Highly efficient CRISPR-mediated gene editing in a rotifer.

Rotifers have been studied in the laboratory and field for over 100 years in investigations of microevolution, ecological dynamics, and ecotoxicology. In recent years, rotifers have emerged as a model system for modern studies of the molecular mechanisms of genome evolution, development, DNA repair, aging, life history strategy, and desiccation tolerance. However, a lack of gene editing tools and transgenic strains has limited the ability to link genotype to phenotype and dissect molecular mechanisms. To facilitate genetic manipulation and the creation of reporter lines in rotifers, we developed a protocol for highly efficient, transgenerational, CRISPR-mediated gene editing in the monogonont rotifer Brachionus manjavacas by microinjection of Cas9 protein and synthetic single-guide RNA into the vitellaria of young amictic (asexual) females. To demonstrate the efficacy of the method, we created knockout mutants of the developmental gene vasa and the DNA mismatch repair gene mlh3. More than half of mothers survived injection and produced offspring. Genotyping these offspring and successive generations revealed that most carried at least 1 CRISPR-induced mutation, with many apparently mutated at both alleles. In addition, we achieved precise CRISPR-mediated knock-in of a stop codon cassette in the mlh3 locus, with half of injected mothers producing F2 offspring with an insertion of the cassette. Thus, this protocol produces knockout and knock-in CRISPR/Cas9 editing with high efficiency, to further advance rotifers as a model system for biological discovery.

Zebrafish pigment cells develop directly from persistent highly multipotent progenitors.

Neural crest cells are highly multipotent stem cells, but it remains unclear how their fate restriction to specific fates occurs. The direct fate restriction model hypothesises that migrating cells maintain full multipotency, whilst progressive fate restriction envisages fully multipotent cells transitioning to partially-restricted intermediates before committing to individual fates. Using zebrafish pigment cell development as a model, we show applying NanoString hybridization single cell transcriptional profiling and RNAscope in situ hybridization that neural crest cells retain broad multipotency throughout migration and even in post-migratory cells in vivo, with no evidence for partially-restricted intermediates. We find that leukocyte tyrosine kinase early expression marks a multipotent stage, with signalling driving iridophore differentiation through repression of fate-specific transcription factors for other fates. We reconcile the direct and progressive fate restriction models by proposing that pigment cell development occurs directly, but dynamically, from a highly multipotent state, consistent with our recently-proposed Cyclical Fate Restriction model.