Two-step protocol for extrachromosomal array integration by MosTI in C. elegans using Cas9 protein

Abstract

Transgenic Caenorhabditis elegans harboring large extrachromosomal arrays (several megabases) are relatively easy to generate, but suffer from variable and mosaic expression. Here, we describe a detailed protocol for stable transgene expression by chromosomal integration of arrays at safe-harbor landing sites or at the endogenous unc-119 locus. Our strategy is based on two rounds of injection. The first injection is a DNA-based injection to generate transgenic animals carrying extrachromosomal arrays. The second injection contains Cas9 protein and sgRNA for site-specific array integration at high frequency (44% of injected animals). Although this strategy necessitates two rounds of injections, the advantage is that no Cas9/sgRNA complex is co-integrated. Additionally, the use of an intermediate strain with a fully-formed extrachromosomal inherited array before integration favors integration of large DNA fragments.