Biallelic genome modification in F(0) Xenopus tropicalis embryos using the CRISPR/Cas system

Abstract

Gene inactivation is an important tool for correlation of phenotypic and genomic data, allowing researchers to infer normal gene function based on the phenotype when the gene is impaired. New and better approaches are needed to overcome the shortfalls of existing methods for any significant acceleration of scientific progress. We have adapted the CRISPR/Cas system for use in Xenopus tropicalis and report on the efficient creation of mutations in the gene encoding the enzyme tyrosinase, which is responsible for oculocutaneous albinism. Biallelic mutation of this gene was detected in the F0 generation, suggesting targeting efficiencies similar to that of TALENs. We also find that off-target mutagenesis seems to be negligible, and therefore, CRISPR/Cas may be a useful system for creating genome modifications in this important model organism.

Keywords: CRISPR/Cas; TALENs; gene knockouts; gene targeting; zinc finger nucleases.

Figure 1. CRISPR/Cas targeting of the tyrosinase locus in Xenopus tropicalis

A) Cas9-bound gRNA:DNA target region on the tyr gene. The 5'→3' orientation of the transcription unit is from right to left. Overlap with the TALEN binding sites (boxed in yellow) from Ishibashi et al. (2012) is shown. B) Right panel contains examples of stage 41 embryos injected with hCas9 and tyr gRNA. These tadpoles exhibiting typical oculocutaneous albinism found in most of the embryos. A subset retained more pigment but significantly less than wild-type levels (not shown). Left panel is a wild-type control sibling from the same clutch.

Figure 2. The range of albino phenotypes in froglets after targeting tyrosinase

Top left panel: A wild-type frog pigmentation pattern as comparison. The extent of mosaicism is highly variable. Scale bar in lower right panel indicates 1cm.

Figure 3. A catalog of the mutations found in a single albino CRISPR/Cas embryo

The wild-type sequence (parental) is shown at the top. Nineteen PCR product sequences are shown. Red highlighted bases indicate the target site with the PAM sequence in black bold and underlined. The nature of the mutations is indicated in the right column. Δ, deletion; sub, substitutions; WT, wild-type.