A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases
- Shimin Lim†1,
- Yin Wang†2, 3,
- Xueyao Yu4,
- Yian Huang5,
- Mark S Featherstone1 and
- Karuna Sampath1, 2, 3Email author
© Lim et al.; licensee BioMed Central Ltd. 2013
Received: 15 March 2013
Accepted: 1 July 2013
Published: 1 July 2013
Precise and effective genome-editing tools are essential for functional genomics and gene therapy. Targeting nucleases have been successfully used to edit genomes. However, whole-locus or element-specific deletions abolishing transcript expression have not previously been reported. Here, we show heritable targeting of locus-specific deletions in the zebrafish nodal-related genes squint (sqt) and cyclops (cyc). Our strategy of heritable chromosomal editing can be used for disease modeling, analyzing gene clusters, regulatory regions, and determining the functions of non-coding RNAs in genomes.
Genome editing tools such as transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) have revolutionized the fields of biotechnology, gene therapy and functional genomic studies in many organisms [1–4]. However, engineering large chromosomal deletions in vertebrates has been largely restricted to mice, where the typical strategy used is gene targeting, and subsequently, specific regions are engineered by site-specific recombination systems such as the Cre/Lox or Flp/FRT systems . Although site-specific recombination has been used successfully to analyze the functions of genes involved in embryonic development, cancer and other diseases, this strategy is time, labor and resource intensive. Hence, rapid and facile methods to engineer chromosomes are of immense value.
Analysis of regulatory elements in the genome, and determining the activity and functions of gene clusters require generation of chromosomal lesions. Although large chromosomal lesions have been generated by gamma ray treatment and other methods, these lesions are often accompanied by complex rearrangements affecting several loci, which is a limitation for precise analysis of specific genomic regions or regulatory elements. In addition, the size and position of the re-arrangements cannot be predetermined by these methods [6, 7]. Therefore, precise and easy techniques to generate segmental mutations at desired locations on chromosomes would be useful for analysis of gene clusters and large regulatory regions in the genome.
Recent genome-wide transcriptome analyses in cells and organisms have identified several non-coding and novel coding RNAs. However, determining their functions requires the generation of RNA-null alleles . The TALEN and ZFN technologies have been used successfully in many organisms to generate small insertion and deletion mutations at target sites of specific genes [3, 9–11]. Large chromosomal deletions and inversions have been shown in cell lines using ZFNs, and deletions using two pairs of TALENs have been generated in silkworm, swine fibroblasts and zebrafish [12–16]. So far, however, heritable chromosomal deletions that specifically abolish expression of a transcript have not been reported with these nucleases in any organism. Thus, rapid and easy methods to generate whole-locus, element-specific or transcript-specific deletions would greatly facilitate functional genomic studies.
Here, we report a simple, effective and rapid strategy to generate a whole locus deletion in zebrafish, by the simultaneous use of two pairs of TALENs or TALEN pairs in conjunction with ZFN pairs, that we used successfully to precisely delete the nodal-related gene sqt and generate sqt RNA-null alleles. We also report targeted deletions in a second zebrafish nodal gene, cyclops (cyc), for which gamma ray- and chemically induced chromosomal rearrangements and point mutations were reported, but a precise locus-specific deletion was not available [6, 17–23]. Our strategy of heritable chromosomal editing can potentially be applied for functional genomic studies in a variety of organisms.
Mutation frequencies induced by single TALEN pairs
Mutation frequency by T7E1 in individual embryos
Mean ± SEM
16.2 ± 1.5%
25.5 ± 3.2%
9.1 ± 1.0%
4.5 ± 0.7%
31.9 ± 3.0%
54.0 ± 3.2%
Frequency of deformities and lethality in egfp TAL-injected embryos
12.5 pg egfp5TAL
25 pg egfp5TAL
12.5 pg egfp3TAL
25 pg egfp3TAL
12.5 pg egfp5TAL+egfp3TAL
25 pg egfp5TAL+egfp3TAL
PCR with primers flanking the TALEN sites (Figure 1a, blue and magenta triangles) shows an approximately 250 bp fragment in all injected embryos (n = 23), compared to a 854 bp wild-type egfp fragment, indicating excision of intervening sequences in some cells of injected embryos (Figure 1d). Sequencing of PCR products from individual embryos showed large as well as small deletions, likely due to mosaicism of the injected nuclease RNA pairs, and non-homologous end joining events (Figure 1e; Figure S1B,C,E in Additional file 1). Comparison of sequences of single TALEN versus double TALEN pair injections shows lower deletion frequency with single TALEN pair injections, presumably because small deletions induced by single nuclease pairs are repaired more efficiently than the larger lesions induced by multiple TALEN pairs (Figure S1A-E,H in Additional file 1). Moreover, the majority of mutant alleles from double TALEN injections showed complete deletions (Figure S1B,C,E in Additional file 1). These results show that defined large deletions that disrupt target gene expression can be generated easily via the combinatorial action of multiple TALENs.
Frequency of cyclopia and mid-line defects in cyc and sqt nuclease-injected embryos
Cyclopia and midline defects
6.25 pg cyc5TAL+cyc3TAL
12.5 pg cyc5TAL+cyc3TAL
25 pg cyc5TAL+cyc3TAL
25 pg sqtZFN2
50 pg sqtZFN2
25 pg sqt5TAL
50 pg sqt5TAL
25 pg sqt3TAL
50 pg sqt3TAL
25 pg sqt5TAL+sqtZFN2
25 pg sqt5TAL+sqt3TAL
Germ-line transmission frequency of cyc and sqt nuclease-induced lesions in zebrafish
Number of F0 screened
Number of mutant F0s
sqt5TAL + sqt3TAL
6 (whole locus deletions)
sqt5TAL + sqtZFN2
2 (TSS deletions)
1 (4 bp insertion)
cyc5TAL + cyc3TAL
10 (9 founders with TSS deletions, and 1 with a non-TSS 151 bp deletion and a deletion + inversion + insertion)
We then ascertained if the sqt TSS and whole locus deletions actually abolish sqt RNA expression in mutant embryos. We also determined if adjacent genomic regions and elements were affected by the sqt deletions, by examining expression of neighboring genes (eif4ebp1, htr1ab, and rnf180; Figure 3k) at appropriate stages. RT-PCR analyses to detect expression of immediate flanking loci show that their transcription is unaffected in the sqt sg27 TSS deletion mutant embryos (Figure 3l). By contrast, expression of sqt RNA is significantly reduced in embryos heterozygous for the sqt sg27 TSS deletion mutation, and is not detected in homozygous sqt sg27 embryos (Figure 3l). Similarly, sqt RNA expression is not detected in homozygous sqt sg32 whole-locus deletion mutant embryos, whereas flanking gene expression is unaffected (Figure 3m). Thus, our sqt deletions do not affect neighboring transcriptional units and these deletions are bona fide sqt RNA-null alleles.
Mutation frequency of double nuclease pairs versus homology directed repair
Percentage of positive founders
Founders screened (n)
Gupta et al. 2013 
Gupta et al. 2013 
Zu et al. 2013 
Bedell et al. 2012 
Bedell et al. 2012 
Chromosomal deletions can be used for analyzing gene clusters and regulatory regions, and for determining the functions of non-coding as well as coding RNAs in the genome. In support of this possibility, our sqt sg27 TSS deletion that is predicted to excise the TSS elements and sqt sg32 whole-locus deletion indeed result in mutant embryos that are sqt RNA-null. Furthermore, zygotic sqt sg27 and sqt sg32 deletion mutant embryos manifest phenotypes that are consistent with the previously identified sqt mutations. Thus, this strategy can be used effectively to investigate the roles of all 'functional' RNAs in the genome.
The various targeting nucleases have different constraints pertaining to target sites. For instance, TALENs prefer a 5' T nucleotide, whereas CRISPR/Cas9 requires a GG dinucleotide for targeting. The spacer requirements for the various nucleases are also different, and targetable sites for the different nucleases likely occur at different frequencies in genomes [31, 32]. Therefore, using the combinatorial action of various nucleases can facilitate generation of defined deletions at desired locations with higher efficacy. Moreover, some TALEN and ZFN sites (for instance, our sqt ZFN target sites) are just not targeted efficiently for reasons that are still unclear. Hence, the ability to use multiple targeting nucleases in various combinations offers additional flexibility and alternative approaches to engineer chromosomes than is possible with individual nuclease pairs. The efficiency and precision of the deletion events can be improved further by using nuclease variants such as the 'GoldyTALEN' system .
Our simple, facile and efficient strategy is largely PCR-based, and, therefore, can be used with modest resources to generate deletion mutants for investigating functional elements in the genome. Finally, this approach of generating large, defined heritable deletions by simultaneously targeting two discrete regions on a single chromosome can potentially also be deployed with RNA-guide mediated or other emerging DNA cleavage methods [32–34] to enhance the toolkit for heritable chromosomal engineering in a variety of organisms.
Targeted and heritable chromosomal deletions can be rapidly generated in a whole organism by using the combinatorial action of targeting nucleases. Multiple nuclease pairs are apparently more effective than single nuclease pairs in generating targeted deletions. Whole-locus as well as TSS element-specific deletions were generated efficiently by this method, and stably transmitted through the germ-line. The deletion mutations result in transcript-null alleles that manifest embryonic mutant phenotypes, demonstrating functional consequences of the chromosomal lesions. This simple, facile and efficient strategy can be used with modest resources. Thus, our strategy can be used to generate disease models, and for analysis of gene clusters, regulatory regions and functional RNAs in the genomes of a variety of organisms.
Materials and methods
Generation of plasmids encoding TALENs and ZFNs
The egfp, sqt and cyc TALENs target sites were designed using an online tool . To check for unique targeting sites, BLAST and UCSC BLAT search was performed with the zebrafish genome assembly (Zv9) using the target site sequences. The TAL effector repeats were constructed from four TAL effector single unit vectors (pA, pT, pGNN and pC) using the 'unit assembly' method . Plasmids encoding sqtZFN1 and sqtZFN2 nuclease pairs were obtained from ToolGen, Inc. (Seoul, South Korea). The TALEN and ZFN target sites for egfp, cyc and sqt are shown in Figures 1a and 2a and sequences are listed in Table S1 in Additional file 1.
TALEN and ZFN capped mRNA synthesis
Using the Ambion® SP6 mMESSAGE mMACHINE kit (Life Technologies, Carlsbad, California, United States of America), capped TALEN mRNAs were transcribed in vitro from 1.0 µg of the respective NotI linearized TALEN expression vectors. To synthesize capped ZFN mRNAs, sqt-specific ZFN plasmids were linearized with XhoI and transcribed using T7 RNA polymerase (Promega, Fitchburg, Wisconsin, United States of America). RNA was purified by phenol-chloroform precipitation and dissolved in RNase-free water.
Microinjection of capped TALEN and ZFN mRNA into zebrafish embryos
All experiments using animals were performed in accordance with institutional animal care and use guidelines. For sqt and cyc experiments, embryos from wild type (AB) fish were used for injections. For egfp TALEN experiments, embryos from Tg (Ds DELGT4) sg310 homozygous males mated with wild-type AB females were used. Tg (Ds DELGT4) sg310 transgenic fish harbor a Ds transposon-mediated enhancer trap insertion on chromosome 21. Various dosages and combinations of nuclease RNAs were tested to determine the toxicity, and the maximum dose that yielded less than 50% lethality was used (Table 2). For testing single TALEN or ZFN pairs, either 12.5, 25 or 50 pg of each mRNA was injected into one-cell stage zebrafish embryos. Higher lethality rates and abnormal embryos were observed with the cyc TALEN pairs and, therefore, cyc5TAL and cyc3TAL mRNAs were introduced at 6.25, 12.5 or 25 pg doses per embryo. For double TALEN pair or TALEN+ZFN experiments, a cocktail of either 12.5 or 25 pg of each mRNA was injected into one-cell stage zebrafish embryos. Injected embryos were examined at prim-5 stage under a Leica MZ12.5 stereomicroscope. PCR products from individual embryos injected with each single nuclease pair were tested by the T7E1 assay and sequencing to assess the efficacy of each nuclease pair. Ten embryos that were morphologically normal were selected and processed for PCR and sequencing. The remaining embryos were raised to adulthood to determine the germ-line transmission rates.
PCR and sequence analyses
To detect deletions in founder (F0) embryos, at least 10 TALEN- and ZFN-injected embryos were individually lysed at 24 hpf in 20.0 µl of DNA extraction buffer (10 mM Tris pH 8.2, 10 mM EDTA, 200 mM NaCl, 0.5 % SDS, 100 µg/ml proteinase K) for 5 h at 55°C, followed by heat inactivation of proteinase K at 65°C for 10 minutes. Genomic DNA was diluted five-fold using 1× TE Buffer (pH 8.0), and 2 µl aliquots were used in 20 µl PCR reactions. For single nuclease pair experiments, fragments containing 100 to 150 bp upstream and downstream of the expected target sites were amplified with Go Taq polymerase (Promega). For double TALEN or TALEN+ZFN experiments, primers annealing to regions 100 to 150 bp upstream of 5' TALEN and downstream of the 3' TALEN or ZFN target sites were used in PCR from genomic DNA template using Phusion® High-Fidelity polymerase (New England Biolabs, Ipswich, Massachusetts, United States of America) following the manufacturer's instructions (the primers used are listed in Table S2 in Additional file 1). Five microliter aliquots of products from ten single embryo PCRs were pooled, gel purified to remove primer dimers and cloned into either Promega pGEM®-T easy TA cloning vector or Fermentas pJET1.2 blunt end cloning vector, and transformed using XL1-blue heat-shock competent bacterial cells. At least 48 bacterial colonies were picked for screening by PCR. PCR products were diluted three-fold, and 1 μl was used directly for sequencing using the same primer pairs. Sequences were analyzed by comparison to the Zv9 Zebrafish Genome Assembly.
T7E1 assay to detect indels induced by single nuclease pairs
Five microliter aliquots of single embryo PCR products were diluted to 20 μl in 1× NEB Buffer 2, denatured at 95°C for 5 minutes, slowly cooled to room temperature to allow annealing and formation of hetero-duplexes. The individual preps were then treated with 5 units of T7E1 (New England Biolabs) for 30 minutes at 37°C. Digested products were separated on a 3.5% agarose/1×TBE gel and band intensity analyzed using ImageJ (NIH) to calculate mutation frequencies .
Genotyping of F1s
To assess the germ-line transmission rates, injected F0 fish were raised to adulthood, and mated either with siblings or wild-type fish to obtain F1 progeny. For genotyping sqt nuclease- or cyc TAL-injected embryos, PCR was performed using primers listed in Table S2 in Additional file 1, and Taq polymerase (Promega). PCR amplicons were electrophoresed on a 2% agarose gel. To screen for germ-line transmission events at the endogenous sqt locus, we analyzed progeny from pairwise mating of founders. Single embryos from six founder fish (three pairs) were screened per 96-well plate. At least 30 embryos (24 hpf) from each mating were collected, lysed and analyzed by PCR using the same primer pairs as used for the transient assays. This number allowed efficient detection of germ-line transmission events (whose frequency ranged from 3 to 10%), and recovery of the mutation. Bands of aberrant sizes were either sequenced directly or after cloning into the pGEM®-T easy vector system. F1 progeny of positive F0s were raised to adulthood, and heterozygous carriers for the deletions were identified by fin-clipping and routine genotyping PCR analysis, using primers listed in Table S2 in Additional file 1. The sqt sg7 ZFN1-induced allele harbors a 4 bp insertion in exon2 (chr21: 19839892-19839896; Figure S5 in Additional file 1). The sg7 mutation is predicted to result in a frame-shift after amino acid 143 in Sqt protein and premature termination after amino acid residue 146. Homozygous sqt sg7 embryos express sqt RNA . The sqt sg27 mutants harbor an indel (chr21: 19838727-1983870; Figure S5 in Additional file 1) and lack the transcriptional start sequences, and the lesion in sqt sg32 is a whole locus deletion of 2.1 kb on chromosome 21 (Figure S5 in Additional file 1). For analyzing germ-line transmission rates of cyc deletions, we collected progeny from pairwise mating of founders in pools of five embryos since the somatic mutation frequency for the cyc TALENs was higher than that for sqt. At least ten pools from each successful mating were collected and used in PCRs to ensure that founders with mutant clone sizes larger than 2% were identified. Subsequently, founders that yielded mutations were mated with wild-type (AB) fish. At least 22 single embryos from each mating were collected for PCR and sequencing to confirm and determine the germ-line transmission rate. (For a list of primers, see Table S2 in Additional file 1.)
Using TRIzol reagent (Invitrogen, Carlsbad, California, United States of America), both genomic DNA and total RNA were extracted from single 30% epiboly stage and 2 dpf (for htr1ab expression) embryos obtained from heterozygous sqt sg27/+ and sqt sg32/+ crosses. For genotyping, 50 ng of genomic DNA was used as template in 20 µl PCR reactions. For first-strand cDNA synthesis, 250 ng of total RNA was used in a pdN6-primed reaction using SuperScript® II Reverse Transcriptase (Life Technologies). First-strand cDNA (1 µl) was used in 20 µl PCR reactions to detect expression of sqt, ring finger protein (rnf180), 5-hydroxytryptamine (serotonin) receptor 1A b (htr1ab), eukaryotic translation initiation factor 4E binding protein 1 (eif4ebp1) and control actin (act), using the primers listed in Table S3 in Additional file 1.
Embryos were manually de-chorionated using fine forceps and mounted in 2.5% methylcellulose on a depression slide. DIC images were captured using a monochrome CoolSNAP HQ camera (Photometrics, Tucson, Arizona, United States of America) fitted on a Zeiss Axioplan2 upright microscope. The egfp TALEN injected and un-injected Tg (Ds DELGT4) sg310 embryos were manually de-chorionated and mounted in 1.5% low melting agarose (Bio-Rad, Hercules, California, United States of America) on tissue culture dishes with cover-slip bottoms (World Precision Instruments, Inc. FluoroDish FD3510-100, Sarasota, Florida, United States of America). Images were captured using a Leica SP5 inverted confocal system.
enhanced green fluorescent protein
polymerase chain reaction
T7 endonuclease I
transcription activator-like effector nuclease
transcriptional start site
zinc finger nuclease.
We thank members of the Sampath laboratory, Mohan Balasubramanian, Zhang Bo and Tom Carney for suggestions; Cherish Tay and Helen Quach for technical support; TLL core facilities; YH acknowledges the NUS High School Advanced Research Attachment Program; SL and MF are supported by SBS, NTU; YW is supported by TLL; work in the laboratory of KS is supported by TLL.
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