reverse genetic approaches in zebrafish

Available online at www.sciencedirect.com

Journal of Genetics and Genomics 39 (2012) 421e433

JGG
REVIEW
Reverse Genetic Approaches in Zebrafish

Peng Huang a, Zuoyan Zhu a, Shuo Lin b,*, Bo Zhang a,*

aKey Laboratory of Cell Proliferation and Differentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, ChinabDepartment 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 beendeveloped for studying gene function in this useful organism. Morpholino is the most widely used reagent to knock down target geneexpression post-transcriptionally. For a long time, targeted genome modification has been heavily relied on large-scale traditional forwardgenetic 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 fingernucleases) 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 approachesfor 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

Zebrafish (Danio rerio) has become a powerful modelorganism not only for the study of vertebrate development, butalso for human disease modeling as well as for drug discoverythrough small molecule screening at individual level. It is alsowell established that genetic studies in zebrafish can provideinsights into the function of vertebrate genes and lead to betterunderstanding of human diseases. During early period inzebrafish research, mutagenesis methods relied primarily onforward 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: [email protected] (S. Lin); [email protected] (B. Zhang).

1673-8527/$ - see front matter Copyright � 2012, Institute of Genetics and Develop

Published by Elsevier Limited and Science Press. All rights reserved.

http://dx.doi.org/10.1016/j.jgg.2012.07.004

identify genes and pathways involved in different develop-mental processes. These forward genetic studies have beenconducted using irradiation, murine leukemia virus (MLV) andchemical mutagens such as the DNA alkylating agent N-ethyl-N-nitrosourea (ENU) (Fig. 1) (Chakrabarti et al., 1983; Walkerand 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 positionalcloning. Applications of forward genetic tools in zebrafishhave been summarized in several excellent reviews (Pattonand Zon, 2001; Lieschke and Currie, 2007; Lawson andWolfe, 2011). However, forward genetic screens are intrinsi-cally limited in their effectiveness to isolate mutations of everysingle gene due to functional redundancy between differentgenes and the need to have measurable phenotypes. This isespecially noteworthy for zebrafish since it has undergone an

mental Biology, Chinese Academy of Sciences, and Genetics Society of China.

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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 withoutguarantees to identify the responsible mutated gene. It istherefore also desirable to first identify genes of interest andsubsequently obtain their mutations followed by the evaluationof the phenotypic effects of these mutations (i.e., “reverse”genetics).

Genome sequencing projects of model organisms such aszebrafish could document each transcript with protein-codingand non-coding genes within genomes. With zebrafishgenome sequencing project nearly complete, it is now theo-retically possible to generate mutations in all zebrafish genesto systematically evaluate their functions, which requirespowerful reverse genetics tools. Unlike mice and Drosophila,targeted gene knock-out through homologous recombination(HR) is still unavailable in zebrafish. The existence ofembryonic stem (ES) cells in zebrafish is still an open ques-tion. Instead, several alternative reverse genetics strategies forloss-of-function studies have been developed, some of whichwere derived from forward genetics approaches, such asmutagenesis screens based on retrovirus or transposons, Tar-geting Induced Local Lesions IN Genomes (TILLING) (Linet al., 1994; Kawakami et al., 2000; Wienholds et al., 2002).In addition, antisense morpholino (MO) oligonucleotideanalogs can be used to disrupt target gene expression withoutchanging its genetic information (Nasevicius and Ekker,2000). Recently, engineered endonucleases (EENs),including zinc finger nuclease (ZFN) and transcription

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

Note that the whole issue for Volume 123 of Development (1996) was dedicated to

based on ENU, therefore, no author names were mentioned in the corresponding b

TALEN, transcription activator-like effector nuclease; TILLING, Targeting Induce

activator-like effector nuclease (TALEN), are emerging asa 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). Inthis review, we summarize the major reverse genetics toolsapplicable for loss-of-function studies in zebrafish andparticularly the recent developments and improvements ofeach strategy (Fig. 2). We also anticipate the future directionsand open questions towards reverse genetic approaches, whichare still unavailable but crucial for zebrafish research.

2. HUNTING FOR TARGET GENES

The first step towards reverse genetic study is to finda target gene or a group of target genes for mutagenesis. Thereare several ways to achieve this purpose, including searchingfor novel and functional unknown genes through bio-informatics analysis and data mining from available cDNAand genomic information, picking up potentially importantgenes whose homologs have only been studied in lowerorganisms such as Drosophila and Caenorhabditis elegans, orscreening for tissue-specifically expressed genes by in situhybridization 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 weonly briefly summarize the gene hunting strategies based onTol2 transposon as examples.

sh.

reporting the results from two major large-scale chemical mutagenesis screens

ox in this figure. EMS, ethyl methanesulfonate; ENU, N-ethyl-N-nitrosourea;

d Local Lesions IN Genomes; ZFN, zinc finger nuclease.

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.

423P. Huang et al. / Journal of Genetics and Genomics 39 (2012) 421e433

Transposons could integrate into genome sequencerandomly and efficiently, and thus are widely used to generatetransgenic fish lines. In addition to transgenesis, transposonscontaining various sequence cassettes have also been used forgenetic screens of different purposes. Several transposons havebeen shown to be functional in zebrafish, including Tol1, Tol2,Sleeping Beauty and Ac/Ds elements, while Tol2 transposon ismore frequently used due to its high efficiency and ease tomanipulate (Kawakami et al., 2000; Davidson et al., 2003;Emelyanov et al., 2006; Koga et al., 2008). Several large-scalegenetic screens based on Tol2 transposon, which are mainlyenhancer trap and gene trap, have been carried out anda collection of transgenic fish lines with known insertion sitesare 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 interestthrough the analysis of the expression pattern of the reportergene and genomic sequences flanking the insertion sites.Enhancer trap lines usually do not disrupt endogenous geneswhile the genes identified from gene trap are generally alreadymutation alleles. Strictly speaking, since gene trap couldreveal genes and their mutations at the same time, it is moreaccurate to be considered as a mixed strategy of forwardgenetics and reverse genetics approaches. Curiously, our andothers’ results suggest that Tol2 transposon tends to insert intotranscribed 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 inzebrafish (Kondrychyn et al., 2009).

Recently, three groups reported strategies to achieveconditional gene inactivation with various gene-trap cassettesusing transposons, where one can simultaneously obtaina 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 cassettewith a terminator that can disrupt the trapped protein. Sincethe mutagenic cassette contains splicing acceptor elementsflanked by two loxP sites, the trapped genes can be rescued byCre recombinase or MOs targeting the splicing sites.Maddison et al. (2011) used a Tol2 transposon based Flip andExcision (FlEx) module which contains a mCherry-EGFP dualreporter and loxP and FRT elements. By combination withtissue and temporal specific expressed Cre or Flp transgenicfish lines, FLEx module could invert and conditionally inac-tivate target gene expression (Maddison et al., 2011). Similarto protein-trap strategy used in Drosophila, Trinh et al. (2011)performed another screening using Tol2 transposon and Ac/Dselements with a “FlipTrap” cassette, which contains a Citrinereporter flanked by a splicing acceptor and donor. In combi-nation with transgenic fish lines carrying Cre recombinase, theCitrine reporter and splicing donor elements can be flipped-outin a tissue and temporal manner, which could generate


a truncated protein of the trapped gene (Trinh et al., 2011).Although alleles generated with these strategies are notcomplete knock-outs and genes of interest cannot be directlytargeted, they provide useful resources to investigate genefunction in a conditional manner, especially when HR iscurrently unavailable in zebrafish.

3. GENE KNOCK-DOWN VIA PHENOCOPY

Zebrafish is originally discovered as an ideal embryologicalmodel to study embryogenesis due to its transparency fromvery beginning, and soon also considered to be a goodorganism for genetic screens due to its capability of producinglarge quantity of offspring. However, it had been a big chal-lenge to explore reverse genetic study in zebrafish, and it isonly until recently that bona fide direct site-specific genetargeting methods are gradually established. Before that, mostloss-of-function studies have heavily relied on knocking-downtarget gene expression via MOs. MOs are a kind of artificiallysynthesized oligonucleotide analogs which use morpholinerings to replace the ribose backbone of nucleic acids(Summerton and Weller, 1997). The replacement with analo-gous structures enables MOs to be resistant to nucleasedigestion in addition to its enhanced binding activity to theircomplementary RNA sequences in cells (Summerton, 1999).By this way, MOs can exert their effects post-transcriptionallyby preventing target genes from expression either by poising/attenuating protein translation or by disrupting normalsplicing. Using an antisense MO complementary to the 50-UTR (untranslated region) sequence, the translation of maturemRNA could be blocked in the cytoplasm, while by binding toa region spanning splicing donor or splicing accepter site,MOs could disrupt normal splicing of pre-mRNA in thenucleus. 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 ismore accurate to be classified as phenocopy rather thana fundamental reverse genetics approach.

To study gene functions during early development, MOs areusually injected directly into zebrafish embryos at 1e2 cellstage. However, other delivery methods have also beendeveloped and used for different purposes, such as injection ofMOs into the yolk at mid-blastula stages to specifically knock-down genes in the dorsal forerunner cells, and delivering MOsby electroporation to the fin or retina in adult fish to study themechanisms of regeneration (Amack and Yost, 2004;Thummel et al., 2006, 2011). The applications of MOs havealso been extended to extra modifications. Fluorescein labeledMOs allow examinations of the cell populations whose targetgene expression has been inhibited (Hyde et al., 2012). CagedMOs or photo-MOs can be activated or inactivated underultraviolet (UV) light and used for conditional gene knock-down in a defined tissue and temporal manner (Shestopalovet al., 2012; Tallafuss et al., 2012).

Unlike genetic mutants, embryos injected with MOs do notneed to be analyzed by applying Mendel’s law of segregationnor 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 duplicationevent of zebrafish genome occurs during teleost evolution,many genes might show functional redundancy with theirparalogs. In this case, another advantage of MOs is thatresearchers can knock-down a number of genes at the sametime by injection of a mixture of corresponding MOs, which israther complicated and time-consuming to achieve through thebreeding between genetic mutants.

MO knocking-down is very convenient but also has obviouslimitations. With the increase of cell numbers, the amount ofMOs 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 effectivewithin the first 5 days of development when injected into onecell stage embryos (Eisen and Smith, 2008). In addition, MOstend to activate p53 pathway and therefore lead to non-specificphenotypes of embryonic defects (Robu et al., 2007).Although it can be overcome by co-injection of anti-p53 MOsor performing all the experiments at p53 mutant background,sometimes it is still unconvincing, especially when the targetgenes have functional interactions with p53 pathway. Last butnot least, sometimes it is difficult to evaluate the true effectand specificity of MO knocking-down; therefore, properly andcarefully designed control experiments and rescue experi-ments are crucial before reaching any conclusion.

RNA interference (RNAi) is another powerful technique forpost-transcriptional gene silencing studies, which has beensuccessfully used in many species. Unfortunately, this tech-nique is still not well established in zebrafish though certainsuccesses 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 beable to down regulate endogenous gene expression in a definedspatial and temporal manner, which opened up the possibilityto do conditional gene knock-down studies in zebrafish (Donget al., 2009).

4. REVERSE GENETICS TOOLS BASED ONGENOME-WIDE LARGE-SCALE SCREENING

4.1. Retrovirus mediated insertional mutagenesis

A pseudo-typed retrovirus, which was derived from MLVwith its envelope protein replaced by the glycoprotein G fromvesicular stomatitis virus (VSV), has been shown to be able toinfect zebrafish embryos and transmit efficiently throughgermline following insertion into the genomes of zebrafish(Lin et al., 1994). As in human cells, this modified MLVprefers to integrate into the genomic regions close to tran-scription start sites in zebrafish genome, which makes it anideal alternative mutagen besides ENU (Wu et al., 2003; Wanget al., 2007). A unique advantage of retroviral mutagenesisover chemical mutagens is that it allows rapid identification ofthe mutated genes. By using the retroviral sequence asa molecular “tag” for mapping, the genomic sequence flanking

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 PCRor 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 forwardgenetics strategy (Amsterdam, 2003). Essentially, 1000e2000cell stage zebrafish embryos are infected with purified andhighly concentrated retroviruses through micro-injection.These embryos are raised to adulthood as founders andincrossed or outcrossed to get F1 families. Individual F1 fishwith high proviral insertions is selected by Southern blot.Different F2 pools are generated by F1 incross. Multiple pairsare mated in each F2 pool. If a visible phenotype is observed inF3 embryos (usually within the first 5 days of developmentafter 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, 315genes 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 sharefunctional redundancy or do not create visible phenotypesduring early development are usually ignored. Also, thisstrategy is not practical for saturation mutagenesis screen due toits demanding for intensive labor and large space to maintainnumerous fish. With the development of zebrafish genomesequence project, the genomic sequence is largely known.Based on this information, researchers designed a new strategyto achieve high throughout retroviral insertional mutagenesisby employing cryopreservation of sperms from F1 fish incombination with identification of all the proviral insertions inthe corresponding male fish instead of phenotype screening intheir offspring (Wang et al., 2007). In this so-called secondgeneration retroviral insertional mutagenesis strategy, sperms


of each F1 male are stored as frozen stocks and the genomicDNA is extracted from tail tissue in order to identify the cor-responding proviral insertions. By this way, a proviral insertionlibrary can be generated and indexed, irrespective of anyphenotypic effect. Any desired insertion (i.e., potential muta-tion) could readily be recovered through in vitro fertilization ofthe frozen sperm sample carrying the integrations of interest(Fig. 3) (Wang et al., 2007). Another obvious advantage for thisstrategy is the release from overload of fish tanks and costs bothfor the screening process and for the subsequent maintenance offish lines carrying proviral insertions. As a proof of theory,a pilot experiment has been carried out since 2005 and morethan 500 gene hits (i.e., proviral insertions within introns orexons) have been identified in less than 5 years (Wang et al.,2007). Since researchers can select their genes of interestfrom the list of insertion sites before going for phenotypeanalysis, this strategy in a way should be considered as a hybridbetween forward and reverse genetic approaches. Since thisstrategy does not produce any bias towards insertion siteidentification, one can estimate for the first time the pattern ofretroviral insertions at organism level. Interestingly, about 40%of the proviral insertions landed in putative gene loci (intronsand exons), with w50% of intron insertions located in the 1stintrons, which is consistent with the observation in mammalianin vitro cultured cells (Wu et al., 2003; Wang et al., 2007).Inbreeding fish carrying same proviral integrations in differentgenes demonstrated that the mRNA transcript levels werereduced by 70% or more in approximately 50% of the gene“hits”, with the highest probability for mutation occurring if theintegration was in an exon or the 1st intron. This strategytherefore produces one mutagenic event from every fiveproviral integrations. In addition, the mapping occurs in the F1generation, thus making recovery of the mutations compara-tively simple and more reliable. Again, this strategy also has itsintrinsic disadvantages, among which mapping insertion sitesbecomes the most labor-intensive step. Recently, in order toimprove the mapping efficiency of proviral insertion sites aswell as reduce the sequencing costs, a third generation strategyis conceived, which incorporates the next-generation high-throughput sequencing technology by pooling the genomicDNA samples from several hundred F1 fish into one sequencingreaction with “barcode” labeling (Fig. 3).

4.2. Gene knock-out via TILLING

TILLING is a method that is used to identify individualorganisms harboring point mutations in a defined gene froma library of potential mutants generated through chemicalmutagenesis. First established in Arabidopsis and soon adoptedinto zebrafish, TILLING has been demonstrated to be an effec-tive approach to identify mutations within a sufficiently bigpopulation 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 lowcost next-generation sequencing and whole exome enrichmenttechnologies were incorporated to facilitate TILLINGmethod toidentify large numbers of alleles rapidly from mutant libraries.

Since TILLNG requires large mutant libraries as well assignificant DNA sequencing capacity in order to identifya desired mutation, it is labor-intensive and generally notfeasible for a regular laboratory. Therefore, a zebrafishmutation project has been organized by Sanger Institute usingTILLING method (http://www.sanger.ac.uk/Projects/D_rerio/zmp/). Researchers could submit an inquiry for desiredmutations by simply providing required information of theirgenes of interest. To date, mutations for 6092 genes (about23% of all zebrafish genes) have been identified by thisproject. All the mutation lines can be searched and requestedby researchers. However, since the mutation frequency ofENU is about 1 per 105 bp, genes consisting of only smallexons are difficult to be mutated using ENU based TILLINGmethod (Moens et al., 2008; de Bruijn et al., 2009).

5. GENE TARGETING BY SITE-SPECIFIC GENOMEMODIFICATION

5.1. Gene targeting via ZFNs

Transcription factors such as those from zinc finger proteinfamily 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 successfulexamples for such a purpose. ZFN is a chimeric protein con-taining a DNA binding domain comprising a C2H2 type zincfinger array (ZFA) mainly based on the backbone of ZIF268,a human or mouse zinc finger protein, and a cleavage domainderived from Fok I, a bacterial non-specific endonuclease(Urnov et al., 2010). The alpha helix domain of each zincfinger 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 DNAsequence. Since Fok I has to dimerize to become fully func-tional, ZFNs usually function in pairs, where they could cleavethe spacer sequence between the two ZFN binding sites in thechromosomal DNA and induce double strand breaks (DSBs) atits target locus. The DSB could be repaired through error-prone non-homologous end joining (NHEJ) or homologoustemplate dependent HR pathway, leading to either indelmutations or DNA replacement (Urnov et al., 2010). Anotherapplication for genome modification with ZFNs is the induc-tion of large genomic deletions, where two pairs of ZFNs wereintroduced into the same cell or organism and the two DSBsgenerated by the two ZFN pairs could join together, resultingin the deletion of the genomic region between the twodistantly separated ZFN target sites (Lee et al., 2010). So far,ZFN technology has been successfully applied in in vitrocultured cells as well as many model organisms includingzebrafish (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; Yuet al., 2011).

http://www.sanger.ac.uk/Projects/D_rerio/zmp/

http://www.sanger.ac.uk/Projects/D_rerio/zmp/


In zebrafish, target genes are mainly disrupted by intro-ducing indels generated through NHEJ pathway, while ZFNmediated HR or large genomic deletion has not been reported(Fig. 4). To date, dozens of zebrafish mutations have beenobtained through ZFNs since the first reports on successfulgene targeting through ZFNs in zebrafish in 2008 (Doyonet al., 2008; Meng et al., 2008; Foley et al., 2009; Siekmannet al., 2009; Ben et al., 2011; Gupta et al., 2011, 2012; Zhuet al., 2011; Sander et al., 2011a, 2011b).

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

A pair of mRNAs encoding ZFNs or TALENs is injected into zebrafish embryos. A

digestion, CEL I or T7 endonuclease digestion, sequencing analysis, melting curve

mutations are identified from F1 embryos obtained by outcross of founders with wild t

genotyping. Phenotype is analyzed in F2 through incrossing heterozygous F1 fish. Eac

show 9 repeats on each side for simplicity. TALE, transcription activator-like effecto

* represents bands of DNA fragments carrying potential mutations.

ZFNs represent the first available direct site-specific genetargeting method in zebrafish. It brought out the possibility toknock-out any desired zebrafish gene. However, selection ofZFAs with highly efficient and specific DNA binding activityremains a big challenge and becomes the limiting step in ZFNapplications. Commercial service for ZFN screening andassembly is available but the cost is considerably high. To getZFAs with high activities, several methods have been devel-oped, either via direct assembly of known zinc fingers or using

fter evaluation of the efficiency by one of the four assays (restriction enzyme

analysis), the mosaic embryos are raised to adulthood as founders. Individual

ype fish. F1 embryos are raised to adulthood and carriers are identified by fin clip

h TALEN is usually composed of more than 12 TALE repeat units; here we only

r; TALEN, TALE nuclease; ZFA, zinc finger array; ZFN, zinc finger nuclease.


screening strategies. Modular assembly (MA) is one of theearliest methods used to generate functional ZFAs, whichdirectly ligates identified zinc fingers that recognize differenttriplet DNA combinations. Since MA does not considercontext-dependent effects of the nucleotide sequence for thetarget site recognition by zinc fingers, it has a relatively highfailure rate, especially when the target sites are devoid of orpoor in GNN (N represents any nucleotide) nucleotide triplets(Ramirez et al., 2008). Oligomerized pool engineering(OPEN) method employs a selection system based on bacterialtwo-hybridization (Maeder et al., 2008), while Meng et al.(2008) used a bacterial one-hybrid selection system, whichwas first developed for the identification of target sites fortranscription factors. Numerous functional ZFAs have beengenerated using these two selection systems. Although theZFNs obtained by these two methods show higher successrates than MA, both strategies are labor-intensive and requireconstructions of big libraries as well as expertise. Severaldatabases 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 ZFAarchives, an improved screening-independent method calledContext-dependent assembly (CoDA) was reported (Sanderet al., 2011b). Comparing with MA, CoDA resolves thecontext-dependent effects by selecting N- and C-terminal zincfingers from identified ZFAs that contain a common middlezinc finger. Fifty percent ZFNs generated with CoDA methodshowed sufficient in vivo activities (Sander et al., 2011b).Nevertheless, targeting any desired locus in a genome is stilldifficult using ZFNs, which limits its broad application.

5.2. Gene targeting via TALENsdthe most promisingreverse genetic tool

TALE proteins are mainly found in Xanthomonas, a plantpathogen. The DNA binding domain of TALE proteins iscomposed of several tandem repeat units, while each repeat isresponsible to recognize and bind to a single nucleotide in thetarget site (Boch et al., 2009; Moscou and Bogdanove, 2009).The major and classical TALE repeat unit contains 34 aminoacid residues where the two at positions 12 and 13 are calledRVD (repeat variable di-residue), which determines the speci-ficity of the repeat unit to recognize and bind to its targetnucleotide. The most representative RVDs, i.e., the mostfrequently occurred RVDs in naturally existing TALE proteins,which correspond for the recognition of each of the fournucleotides A, T, C and G, are NI (Asn Ile), NG (Asn Gly), HD(His Asp), NN (Asn Asn)/NK (Asn Lys), respectively. Twomost recent publications report that NH (Asn His) hasa comparable activity but higher specificity than NN to recog-nize nucleotide G (Cong et al., 2012; Streubel et al., 2012). Inaddition, an interesting and mysterious feature for the target siterecognition of TALE proteins is that they require a T as thenucleotide immediately upstream of the 50 end of its bindingsite. Nevertheless, comparing with zinc finger proteins, theprinciple for the target nucleotide recognition and binding forTALE is much simpler and predictable, which makes it

a promising alternative sequence-specific DNA binding domainto ZFA (Boch et al., 2009; Moscou and Bogdanove, 2009).

To achieve better specificity, one usually needs to constructTALENs each targeting more than 12 bp, and theoretically thelonger the better. However, due to its highly repetitive feature,it is difficult to construct long TALE repeats. Several methodshave been developed to resolve this issue and most are basedon Golden Gate cloning strategy, which employs type IIsrestriction enzymes and theoretically can ligate up to dozensof repeats through a single ligation reaction (Fig. 5) (Christianet 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 “UnitAssembly”, which took advantages of a pair of isocaudomersNhe I and Spe I. Using four single-unit vectors as the startingmaterial, which contain the coding sequence of alternativeunits recognizing A, T, C or G, TALE repeats can be easilyassembled by standard molecular cloning method of restrictionenzyme digestion and ligation with any and unlimited numberof repeats at any desired order (Fig. 5) (Huang et al., 2011).More recently, two high-throughput methods for TALE repeatsassembly called FLASH (fast ligation-based automatablesolid-phase high-throughput) and ICA (iterative cappedassembly) were reported, which bypass the time-consumingintermediate steps of bacterial culture and amplification byusing solid-phase magnetic beads for enzyme digestion andligation and allow researchers to construct large numbers ofTALENs rapidly (Fig. 5) (Briggs et al., 2012; Reyon et al.,2012). However, the advantages and disadvantages of allthese different TALEN construction methods still need furtherinvestigation.

Although TALEN targeting as a technique was reportedonly less than 2 years ago, it is soon broadly applied in in vitrocultured 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 ina similar way as ZFNs, and they could also induce indelmutations to disrupt the sequence in the spacer of TALENtarget sites (i.e., the region between the two TALEN bindingsites), but usually with higher targeting efficiency and loweroff-target effects (Hockemeyer et al., 2011; Huang et al., 2011;Li et al., 2011b). Therefore, a unique restriction enzyme site inthe spacer region is very convenient for the detection ofTALEN efficiency. After PCR amplification of the sequenceflanking the target site and digestion with the correspondingenzyme, the efficiency of TALENs can be estimated bymeasuring and comparing the density and ratio of the undi-gested band (Fig. 4). In addition, TALEN efficiency could berevealed directly by sequencing after PCR amplification orevaluated 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 inzebrafish have been constructed in our laboratory and theoverall success rate is above 70%, which is similar to a recent

http://bindr.gdcb.iastate.edu/ZiFDB/

http://bindr.gdcb.iastate.edu/ZiFDB/

http://web.iitd.ac.in/%7Esundar/zifbase/

http://web.iitd.ac.in/%7Esundar/zifbase/

http://eendb.zfgenetics.org/

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 pairstested, more than half of them showed >30% mutagenizingactivity in founder embryos, some of which even reached theefficiency to nearly 100%, as detected by restriction enzymedigestion assay (unpublished data), suggesting that TALEN isa promisingly efficient and easy technology for gene targetingin zebrafish. However, about 30% TALEN pairs did not showdetectable mutagenizing activity in zebrafish and the reason isstill unclear. In addition to the unpredictable and variableefficiency, another limitation for the applications of TALENsin zebrafish is that they can only induce indel mutations butnot precise modification of the target site at present, though webelieve this limitation is only temporary and will soon beovercome in the near future. Nevertheless, the ease in findingsuitable target sites, the high success rate, high efficiency andspecificity of TALEN targeting confer this relatively newreverse genetic strategy to be the most promising technique tobe able to target virtually any genes of interest in any species.It will not be surprising if TALEN eventually becomes themost powerful and popular site-specific mutagenesis method

for zebrafish, and perhaps, for other organisms as well. Thissituation reminds us of the discovery of RNA interference(RNAi) about 14 years ago, which has achieved a strongimpact to the field of study on gene functions shortly after. Webelieve 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 geneticstools will greatly enhance the advantages and utility ofzebrafish as a vertebrate model organism. With the expansionof zebrafish genetic toolkit, ingenious reverse geneticsmethods will certainly turn zebrafish mutagenesis intoa convenient and important process. Apart from point muta-tions isolated from TILLING, insertional mutations identifiedfrom retrovirus or Tol2 transposon mutagenesis screening andindel mutations induced by EENs (ZFNs or TALENs), thereare other types of mutations and mutagenesis methods whichare of equal or even more importance comparing with the


currently available ones. Large genomic fragment deletion andHR based gene knock-in and conditional knock-out are amongthe most interesting ones.

There are at least two potential approaches to achieve largegenomic deletions. Although only indel mutations have beenreported for TALEN targeting in zebrafish so far, ourpreliminary data showed that TALENs also hold promise toinduce large genomic deletions when using two pairs ofTALENs separated within a reasonable distance (Fig. 2 andunpublished data). In addition to disrupting genes by inser-tions, transposon can also be used for mutagenesis uponexcision. Deletion of large genomic sequence by inducing Pelement imprecise excision at a defined locus has been widelyused in Drosophila as a powerful, and during certain period oftime, the only method for the study of gene function andregulation through loss-of-function mutations. We found thatTol2 transposon excision can also induce large chromosomaldeletions in zebrafish (Fig. 2 and unpublished data). Consid-ering the availability of large and still increasing numbers oftransgenic zebrafish carrying Tol2 insertions, this strategycould become an alternative gene targeting method and couldbe used for the complete disruption/removal of a single geneor a cluster of genes simultaneously. This is especially usefulfor loss-of-function studies of non-coding RNA genes sincepoint mutations or small indel mutations might not be enoughto disrupt the function of these non-coding RNAs which do nothave a predictable open reading frame.

Other advanced genetic tools such as conditional knock-out and precise gene knock-in/replacement are widely usedin mouse and Drosophila and proved to be very useful.Although some preliminary work has been performed, genetargeting through HR is still not established in zebrafish(Hagmann et al., 1998; Cui et al., 2003; Wu et al., 2006). Thenext, and most likely also the last and the biggest, challengefor zebrafish reverse genetics studies is therefore to achieveHR 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 incultured cells or at organism level. By applying ZFNs toinduce DSBs, gene targeting through HR has been achievedin Drosophila, mouse and rat (Bibikova et al., 2003; Cuiet al., 2011). Similarly, TALENs have been shown to beable to induce HR in human iPSC and ES cells (Hockemeyeret al., 2011). Single strand oligonucleotide template has beenshown to be more efficient than traditional double-strandedtemplate as donors (Chen et al., 2011). Blocking DNAligase 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 tocreate nicks at the target site. These modified ZFNs couldincrease the relative frequency of HR to NHEJ, though theoverall 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, webelieve that gene targeting through HR, the last missingreverse genetics tool in zebrafish, will be ultimately achievedin 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 technicalsupport. This work was partially supported by the grants fromNational Program on Key Basic Research Project (973program) (Nos. 2012CB945101 and 2011CBA01000) andNational Natural Science Foundation of China (NSFC) (Nos.31110103904 and 30730056).

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reverse genetic approaches in zebrafish

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