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bioRxiv · 10.1101/2025.11.11.687718

High-efficiency targeted integration of extrachromosomal arrays in C. elegans using PhiC31 integrase

Rich, M. S.·Pellow, R.·Hefel, A.·Rog, O.·Jorgensen, E. M.
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Abstract

Extrachromosomal arrays are unique chromosome-like structures created from DNA injected into the C. 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 non-mutagenic 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 RNP, 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.

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Rich, M. S., Pellow, R., Hefel, A., Rog, O., Jorgensen, E. M.. 2025-11-12. High-efficiency targeted integration of extrachromosomal arrays in C. elegans using PhiC31 integrase. https://doi.org/10.1101/2025.11.11.687718

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