Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

Abstract

Among tetrapods, only urodele salamanders, such as the axolotl Ambystoma mexicanum, can completely regenerate limbs as adults. The mystery of why salamanders, but not other animals, possess this ability has for generations captivated scientists seeking to induce this phenomenon in other vertebrates. Although many recent advances in molecular biology have allowed limb regeneration and tissue repair in the axolotl to be investigated in increasing detail, the molecular toolkit for the study of this process has been limited. Here, we report that the CRISPR-Cas9 RNA-guided nuclease system can efficiently create mutations at targeted sites within the axolotl genome. We identify individual animals treated with RNA-guided nucleases that have mutation frequencies close to 100% at targeted sites. We employ this technique to completely functionally ablate EGFP expression in transgenic animals and recapitulate developmental phenotypes produced by loss of the conserved gene brachyury. Thus, this advance allows a reverse genetic approach in the axolotl and will undoubtedly provide invaluable insight into the mechanisms of salamanders' unique regenerative ability.

Keywords: Axolotl; CRISPR; Mutagenesis; Regeneration.

Fig. 1.

Efficient targeted mutagenesis of EGFP in Tg(CAGG:EGFP) transgenic axolotls. (A) Distribution of phenotypes in axolotl (Ambystoma mexicanum) embryos from a mating between a wild-type and hemizygous Tg(CAGG:EGFP) animal injected with various concentrations of cas9 mRNA and an EGFP-directed sgRNA at 3 weeks post-injection, as assessed by fluorescence microscopy. Whereas ∼50% of embryos injected with lower concentrations of cas9 and sgRNA displayed normal EGFP expression, those injected with the highest concentrations of cas9 and sgRNA displayed mosaic EGFP expression. Any animal exhibiting clones of EGFP-negative cells was classified as ‘partial EGFP’. The survival rate in all cas9 and sgRNA-injected embryos did not differ from that of embryos injected with nls-EGFP mRNA only (right column). (B-E′) Whereas 6-month-old uninjected Tg(CAGG:EGFP) animals display strong uniform EGFP expression in both their heads (B′) and tails (C′), siblings injected with EGFP-directed sgRNA and cas9 (D,E) display a dramatic loss of EGFP, with one individual with only EGFP-positive cells apparent in the head (D′) and another with EGFP expression only in the tail (E′). (B-E) Brightfield; (B′-E′) EGFP fluorescence. (F) All 34 sequences of cloned PCR products of the EGFP locus in a single animal injected with an EGFP-directed sgRNA and cas9 that displayed no apparent EGFP expression contain indels at the targeted site (yellow). The size of each deletion (–) or insertion (+; in red) and frequency of occurrence among clones are indicated.

Fig. 2.

Efficient targeted mutagenesis of brachyury ortholog. (A,B) Whereas animals injected with nls-EGFP mRNA display no phenotype at 21 days post-fertilization (A), their siblings injected with sgRNA directed against a putative brachyury ortholog and cas9 display a dramatic shortening along the anteroposterior axis (B). (C) This phenotype persists at 7 months post-fertilization, as an sgRNA-injected animal (bottom) shows a severe reduction in length of the posterior body compared with an nls-EGFP-injected sibling (top). (D) Sequencing of the targeted region from two brachyury RGN-injected 21-day-old whole larvae displaying a severe phenotype as in B reveal a high indel frequency at the targeted site. (E) Sequencing of the targeted region from DNA extracted from fin clips of two brachyury RGN-injected 5-month-old animals displaying a severe phenotype as in C reveals a high indel frequency at this site.

Fig. 3.

Efficient targeted mutagenesis at the bambi locus. (A) Sequencing of the targeted region of the bambi locus from three bambi RGN-injected 5-day-old whole embryos reveals a high indel frequency at the targeted site. (B) Sequencing of the targeted region of the bambi locus from DNA extracted from fin clips of three bambi RGN-injected 1-month-old embryos reveals a high indel frequency at the targeted site. (C) A sibling control (left) and the three bambi mutant embryos with the reported (see B) A, B and C genotypes (left to right, respectively), at 4 months.