Abstract
The introduction of exogenous genes in single-copy at precise genomic locations is a powerful tool that has been widely used in the model organism Caenorhabditis elegans. Here, we have streamlined the process by creating a rapid, cloning-free method of single-copy transgene insertion we call Mos1 element-mediated CRISPR integration (mmCRISPi). The protocol combines the impact of Mos1 mediated single-copy gene insertion (mosSCI) with the ease of CRISPR/Cas9 mediated gene editing, allowing in vivo construction of transgenes from linear DNA fragments integrated at defined loci in the C. elegans genome. This approach was validated by defining its efficiency at different integration sites in the genome and by testing transgene insert size. The mmCRISPi method benefits from in vivo recombination of overlapping PCR fragments, allowing researchers to mix-and-match between promoters, protein-coding sequences, and 3′ untranslated regions, all inserted in a single step at a defined Mos1 loci.
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