Rgma and Neo1 LOF tools. A, Schematic representation of rgma, neo1a and neo1b loci showing exons (Ex, black boxes) and 5′ and 3′ untranslated regions of the genes (5′UTR and 3′UTR) and target sites for MOs (green bars) and gRNAs (magenta bars). B–G, High-resolution melt analysis of indels in embryos co-injected with HF-nCas9 and gRNAs against rgma exons 2 (B) and 3 (C), neo1a exons 3 (D) and 8 (E), and neo1b exons 3 (F) and 8 (G). Each line indicates relative fluorescence units (RFUs) over a range of temperatures corresponding to individual uninjected (control) 24 h post-fertilization (hpf) embryos (black graph lines) or Cas9/gRNA-coinjected embryos that accumulate mosaic indels in somatic and germ line cells (magenta graph lines). H, Alignment of the neo1a MO2 target sequence to the neo1a and neo1b transcripts. The underlined residues indicate the translational start site. The nucleotides in bold are not conserved. I, Schematic of Neo1a (top) and predicted protein product of neo1aδ1133 mRNA lacking the NeoICD (bottom). S, Signal peptide; IgG, immunoglobulin domain; FN, fibronectin domain; TM, transmembrane domain. J, K, Western blot analysis of Rgma protein levels in embryos injected with multiplexed rgma gRNAs with Cas9 (J) and MO2 (K), compared with uninjected controls and std MO-injected embryos, respectively. L, M, Western blot analysis of Neo1 protein levels in embryos injected with multiplexed neo1a and neo1b gRNAs with Cas9 (L) and neo1a MO2 (L), compared with uninjected controls and std MO-injected embryos, respectively. N, Detection of the FLAG epitope by Western blot in control (uninjected) and neo1aδ1133-FLAG mRNA-injected embryos. Each lane was loaded with 5 μg of total protein with Gapdh used as a loading control (J–N).
