The integration of transgenes in Caenorhabditis elegans has traditionally utilized transposable elements or bombardment, with current protocols adopting CRISPR-based integration. CRISPR also allows for the creation of custom-designed integration loci. Here, we take advantage of this experimental freedom to design such loci, or 'landing pads,' to facilitate transgene integration. These landing pads are designed for integration-specific selection, thereby avoiding the selective benefit of array formation and consequent false positives, eliminating the need for anti-array selection or co-CRISPR methodologies. Within the genome, landing pads are built with a partial Hygromycin B resistance gene missing the promoter and the 5'coding sequence. In addition, a synthetic unique guide RNA target sequence is present upstream for targeting by Cas9. Six synthetic guides were pre-tested to choose an optimal guide target, and four were identified with similar integration efficiencies. Donor homology plasmids are constructed with the transgenic cargo plus the
rps-0p::HYGR 5' coding sequence. As the Hygromycin B resistance gene has been split, there is no selectable advantage in the array. Only upon integration is the Hygromycin B resistance restored. A landing pad locus was initially constructed on Chr. II, and we recently expanded the toolkit, creating landing pad strains and plasmid donors for Chr. I and III. For each landing pad/donor pair, the Hygromycin B resistance gene is flanked by unique Lox sites, allowing for optional removal of the resistance gene without inducing interchromosomal rearrangements. Additional donor plasmids have been constructed to allow for self-excision of the resistance gene, similar to Dickinson et al. 2015. In addition to streamlining the integration process, we bypassed the requirement for pre-constructed plasmids. It is known that C. elegans can stitch together linear dsDNA fragments with homology-directed repair (HDR), which has been used for clone-free transgenesis. We tested two and six-part transgenic insertions for
sqt-1(
e1350) and found functionally integrated transgenes for both. We couple this strategy with our synthetic landing pads to streamline the transgenesis process from PCR to confirmed single copy integration in as little as five days. Overall, the combined approach provides an economical and rapid method to generating single-copy transgenes for C. elegans.