(A) Schematic diagram to indicate positions of primer binding sites (blue arrows), with the foreign gene cassette as the forward primer (Smω1X6–6 stop codons cassette-F) paired with three discrete reverse primers, Smω1-R1, Smω1-R2 and Smω1-R3 from the ω1 locus and a primer pair for target amplicon NGS library amplification; miSeq-F and miSeq-R. The control PCR amplicon was generated using the Smω1-control-F and –R primers. The green box shows the location of 5’ and 3’ homology arms, the red box and arrow indicate the stop codon bearing transgene. (B) PCR products visualized in ethidium bromide-stained agarose gel demonstrating Cas9-catalyzed target site-specific insertional mutagenesis in exon 6 of the ω1 gene. Evidence for transgene knocked-in into programmed target site revealed by amplicons of the expected sizes in lanes R1, R2 and R3, of 184, 285 and 321 bp, respectively (arrows at left) spanned the mutated site in the genomic DNAs pooled from schistosome eggs, including a positive control flanking the insert site (991 bp). The control DNA result shown in this gel was isolated from heat-inactivated-pLV-ω1X6 virions and ssODN treated LE. Similar findings were obtained when programmed gene editing was executed by lentiviral virion-delivered Cas9 and ω1-gRNA transgenes and by ribonucleoprotein complex (RNP) delivered by square wave electroporation (supporting information). The non-KI control groups (sgRNA only, heat-inactivated pLV-ω1X6 virions only, ssODN only) showed no amplicons by stop cassette-KI primers with R1, R2 or R3. (C) Multiple sequence alignments confirmed the presence of the 24 nt transgene inserted precisely into exon 6 of ω1 locus from KI-R1, -R2 and -R3 fragments compared with ω1 wild type (WT). The white box on ω1-WT indicates the absence of the transgene sequence and white boxes on KI-R1, -R2 and –R3 fragments show locations of substitutions relative to the other ω1 copies (Smp_184360): 2 bp (AT to CC) mismatches at positions 253–254 nt. All three contained the (knock-in) insertion sequence (white box), which confirmed targeted mutation of the ω1 gene. (D–F) Illumina deep sequence analysis of amplicon libraries revealed Cas9 induced on-target repair of programmed gene mutation of the ω1 locus by deletions, insertions, and substitutions by CRISPResso analysis. D; position dependent deletion size (deletion site, X-axis; deletion size, Y-axis); the deletions varied in length from point mutations to >20 bp adjacent to the DSB. The dotted line indicates the predicted position of the programmed double-stranded break. (E), frequency of frameshift versus in-frame mutations reported by CRISPResso. The pie charts show the fraction of all mutations (indels and substitutions) in the coding region (positions 42–179) of the amplicon predicted to induce frameshifts, that is indels of 1–2 bp, or multiples thereof. Top graph corresponds to sample 2 (eggs only control) (Supplementary file 3), bottom graph corresponds to sample 9 (eggs exposed to virions and ssODN, that is CRISPR/Cas9-treated) (Supplementary file 3). Findings for control and treated samples are provided in Supplementary File 3. (F), Frequency distribution of insertions of the knock-in transgene. Number of amplicon reads containing an insertion of the knock-in sequence (with ≥75% identity to it) is shown in the Y-axis, and the position of the insertion relative to the reference amplicon is shown on the X-axis. The programmed Cas9 scission lies between positions 102 and 103. Samples 3, 7 and 9 are independent amplicon libraries (technical replicates) made from the same sample of genomic DNA pooled from six biological replicates exposed to virions and ssODN. The insert shows a sequence logo, created using WebLogo (Crooks et al., 2004), of the sequences of the 3826 sequence reads from samples 3, 7 and 9, with insertions of 24 bp at position 102; most matched the donor template, TAAGTGACTAGGTAACTGAGTAGC.