Development of Highly Efficient CRISPR-Mediated Gene Editing in the RotiferBrachionus manjavacas

Abstract

AbstractRotifers have been studied in the laboratory and field for over 100 years and are an emerging modern model system for investigation of the molecular mechanisms of genome evolution, development, DNA repair, aging, life history strategy, and desiccation tolerance, and have a long been used in studies of microevolution, ecological dynamics, and ecotoxicology. 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, we developed a protocol for highly efficient, transgenerational, CRISPR-mediated gene editing in the monogonont rotiferBrachionus manjavacasby 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 genevasaand the DNA mismatch repair genemlh3. More than half of mothers survived injection and produced offspring. Genotyping these offspring and successive generations revealed that most carried at least one CRISPR-induced mutation, with many apparently mutated at both alleles or mosaic. In addition, we achieved precise CRISPR-mediated knockin of a stop codon cassette in themlh3locus, with half of injected mothers producing 33% or more F2 offspring with an insertion of the cassette. These results demonstrate the efficacy of the CRISPR/Cas9 system in rotifers to provide insight into the function of specific genes and further advance rotifers as a model system for biological discovery.