Extended Data Figure 6: Continuous indel generation by breeding.
a. Strategy for generating lineage traced mice through breeding. The target site and guide array cassette are integrated into mouse zygotes as in Figure 2a using C57Bl/6J sperm to generate P0 breeder mice, which are capable of transmitting high copy genomic integrations of the technology. Then, P0 animals are crossed with homozygous, constitutively expressing Cas9 transgenic animals to enable continuous cutting from fertilization onwards in F1 progeny. Shown is Sibling 2 of a cross between a P0 male and a Cas9:EGFP female.
b. Bar charts showing the degree of mutation (% cut, red) for a P0 male (top row) and 4 F1 offspring generated by breeding with a Cas9:EGFP female prior to weaning (21 days post partum). Each row represents a mouse and each column represents a target site. Each sibling inherits its own subset of the 23 parental target site integrations, and demonstrates different levels of mutation throughout gestation and maturation.
c. Indel frequencies for the 10 most frequent indels from 3 siblings in a common target site integration (column 1 in b). Each mouse shows a large diversity of indels and the different frequencies observed in each animal demonstrates an independent mutational path.