Single gene disruption targeting genes involved in NC development in X.
tropicalis embryos. a Schematic diagram of sgRNA-guided Cas9 nuclease interacting with the DNA target site. The red letters indicate the target site, the green letters indicate the PAM and the blue letters indicate the sequence of tracer RNA. b Schematic drawing of Cas9 target site (protospacer). The protospacer adjacent motif (PAM) 3′ to the protospacer is highlighted in red. The seed region which consists of 12 nucleotides immediate 5′ to PAM is shown in blue. c T7E1 assay of somatic mutations induced by pax3, snai1, ctnnb, sox9, or Ap2α sgRNAs and Cas9 nuclease. d Somatic mutation rate calculated from T7E1 assay in (c). e DNA sequences targeted by sgRNAs at the indicated gene loci and representative somatic mutations induced in X. tropicalis embryos. PAM is underlined and highlighted in green. Deleted sequences are highlighted in gray with red dashes while insertions are indicated by lowercase letters in blue. The parentheses enclosed the number of deleted or inserted base pairs, while the square brackets show the frequencies of the mutation in the sequenced samples