Proc Natl Acad Sci U S A
April 26, 2011;
Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases.
The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin
gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin
gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus.
Proc Natl Acad Sci U S A
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Disruption of the eGFP transgene in X. tropicalis using ZFNs. (A�C) Uninjected tadpoles (U.C.). (D�F) Tadpoles injected with 20 pg of eGFP ZFN mRNA and 200 pg mCherry RNA (to monitor injection). (G�I) Heterozygous eGFP tadpoles injected with 50 pg eGFP ZFN mRNA and 200 pg mCherry RNA (tracer). (A, D, and G) Brightfield. (B, E, and H) eGFP expression of tadpoles in A, D, and G, respectively. (C, F, and I) Enlarged view of eGFP expression in B, E, and H, respectively. (J) Cel-1 digestion of eGFP amplicons. Bands migrating at 345 bp are full-length amplicons; Cel-1 cleavage products migrate at 246 and 99 bp. The fractions of modified chromatids detected by Cel-1 are quantified as percentage NHEJ. UC, uninjected control. (K) Sequence alignment of ZFN-induced mutant eGFP transgene alleles from tadpoles injected with 50 pg ZFN mRNA. Red nucleotides indicate insertions and dashes represent deletions. Horizontal bold lines at top indicate ZFN-binding sites.
Expression of ZFNs targeting noggin in X. tropicalis. (A) Optimization of ZFN delivery in X. tropicalis. EL+KK: ZFNs with the EL and KK modifications in the FokI domain (8); numbers represent different noggin ZFN pairs (Table S1). WT, ZFNs with wild-type Fok1 nuclease domains. PA−, ZFN transcripts lacking a polyadenylation signal. UC, uninjected control. (B) Comparison of different noggin ZFN pairs in the yeast activity assay and in injected tadpoles. Tadpoles were injected with 100 pg ZFN mRNA. Yeast activity values are represented as a percentage relative to ZFNs targeting the human CCR5 gene (9). Activity in tadpoles is calculated as the percentage of mutant amplicons sequenced from injected embryos. ND, no data. (C) Western blot for FLAG-tagged ZFN proteins.
ZFN-driven editing of the noggin locus in X. tropicalis. (A) Somatic mutations in noggin detected by Cel-1. Bands migrating at 450 bp are full-length noggin amplicons. Bands migrating at 300 and 150 bp are Cel-1 digestion products. (B) Sequence alignment of noggin alleles induced by indicated ZFN pairs. Red nucleotides indicate insertions and dashes represent deletions. Horizontal bold lines at top indicate ZFN-binding sites. EL+KK, ZFNs with the EL and KK modifications in the FokI domain (8); numbers represent different noggin ZFN pairs (Table S1).
Germ-line transmission of ZFN-induced noggin mutations. (A) Cel-1 digests of noggin amplicons from sibling heterozygous mutant and homozygous wild-type F1 tadpoles produced from three mutant line founders. Bands migrating at 450 bp are full-length noggin amplicons. Bands migrating at 300 and 150 bp are Cel-1 digest products. (B) Sequence alignments of the targeted noggin locus from Cel-1�positive F1 mutants. Genomic and translated sequences are shown for each mutant line. Asterisk indicates a stop codon. Red nucleotides and amino acids indicate insertions and dashes represent deletions. (C) Schematic of synthetic RNA injections into ventral vegetal blastomeres of four-cell-stage embryos to test functionality of the induced mutant noggin alleles. (D) Quantification of secondary axis induction following wild-type or mutant noggin RNA injection. Bars represent results of two (5 pg) or three (10 pg) independent experiments (�SD). Black bars show 5-pg RNA injections; white bars show 10-pg RNA injections. Two asterisks indicate significantly different (P < 0.01) from uninjected controls. (E, F, G, and G′) Uninjected control embryos. (H, I, J, and J′) Embryos injected with 10 pg of 4-bp insertion mutant noggin and 200 pg LacZ RNA. (K�M) Embryos injected with 10 pg of 12-bp deletion mutant noggin and 200 pg LacZ RNA. (N�P) Embryos injected with 10 pg of 3-bp insertion mutant noggin and 200 pg LacZ RNA. (Q�S) Embryos injected with 10 pg wild-type noggin and 200 pg LacZ RNA. (E, H, K, N, and Q) Dorsal view of stage 19. (F, I, L, O, and R) Dorsal view of stage 28. (G, J, M, P, and S) Embryos stained with 12/101 antibody. (G, J, and M) Lateral view. (G′, J′, P, and S) Dorsal view. Arrows show weak ectopic dorsal axis induction.