Efficient generation of a single-copy eft-3p::TIR1::F2A:: BFP::AID*::NLS allele in the C. elegans ttTi5605 insertion site through recombination-mediated cassette exchange.

ABSTRACT

The auxin-inducible degron (AID) system is a widely used system to conditionally deplete proteins. Using CRISPR/Cas9-based genome editing in C. elegans, we recently generated a set of single-copy, tissue-specific and pan-somatic TIR1-expressing strains carrying a BFP reporter inserted in single-copy into two commonly used, well-characterized genetic loci. However, we were unable to obtain a strain carrying a pan-somatic eft-3pTIR1F2ABFPAID*NLS transgene inserted into the chromosome II ttTi5605 insertion site. Using recombination-mediated cassette exchange (RMCE) we were able to efficiently obtain this knock-in. The resulting strain displayed equivalent depletion of an AID*GFP reporter compared to our previously generated eft-3pTIR1F2ABFPAID*NLS transgene knocked into the chromosome I ttTi4348 insertion site. This work highlights the power of RMCE for generating new reagents for the AID system and provides an eft-3pTIR1F2ABFPAID*NLS allele on chromosome II which will simplify genetic crossing schemes when using the AID system.