Sequence-Based Mapping and Genome Editing Reveal Mutations in Stickleback<i>Hps5</i>Cause Oculocutaneous Albinism and the<i>casper</i>Phenotype

Abstract

Here, we present and characterize the spontaneous X-linked recessive mutation <i>casper</i>, which causes oculocutaneous albinism in threespine sticklebacks (<i>Gasterosteus aculeatus</i>). In humans, Hermansky-Pudlak syndrome results in pigmentation defects due to disrupted formation of the melanin-containing lysosomal-related organelle (LRO), the melanosome. <i>casper</i> mutants display not only reduced pigmentation of melanosomes in melanophores, but also reductions in the iridescent silver color from iridophores, while the yellow pigmentation from xanthophores appears unaffected. We mapped <i>casper</i> using high-throughput sequencing of genomic DNA from bulked <i>casper</i> mutants to a region of the stickleback X chromosome (chromosome 19) near the stickleback ortholog of Hermansky-Pudlak syndrome 5 (<i>Hps5</i>). <i>casper</i> mutants have an insertion of a single nucleotide in the sixth exon of <i>Hps5</i>, predicted to generate an early frameshift. Genome editing using CRISPR/Cas9 induced lesions in <i>Hps5</i> and phenocopied the <i>casper</i> mutation. Injecting single or paired <i>Hps5</i> guide RNAs revealed higher incidences of genomic deletions from paired guide RNAs compared to single gRNAs. Stickleback <i>Hps5</i> provides a genetic system where a hemizygous locus in XY males and a diploid locus in XX females can be used to generate an easily scored visible phenotype, facilitating quantitative studies of different genome editing approaches. Lastly, we show the ability to better visualize patterns of fluorescent transgenic reporters in <i>Hps5</i> mutant fish. Thus, <i>Hps5</i> mutations present an opportunity to study pigmented LROs in the emerging stickleback model system, as well as a tool to aid in assaying genome editing and visualizing enhancer activity in transgenic fish.