Figure 2. Programmed chromosomal break at ω1 locus repaired by homologous recombination from donor template.

(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.

Figure 2—figure supplement 1. The nucleotide sequence of the Smp_193860 copy, and indicates the UTR (green), coding exons (blue) and introns (red).

Figure 2—figure supplement 2. Variation among copies of ω1 in the reference genome of S.mansoni.

(A) An alignment assembled using CLUSTALW (Larkin et al., 2007) of the MiSeq amplicon region in the ω1 genes Smp_179960, Smp_184360, Smp_193860 and Smp_168300 using the sequences from the S. mansoni genome, assembly V5. The MiSeq primers (highlighted in blue) and sgRNA (red) were designed based on Smp_184360. Residues highlighted in green show where Smp_168300 differs from the other three genes. (B) An alignment made using CLUSTALW (Larkin et al., 2007) of the coding region of the amplicon sequence found in the gene Smp_193860 in the S. mansoni V7 assembly, the predicted alternative sequence for the variant having ‘TT’ at position 60–61 and ‘TA’ at 152–153 in the amplicon (‘var’), and the translations of two mDNAs retrieved from NCBI (top BLASTP hits in the NCBI protein database). Note that this corresponds to a part only of the ω1 T2 ribonuclease. The CAS-2 catalytic region is highlighted in red (Fitzsimmons et al., 2005). The mRNA XP_018647488.1 has an alternative splicing event at the start of the exon included in the amplicon (shaded grey), so that it only matches the region from ‘KHEFEK…’ (in the CAS-2 catalytic region) onwards. (C) An alignment of the coding region of the amplicon sequence in schistosome ω1 homologues (family 873078), excluding diverged members from S. japonicum as well as the diverged S. haematobium gene B_00112. The region with sequence similarity to the alternative splice-form is in shaded grey. Note that the Smp_193860 sequence used in this alignment is the version in the S. mansoni draft genome V5 gene set, which has the Q->K substitution at 152–153.

Figure 2—figure supplement 3. Programmed HDR-catalyzed knock-in of transgene into ω1, exon 6.

The PCR products visualized in ethidium bromide-stained agarose gel demonstrated Cas9-catalyzed target site-specific insertional mutagenesis in exon 6 of the ω1 gene. The 24 nt transgene knocked-in into target site indicated in lanes R1, R2 and R3 of 321, 285 and 184 bp, respectively (arrows at left) spanning the mutated site in the genomic DNAs pooled from schistosome eggs, including a positive control flanking the insert site (991 bp). Lanes 1–5 show the amplicons from the control groups; medium only, sgRNA only, cas9 only, ssODN only and heat-inactivated pLV-ω1X6 + ssODN. Lanes 6–9 show the amplicons from experiment groups; RNP, RNP and ssODN (KI), pLV-ω1X6 virions, and pLV-ω1X6 virions and ssODN (KI). Programmed knock-in was detected only in the KI experimental groups, as shown in lanes 7 and 9 where the transgene-specific amplicon obtained with the R1, R2 and R3 primers was present. This product was not amplified by PCRs where DNAs from control and other experimental groups served as the template. Amplicons of 991 bp were obtained with all genomic DNAs, confirming the integrity of the genomic DNAs and the control amplicon primer pair.

Figure 2—figure supplement 4. Estimated relative copy number of ω1.

Copy number estimated by qPCR in genomic DNAs isolated from CRISPR/Cas9-treated eggs or controls. The relative copy number of the LE as calibrator sample = 1. Means ± SD of control (wt LE), LE treated- Cas9 only, LE treated-ssODN only, LE treated-heat-inactivated pLV-ω1X6 virions, LE treated-heat-inactivated pLV-ω1X6 virions and ssODN, LE treated- pLV-ω1X6 virions, LE treated-pLV-ω1X6 virions and ssODN.