Protocol for the continuous generation of single-copy MosTI insertions in C. elegans from a multiplex mix.

Precise expression of transgenes in C. elegans can be used to understand gene regulation, control cells, or as a starting point for genetic screens. Inserting singlecopy transgenes into well-defined safe-harbor locations is useful when consistent expression levels are required (e.g., to compare expression from different transgenes) or when expression is desired in germ cells. Here, we describe a detailed protocol for the simultaneous injection of several different plasmids ("multiplex mix"). Heat-shock inducible Cas9 expression allows the insertion of single-copy transgenes from the multiplex mix using Modular Safe-harbor Transgene Insertion (MosTI). The protocol allows the generation of many different transgene inserts over several generations from a single injection. In principle, the method can be used to screen a collection of plasmids for a desirable property, such as expression in a particular tissue. Only a few extra-chromosomal strains are required, which makes the protocol particularly appropriate for researchers with limited experience with C. elegans micro-injection.