Sequencing and NHEJ alleles analysis of DsTdc2^CRISPR SWD.

(A) PCR products sequencing chromatograms of wild type (WT) SWD, microinjected G0, G1 heterozygotes DsTdc2CRISPR, and G2 resistant mutant at the gRNA1 target site. (B) NHEJ analysis of PCR products from G1 heterozygotes DsTdc2CRISPR, and G2 mutants at the gRNA1 and gRNA3 target sites. GRNA Target sites are indicated in green, PAM in yellow, and dashed lines represent the deleted bases. Letter N represent different types of NHEJ. The numbers on the right represent the count of base deletions. (TIF)</p

Maximum likelihood estimates of female drive heterozygote fitness.

Maximum likelihood estimates of female drive heterozygote fitness.</p

Gene structure of the SWD <i>vasa</i>.

(A) Gene structure of the SWD vasa (A). (B) Dsvasa promoter used in this study. (TIF)</p

Sequencing and NHEJ alleles analysis of mutants mediated by <i>vasa</i>-Cas9(X).

(A) Heterozygous G1 master female mediated by vasa-Cas9(X) were sterile due to egg retention. (B) PCR product sequencing chromatograms of WT, heterozygous master G1 and G2 resistant mutant at the gRNA3 target site. (C) NHEJ analysis of PCR products from G2 resistant mutant of G1 master female mediated by vasa-Cas9(X) at three gRNA target sites. (D) Statistics of resistance alleles in G2 progeny (from two different males, left and right pie charts) mediated by vasa-Cas9(X). Specifically, 65 and 59 of their G2 progeny were sampled and analyzed. (TIF)</p

Establishment of a homing gene drive system targeting female essential gene <i>Tyrosine decarboxylase 2</i> (<i>DsTdc2</i>) in SWD.

(A) Gene structure of DsTdc2 and three gRNA sequences. (B) Schematic representation of the HDR knock-in construct of gene drive donor plasmid specifically recognizing DsTdc2 gene and the corresponding target locus. Homology arms used to act HDR are on both sides of the U6-gRNA, vasa-Cas9 and PUb-DsRed. U6-gRNA and vasa-Cas9 are components of the fGD system and allow for a double strand break at target site. PUb-DsRed is a marker constructed to score the transgene flies. (C) Natural and red fluorescence images of male and female DsTdc2CRISPR. As the marker indicated, red fluorescence was observed all over the mutants under excitation light of 510 nm-560 nm. (D) Molecular confirmation of the correct integration of the HDR-mediated event to generate gene drive SWD. Primer pairs used in diagnostic PCR are shown in (B). The chromosome carrying the gene drive was named as GD.</p

Sequencing and NHEJ alleles analysis of mutants mediated by <i>nos</i>-Cas9(II) and <i>nos</i>-Cas9(III).

(A) PCR products sequencing chromatograms of wild type w1118, heterozygous master G1 mediated by nos-Cas9(III) and G2 resistant mutant at the gRNA3 target site. (B) NHEJ analysis of PCR products from G2 resistant mutant mediated by nos-Cas9(III) at three gRNA target sites. Base insertions are represented by green uppercase letters, while base substitutions are indicated in red. The numbers on the right represent the count of base deletions, insertions or substitutions. (C) Statistics of resistance alleles in G2 progeny mediated by nos-Cas9(II) and nos-Cas9(III). As for a G1 male mediated by nos-Cas9(II), 75 of their G2 offspring were randomly sampled and analyzed. And for two G1 females mediated by nos-Cas9(III), 38 and 37 of their G2 offspring were tested. (TIF)</p

An oviposition defect of the DsTdc2^CRISPR can be compensated by octopamine and dynamics of the spread of the gene drive allele in cage trials.

(A) Morphological analysis of WT female SWD, homozygous female DsTdc2CRISPR and their ovaries. Abdominal enlargement was observed in homozygous female DsTdc2CRISPR due to egg retention caused by disruption of the Tdc2 gene. After the ovaries were dissected, it was found that the eggs of the homozygous female DsTdc2CRISPR were trapped in the ovaries. (B) Reproductive phenotype of WT, heterozygous and homozygous DsTdc2CRISPR with or without the addition of octopamine. Oviposition defect of homozygotes compared to WT was analyzed using unpaired two-tailed t-test, n1 = 6, n2 = 3, Pt-test, n1 = 3, n2 = 5, Pth generation of Cage 1 and 2, and the 7th generation of Cage 3. Ten flies without DsRed were randomly chosen for genome typing. The rectangular squares represent the proportion of fluorescence flies in each generation. And the pie charts illustrate the editing situation at the gRNA cleavage sites in 10 randomly-chosen DsRed-negative flies separately.</p

Genetic polymorphism analysis.

(A) Genetic polymorphism analysis of the homology arm and gRNA targeted sequences in the DsTdc2 gene in the 26 heterozygous DsTdc2CRISPR males. The same nucleotide sequences are noted in gray. Different nucleotide sequences are noted in green and yellow. Three gRNAs are shown in the Fig, and the left and right homology arms are indicated. Among the 26 flies tested, there were 16 of the first type, 1 of the second type, 4 of the third type, 3 of the fourth type, and 2 of the fifth type. (B) The inheritance rates of five types of flies. (TIF)</p

SGD systems targeting female essential gene <i>Tdc2</i> were constructed and validated in <i>D</i>. <i>melanogaster</i>.

(A) and (B) Schematic representation of the HDR knock-in construct of gene drive donor plasmid specifically recognizing DmTdc2 gene and the corresponding target locus. The plasmid contains homology arms of both ends of the cut site in order to act homology-directed repair, multi-gRNAs and Cas9 cassettes (with the ubiquitous promoter of hsp70 or Actin5C) to form fGD systems, and the marker construct (IE1-DsRed for red fluorescent body or 3×P3-DsRed for red fluorescent eyes) aiming to score the transgenic flies. (C) and (D) Outline of the genetic cross schemes used to demonstrate the driving efficiency of the sGD systems. The gene drive allele and Cas9 endonuclease gene are located in either different or same chromosomes. The conversion event at the sGD locus is shown with a yellow triangle in G1 individuals. (E) and (F) For DmTdc2hsp70 and DmTdc2Actin5C, G1 flies were separately single-pair mated to w1118 and assessed for inheritance of the gene drive construct. The introduced Cas9 protein was marked as +. Data analyzed using one-way ANOVA (P<0.0001) followed by Tukey’s multiple comparison tests (letters). Note that points marked as 0 drive inheritance represent drive flies that had no offspring.</p

Sequencing and NHEJ alleles analysis of mutants mediated by Act5C-Cas9(II).

(A) PCR products sequencing chromatograms of WT, heterozygous DmTdc2Actin5C, and G2 resistant mutant at the gRNA3 target site. (B) Statistics of resistance alleles in G2 progeny mediated by endogenous Act5C-Cas9(II). As for two DmTdc2Actin5C G1 males, 77 and 61 of their G2 offspring were randomly sampled and analyzed. And for two DmTdc2Actin5C G1 females, 34 of their G2 offspring were tested. (TIF)</p