Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs) - PubMed (original) (raw)

Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs)

Yong Lei et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F(1) frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Schematic drawing of TALENs and ZFNs and sequences of somatic mutations induced in X. tropicalis G0 embryos. (A) Schematic drawing of TALEN with 15.5 RVDs located between 136-aa N-terminal and 63-aa C-terminal regions. The ELD and KKR Fok I nuclease domains are linked to the C terminus of the TALE monomer. RVDs are shown in red and Fok I domains in blue. (B) Schematic drawing of TALENs (Upper) and ZFNs (Lower) that target ptf1a/p48. Recognition sequences are highlighted in yellow. (C) DNA sequences targeted by noggin-, ptf1a/p48-, or ets1-TALENs, and somatic mutations induced in Xenopus embryos. Sequenced mutations are listed. The largest forward deletion (400 bp) and the largest backward deletion (403 bp) were found in ets1 of X. tropicalis. (D) noggin- and ptf1a/p48- ZFNs targeting sites and somatic mutations induced by these pairs of ZFNs. In C and D, mutated regions are marked in gray, with red dashes indicating deletions (Δ) and lowercase letters in red indicating insertions (+). The numbers in parentheses show the number of deleted or inserted base pairs, whereas numbers in square brackets show the frequencies of the mutation in the sequenced samples.

Fig. 2.

Fig. 2.

Mutagenesis detection by PCR. (A) Schematic drawing of primer pairs used for detecting mutagenesis by PCR (Materials and Methods). (B) A representative DNA gel displaying results of colony PCR with the two primer pairs to detect mutagenesis in X. tropicalis embryos injected with ets1-TALENs. In the upper panel, the ∼200-bp fragments amplified with primer pair 1, 3, indicates the presence of the targeted sequence in the plasmids. In the lower panel, the absence of PCR products for primer pair 2, 3, indicates the presence of targeted mutations, whereas a ∼140-bp fragment is amplified from wild-type sequences (indicated by arrows).

Fig. 3.

Fig. 3.

Frequencies of mutations and abnormal embryos induced by the indicated TALENs or ZFNs in Xenopus. Mutation frequencies were assayed as shown in Fig. 2. Only the mutation ratios induced by lower doses of ZFNs are shown because higher doses (200, 500, and 800 pg) resulted in dead or malformed embryos. (A–C) Frequencies of targeted mutagenesis induced by TALENs or ZFNs for the genes and at the doses indicated in the panels. All data refer to X. tropicalis except in C where X. laevis results are also shown. (D–F) Percentage of normal, abnormal, and dead X. tropicalis embryos injected with the indicated doses of TALEN or ZFN mRNAs. The injected embryos were inspected at 48 h postfertilization (about stage 41). (G) Overall morphology of X. tropicalis embryos injected with TALEN mRNAs directed against the indicated gene at stage 41. Curled axis, repression of head structures including eyes, and loss of pigments were observed. Such abnormal tadpoles usually could not complete metamorphosis.

Fig. 4.

Fig. 4.

Phenotypes of ptf1a/p48-TALEN targeted X. tropicalis. (A and B) Anatomical analysis revealed visceral abnormalities such as much reduced pancreas in ptf1a/p48-TALEN-injected G0 froglets. The pancreas is outlined by dashed lines. (C) DNA sequencing of genomic DNA extracted from hindlimb tissue dissected from a froglet showing a phenotype similar to that in B confirmed the gene disruption at the ptf1a/p48 locus (20/23) (du, duodenum; st, stomach). (D–F) Whole-mount in situ hybridization of pancreas marker pdip in X. tropicalis tadpoles injected with the indicated TALEN mRNAs. ptf1a/p48-TALENs, but not ets1-TALENs, induced repression of pancreas bud formation. (D) Uninjected control embryos, (E) ptf1a/p48-TALEN-injected embryos (800 pg), and (F) ets1-TALEN-injected embryos (800 pg). (G) Summary of the phenotypes shown in D–F. The TALEN mRNAs were injected into the animal pole region at the one-cell stage; embryos were analyzed at stage 40.

Fig. 5.

Fig. 5.

Sequence and frequency of mutant alleles inherited in F1 embryos. (A and B) DNA sequencing confirmed germ-line transmission of ets1 and ptf1a/p48 mutants induced by the TALENs. Disrupted sequences are shaded in gray. Red dashes indicate deletions (Δ). The numbers of deleted base pairs are shown in parentheses, and the number of embryos showing the particular mutation is given in square brackets. (C and D) The percentage of wild-type and the TALEN-disrupted alleles in F1 embryos derived from ets1- or ptf1a/p48-TALEN-targeted frogs, determined either by colony PCR (ets1-TALEN-targeted F1 embryos) or DNA sequencing (ptf1a/p48-TALEN-targeted F1 embryos). Twenty F1 embryos for ets1 from three crosses and 15 F1 embryos for ptf1a/p48 from two crosses of G0 by wild type were examined. The n in C and D indicates the number of bacterial clones that were examined after TA cloning.

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