Homology-Directed Repair by CRISPR–Cas9 Mutagenesis inXenopusUsing Long Single-Stranded Donor DNA Templates via Simple Microinjection of Embryos

Abstract

We describe a step-by-step procedure to perform homology-directed repair (HDR)-mediated precise gene editing inXenopusembryos using long single-stranded DNA (lssDNA) as a donor template for HDR in conjunction with the CRISPR–Cas9 system. A key advantage of this method is that it relies on simple microinjection of fertilizedXenopuseggs, resulting in high yield of healthy founder embryos. These embryos are screened for those animals carrying the precisely mutated locus to then generate homozygous and/or heterozygous mutant lines in the F1generation. Therefore, we can avoid the more challenging “oocyte host transfer” technique, which is particularly difficult forXenopus tropicalis, that is required for an alternate HDR approach. Several key points of this protocol are (1) to use efficiently active single-guide RNAs for targeting, (2) to use properly designed lssDNAs, and (3) to use 5′-end phosphorothioate-modification to obtain higher-efficiency HDR.