Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion-"Xshredding" and "X-poisoning"-and dissected their target-site requirements and repair dynamics. X-shredding resulted in sex distortion when Cas9 endonuclease activity occurred during the meiotic stages of spermatogenesis but not when Cas9 was expressed from the stem cell stages onwards. Our results suggest that X-shredding is counteracted by the NHEJ DNA repair pathway and can operate on a single repeat cluster of non-essential sequences, although the targeting of a number of such repeats had no effect on the sex ratio. X-poisoning by contrast, i.e. targeting putative haplolethal genes on the X chromosome, induced a high bias towards males (>92%) when we directed Cas9 cleavage to the X-linked ribosomal target gene RpS6. In the case of X-poisoning sex distortion was coupled to a loss in reproductive output, although a dominant-negative effect appeared to drive the mechanism of female lethality. These model systems will guide the study and the application of sex distorters to medically or agriculturally important insect target species.

Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion -"X-shredding" and "X-meddling" -and dissected their target-site requirements and repair dynamics. X-shredding relies on sufficient meiotic activity of the endonuclease to overpower DNA repair and can operate on a single repeat cluster of non-essential sequences. X-meddling by contrast, i.e. targeting putative haplolethal X-linked genes, induced a bias towards males that is coupled to a loss in reproductive output, although a dominant-negative effect may drive the mechanism of female lethality. Our model system will guide the study and the application of sex distorters to medically or agriculturally important insect target species. 1978, Newton M.E. 1976). These findings inspired the generation of artificial distorter traits (Windbichler, Papathanos, and Crisanti 2008) first by using His-Cys box homing endonucleases and subsequently RNAguided endonucleases. In the malaria vector Anopheles gambiae (A. gambiae) autosomal I-PpoI (Galizi et al. 2014) or CRISPR/Cas9-bearing transgenes (Galizi et al. 2016) were used to target sequences on the Xchromosome during male meiosis. Cage experiments using such distorter traits induced extreme male-biased sex-ratios and population collapse confirming the potential of this system for genetic control. Mechanistically, the nature of the target locus for which successful X-shredding was demonstrated suggested a possible coalescence of different effects in the mosquito system. The target sites are situated within the Anopheles gambiae 28S rDNA cluster which simultaneously represents (i) a high-copy number repeat on the X-chromosome, (ii) an essential gene for ribosome biogenesis and function, (iii) the nucleolar organizing region of the cell as well as (iv) a sequence adjacent to the centromere of the X-chromosome and (v) the predicted pseudo-autosomal region of the X-chromosome mediating pairing with the Y chromosome during meiosis (Hall et al. 2015). The possible conflation of effects induced by X-shredding in the mosquito has been a source of uncertainty regarding the potential to transfer this paradigm to other important pest species.In particular, the X-shredding approach has not been clearly delineated from a related, recently-proposed strategy, based on the targeting of X-linked haploinsufficient genes (commonly ribosomal genes) with the intent to induce female lethality (Burt and Deredec 2018). To delineate this mechanism, which is assumed not to alter gamete production and to come into effect only in the developing progeny, we refer to it as Xmeddling. X-meddling would also generate a male biased progeny but would be expected to lead to a sig...

No abstract

No abstract