Figure 6

CNS1 CRISPR editing inhibits endogenous hnf1b pronephric expression in Xenopus tropicalis embryos. (a) GRNAa and gRNAb efficiently edit Xenopus tropicalis embryo DNA in two different locations in CNS1. Embryos were injected at the 1-cell stage with Cas9 protein and gRNAa or gRNAb and cultured until neurula stage. The target sequences of gRNAa and gRNAb are depicted in Fig. 1 (see also “Methods” section). Top panels: chromatogram showing CRISPR editing by gRNAa and gRNAb in single embryos. Lower panels: TIDE analysis of sequence trace degradation at the expected Cas9 site of DNA cleavage. Percentage of CNS1 sequence containing insertions and deletions (Indels) represented as the mean from four embryos. The use of gRNAa and gRNAb resulted in 51.3% and 71.7% editing efficiency, respectively. (b) The use of gRNAa and gRNAb together efficiently leads to nucleotide sequence deletion into CNS1. Embryos were injected at the 1-cell stage with Cas9 protein, together with gRNAa, gRNAb or both. At tailbud stage, their DNA were analysed by PCR using primers allowing amplification of a DNA fragment containing the entire CNS1. Electrophoresis of amplified fragments from single embryos. A band corresponding to the wild type sequence is observed in all embryos. An additional lower band is present in 3 out of 5 embryos co-injected with gRNAa and gRNAb, indicating that a deletion occurred (c) Hnf1b pronephric expression in gRNAa, gRNAb and gRNAa–gRNAb CRISPR embryos analyzed by in situ hybridization at tailbud stage 28. Embryos were classified into three groups according to their hnf1b pronephric expression (normal, slightly diminished, strongly diminished). The histogram shows the percentage of embryos for each group. Average values from four independent experiments. Statistical significance was determined using Fisher’s exact test (comparison to the uninjected control embryos). P-values are annotated on the figure. n indicates the total number of analysed embryos. (d) Lhx1 pronephric expression in gRNAa, gRNAb and gRNAa-gRNAb CRISPR embryos analyzed by in situ hybridization at tailbud stage 28. No significant effect on lhx1 expression is observed. n indicates the total number of embryos analysed from three independent experiments.