Fig 2: Detection of the hybrid chromosome containing the 1q/19p arms. (A) Schematic of the PCR assay that specifically detects the hybrid chromosome containing the 1q/19p arms (the 1q/19p product). To ensure reaction specificity, primer pairs recognizing 1q and 19p (arrows) and an internal Taqman probe recognizing 1q sequences have been used. INDELs, insertions and deletions of several nucleotides at the DSB repair site. Note that the internal probe is designed to recognize sequences outside the anticipated Cas9-cutting site and INDEL region. (B) Droplet digital PCR (ddPCR) analysis of the 1q/19p product in HEK 293T cells collected on different days after CRISPR/Cas9 introduction. CCR5, internal control to normalize the copy number of input genomic DNA. The percentage shows the fraction of 1q/19p positive droplets of each sample. (C) Electrophoresis of the 1q/19p product. Arrow, the expected PCR product. DNA ladder, 1KB Plus (NEB #N3200S). (D-E) Automatic sequencing analysis of the 1q/19p product using (D) the 1q primer and (E) the 19p primer. Red arrowheads, Cas9 cutting sites.

Fig 3: Causal structural variation that controls soybean seed coat pigmentation. a Sequence comparison between W05 genome and Wm82 bacterial artificial chromosome (BAC) sequences at the I locus region. CHS genes and subtilisin gene/gene fragments are indicated with blue and orange, respectively. b Top panel: cartoon shows the exon structure of the subtilisin gene fragment (orange), the CHS1 gene (blue), and the Expressed Sequence Tag (EST) sequence Gm-c1069–6017. Positions of primers designed for PCR amplification of subtilisin-anti-CHS1 chimeric transcript are indicated with black arrows. Bottom panel: PCR amplification of the subtilisin-anti-CHS1 chimeric transcript. Experiment was repeated at least twice with independent samples. Marker: 1 Kb Plus DNA ladder (NEB, cat. N3200S). NTC, no template control. GmACT11 is used as a housekeeping control. Unprocessed gel image is provided in Source Data file. c Proposed model for the generation of siRNAs originated from a large structural rearrangement in the I locus. CHS genes and the subtilisin gene/gene fragments are illustrated as blue and orange, respectively. Arrowheads indicated the direction of transcription that causes the formation of double-stranded RNA. Cluster A and B are named according to a previous report23. IR-CHS gene cluster: inverted repeat of CHS gene cluster