Fig. 3: Functionality of SGyA as a genetic sex-sorter termed SELECT using a single gRNA targeting PolG2.
From: Exploiting a Y chromosome-linked Cas9 for sex selection and gene drive
In a, c, e, g Heterozygous U6.3-gRNA#1PolG2 females were crossed to a male that was either WT, SGyA, or autosomal Cas9. Below the cross is the outcome of the F1 progeny. Each F1 fly with a corresponding genotype is associated with either X’, X”, X”’, or X”” labels. The gRNA transgene is marked with a non-fluorescent orange-red eye maker. a No lethal phenotypes were seen in negative control crosses. c In autosomal Cas9 and gRNA crosses, both F1 transheterozygous males and females were not recovered due to the lethal effects of PolG2 cleavage. e In X-linked Cas9 crosses, both F1 transheterozygous males and females were not recovered. Only individuals without gRNA survived. g In SGyA crosses, only F1 transheterozygous males inheriting both SGyA and the U6.3-gRNA#1PolG2 transgene resulted in lethality (as indicated with red “X”). Females survived. b, d, f, h Homozygous U6.3-gRNA#1PolG2 females were outcrossed to a male that was either WT, SGyA, or autosomal Cas9. b No lethal phenotypes were seen in negative control crosses. d No surviving offspring were recovered in autosomal Cas9 crosses. f All F1 U6.3-gRNA#1PolG2 males survived and F1 females perished in X-linked-Cas9 crosses. h All F1 transheterozygous males resulted in lethality while F1 females survived in SGyA outcrosses. i Mechanism and schematic depicting lethal mosaicism in progeny from paternal vs maternal inheritance of Cas9. Lethality is only observed in males which inherit Y-linked Cas9. However, all progeny perish when inheriting maternal Cas9 due to maternally deposited Cas9. Red shaded boxes in Punnett square represent lethality. Mosaic skulls represent lethal mosaicism. Instances, where this symbol is seen, represents no progeny was recovered. Blue and pink bars represent the percentage of males and females, respectively. Black bars represent the standard error of the mean(SEM). Ten replicate crosses were set up per experiment. A two-tailed unpaired student’s t test is used to determine the significance of percentages compared with WT. (refer to Table 2 for ANOVA comparisons) ****p < 0.0001; **p < 0.005; *p < 0.05. N.s. no significance. Source data is provided as a Source Data file.
