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.