Journal: Nature Communications

Article Title: Engineered reproductively isolated species drive reversible population replacement

doi: 10.1038/s41467-021-23531-z

Figure Lengend Snippet: a Homozygous dCas9-VPR flies were crossed bidirectionally to homozygous sgRNA flies. Shaded gray background indicates expected lethal crosses. b Homozygous dCas9-VPR individuals were crossed bidirectionally to heterozygous spCas9/sgRNA individuals. Surviving individuals were repeatedly inbred to generate SPECIES. c , d Plots depicting % progeny survival from the crosses depicted in A and B. N = 3 biologically independent replicates for each gRNA combination (eight total combinations). Middle lines indicate mean. Gray dots indicate homozygous sgRNA and heterozygous spCas9/sgRNA flies crossed to wildtype flies. Light and dark blue dots indicate homozygous sgRNA and heterozygous spCas9/sgRNA flies crossed to homozygous dCas9-VPR mothers and fathers, respectively. Purple stars indicate the presence of maternally deposited Cas9. Source data are provided as a Source Data file.

Article Snippet: The resulting plasmid was then digested with PacI, and the following components were cloned into generate the final dCas9-expressing vectors: the Ubiquitin-63E promoter fragment amplified with primers 986.C9 and 986.C10 from D. melanogaster genomic DNA and a dCas9-VPR fragment amplified from Addgene plasmid #78898 with primers 986.C11 and 986.C12 to generate plasmid OA-986B (Addgene #124999); the bottleneck promoter fragment amplified with primers 986.C13 and 986.C14 from D. melanogaster genomic DNA and a dCas9-VPR fragment amplified from Addgene plasmid #78898 with primers 986.C15 and 986.C12 to generate plasmid OA-986C (Addgene #125000); the Ubiquitin-63E promoter fragment amplified with primers 986.C9 and 986.C16 from D. melanogaster genomic DNA and a dCas9-VP64 fragment amplified from Addgene plasmid #78897 with primers 986.C17 and 986.C18 to generate plasmid OA-986D (Addgene #125001); and the bottleneck promoter fragment amplified with primers 986.C13 and 986.C19 from D. melanogaster genomic DNA and a dCas9-VP64 fragment amplified from Addgene plasmid #78897 with primers 986.C20 and 986.C18 to generate plasmid OA-986E (Addgene #125002).

Techniques:

Journal: Nature Communications

Article Title: Engineered reproductively isolated species drive reversible population replacement

doi: 10.1038/s41467-021-23531-z

Figure Lengend Snippet: a Crosses used to determine embryo and adult viability. (Cross #1) Homozygous dCas9-VPR;sgRNA “SPECIES” females crossed to homozygous dCas9-VPR;sgRNA SPECIES males. (Cross #2) Homozygous dCas9-VPR;sgRNA SPECIES females crossed to wildtype (WT) males. (Cross #3) WT females crossed to homozygous dCas9-VPR;sgRNA SPECIES males. Gray shaded background indicates expected lethal crosses. (Cross #4) WT females crossed to WT males. Each cross type was performed for all eight generated SPECIES (Fig. S3). b Schematic detailing the methods of determining embryo and adult survival compared to WT. c % embryo and adult survival was calculated and plotted. The number below each x -axis group indicates cross number (#1–4). N = 3 biologically independent replicates for each cross number. ✝ indicates that embryos did not survive past L1/L2 stages. Unpaired two-tailed t -tests were performed for each SPECIES compared to WT (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). Middle lines indicate mean, while error bars represent standard deviation. The color of the dots represent each species as indicated in figure key. d Embryo (left graph) and adult (right graph) Cohen’s d effect sizes compared to WT. N = 6 observations per cross number per SPECIES (effect size based on mean comparison between three replicates of each experimental cross (#1–3) to control cross (#4)). Error bars represent 95% confidence interval. The color of the dots represent each species as indicated in figure key. Source data are provided as a Source Data file.

Article Snippet: The resulting plasmid was then digested with PacI, and the following components were cloned into generate the final dCas9-expressing vectors: the Ubiquitin-63E promoter fragment amplified with primers 986.C9 and 986.C10 from D. melanogaster genomic DNA and a dCas9-VPR fragment amplified from Addgene plasmid #78898 with primers 986.C11 and 986.C12 to generate plasmid OA-986B (Addgene #124999); the bottleneck promoter fragment amplified with primers 986.C13 and 986.C14 from D. melanogaster genomic DNA and a dCas9-VPR fragment amplified from Addgene plasmid #78898 with primers 986.C15 and 986.C12 to generate plasmid OA-986C (Addgene #125000); the Ubiquitin-63E promoter fragment amplified with primers 986.C9 and 986.C16 from D. melanogaster genomic DNA and a dCas9-VP64 fragment amplified from Addgene plasmid #78897 with primers 986.C17 and 986.C18 to generate plasmid OA-986D (Addgene #125001); and the bottleneck promoter fragment amplified with primers 986.C13 and 986.C19 from D. melanogaster genomic DNA and a dCas9-VP64 fragment amplified from Addgene plasmid #78897 with primers 986.C20 and 986.C18 to generate plasmid OA-986E (Addgene #125002).

Techniques: Generated, Two Tailed Test, Standard Deviation