Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases

Results

Xenopus eggs are large and easily manipulated (13), offering the opportunity to deliver ZFNs via injection of mRNA, a method that has been successful in bringing about ZFN-driven gene disruption in other organisms (10, 11).

To develop conditions for gene disruption in X. tropicalis, we made use of transgenic animals carrying a single-copy GFP transgene (14). Wild-type X. tropicalis eggs were fertilized with sperm from a homozygous GFP transgenic male. The resulting heterozygous embryos were injected with mRNA-encoding ZFNs that target the eGFP coding region (15). Uninjected tadpoles express GFP robustly in the somites, lens, and head musculature (Fig. 1 A and B). Injection of 20 pg eGFP ZFN RNAs led to mosaic loss of fluorescence in otherwise healthy tadpoles (Fig. 1 E and F). At a higher dose of ZFNs, most cells had lost fluorescence, suggesting efficient somatic mutation of the transgene (Fig. 1 H and I).

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Fig. 1.

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.

To determine whether loss of fluorescence resulted from a ZFN-induced mutation in the eGFP transgene, we first genotyped the target locus using an assay based on the mismatch-sensitive endonuclease, Cel-1 (8). This analysis (Fig. 1J) demonstrated that ZFN-treated, but not control, tadpoles had acquired a DNA sequence alteration in the stretch targeted by the ZFNs. Sequencing subsequently revealed that individual tadpoles often carried multiple distinct indels ranging from 5 to 20 bp centered over the ZFN recognition site (Fig. 1K), a signature of mutagenic NHEJ. Taken together, these experiments show that ZFN mRNA injection into the two-cell embryo yields tadpoles without detectable developmental defects that exhibit both genetic and phenotypic mosaicism for the ZFN-targeted locus and trait, respectively.

To determine whether this approach can be used to disrupt an endogenous gene, we designed ZFNs that target the noggin locus. Noggin is a bone morphogenetic protein (BMP) antagonist that contributes to dorsal/ventral patterning during gastrulation in Xenopus (16). Although its function in later development has been studied in human patients (17) and mice (18–21), its developmental role in nonmammalian vertebrates remains poorly understood. Noggin is expressed at stages when knockdown via morpholinos is not effective. Therefore, mutant alleles of the endogenous noggin gene are required to probe its role throughout amphibian development.

We designed a panel of ZFNs targeting noggin, screened them in a budding yeast proxy system (22), and cloned the ZFNs into an expression construct that allows the synthesis of an efficiently translated mRNA with a stabilizing polyadenylation signal (23). Embryos were injected in the animal pole, and mRNA was deposited in the center of each blastomere at the two-cell stage and raised to stage 40, and DNA was isolated from tadpoles that exhibited broad mCherry (i.e., tracer) expression. Use of ZFNs carrying a wild-type FokI endonuclease domain yielded a significant fraction of embryos with developmental defects (Fig. 2A). However, this was alleviated by expressing the same zinc-finger DNA recognition domains fused to the obligate heterodimer forms of FokI, in which point mutations are made in the nuclease domain that prevent homodimers from forming a functional nuclease (8). These mutations, E490K, I538K and Q486E, I499L, are made in the FokI domain of the left and right ZFN, respectively (referred to as EL+KK). Even at the highest tested doses of such ZFN mRNA, more than 60% of the injected embryos developed normally (Fig. 2A).

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Fig. 2.

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.

To optimize the delivery of ZFNs, we also tested whether unstable, nonadenylated mRNA, which would be translated early and deliver a transient burst of ZFN, might be superior to the extended expression of ZFNs from transcripts that are cleaved and polyadenylated after injection (23). As expected, the nonadenylated transcripts led to expression of considerably less protein (Fig. 2C and Fig. S1), but even at higher doses did not induce noggin gene disruption at a frequency measurable by Cel-1. Thus, we conclude that the provision of ZFNs from stable polyadenylated mRNA is superior for effective genome editing in Xenopus.

Because it is difficult to predict a priori the extent to which ZFN overexpression may cause embryonic defects, we tested a panel of ZFNs in a yeast-based single-strand annealing assay (22) and in embryos (Fig. 2B and Table S1). Six ZFN pairs shown to have activity in the yeast assay were chosen for testing in Xenopus. These ZFNs were well tolerated in tadpoles and yielded efficient genome editing in Xenopus embryos as measured by sequencing noggin amplicons from injected tadpoles.

Because no Xenopus strains carrying noggin mutations exist, we were unable to screen for phenotypes on a heterozygous background (22) and instead screened the ZFNs for activity by genotyping the targeted region using the Cel-1 endonuclease (Fig. 3A). All of the ZFNs that were well tolerated produced targeted gene disruption. Direct sequencing of the nuclease-targeted region in tadpoles injected with ZFNs revealed a broad panel of insertions and deletions ranging in size from 5 to 195 bp (Fig. 3B), with frequencies of mutant amplicons from 10% to 47%. Such high rates of somatic mutagenesis suggested that tadpoles might also carry mutations in the germ line, allowing the establishment of lines carrying unique noggin alleles, a possibility we investigated next.

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Fig. 3.

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).

We injected wild-type embryos at the two-cell stage with 100 pg of mRNA encoding ZFNs targeting noggin that were tolerated by more than 60% of the injected embryos (Fig. 2B). Successful somatic genome editing in tadpoles and froglets was confirmed by isolating genomic DNA from tail or toe clips, respectively, and genotyping the noggin locus by Cel-1 (Table 1). Injected embryos were raised to sexual maturity and outcrossed to wild-type animals. Offspring from this cross were raised to tadpole stage 40, lysed, and analyzed via Cel-1 for mutations in the noggin locus. Genotyping offspring from a cross using a ZFN-treated male founder revealed that 3 of 18 tadpoles had inherited a ZFN-induced Δ12 allele of noggin (Fig. 4 A and B). A second male founder produced 6 of 50 embryos heterozygous for a ZFN-induced 3-bp insertion allele (Fig. 4 A and B), and a third male produced 12 of 50 tadpoles heterozygous for a 4-bp insertion. This ZFN-induced noggin frameshift allele results in a premature stop codon at position 55. This is likely to behave as a null allele because it lacks most of the BMP-binding residues and all of the residues required for dimerization (24).

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Fig. 4.

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.

The recovery of these mutations in the offspring of adult animals raised from injected embryos demonstrates that ZFN-induced alleles of an endogenous gene can be transmitted to the next generation. Significantly, the parent and heterozygous tadpoles carrying mutant alleles were indistinguishable from wild-type siblings, indicating that this is an effective approach to establishing lines of animals carrying mutant alleles of investigator-specified genes. To test whether ZFN-induced mutagenesis caused off-target mutations, we made gynogenotes from noggin ZFN-injected females that lacked mutant noggin alleles in their germ line. These were siblings of the germ-line mutated males from clutches that showed a high frequency of somatic mutation. Gynogenesis diploidizes activated eggs by preventing the extrusion of the second polar body and serves to homozygose recessive mutations (25). Although off-target mutations would result in high frequencies of mutant embryos in gynogenotes produced from these females, we did not detect such mutations. Indeed, 87% of 192 gynogenotes were indistinguishable from wild-type tadpoles, and the remaining 13% had various mediolateral and dorsoventral defects, consistent with reported phenotypes and frequencies from young wild-type females (26). This result suggests that the potential for confounding off-target mutations in founder animals is negligible.

To test whether the ZFN-induced alleles result in loss of function, we cloned the full-length mutant alleles and synthesized corresponding mRNA for injection. Noggin induces ectopic dorsal tissue when expressed on the ventral side of Xenopus embryos (16), and this serves as an excellent test for noggin function. Amplified alleles were transcribed in vitro and 5 or 10 pg was injected into the ventral vegetal blastomeres of four-cell-stage X. laevis embryos (Fig. 4C). Embryos were cultured to stage 28 and scored for the presence or absence of an ectopic axis (Fig. 4D). The 4-bp insertion allele failed to induce any ectopic dorsal tissues in embryos (Fig. 4 H, I, J, and J′), consistent with a frameshift mutation. LacZ mRNA was used as a tracer and showed that injected cells populated the ventral posterior of injected tadpoles. The Δ12 allele induced ectopic axes in 8% and 16% of embryos when injected with 5 and 10 pg mutant noggin RNA, respectively. Interestingly, the induced axes in these embryos were underdeveloped, suggesting that this allele functions as a hypomorph (Fig. 4M, arrows). Finally, the 3-bp insertion allele was indistinguishable in activity from wild-type noggin (Fig. 4 N–P). These results demonstrate that specific loss-of-function mutant lines of X. tropicalis can be generated via targeted ZFN mutagenesis. Furthermore, the mutations in noggin form the basis of an allelic series that will be useful to probe noggin function in later development.