Major methodologies for mutation detection. (a) Sequence decoding from Sanger sequencing. An example of a Sanger sequencing read was shown to illustrate the significant decrease of read quality from the predicted CRISPR cut site (PAM position labeled by magenta). This is due to the inclusion of the mutated DNA (decoded as the bottom sequence) with the wild-type DNA sequence (decoded as the top sequence). Underlined sequence reveals identical nucleotides between the wild-type and mutant sequences, which indicates the major mutation is a 3-nucleotide (TAG) deletion. (b) Recognizing mismatched dsDNA using the single-stranded specific nucleases. Mixed sequences with local sequence polymorphisms (CRISPR-induced indel mutations) form a mismatch when rehybridizing. The result from the mismatch-recognizing nuclease assay is visualized using fragment analysis as a digital nucleic acid size profile. (c) High Resolution Melting Analysis. (d) PAGE electrophoresis of a DNA hybrid.