Fig. 3.

Isolation and characterization of Volvox VSR1 edited mutants and rescued strains. (A) Diagram of Volvox VSR1 locus showing two different editing sites in detail including the PAM site that specifies cleavage by targeted Cas9 guide RNA RNP complexes. The vsr1-1 insertion/deletion allele is shown below in blue lettering. Dark boxes represent exons. Start and termination codons are also shown. (B–D) Micrographs of male vsr1-1 mutant taken from vegetative cultures (B), from post-embryonic sexual males with uncleaved presumptive androgonidia with an example shown by a white arrowhead (C), and from a stage when the presumptive androgonidia had undergone vegetative embryogenesis to produce vegetative juveniles (black arrowhead) instead of sperm packets (D). (E–G) Micrographs of a wild-type male strain taken at equivalent stages as the vsr1 mutant in panels B–D, respectively. A mature male with sperm packets (G) is shown with a magnified sperm packet in the Inset. (H) Rescued vsr1-1::SF-VSR1 male strain at sexual maturity with the restoration of sperm packets. A magnified sperm packet is shown in the Inset. All (Scale bars are 50 µm.) (I) Immunoblot detection of FLAG epitope–tagged VSR1 in a rescued vsr1-1 strain. Samples were taken from vegetative (veg) and from early (48 h) and middle (59 h) stages of sexual (sex) development as depicted in SI Appendix, Fig. S1. (J) Wild-type sexual female with eggs. (K) Sexually induced vsr1-1 female mutant spheroids with egg-like cells. (L) Fertilized wild-type female with maturing zygotes. Inset in L shows a magnified view of a zygote. (M) Post-mating vsr1-1 sexual females with fertilization pore at sperm entry point (Inset) but no zygotes. The egg-like cells behave as unfertilized vsr1-1 mutants and undergo vegetative embryogenesis.