Figure 8.

Efficient co-conversion marker to edit genomes of diverged nematode species. Mutations in the highly conserved gene ben-1, which encodes β-tubulin, confer resistance to benomyl, a drug that binds to β-tubulin, inhibits microtubule polymerization, and causes genetically wild-type animals to be slow growing and paralyzed. Loss-of-function mutations in ben-1 confer dominant benomyl resistance in both C. elegans and C. briggsae. (A) In C. elegans, the repair template created an XbaI restriction site adjacent to the PAM by converting an AA to CTAGAG, resulting in an in-frame stop codon that caused premature translation termination. (B) In C. briggsae, the repair template created an NdeI site by inserting a T adjacent to the PAM, causing an in-frame translation termination stop codon. In both species, the repair template altered protospacer sequences adjacent to the PAM, preventing the ben-1 mutants from being cleaved by Cas9. Successful editing of C. elegans sex-1 and C. briggsae rpb-1 established ben-1 as an effective co-conversion marker. WT, wild type.