A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters - Fig 4
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(A) gRNA target sites within presumptive haploinsufficient genes on the X-chromosome. Schematic representation of RpS5a and RpS6 gene organization. Both genes encode for a small ribosomal subunit protein (RpS). As illustrated in the figure, the RpS5a gRNAs, RpS5a_1 and _2, map in the fourth exon shared by all four transcripts of the gene and the RpS6 gRNAs, RpS6_1 and 2, map in the third exon of two transcripts in the corresponding gene. The figure shows the 234 bp fragment (with Illumina Sequence adapters on both sides as grey boxes) surrounding the RpS6_2 gRNA target site that was used for amplicon sequencing. Blue boxes indicate coding sequences, white boxes indicate UTR regions and PAMs are indicated in red. (B) Efficiency of single gRNAs or combinations of gRNAs for X-poisoning. Shown is the frequency of males in the progeny from βtub85D-cas9 males combined with four gRNA lines and crossed to wild type w females. Individual gRNAs, RpS5a_1 or RpS5a _2 (red), and RpS6_1 or RpS6_2 (orange) are compared to double gRNA arrays co-expressing RpS6_2 + RpS5a_1 (green) or RpS6_2 + RpS6_1 (purple) as well as a combination of both RpS6_2 and Muc14a_6 transgenes (pink). As a control, crosses from βtub85D-cas9/+ (no gRNA, grey) fathers are shown. Each dot represents the percentage of F1 males from a cross between one male and three females. n is the number of individuals (males + females) in the F1 progeny. Black bars show means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. **p < 0.01, ***p< 0.001 and ****p< 0.0001. All crossing data can be found in S7 Table. (C) Developmental survival analysis of the F1 progeny of RpS6_2/βtub85D-cas9, w or βtub85D-cas9/+ males crossed to w females. Left columns: embryos to pupae survival rate; central columns: pupae to adults survival rate and right columns: fraction of males and females in adults. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. **p < 0.01, ***p< 0.001 and ****p< 0.0001. All crossing data can be found in S8 Table. (D) CRISPR induced target site mutations within the RpS6 gene analysed by pooled amplicon sequencing of surviving female progeny. On top, the wild type DNA sequence spanning the RpS6_2 gRNA target site in the RpS6 ribosomal gene is shown with the gRNA binding position (underline), the cut site (red arrowhead), the PAM (bold nucleotides) as well as the encoded amino acids (blue). Target site variants identified in pools of F1 females from RpS6_2/βtub85D-cas9 fathers or RpS6_2 RpS5a_1/βtub85D-cas9 fathers are shown below. Dashed lines correspond to nucleotide deletions, green coloured bases represent insertions. Predicted amino-acid substitutions (>) are indicated to the right.