Simple embryo injection of long single-stranded donor templates with theCRISPR/Cas9 system leads to homology-directed repair inXenopus tropicalisandXenopus laevis

We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology-directed repair (HDR)-mediated gene editing in the twoXenopusspecies used most frequently for research:X. tropicalisandX. laevis. We have used long single-stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. First,X. tropicalis tyrmutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte-specific geneslc45a2.LofX. laevisto label it with theSuperfolder greenFP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of thetyrmutation (X. tropicalis) and tagging in the appropriate pigment cell-specific manner ofslc45a2.LwithsfGFP(X. laevis).