In vivo genome editing using a high-efficiency TALEN system

Abstract

The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.

Figure 1. Second-generation GoldyTALEN scaffold improves genome-editing efficacy

a, A schematic showing the layout of TALEN target sites. TALENs were targeted to flanking sequences surrounding a restriction enzyme site for easy screening through introduction of a restriction fragment length polymorphism. b, Relative activity of the GoldyTALEN and pTAL scaffolds at two loci, ponzr1 and crhr1. Under each lane is the percent uncut DNA of a single larva, illustrating the increased activity of GoldyTALEN. c, Whisker plots of the percent uncut DNA demonstrates TALEN cutting efficiency at two loci. ponzr1 TALENs demonstrate a significant (p < 10⁻¹⁶), 6-fold increase in activity using GoldyTALEN. crhr1 TALENs also demonstrate a significant (p < 10⁻⁹), 15-fold increase in activity. n = number of embryos screened, mdn = the median percent cut. d, The ponzr1 GoldyTALENs were more active in a cell-free restriction enzyme digestion assay. ponzr1 DNA is labeled in both uncut and cut forms. − ctrl = negative control.

Figure 2. Increased TALEN efficiency results in biallelic gene targeting

a, GoldyTALENs were designed against the moesina, ppp1cab and cdh5 genes. All three gene targets contained a restriction enzyme site within the spacer region between the TALEN binding sites. Injection of GoldyTALEN mRNAs demonstrated a nearly complete loss of the restriction enzyme site in the amplicons of somatic tissue. Each lane is the amplification product from a group of 10 embryos. Mutant seq (%) = percentage of amplicons that carry mutant sequences as determined by sequencing 10 clones (Supplementary Fig. 4). b–d, Injection of cdh5 GoldyTALENs (d) phenocopies the MO-based loss-of-function phenotype (c). Brightfield images (top panels) show pronounced cardiac edema (arrows) in both GoldyTALEN (d)- and MO (c)-injected larvae at 2 days post fertilization. Using the Tg(fli1-egfp)^y1 line, the intersomitic vessels were visualized (bottom panels) and show a loss of lumen formation (white arrow) in both the MO (c)- and GoldyTALEN (d)-injected larvae. The Tg(gata1:dsred)^sd2 line revealed reduced circulation in GoldyTALEN- and MO-injected larvae, demonstrated by the increase in red fluorescence in the confocal images (see Movies 1–3).

Figure 3. Targeted genome editing using GoldyTALENs

a, A schematic of the ponzr1 locus with the ssDNA sequence used to introduce a targeted exogenous EcoRV sequence into the genome in vivo. The left and right TALEN binding sites are shown in red and orange, respectively, and the spacer region is in blue. b, A representative gel demonstrating germline transmission of the HDR-based EcoRV sequence in 3 out of 4 fin tissue-positive fish. c, Sequence analysis of the three germline-transmitting lines. The first fish transmitting HDR-based genome changes through the germline (#1) yielded 7 out of 96 embryos with an incorporated EcoRV site. The genomes of all 7 embryos showed the same modified sequence. The second founder fish (#2) yielded 7 out of 46 embryos with EcoRV incorporation. All 7 embryos showed precise HDR-based addition of the EcoRV sequence. The third fish with germline transmission (#3) yielded 5 out of 18 embryos with an incorporated EcoRV site, and showed a mosaic germline as demonstrated by offspring with three different modified sequences. One embryo included precise HDR-based EcoRV addition. The other 4 embryos contained sequence insertions on the 5′ end with two embryos each harboring the specific sequences changes.

Figure 4. Germline mloxP integration into the crhr2 locus

a, A diagram of the TALEN target sites with the mloxP ssDNA oligo. The left and right TALEN target sequences are red and orange, respectively, the spacer region is blue, and the oligo’s right homology arm is in purple. The mloxP sequence is underlined. b, Germline screening of the crhr2 locus. 53 adult fish were prescreened via fin biopsy. Of those prescreened, 20 demonstrated mloxP maintenance. 16 F0s were outcrossed with 2 showing germline transmission. 42 unscreened F0s were outcrossed and 4 demonstrated germline transmission. c, Sequence confirmation of three mloxP germline fish. One fish demonstrated precise germline HDR while two showed indels. In #NS24, we saw the reverse complement of the mloxP was noted (shaded in grey).

Figure 5. in vivo TALEN-induced genome editing outcomes

TALENs efficiently create double-stranded breaks in chromosomal DNA and catalyze three major outcome classes. First, error-prone NHEJ produces an indel in and near the spacer region of the TALEN binding site. If a complementary ssDNA oligonucleotide is also added, two different outcomes are noted. First, HDR precisely uses the exogenous sequence information in the ssDNA to add sequence at the cut site. Alternatively, ssDNA acts as a primer for 3′ integration of the oligonucleotide but the 5′ end undergoes error-prone NHEJ.