A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

Abstract

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.

Engineered sex distortion in the global agricultural pestCeratitis capitata

“…In both the An. gambiae and D. melanogaster this promoter drives expression of genes during the primary spermatocyte stage at the onset of meiosis I and endonuclease activity at this stage has been demonstrated to cause shredding of the X-chromosome and to interfere with its transmission to progeny 7,8 . For the expression of the gRNA we identified a putative U6 gene using BLAST of the Drosophila U6snRNA sequence (GenBank accession no.…”

“…X-shredding interferes with the transmission of the X-chromosome by inducing multiple DNA double strand breaks during male meiosis. More recently, the advent of CRISPR/Cas9 enabled the design of RNA-guided X-shredders that have now successfully been demonstrated in Anopheles gambiae 7 and Drosophila melanogaster 8 . As an alternative approach the male-determining genes or M-factors that have recently been identified in several insect species could also possibly be used to engineer effective sex conversion traits 9–13 .…”

“…Finally, post-zygotic genetic sex distorters that act in and are transmitted via the male germline could also be linked to the Y-chromosome and would obtain a level of persistence in the population. Such post-zygotic sex ratio distorters have recently been demonstrated in Drosophila melanogaster via the targeting of X-linked haplolethal genes during male meiosis 8 .…”

Advances and challenges in genetic technologies to produce single-sex litters

“…One method to selectively destroy X-carrying sperm is by X-shredding or X-meddling. Although this technique was shown to be highly efficient in mosquitos [90,93], it was variable in Drosophila [95]. An important consideration of harnessing X-shredding in other nonmosquito species is the availability of X-targets because this is likely to strongly influence the success rate.…”

“…Faluso and colleagues expanded on the Galizi and colleagues [90,93] studies by modelling X-shredding and a new strategy called X-meddling ( Fig 1B) in Drosophila [95]. They produced germline-expressed Cas9 endonuclease lines and bred them with engineered sgRNA-encoding lines, targeting multiple repeat sequences on the X chromosome (for X-shredding) and putative haplo-insufficient genes on the X chromosome (for X-meddling).…”