Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.

doi: 10.1111/gtc.12379

Figure Lengend Snippet: Figure 4 Genotyping of founders with different phenotypes with RFLP analyses. (A) A schematic of restriction enzyme-RFLP (RE-RFLP) and RNA-guided engineered nuclease-RFLP (RGEN-RFLP) analysis. (B) Estimation of somatic mutation rates of the target alleles in each phenotype. PCR products of tyr and ltk were cleaved by Cas9 protein with sgRNAs in vitro. PCR prod- ucts of slc45a2 were cleaved by BfaI. Cleaved fragments were analyzed by electrophoresis (MultiNA system; Shimadzu). Upper and lower images show Cas9 protein or BfaI (+) and () gel images converted from electropherograms. PCR products of target regions are shown by black arrowheads. Colored arrowheads indicate cleaved products by Cas9 or BfaI. Some extra bands were observed and thought to be large deletions and insertions. The mutation rates were calculated from the molarity of the uncleaved bands (black arrowheads and other extra bands) and the larger cleaved fragments (blue arrowheads). Wt, wild type; W, weak phe- notype; M, moderate phenotype; S, severe phenotype; PS, severe phenotype by Cas9 protein injection; H, half phenotype; F, full phenotype. Full scan images of RFLP analyses are shown in Figs S5–S8 in Supporting Information.

Article Snippet: Preparation and injection of Cas9 and sgRNA Humanized codon hspCas9 cDNA from pX330 (Addgene, #42230; Cong et al. 2013) containing two NLSs was subcloned into a modified pCS2+ vector (pCS2+-T7/hspCas9; Fig. S12 in Supporting Information).

Techniques: Mutagenesis, In Vitro, Electrophoresis, Injection

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.

doi: 10.1111/gtc.12379

Figure Lengend Snippet: Figure 7 Example of target gene analysis according to our workflow. We designed an sgRNA targeting activin receptor-like kinase 2 (alk2). (A) Representative phenotypes produced by Cas9 mRNA and sgRNA (upper panel). Most phenotypes exhibited a partial loss of ventral fin tissue accompanied by the hypertrophic anus. Normal means no alteration in the ventral fin or anus. The total numbers of individuals are shown at the top of each graph. In vitro fertilization (IVF) indicates control embryos without injection. Frequencies of alk2-disrupted phenotypes from three independent experiments are shown in under panel. (B) RGEN-RFLP analy- sis of somatic mutation rates. (C) Sanger DNA sequencing of somatic mutation alleles from five phenotypes. In total, 107 clones were sequenced. (D) Off-target analysis using heteroduplex mobility assay (HMA). Wt, wild type; P, phenotype.

Article Snippet: Preparation and injection of Cas9 and sgRNA Humanized codon hspCas9 cDNA from pX330 (Addgene, #42230; Cong et al. 2013) containing two NLSs was subcloned into a modified pCS2+ vector (pCS2+-T7/hspCas9; Fig. S12 in Supporting Information).

Techniques: Produced, In Vitro, Control, Injection, Mutagenesis, DNA Sequencing, Clone Assay