in cells (Summerton, 1999).
By this way, MOs can exert their effects post-transcriptionally
by preventing target genes from expression either by poising/
attenuating protein translation or by disrupting normal
splicing. Using an antisense MO complementary to the 50 -
UTR (untranslated region) sequence, the translation of mature
mRNA could be blocked in the cytoplasm, while by binding to
a region spanning splicing donor or splicing accepter site,
MOs could disrupt normal splicing of pre-mRNA in the
nucleus. MOs could also be used to block microRNA func-
tions by binding to the mature microRNA or primary micro-
RNA (Eisen and Smith, 2008). In the narrow sense, MO is
more accurate to be classified as phenocopy rather than
a fundamental reverse genetics approach.
To study gene functions during early development, MOs are
usually injected directly into zebrafish embryos at 1e2 cell
stage. However, other delivery methods have also been
developed and used for different purposes, such as injection of
MOs into the yolk at mid-blastula stages to specifically knock-
down genes in the dorsal forerunner cells, and delivering MOs
by electroporation to the fin or retina in adult fish to study the
mechanisms of regeneration (Amack and Yost, 2004;
Thummel et al., 2006, 2011). The applications of MOs have
also been extended to extra modifications. Fluorescein labeled
MOs allow examinations of the cell populations whose target
gene expression has been inhibited (Hyde et al., 2012). Caged
MOs or photo-MOs can be activated or inactivated under
ultraviolet (UV) light and used for conditional gene knock-
down in a defined tissue and temporal manner (Shestopalov
et al., 2012; Tallafuss et al., 2012).
Unlike genetic mutants, embryos injected with MOs do not
need to be analyzed by applying Mendel’s law of segregation
nor confirmation by genotyping. MOs can be used in
combination with other genetic manipulations, such as injec-
tion into a transgenic fish line labeled with certain fluores-
cence reporters/markers. Since the additional duplication
event of zebrafish genome occurs during teleost evolution,
many genes might show functional redundancy with their
paralogs. In this case, another advantage of MOs is that
researchers can knock-down a number of genes at the same
time by injection of a mixture of corresponding MOs, which is
rather complicated and time-consuming to achieve through the
breeding between genetic mutants.
MO knocking-down is very convenient but also has obvious
limitations. With the increase of cell numbers, the amount of
MOs per cell is gradually diluted along with embryo devel-
opment. Needless to say, phenocopy by MOs is not heritable,
and it has been shown that MOs are only sufficiently effective
within the first 5 days of development when injected into one
cell stage embryos (Eisen and Smith, 2008). In addition, MOs
tend to activate p53 pathway and therefore lead to non-specific
phenotypes of embryonic defects (Robu et al., 2007).
Although it can be overcome by co-injection of anti-p53 MOs
or performing all the experiments at p53 mutant background,
sometimes it is still unconvincing, especially when the target
genes have functional interactions with p53 pathway. Last but
not least, sometimes it is difficult to evaluate the true effect
and specificity of MO knocking-down; therefore, properly and
carefully designed control experiments and rescue experi-
ments are crucial before reaching any conclusion.
RNA interference (RNAi) is another powerful technique for
post-transcriptional gene silencing studies, which has been
successfully used in many species. Unfortunately, this tech-
nique is still not well established in zebrafish though certain
successes have been reported (Zhao et al., 2001; Su et al.,
2008a, 2008b; Dong et al., 2009). Recently, a microRNA-
based shRNA (mir-shRNA) strategy has been shown to be
able to down regulate endogenous gene expression in a defined
spatial and temporal manner, which opened up the possibility
to do conditional gene knock-down studies in zebrafish (Dong
et al., 2009).
4. REVERSE GENETICS TOOLS BASED ON
GENOME-WIDE LARGE-SCALE SCREENING
4.1. Retrovirus mediated insertional mutagenesis
A pseudo-typed retrovirus, which was derived from MLV
with its envelope protein replaced by the glycoprotein G from
vesicular stomatitis virus (VSV), has been shown to be able to
infect zebrafish embryos and transmit efficiently through
germline following insertion into the genomes of zebrafish
(Lin et al., 1994). As in human cells, this modified MLV
prefers to integrate into the genomic regions close to tran-
scription start sites in zebrafish genome, which makes it an
ideal alternative mutagen besides ENU (Wu et al., 2003; Wang
et al., 2007). A unique advantage of retroviral mutagenesis
over chemical mutagens is that it allows rapid identification of
the mutated genes. By using the retroviral sequence as
a molecular “tag” for mapping, the genomic sequence flanking
 P. Huang et al. / Journal of Genetics and Genomics 39 (2012) 421e433 425

Fig. 3. Strategies of retrovirus based insertional mutagenesis.

Zebrafish embryos are injected at 1000e2000 cell stage with the retrovirus preparation and raised to adulthood as founders. F1 families are collected by inbreeding or
outbreeding of founders. For the first generation strategy of mutagenesis, the number of proviral insertions is estimated in each F1. F2 pools are collected through
inbreeding F1 families with high insertion numbers. Phenotype analysis is carried out in F3 embryos. In the strategies for the second and third generations, sperms
from each F1 male are collected and archived, and the corresponding genomic DNA was extracted from fin clips and the proviral insertion sites were identified
through LM-PCR and sequencing. Desired insertions can be restored by index to the cryopreserved sperms and in vitro fertilization. LM-PCR, linker-mediated PCR.

the insertion site can be easily identified through inverse PCR
or linker-mediated PCR (LM-PCR). Since the original muta-
genesis strategy using retrovirus in zebrafish is a phenotype-
based screen, it is more accurate to be classified as a forward
genetics strategy (Amsterdam, 2003). Essentially, 1000e2000
cell stage zebrafish embryos are infected with purified and
highly concentrated retroviruses through micro-injection.
These embryos are raised to adulthood as founders and
incrossed or outcrossed to get F1 families. Individual F1 fish
with high proviral insertions is selected by Southern blot.
Different F2 pools are generated by F1 incross. Multiple pairs
are mated in each F2 pool. If a visible phenotype is observed in
F3 embryos (usually within the first 5 days of development
after fertilization), the retroviral insertion site and the corre-
sponding disrupted gene can be identified and mapped by PCR
(Fig. 3) (Amsterdam et al., 1999). Using this approach, 315
genes essential for zebrafish development have been
successfully identified until 2004 (Amsterdam et al., 1999,
2004; Golling et al., 2002).
However, there are certain limitations for this phenotype-
based mutagenesis screen. For example, genes that share
functional redundancy or do not create visible phenotypes
during early development are usually ignored. Also, this
strategy is not practical for saturation mutagenesis screen due to
its demanding for intensive labor and large space to maintain
numerous fish. With the development of zebrafish genome
sequence project, the genomic sequence is largely known.
Based on this information, researchers designed a new strategy
to achieve high throughout retroviral insertional mutagenesis
by employing cryopreservation of sperms from F1 fish in
combination with identification of all the proviral insertions in
the corresponding male fish instead of phenotype screening in
their offspring (Wang et al., 2007). In this so-called second
generation retroviral insertional mutagenesis strategy, sperms
426 P. Huang et al. / Journal of Genetics and Genomics 39 (2012) 421e433
of each F1 male are stored as frozen stocks and the genomic
DNA is extracted from tail tissue in order to identify the cor-
responding proviral insertions. By this way, a proviral insertion
library can be generated and indexed, irrespective of any
phenotypic effect. Any desired insertion (i.e., potential muta-
tion) could readily be recovered through in vitro fertilization of
the frozen sperm sample carrying the integrations of interest
(Fig. 3) (Wang et al., 2007). Another obvious advantage for this
strategy is the release from overload of fish tanks and costs both
for the screening process and for the subsequent maintenance of
fish lines carrying proviral insertions. As a proof of theory,
a pilot experiment has been carried out since 2005 and more
than 500 gene hits (i.e., proviral insertions within introns or
exons) have been identified in less than 5 years (Wang et al.,
2007). Since researchers can select their genes of interest
from the list of insertion sites before going for phenotype
analysis, this strategy in a way should be considered as a hybrid
between forward and reverse genetic approaches. Since this
strategy does not produce any bias towards insertion site
identification, one can estimate for the first time the pattern of
retroviral insertions at organism level. Interestingly, about 40%
of the proviral insertions landed in putative gene loci (introns
and exons), with w50% of intron insertions located in the 1st
introns, which is consistent with the observation in mammalian
in vitro cultured cells (Wu et al., 2003; Wang et al., 2007).
Inbreeding fish carrying same proviral integrations in different
genes demonstrated that the mRNA transcript levels were
reduced by 70% or more in approximately 50% of the gene
“hits”, with the highest probability for mutation occurring if the
integration was in an exon or the 1st intron. This strategy
therefore produces one mutagenic event from every five
proviral integrations. In addition, the mapping occurs in the F1
generation, thus making recovery of the mutations compara-
tively simple and more reliable. Again, this strategy also has its
intrinsic disadvantages, among which mapping insertion sites
becomes the most labor-intensive step. Recently, in order to
improve the mapping efficiency of proviral insertion sites as
well as reduce the sequencing costs, a third generation strategy
is conceived, which incorporates the next-generation high-
throughput sequencing technology by pooling the genomic
DNA samples from several hundred F1 fish into one sequencing
reaction with “barcode” labeling (Fig. 3).
4.2. Gene knock-out via TILLING
TILLING is a method that is used to identify individual
organisms harboring point mutations in a defined gene from
a library of potential mutants generated through chemical
mutagenesis. First established in Arabidopsis and soon adopted
into zebrafish, TILLING has been demonstrated to be an effec-
tive approach to identify mutations within a sufficiently big
population of ENU-mutagenized zebrafish (Wienholds et al.,
2002). At first, CEL I enzyme is used to identify point muta-
tions induced by ENU (Wienholds et al., 2002). Later on, the low
cost next-generation sequencing and whole exome enrichment
technologies were incorporated to facilitate TILLING method to
identify large numbers of alleles rapidly from mutant libraries.
Since TILLNG requires large mutant libraries as well as
significant DNA sequencing capacity in order to identify
a desired mutation, it is labor-intensive and generally not
feasible for a regular laboratory. Therefore, a zebrafish
mutation project has been organized by Sanger Institute using
TILLING method (http://www.sanger.ac.uk/Projects/D_rerio/
zmp/). Researchers could submit an inquiry for desired
mutations by simply providing required information of their
genes of interest. To date, mutations for 6092 genes (about
23% of all zebrafish genes) have been identified by this
project. All the mutation lines can be searched and requested
by researchers. However, since the mutation frequency of
ENU is about 1 per 105 bp, genes consisting of only small
exons are difficult to be mutated using ENU based TILLING
method (Moens et al., 2008; de Bruijn et al., 2009).
5. GENE TARGETING BY SITE-SPECIFIC GENOME
MODIFICATION
5.1. Gene targeting via ZFNs
Transcription factors such as those from zinc finger protein
family could recognize and bind to specific DNA sequences.
Researchers have tried hard to adopt these properties in site-
specific genome modifications since the discovery of tran-
scription factors. ZFN has become one of the first successful
examples for such a purpose. ZFN is a chimeric protein con-
taining a DNA binding domain comprising a C2H2 type zinc
finger array (ZFA) mainly based on the backbone of ZIF268,
a human or mouse zinc finger protein, and a cleavage domain
derived from Fok I, a bacterial non-specific endonuclease
(Urnov et al., 2010). The alpha helix domain of each zinc
finger recognizes and binds to a specific 3 bp DNA sequence.
Each ZFA usually contains three to six zinc finger motifs,
which could recognize a total of 9e18 bp target DNA
sequence. Since Fok I has to dimerize to become fully func-
tional, ZFNs usually function in pairs, where they could cleave
the spacer sequence between the two ZFN binding sites in the
chromosomal DNA and induce double strand breaks (DSBs) at
its target locus. The DSB could be repaired through error-
prone non-homologous end joining (NHEJ) or homologous
template dependent HR pathway, leading to either indel
mutations or DNA replacement (Urnov et al., 2010). Another
application for genome modification with ZFNs is the induc-
tion of large genomic deletions, where two pairs of ZFNs were
introduced into the same cell or organism and the two DSBs
generated by the two ZFN pairs could join together, resulting
in the deletion of the genomic region between the two
distantly separated ZFN target sites (Lee et al., 2010). So far,
ZFN technology has been successfully applied in in vitro
cultured cells as well as many model organisms including
zebrafish (Bibikova et al., 2003; Porteus and Baltimore, 2003;
Lombardo et al., 2007; Doyon et al., 2008; Meng et al., 2008;
Foley et al., 2009; Geurts et al., 2009; Shukla et al., 2009;
Townsend et al., 2009; Dong et al., 2011; Yang et al., 2011; Yu
et al., 2011).
 P. Huang et al. / Journal of Genetics and Genomics 39 (2012) 421e433 427

In zebrafish, target genes are mainly disrupted by intro-
ducing indels generated through NHEJ pathway, while ZFN
mediated HR or large genomic deletion has not been reported
(Fig. 4). To date, dozens of zebrafish mutations have been
obtained through ZFNs since the first reports on successful
gene targeting through ZFNs in zebrafish in 2008 (Doyon
et al., 2008; Meng et al., 2008; Foley et al., 2009; Siekmann
et al., 2009; Ben et al., 2011; Gupta et al., 2011, 2012; Zhu
et al., 2011; Sander et al., 2011a, 2011b).
ZFNs represent the first available direct site-specific gene
targeting method in zebrafish. It brought out the possibility to
knock-out any desired zebrafish gene. However, selection of
ZFAs with highly efficient and specific DNA binding activity
remains a big challenge and becomes the limiting step in ZFN
applications. Commercial service for ZFN screening and
assembly is available but the cost is considerably high. To get
ZFAs with high activities, several methods have been devel-
oped, either via direct assembly of known zinc fingers or using

Fig. 4. Gene targeting via engineered endonuclease ZFNs or TALENs.

A pair of mRNAs encoding ZFNs or TALENs is injected into zebrafish embryos. After evaluation of the efficiency by one of the four assays (restriction enzyme
digestion, CEL I or T7 endonuclease digestion, sequencing analysis, melting curve analysis), the mosaic embryos are raised to adulthood as founders. Individual
mutations are identified from F1 embryos obtained by outcross of founders with wild type fish. F1 embryos are raised to adulthood and carriers are identified by fin clip
genotyping. Phenotype is analyzed in F2 through incrossing heterozygous F1 fish. Each TALEN is usually composed of more than 12 TALE repeat units; here we only
show 9 repeats on each side for simplicity. TALE, transcription activator-like effector; TALEN, TALE nuclease; ZFA, zinc finger array; ZFN, zinc finger nuclease.
* represents bands of DNA fragments carrying potential mutations.
428 P. Huang et al. / Journal of Genetics and Genomics 39 (2012) 421e433
screening strategies. Modular assembly (MA) is one of the
earliest methods used to generate functional ZFAs, which
directly ligates identified zinc fingers that recognize different
triplet DNA combinations. Since MA does not consider
context-dependent effects of the nucleotide sequence for the
target site recognition by zinc fingers, it has a relatively high
failure rate, especially when the target sites are devoid of or
poor in GNN (N represents any nucleotide) nucleotide triplets
(Ramirez et al., 2008). Oligomerized pool engineering
(OPEN) method employs a selection system based on bacterial
two-hybridization (Maeder et al., 2008), while Meng et al.
(2008) used a bacterial one-hybrid selection system, which
was first developed for the identification of target sites for
transcription factors. Numerous functional ZFAs have been
generated using these two selection systems. Although the
ZFNs obtained by these two methods show higher success
rates than MA, both strategies are labor-intensive and require
constructions of big libraries as well as expertise. Several
databases have collected identified ZFAs (http://bindr.
gdcb.iastate.edu/ZiFDB/, http://web.iitd.ac.in/wsundar/zifbase/
and http://eendb.zfgenetics.org/). Utilizing the verified ZFA
archives, an improved screening-independent method called
Context-dependent assembly (CoDA) was reported (Sander
et al., 2011b). Comparing with MA, CoDA resolves the
context-dependent effects by selecting N- and C-terminal zinc
fingers from identified ZFAs that contain a common middle
zinc finger. Fifty percent ZFNs generated with CoDA method
showed sufficient in vivo activities (Sander et al., 2011b).
Nevertheless, targeting any desired locus in a genome is still
difficult using ZFNs, which limits its broad application.
5.2. Gene targeting via TALENsdthe most promising
reverse genetic tool
TALE proteins are mainly found in Xanthomonas, a plant
pathogen. The DNA binding domain of TALE proteins is
composed of several tandem repeat units, while each repeat is
responsible to recognize and bind to a single nucleotide in the
target site (Boch et al., 2009; Moscou and Bogdanove, 2009).
The major and classical TALE repeat unit contains 34 amino
acid residues where the two at positions 12 and 13 are called
RVD (repeat variable di-residue), which determines the speci-
ficity of the repeat unit to recognize and bind to its target
nucleotide. The most representative RVDs, i.e., the most
frequently occurred RVDs in naturally existing TALE proteins,
which correspond for the recognition of each of the four
nucleotides A, T, C and G, are NI (Asn Ile), NG (Asn Gly), HD
(His Asp), NN (Asn Asn)/NK (Asn Lys), respectively. Two
most recent publications report that NH (Asn His) has
a comparable activity but higher specificity than NN to recog-
nize nucleotide G (Cong et al., 2012; Streubel et al., 2012). In
addition, an interesting and mysterious feature for the target site
recognition of TALE proteins is that they require a T as the
nucleotide immediately upstream of the 50 end of its binding
site. Nevertheless, comparing with zinc finger proteins, the
principle for the target nucleotide recognition and binding for
TALE is much simpler and predictable, which makes it
a promising alternative sequence-specific DNA binding domain
to ZFA (Boch et al., 2009; Moscou and Bogdanove, 2009).
To achieve better specificity, one usually needs to construct
TALENs each targeting more than 12 bp, and theoretically the
longer the better. However, due to its highly repetitive feature,
it is difficult to construct long TALE repeats. Several methods
have been developed to resolve this issue and most are based
on Golden Gate cloning strategy, which employs type IIs
restriction enzymes and theoretically can ligate up to dozens
of repeats through a single ligation reaction (Fig. 5) (Christian
et al., 2010; Morbitzer et al., 2010, 2011; Cermak et al., 2011;
Geibetaler et al., 2011; Li et al., 2011a, 2011b, 2012a;
Mahfouz et al., 2011; Sander et al., 2011a; Reyon et al., 2012).
We developed a simple alternative method called “Unit
Assembly”, which took advantages of a pair of isocaudomers
Nhe I and Spe I. Using four single-unit vectors as the starting
material, which contain the coding sequence of alternative
units recognizing A, T, C or G, TALE repeats can be easily
assembled by standard molecular cloning method of restriction
enzyme digestion and ligation with any and unlimited number
of repeats at any desired order (Fig. 5) (Huang et al., 2011).
More recently, two high-throughput methods for TALE repeats
assembly called FLASH (fast ligation-based automatable
solid-phase high-throughput) and ICA (iterative capped
assembly) were reported, which bypass the time-consuming
intermediate steps of bacterial culture and amplification by
using solid-phase magnetic beads for enzyme digestion and
ligation and allow researchers to construct large numbers of
TALENs rapidly (Fig. 5) (Briggs et al., 2012; Reyon et al.,
2012). However, the advantages and disadvantages of all
these different TALEN construction methods still need further
investigation.
Although TALEN targeting as a technique was reported
only less than 2 years ago, it is soon broadly applied in in vitro
cultured cells as well as living organisms including zebrafish
(Hockemeyer et al., 2011; Huang et al., 2011; Miller et al.,
2011; Sander et al., 2011a; Tesson et al., 2011; Wood et al.,
2011; Cade et al., 2012; Liu et al., 2012; Moore et al., 2012;
Tong et al., 2012; Li et al., 2012b). TALENs function in
a similar way as ZFNs, and they could also induce indel
mutations to disrupt the sequence in the spacer of TALEN
target sites (i.e., the region between the two TALEN binding
sites), but usually with higher targeting efficiency and lower
off-target effects (Hockemeyer et al., 2011; Huang et al., 2011;
Li et al., 2011b). Therefore, a unique restriction enzyme site in
the spacer region is very convenient for the detection of
TALEN efficiency. After PCR amplification of the sequence
flanking the target site and digestion with the corresponding
enzyme, the efficiency of TALENs can be estimated by
measuring and comparing the density and ratio of the undi-
gested band (Fig. 4). In addition, TALEN efficiency could be
revealed directly by sequencing after PCR amplification or
evaluated through other assays such as CEL I or T7 endonu-
clease digestion and melting curve analysis (Fig. 4). So far,
nearly eighty TALEN pairs targeting different genomic loci in
zebrafish have been constructed in our laboratory and the
overall success rate is above 70%, which is similar to a recent
 P. Huang et al. / Journal of Genetics and Genomics 39 (2012) 421e433 429

Fig. 5. Strategies for the construction of TALE repeats.

One-step assembly strategy for TALE repeats construction is based on Golden Gate cloning derived methods, which take advantages of type IIs enzymes and can
theoretically construct TALE repeats by a one-step digestion and ligation reaction, though it can also divide into more than one serial steps. Unit Assembly and
REAL represent the strategy of serial assembly, which can construct TALEs with any and unlimited number of repeat units through repeated cycles of standard
restriction enzyme digestion and ligation steps. Using a strategy involving solid-phase ligation, FLASH and ICA methods bypass the time-consuming trans-
formation and growing of bacteria during intermediate cloning steps and can construct TALE repeats in a high-throughput manner. FLASH, fast ligation-based
automatable solid-phase high-throughput; ICA, iterative capped assembly; REAL, restriction enzyme and ligation; TALE, transcription activator-like effector.

publication (Cade et al., 2012). Among all the TALEN pairs
tested, more than half of them showed >30% mutagenizing
activity in founder embryos, some of which even reached the
efficiency to nearly 100%, as detected by restriction enzyme
digestion assay (unpublished data), suggesting that TALEN is
a promisingly efficient and easy technology for gene targeting
in zebrafish. However, about 30% TALEN pairs did not show
detectable mutagenizing activity in zebrafish and the reason is
still unclear. In addition to the unpredictable and variable
efficiency, another limitation for the applications of TALENs
in zebrafish is that they can only induce indel mutations but
not precise modification of the target site at present, though we
believe this limitation is only temporary and will soon be
overcome in the near future. Nevertheless, the ease in finding
suitable target sites, the high success rate, high efficiency and
specificity of TALEN targeting confer this relatively new
reverse genetic strategy to be the most promising technique to
be able to target virtually any genes of interest in any species.
It will not be surprising if TALEN eventually becomes the
most powerful and popular site-specific mutagenesis method
for zebrafish, and perhaps, for other organisms as well. This
situation reminds us of the discovery of RNA interference
(RNAi) about 14 years ago, which has achieved a strong
impact to the field of study on gene functions shortly after. We
believe TALEN has already started to bring a similar revolu-
tion to the field of reverse genetic studies.
6. CONCLUDING REMARKS
Genome manipulations using powerful reverse genetics
tools will greatly enhance the advantages and utility of
zebrafish as a vertebrate model organism. With the expansion
of zebrafish genetic toolkit, ingenious reverse genetics
methods will certainly turn zebrafish mutagenesis into
a convenient and important process. Apart from point muta-
tions isolated from TILLING, insertional mutations identified
from retrovirus or Tol2 transposon mutagenesis screening and
indel mutations induced by EENs (ZFNs or TALENs), there
are other types of mutations and mutagenesis methods which
are of equal or even more importance comparing with the
430 P. Huang et al. / Journal of Genetics and Genomics 39 (2012) 421e433
currently available ones. Large genomic fragment deletion and
HR based gene knock-in and conditional knock-out are among
the most interesting ones.
There are at least two potential approaches to achieve large
genomic deletions. Although only indel mutations have been
reported for TALEN targeting in zebrafish so far, our
preliminary data showed that TALENs also hold promise to
induce large genomic deletions when using two pairs of
TALENs separated within a reasonable distance (Fig. 2 and
unpublished data). In addition to disrupting genes by inser-
tions, transposon can also be used for mutagenesis upon
excision. Deletion of large genomic sequence by inducing P
element imprecise excision at a defined locus has been widely
used in Drosophila as a powerful, and during certain period of
time, the only method for the study of gene function and
regulation through loss-of-function mutations. We found that
Tol2 transposon excision can also induce large chromosomal
deletions in zebrafish (Fig. 2 and unpublished data). Consid-
ering the availability of large and still increasing numbers of
transgenic zebrafish carrying Tol2 insertions, this strategy
could become an alternative gene targeting method and could
be used for the complete disruption/removal of a single gene
or a cluster of genes simultaneously. This is especially useful
for loss-of-function studies of non-coding RNA genes since
point mutations or small indel mutations might not be enough
to disrupt the function of these non-coding RNAs which do not
have a predictable open reading frame.
Other advanced genetic tools such as conditional knock-
out and precise gene knock-in/replacement are widely used
in mouse and Drosophila and proved to be very useful.
Although some preliminary work has been performed, gene
targeting through HR is still not established in zebrafish
(Hagmann et al., 1998; Cui et al., 2003; Wu et al., 2006). The
next, and most likely also the last and the biggest, challenge
for zebrafish reverse genetics studies is therefore to achieve
HR mediated gene targeting, which is crucial for gene knock-
in as well as conditional mutagenesis studies. Several strat-
egies have been developed to increase the efficiency of HR in
cultured cells or at organism level. By applying ZFNs to
induce DSBs, gene targeting through HR has been achieved
in Drosophila, mouse and rat (Bibikova et al., 2003; Cui
et al., 2011). Similarly, TALENs have been shown to be
able to induce HR in human iPSC and ES cells (Hockemeyer
et al., 2011). Single strand oligonucleotide template has been
shown to be more efficient than traditional double-stranded
template as donors (Chen et al., 2011). Blocking DNA
ligase IV activity may facilitate HR by inhibiting NHEJ
(Ochiai et al., 2012). Instead of DSB, modified ZFNs con-
taining an inactive Fok I cleavage domain could be used to
create nicks at the target site. These modified ZFNs could
increase the relative frequency of HR to NHEJ, though the
overall frequency of HR is lower than traditional ZFN pairs
(Kim et al., 2012; Ramirez et al., 2012; Wang et al., 2012).
With the help of these experience and TALEN technique, we
believe that gene targeting through HR, the last missing
reverse genetics tool in zebrafish, will be ultimately achieved
in the near future.
ACKNOWLEDGEMENTS
We thank X. Tong, Y. Shen, A. Xiao, L. Xu, Z. Wang, Y.
Zu, Y. Hu, Z. Luo and Q. Wu for helpful discussions; Y. Gao,
J. Zhang, Y. Jia, J. Chen, X. Yang and H. Cui for technical
support. This work was partially supported by the grants from
National Program on Key Basic Research Project (973
program) (Nos. 2012CB945101 and 2011CBA01000) and
National Natural Science Foundation of China (NSFC) (Nos.
31110103904 and 30730056).
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