Highly efficient CRISPR-mediated gene editing in a rotifer

Area of Science:

• * Molecular Biology
• * Developmental Biology
• * Genetics

Background:

• * Rotifers are valuable model organisms for studying microevolution, ecology, and toxicology.
• * Previous limitations in gene editing tools hindered genotype-phenotype linkage in rotifers.
• * Advancing rotifer genetics is crucial for dissecting molecular mechanisms of various biological processes.

Purpose of the Study:

• * To develop a highly efficient, transgenerational CRISPR-mediated gene editing protocol for the rotifer Brachionus manjavacas.
• * To establish a method for creating knockout and knock-in mutants in rotifers.
• * To facilitate the creation of reporter lines and advance rotifers as a model system.

Main Methods:

• * Microinjection of Cas9 protein and synthetic single-guide RNA into the vitellaria of young amictic female rotifers.
• * CRISPR-mediated knockout of the developmental gene vasa and the DNA mismatch repair gene mlh3.
• * CRISPR-mediated knock-in of a stop codon cassette into the mlh3 locus.

Main Results:

• * Over 50% of injected mothers survived and produced offspring with CRISPR-induced mutations.
• * Most offspring and subsequent generations carried at least one mutation, with many showing biallelic mutations.
• * Successful knock-in of a stop codon cassette in the mlh3 locus was achieved in F2 offspring.

Conclusions:

• * The developed protocol enables efficient, transgenerational CRISPR/Cas9 knockout and knock-in editing in rotifers.
• * This method significantly advances the utility of rotifers as a model system for biological research.
• * The protocol facilitates genotype-phenotype linkage and the dissection of molecular mechanisms in rotifers.