SapTrap assembly of repair templates for Cas9-triggered homologous recombination with a self-excising cassette

Abstract

The advent of CRISPR/Cas9 technology in C. elegans has enabled an unprecedented level of control over this organism’s genome, which is facilitating research in a wide variety of fields. Several different experimental approaches exist for CRISPR in this organism (Dickinson and Goldstein 2016). One particularly powerful approach uses a drug selection with a Self-Excising Cassette (SEC) to isolate the desired genetically-modified animals following Cas9-triggered homologous recombination (Dickinson et al. 2015). The most labor-intensive step in this procedure is the construction of a plasmid-based repair template for homologous recombination. SapTrap (Schwartz and Jorgensen 2016) is a high-throughput cloning procedure that allows modular assembly of repair templates for CRISPR/Cas9-triggered homologous recombination. The original publication that described SapTrap cloning (Schwartz and Jorgensen 2016) used unc-119(+) as a positive selection marker for isolating recombinant strains. Compared to unc-119 selection, SEC selection is faster; can be used directly in a wild-type background; and eliminates an extra injection step followed by outcrossing to remove the selection markers after strain isolation (Dickinson et al. 2015). Because of these advantages of SEC selection over unc-119, we wished to incorporate the SEC into the SapTrap workflow. Here, we describe a toolkit of plasmids that allow building SEC-containing repair templates via SapTrap cloning.