Figure 6. Assessment of non-autonomous gene drive of the modified host genes.

(A) Homozygous individuals of strains Sco^GFP-CP, ScoG^CFP-AP2, and Aper1-Sco^GFP or the markerless strain ScoG-AP2 were crossed to the vasa-Cas9 strain to assess the homing potential induced by the intronic guide RNAs. As a control, hemizygous individuals lacking Cas9 were crossed to WT. In each case homing was measured by the rate of fluorescent larvae recorded in the progeny with the exception of the markerless strain ScoG-AP2 where it was assessed via PCR genotyping of the progeny. Mean and standard deviation from three biological replicates are plotted and the number n is indicated at the base of the column. All comparisons to the control crosses were significant (p<0.0001, chi-squared test). The data is found in Supplementary file 1 - Supplementary Tables 4 and 5. (B) Amplicons from PCRs over the predicted cut site within the three loci performed on pooled progeny from transhemizygotes were subjected to next generation sequencing. G3 and Cas9 served as controls. Overall number of reads and the percentage of reads with modifications in the quantification window are shown. (C) Predicted classes of modifications represented within the set of modified alleles for each locus.

Figure 6—figure supplement 1. Crossing schemes for assessing the homing rate.

The non-homing vasa-Cas9-line carrying a 3xP3-YFP marker was crossed to the transgenics. If homing in the germline occurs, all progeny from the cross to WT will be heterozygotes. In the case of Sco^GFP-CP (A) and Aper1-Sco^GFP (B), only the larvae without the Cas9 transgene can be considered for the assessment due to an overlap of fluorescent markers in the GFP channel, whereas for ScoG^CFP-AP2 (C) there was no overlap. ScoG-AP2 (D) was screened via PCR.

Figure 6—figure supplement 2. Analysis of rearrangements in ScoG^CFP-AP2 following Cas9 cleavage.

(A) PCR analysis of non-fluorescent offspring from the cross of ScoG^CFP-AP2/Cas9 to WT with primers spanning the locus showed that the majority of individuals carries an insertion but of reduced size. (B) Sequencing result of the upper band identified in the PCR suggests the loss of the intron cassette as well as a recombination and sequence exchange between the GFP and CFP coding sequences.

Figure 6—figure supplement 3. Sanger sequencing of non-fluorescent individuals.

(A) Schematic explaining the difference between the sets of chromosomes subjected to Sanger and amplicon sequencing. (B) Non-fluorescent individuals identified in the homing assay (shown in grey in the first pie chart) were subjected to PCR over the gRNA target side, the products were Sanger sequenced, and the results were categorized (shown in the final pie chart). The corresponding alignments are shown to the right, with the number of occurrences of each particular sequence indicated in the coloured square before the sequence. Only few non-fluorescent individuals were found for Sco^GFP-CP and Aper1-Sco^GFP and they were not genotyped further for the construct. ScoG^CFP-AP2 had to be genotyped by multiplex PCR first, which either gave bands for the WT and the construct or for the WT only. Subsequently only individuals that solely gave the WT band were sequenced. SNPs are indicated with a grey background, deletions are represented by a dash, and the red dashed line indicates the cleavage site.