Targeted integration of extrachromosomal arrays in C. elegans using PhiC31 integrase

Extrachromosomal arrays are unique chromosome-like structures created from DNA injected into the Caenorhabditis elegans germline. However, they are unstable unless integrated into a chromosome. Current methods for integration using x-rays or CRISPR can damage DNA and exhibit low efficiency. We demonstrate that the viral integrase PhiC31, which mediates nonmutagenic recombination between short attB and attP sequences, can be used for extremely efficient and targeted integration of arrays. Arrays were integrated by PhIAT (PhiC31-mediated integration of arrays of transgenes) at attB sites on three chromosomes, including at a fluorescent landing pad. Moreover, integrations can be inserted at any arbitrary site in the genome by simultaneously co-injecting Cas9 ribonucleoprotein, an attB repair template, and the DNA components for the array-thereby providing sites on all six chromosomes. Single injections can integrate arrays ranging in size from 1 to 18 megabases. PhIAT makes it practical to study genomes of other organisms in the nematode; one of our strains incorporates 65% of the yeast genome at a single site in the worm genome. PhIAT will accelerate a shift from unstable extrachromosomal arrays to direct integration of arrays in C. elegans.