Streamlined single shot safe harbor transgene integration in C. elegans using unc-119 rescue.

Abstract

Transgenic animals are an invaluable tool in model organism genetics. The ease of modifying the C. elegans genome through high-copy integration of transgenes facilitates the investigation of diverse and fundamental biological processes. However, generation of new multicopy integrated transgenes is limited by the time and labor cost. Further, many transgenes are integrated using non-specific DNA damaging agents. These DNA damaging agents cause unwanted mutations during the integration process and may have deleterious effects. A recently described method for CRISPR/Cas9-based integration of multicopy transgenes at safe harbor loci using Fluorescent Landmark Interference (FLInt) greatly increases the efficiency of multicopy transgene integration and mitigates issues related to off-target mutagenesis during integration. unc-119 rescue is a simple and widely used phenotypic marker in C. elegans transgenesis and genome engineering. To streamline generation of multicopy transgenes via FLInt, we have generated a set of strains suitable for FLInt-mediated integration of transgenes using rescue of the unc-119 mutant phenotype to select transgenic animals. We demonstrate the utility of this approach and outline a protocol that uses unc-119 rescue as a selection marker for streamlined integration of multicopy transgenes at safe harbor loci.