Mmessage t7 ultra transcription kit

Cas9 mRNA and gRNA were synthesized using mMESSAGE T7 ULTRA Transcription Kit (Invitrogen) and GeneArt Precision gRNA Synthesis Kit (Invitrogen), respectively. Genomic DNA was extracted with Qiagen DNAeasy kit. The sgRNA sequence is: GAGATGGCAAAGACCCCTTC. The IVT was performed according to the manufacturer's instructions. The sgRNAs concentration was adjusted to 50–100 ng/μl and sgRNAs were kept at −80°C until use.
Single‐cell fertilized eggs were obtained by either natural or induced breeding and injected according to previously published protocols with modifications.³³ Briefly, 250 pg Cas9 RNA and 50 pg gRNA were mixed into 5 nl and injected into freshly laid single‐cell‐stage embryos. The screening of F0 mosaic animals was done by both genotyping and phenotype characterization, including immunohistochemistry of limbs/tails. The Yap^−/− F1 animals were produced by crossing between adult F0 animals. The PCR primers for genotyping were PL: CTAGGTGCTGTCTCTCCCG PR: GTTGTGTGGGTTTTGTGAGCA. Before surgery, larval and adult newts were anaesthetized with 0.01% and 0.2% of ethyl‐p‐aminobenzoate (Sigma‐Aldrich), respectively.

Cas9 mRNA and gRNA were synthesized using mMESSAGE T7 ULTRA Transcription Kit (Invitrogen) and MEGAshortscript T7 Transcription Kit (Invitrogen), respectively. Genomic DNA was extracted with Qiagen DNAeasy kit. The PCR products were cloned into TOPO cloning vector (Invitrogen). Individual clones were sequenced with T7 primer (Supplementary Table 9).
Single-cell fertilized eggs were obtained by either natural or induced breeding and injected according to previously published protocols with modifications^{60 (link), 61 (link)}. Briefly, 500 pg Cas9 RNA and 100 pg gRNA were mixed into 5 nl and injected into freshly laid single-cell-stage embryos. The animals were raised according to^{60 (link)}. The screening of F₀ animals was done by both genotyping (see above) and phenotype characterization, including pigmentation analysis and immunohistochemistry of limbs/tails, according to^{39 (link), 40 (link)}. The Pax7^−/− and Tyr^−/− F1 animals were produced by crossing between adult F₀ animals. The larvae or post-metamorphic newts were anaesthetized within 0.01% or 0.1% ethyl-p-aminobenzoate (benzocaine; Sigma) prior to imaging and tissue collection. Newts utilized for this study were processed according to Swedish Board of Agriculture animal ethical regulations (N429/12).