Figure 1 |. Principle of IDAA.

IDAA detects indel mutations elicited by nucleases at their genomic target site (indicated by yellow lightning). In stage 1 of IDAA, the nuclease target site is amplified by genomic PCR using three primers: a locus-specific pair of forward (Fwd) and reverse (Rev) primers and a universal primer (FamFwd) with same sequence as an extension of the Fwd primer and labelled with 6-FAM (green asterisk), which renders the amplicons fluorescent. Amplicons derived from alleles with a deletion or an insertion will be shorter or longer, respectively, than amplicons derived from the wild-type allele, which typically are designed to be 200–450 bp in size. In stage 2, the amplicons are analyzed in a fragment analyzer, such as a commonplace Sanger sequenator. This reveals the size of the amplicons, and thereby the indels contained in them in bp (x-axis) as well as their frequency in relative fluorescence units (RFU) (y-axis). An IDAA profile of indels at the GALNT10 locus in a HEK293 cell pool FACS-edited using CRISPR/Cas9 as outlined in Fig. 3 is shown. The size and frequency of selected indels contained in the amplicons are indicated. IDAA was performed in an ABI 3130 instrument.