Locus-specific integration of extrachromosomal transgenes in C. elegans with the CRISPR/Cas9 system

• A method to generate multi-copy integration lines with the CRISPR/cas9 system.
• Transgenes were integrated into defined loci in the chromosomes of C. elegans.
• Positive/negative selection was applicable to the CRISPR/Cas9 integration system.

We established a method to generate integration from extrachromosomal arrays with the CRISPR/Cas9 system. Multi-copy transgenes were integrated into the defined loci of chromosomes by this method, while a multi-copy transgene is integrated into random loci by previous methods, such as UV- and gamma-irradiation. The effects of a combination of sgRNAs, which define the cleavage sites in extrachromosomes and chromosomes, and the copy number of potential cleavable sequences were examined. The relative copy number of cleavable sequences in extrachromosomes affects the frequency of fertile F1 transgenic animals. The expression levels of the reporter gene were almost proportional to the copy numbers of the integrated sequences at the same integration site. The technique is applicable to the transgenic strains abundantly stored and shared among the C. elegans community, particularly when researchers use sgRNAs against common plasmid sequences such as β-lactamase.