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Amphibians have made many fundamental contributions to our knowledge, from basic biology to biomedical research on human diseases. Current genome editing tools based on the CRISPR-Cas system enable us to perform gene functional analysis in vivo, even in non-model organisms. We introduce here a highly efficient and easy protocol for gene knockout, which can be used in three different amphibians seamlessly: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl. As it utilizes Cas9 ribonucleoprotein complex (RNP) for injection, this cloning-free method enables researchers to obtain founder embryos with a nearly complete knockout phenotype within a week. To evaluate somatic mutation rate and its correlation to the phenotype of a Cas9 RNP-injected embryo (crispant), we also present accurate and cost-effective genotyping methods using pooled amplicon-sequencing and a user-friendly web-based tool.
Fig. 1
A workflow for the crispant assay. Using Cas9 recombinant protein and commercially ordered oligonucleotides for the template DNA of sgRNA synthesis, crispants will be obtained within a week from the design of sgRNAs, without cloning
Fig. 2
Representative phenotypes of crispants. (a) An example of a P. waltl crispant. Wild-type (upper) and Tyr crispant (lower). Complete loss of pigmentation was observed in the founder generation. (b) Siblings from a single experiment. Uninjected control (right) and Tyr crispant (left)
Fig. 3
Detection of CRISPR-Cas-mediated mutagenesis using the heteroduplex mobility assay. The PCR products containing the sgRNA target site from the uninjected control and crispants were electroporated using a microtip. The single band from the uninjected embryo is a homoduplex of the wild-type allele (closed arrowhead). Upshifted bands are heteroduplexes of the wild-type and mutant alleles (brackets) and were only detected in crispants
Fig. 4
Genotyping of crispant using amplicon-sequencing analysis. (a) A bar graph of mutation rate generated by CLiCKAR. Blue and green show frameshift and in-frame mutation rates of Tyr crispant of P. waltl, respectively. (b) Representative mutant alleles, their occupancy rates, frameshift mutation rate, and total read count are shown corresponding to the phenotype in Fig. 3a, calculated by CLiCKAR. Deletions are indicated by dashes. PAM and microhomologous sequences are indicated by red letters and underscore, respectively
