Abstract
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.
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Background
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 [Full size image
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.
Next, to determine if large deletions in endogenous loci and element-specific deletions can be generated effectively, we designed and synthesized one TALEN pair towards sequences approximately 230 bp upstream of the predicted transcriptional start site (TSS), and a second pair targeting sequences within cyc exon 1 (cyc5TAL, chr12: 49,427,780-49,427,835; cyc3TAL, chr12: 49,428,165-49,428,221), spanning a genomic region of approximately 380 bp that encompasses the TSS (Figure 2a). Similarly, to target sqt, we generated one TALEN pair specific to the 5' sequences upstream of the TSS, and a second pair targeting sequences in the 3' UTR of sqt (sqt5TAL, chr21: 19,838,706-19,838,767; sqt3TAL, chr21: 19,840,869-19,840,929; zebrafish genome assembly Zv9). The TALEN target sites span a chromosomal region of approximately 2.16 kb, encompassing the sqt gene. We also used sqt5TAL in conjunction with a ZFN pair targeting sqt exon 1 (sqtZFN2, Figure 2a), to delete a 98 bp genomic region surrounding the TSS (sqtZFN2, chr21: 19,838,905-19,838,934).
Targeted deletions in cyc and sqt by multiple TALEN and ZFN pairs. (a) Schematic representation of the cyc and sqt loci, with positions of the TALEN targeting sites and ZFN targeting sites indicated by black arrows. E1, E2 and E3 indicate cyc or sqt exons 1 to 3. Colored triangles in the both cyc and sqt panels indicate the position of primers used for genoty**. (b) Phenotype of cyc TALEN injected embryo at 24 hpf showing cyclopia. Scale bar, 100 μm. (c) Phenotype of representative sqt nuclease-injected embryo manifesting cyclopia and midline defects. (d) PCR with primers (yellow and black triangles in (a)) spanning the TALEN targeting sites (black arrows in (a)) shows the expected approximately 400 bp truncated cyc (white arrowhead), and 779 bp full-length cyc (black arrowhead) products in ten single embryos injected with cyc TALEN pairs, whereas the full-length product is observed in the un-injected control embryo. All embryos show faint intermediate sized products. No template control is indicated by -g. (e) PCR with primers (red and blue triangles in (a)) spanning the sqt locus show a 2.4 kb product (black arrowhead) for the intact sqt locus, whereas individual embryos with TALEN deletions show an approximately 220 bp complete locus deletion product (white arrowhead) and several other intermediate sized products. (f) PCR with primers spanning the sqt TSS site (red and green triangles in (a)) show a 478 bp full-length wild-type product (black arrowhead), and only one embryo (number 1) shows the expected approximately 300 bp deletion product (white arrowhead). (g) Alignment of wild-type (WT) cyc sequences with mutated PCR amplicons shows various deletions of approximately 400 bp between the targeting sites, accompanied by small insertions (red). (h) Alignment of wild type sqt sequences with mutated PCR amplicons shows various deletions of approximately 2.2 kb between the targeting sites, accompanied by small insertions (red).
To determine the optimal dosage, we microinjected various concentrations of sqt TALEN and ZFN pairs, or cyc TALEN pairs into one-cell zebrafish embryos individually and in combinations, and assessed the cutting efficiency, phenotypes and survival at 24 hpf (Table 3; Figures S1A-D,F-G,I-J and S2 in Additional file 1). Cyclopia and midline defects, phenotypes that are visible in cyc and sqt mutant embryos [23, 25], were found at frequencies ranging from 13 to 40% for cyc and 15 to 25% for sqt, indicating bi-allelic mutations in a proportion of injected F0 embryos (Figure 2b,c, Table 3; Figure S2 in Additional file 1). The efficacy of deletion mutations was assessed by PCR and sequencing from individual 1 dpf embryos (Figure 2d-f; Figure S1A-D,F-G,I-J in Additional file 1). Alignment to wild-type sqt genomic sequences showed that each TALEN and ZFN pair by itself generated small insertions and deletions of varying lengths (Figure S1D,I in Additional file 1), consistent with previous reports using single ZFN or TALEN pairs [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-clip** and routine genoty** PCR analysis, using primers listed in Table S2 in Additional file 1. The sqtsg7 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 sqtsg7 embryos express sqt RNA [37]. The sqtsg27 mutants harbor an indel (chr21: 19838727-1983870; Figure S5 in Additional file 1) and lack the transcriptional start sequences, and the lesion in sqtsg32 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.)
Semi-quantitative RT-PCR
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 sqtsg27/+ and sqtsg32/+ crosses. For genoty**, 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.
Microscopy
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.