Assessment of anti-Myo10 antibody and myo10l1 gRNA efficiency.
(A) Myo10 antibody staining for neurons in embryos injected with myo10l1-eGFP BAC. Rohon-Beard neurons expressing Myo10l1-eGFP (A) are also positive for Myo10 antibody staining (A’). (B) Scale diagram of zebrafish Myo10l1 protein. gRNAs target distinct sites in myo10l1 exons corresponding to specific regions of the protein (red arrowheads). PH = Pleckstrin homology domain, MyTH = Myosin tail homology four domain, FERM = 4.1 protein, Ezrin, Radixin, Moesin domain. (C) Amplification of targeted CRISPR cut target regions containing restriction cut sites. Gel electrophoresis (2% agarose gel) of PCR amplification of each CRISPR cut site from genomic DNA of individual four dpf larvae injected with gRNA triplex cocktail that display significant pLLG truncation or uninjected embryos. Following PCR, amplicons are incubated with restriction enzymes that cut near CRISPR target sites. Uninjected embryos have near complete digest of amplicons at each CRISPR target site, but triplex-injected crispants that display pLLG truncation show substantial amounts of resistance to digest. *=1 kb plus ladder.