Figure 3. Sea lamprey AID^evoCDA1 introduces C→T mutations at high rates in embryos.

(A) Plasmids with different versions of AID used in experiments to mutagenize GFP in the genome fly embryos using 4 gRNAs. We tested AID stemming from different organisms under a plethora of different promoters. For the inducible UASz, we crossed to bam-Gal4 and osk-Gal4 to induce high germline expression. (B) Average mutation rate (defined as C→T or G→A mutations over all mutable positions) in conditions aiming to increase mutation rate in embryos. Mutation rates in the Kc experiment from Figure 2 are shown in the left panel for reference though relative levels of expression of AID between cell culture and embryos are likely not comparable. Propagating fly lines over multiple generations or replacing the pBam promoter for act5C of UASz had no or very little effect on the mutation rate. Only act5C-evoCDA1-nCas9 was successful at producing a high number of mutations in embryos. (C-D) Mismatches in GFP classified by base substitution in adults were act5C-evoCDA1-nCas9 introduced mutations with the 4 gRNAs from the male (C) or female (D) germline (left), and distribution of observed number of mutations per read after subtracting the number of mismatches observed in control samples (right). Red dots mark all “mutable bases” (Cs on FWD gRNAs and Gs on REV gRNAs).