Supplementary Figure 5: Using IDAA to estimate probability of germline transmission of indels from F0 mosaic fish and to identify F1 mutant fish in Danio rerio genome editing.

(a) In zebrafish genome editing, a major challenge is determination of the nature and degree of indel mosaicism and hence, probability of germline transmission of indels from F0 fish derived from one-cell embryos injected with Cas9 and gRNA. IDAA enables easy and rapid evaluation of these variables through analysis of F0 fin DNA, allowing optimal set-up of F0 breeding pairs. IDAA also enables easy identification of indels in subsequent generations through fin DNA analysis. (b) Somatic IDAA profiles of the bambi locus in fin DNA of F0 fish targeted at the one-cell embryo stage via injection of Cas9 and gRNA. F0 male #1 and F0 female #4 were chosen for mating due to the presence of predominant indels indicated by open or closed circles, suggesting high probability of germline transmission of these mutations. (c) IDAA profiles of 200 downstream F1 fish, showing that 4 out of 5 fish harbored the predominant indels identified in the F0 breeding pair, of which 3 were in the biallelic state (F1 #1, #3, #4) and one homozygous for an indel (F1 #4). In this example, IDAA was performed on embryos, but IDAA could also have been performed on fin DNA. The size and frequency of selected indels are indicated. IDAA profiles were generated in an ABI 3130 instrument.