Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Cláudia Pereira; Rita M. Reis; José B. Gama; Dhanya K. Cheerambathur; Ana X. Carvalho; Reto Gassmann

Integration of the transgene was verified by PCR and sequencing. Endogenous GFP tagging of the rod-1 locus was done using CRISPR/Cas9-mediated genome editing, as described [45] . The repair template for gfp::rod-1 included gfp(S65C) with introns, inserted upstream of the rod-1 start codon via a GGRAGS linker, and the homology arms (1046 bp left; 1036 bp right). The PAM site for the guide RNA (5'-CCACAGCTTTTGCTTCGCCT-3') was mutated from AGG to AGA in the repair template. To modify the rod-1 locus using Cas9-triggered homologous recombination, the repair template (50 ng/µL) was co-injected with two separate plasmids, one expressing guide RNA under the U6 promoter (50 ng/µL) and the other expressing Cas9 under the eft-3 promoter (30 ng/µL) into N2 worms [46] .