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. 2024 Jan 30;12:RP90199. doi: 10.7554/eLife.90199

Figure 2. The precision-guided sterile insect technique (pgSIT) cross results in nearly complete female lethality and male sterility.

(A) Schematic maps of targeted genes (box) and gRNAdsx,ix,βTub construct. Red arrows show relative locations of gRNA target sequences (box). gRNAdsx,ix,βTub harbors a 3xP3-tdTomato marker and six gRNAs to guide the simultaneous CRISPR/Cas9-mediated disruption of dsx, ix, and βTub genes. Violet exon Box 5a and 5b represent ♀-specific exons. (B) A schematic of the reciprocal genetic cross between the homozygous Cas9, marked with Opie2-CFP, and homozygous gRNAdsx,ix,βTub to generate the trans-hemizygous F1 (aka. pgSIT) progeny. Relative positions of dsx, ix, and βTub target genes (bar color corresponds to gRNA color), and transgene insertions in the Cas9 (Nup50-Cas9 strain1) and gRNAdsx,ix,βTub#1 strains are indicated in the three pairs of Ae. aegypti chromosomes. To assess the fecundity of generated pgSIT mosquitoes, both trans-hemizygous ♀’s and ♂’s were crossed to the wild-type (WT) ♂’s and ♀’s, respectively. (C) Comparison of the survival, sex ratio, and fertility of trans-hemizygous, hemizygous Cas9 or gRNAdsx,ix,βTub#1, and WT mosquitoes. The bar plot shows means ± standard deviation (SD) (n = 3, all data in Supplementary file 1d). (D) Blood feeding assays using both types of trans-hemizygous intersexes (⚥’s): pgSIT♀Cas9 and pgSIT♂Cas9 ⚥’s. To assess blood feeding efficiency, individual mated ♀’s or ⚥’s were allowed to blood feed on an anesthetized mouse inside a smaller (24.5 × 24.5 × 24.5 cm) or larger cage (60 × 60 × 60 cm), and we recorded the time: (1) to initiate blood feeding (i.e., time to bite); and (2) of blood feeding (i.e., feeding time). The plot shows duration means ± SD over 30 ♀’s or ⚥’s (n = 30) for each genetic background (Supplementary file 1e). (E) Flight activity of individual mosquitoes was assessed for 24 hr using vertical Drosophila activity monitoring (DAM, Supplementary file 1f). The plot shows means ± SD (n=24). (F) Mating assays for fertility of offspring produced via crosses between trans-hemizygous ♂’s that inherited a maternal Cas9 (pgSIT♀Cas9) or paternal Cas9 (pgSIT♂Cas9) and WT ♀’s (Supplementary file 1h). The plot shows fertility means ± SD over three biologically independent groups of 50 WT ♀’s (n = 3) for each experimental condition. Statistical significance of mean differences was estimated using a two-sided Student’s t-test with equal variance (ns: p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001). Source data are provided in Supplementary file 1.

Figure 2.

Figure 2—figure supplement 1. Genetic characterization of independent transgenic strains for CRISPR/Cas9-mediated disruption of dsx, ix, and βTub.

Figure 2—figure supplement 1.

(A) Schematic maps of targeted genes (box) and gRNAdsx,ix,βTub construct. Relative locations of gRNA target sequences are depicted with red arrows (box). gRNAdsx,ix,βTub harbors a 3xP3-tdTomato marker and six gRNAs to guide the simultaneous CRISPR/Cas9-mediated disruption of dsx, ix, and βTub genes. (B) A schematic of the reciprocal genetic cross between the homozygous Cas9, marked with Opie2-CFP, and homozygous gRNAdsx,ix,βTub#1 to generate the trans-hemizygous F1 (aka. precision-guided sterile insect technique [pgSIT]) progeny. Relative positions of dsx, ix, and βTub genes (bar’s color corresponds to gRNA’s color), and transgene insertions in the Cas9 (Nup50-Cas9 strain) and gRNAdsx,ix,βTub#1 strains are indicated in the three pairs of Ae. aegypti chromosomes. (C) Three independent gRNAdsx,ix,βTub strains were generated and assessed by crossing to the Cas9 strain and comparing the pgSIT phenotypes induced in each trans-hemizygous progenies. The survival, sex ratio, and fertility of trans-hemizygous and hemizygous gRNAdsx,ix,βTub mosquitoes were scored for each of three gRNAdsx,ix,βTub strains and compared to the corresponding values found for gRNAdsx,ix,βTub#1 strain. The bar plot shows means ± standard deviation (SD) over triple biological replicates (n = 3, all data can be found in Supplementary file 1d). Statistical significance of mean differences was estimated using a two-sided Student’s t-test with equal variance (ns: p ≥ 0.05). Source data are provided in Supplementary file 1.
Figure 2—figure supplement 2. Determination of transgene copy number for the gRNAdsx,ix,βTub#1 strain using Oxford Nanopore genome sequencing.

Figure 2—figure supplement 2.

tandard box plot depicting the coverage distributions of the three chromosomes and the Cas9 and gRNAdsx,ix,βTub transgenes in trans-hemizygous gRNAdsx,ix,βTub#1/Cas9 (aka. precision-guided sterile insect technique [pgSIT]) mosquitoes. The center line is median, first and third quartiles are the bounds of the box, upper and lower whiskers extend from the box to the largest and lowest observed value, but no further than 1.5* interquartile range (IQR) from the box. Sequencing depths for chromosomes 1, 2, 3 and for Cas9 and gRNAdsx,ix,βTub transgenes were 9.71, 9.47, 9.17, 12.23, 9.50, respectively. Normalized sequencing depths were 1.03, 1.00, 0.97, 1.30, 1.01 consistent with the transgenes being present at single copy. The Nanopore sequencing data have been deposited to the NCBI sequence read archive (SRA) under BioProject ID PRJNA942966 and BioSample ID SAMN33705934.
Figure 2—figure supplement 3. Oxford Nanopore sequencing results validating disruptions in the target sites.

Figure 2—figure supplement 3.

Integrated genome browser snapshots are zoomed in at genome regions containing gRNA target sites at dsx (A), ix (B), and βTub (C) genes in precision-guided sterile insect technique (pgSIT) ♂’s and ♀’s. The direction of DNA strand and scal is indicated above generated reads. Sampled deletion mutations localize to gRNA target sites (yellow overlay) (A).
Figure 2—figure supplement 4. Integrative genome browser snapshot of dsx validating gRNA target disruption in both the DNA and RNA.

Figure 2—figure supplement 4.

Oxford Nanopore reads are aligned on the top and RNAseq reads for the various genotypes indicated are aligned. On the bottom in blue are the gRNA target sites.
Figure 2—figure supplement 5. Integrative genome browser snapshot of ix validating gRNA target disruption in both the DNA and RNA.

Figure 2—figure supplement 5.

Oxford Nanopore reads are aligned on the top and RNAseq reads for the various genotypes indicated are aligned. On the bottom in blue is the gRNA target site.
Figure 2—figure supplement 6. Integrative genome browser snapshot of βTub validating gRNA target disruption in both the DNA and RNA.

Figure 2—figure supplement 6.

Oxford Nanopore reads are aligned on the top and RNAseq reads for the various genotypes indicated are aligned. On the bottom in blue are the gRNA target sites.
Figure 2—figure supplement 7. Transcription profiling and expression analysis of Ae. aegypti liverpool and precision-guided sterile insect technique (pgSIT) mosquito samples.

Figure 2—figure supplement 7.

(A) Principal Component Analysis (PCA) analysis and (B) hierarchical clustering of twelve samples used for RNA sequencing. MA plots showing the differential expression patterns between: (C) pgSIT ♂’s vs wild-type (WT) ♂’s, and (D) pgSIT ⚥’s vs WT ♀’s. Blue bots indicate significantly differentially expressed genes (False Discovery Rate (FDR) <0.05), while non-significantly differentially expressed genes are indicated by gray dots (FDR >0.05). Targeted and transgenic genes are depicted by the larger blue or gray dots.
Figure 2—figure supplement 8. Longevity, fecundity, and developmental times of the generated precision-guided sterile insect technique (pgSIT) mosquitoes.

Figure 2—figure supplement 8.

(A) Survival plots of 20 adult ♀’s or ⚥’s either cohabitated with ♂’s or not, and (B) 20 adult ♂’s either cohabitated with ♀’s or not over three independent experiments (n = 3). Survival means ± standard errors (SE) over days following adult eclosion are plotted. Vertical lines and values present median survivals for each tested group. Mosquito survival curves for each tested group were compared to the curve for wild-type (WT) mosquitoes of the corresponding sex. The departure significance was assessed with the Log-rank (Mantel–Cox) test and is indicated above median values. (C) Female fecundity plots, measured as egg laying and hatching rates, of 60 adult ♀’s or ⚥’s of each tested group (n = 60). (D) Plots of larva-to-pupa and pupa-to-adult developmental times were measured in 60 ♂’s and ♀’s or ⚥’s for each tested group (n = 60). Both point plots in panels (C) and (D) show mean ± standard deviation (SD) (Supplementary file 1g). Statistical significance of mean differences was estimated using a two-sided Student’s t-test with equal variance (ns: p ≥ 0.05, **p < 0.01, and ***p < 0.001). Source data are provided in Supplementary file 1.