Generation of smo mutant fish and genotyping
A single guide RNA (sgRNA) targeting exon 5 of smo (GenBank accession number: NM_131027) (GGCTGATGGGTGGTGCCAA) was designed using the ‘ZebrafishGenomics”’ track on the UCSC Genome Browser. Primers were designed to amplify a 275 bp region flanking the target site for use in all screening and subsequent genotyping steps using the previously described fluorescent PCR assays (Sood et al. 2013). The primer sequences are as follows: smo -E5-Fwd (5’- TGTAAAACGACGGCCAGTTTACTCGCTTATGTCTGGAG) and smo -E5-Rev (5’- GTGTCTTTAACTGAATCTGCATCCACC). Synthesis of target oligonucleotide (Integrated DNA Technologies), preparation of mRNA, microinjection, evaluation of sgRNA activity by CRISPR-STAT and generation of mutant lines were carried out as described previously (Varshney et al., 2016). Briefly, Wildtype embryos (TAB5) were injected with sgRNAs (5pg) and Cas9 mRNA (300pg) and grown to adulthood. Screening for germline transmission of indels was carried out by analysis of 8 embryos from the progeny of each founder fish at 24 hours post fertilization by fluorescent PCR. Progeny of founder fish with desired mutant alleles were grown to adulthood and genotyped to identify heterozygous fish for subsequent phenotype analysis. The mutant alleles were sequenced to determine their predicted effect on the encoded protein.