Figure 2

LKO mutations induced by multiple gRNAs targeting the rnf185 gene in zebrafish. (A) Illustration of gRNAs that are against rnf185 exon 2 and exon 4 based on Ensemble annotation. Red bars with arrows indicate the positions of gRNAs against the rnf185 coding DNA. Red arrows match the gRNA directions. The lengths of illustrated exons are not proportional to the real size of corresponding exons. Black lines and arrows represent primers and their directions. (B) Representatives of positive adult F₁ fish by PCR with primers (Fw 2 and Rv 4) that bind to exons 2 and 4 respectively. Among the 48 fish, 2 were identified to have large deletions between exon 2 and exon 4 (also see Table 1). Each lane is corresponding to an F₁ adult fish. (C) The exon 2 - exon 4 deletion (3,366bp) was confirmed by Sanger sequencing. 3,366bp is the size of the omitted region including the dashes. (D) Predicted zebrafish Rnf185 wildtype and mutant protein based on the mutation positions. Protein domains were based on cBioPortal and SMART annotations. Mutant protein sizes were calculated by the ORF finder.