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 -"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...

Double drives and private alleles for localised population genetic control

“…More powerful constructs can be made by adding an X-shredding sex ratio distorter to the load-inducing construct; these have been most effectively demonstrated in An. gambiae mosquitoes [11,43], but may also work more broadly [44]. In other species there are other ways to distort the sex ratio [45][46][47], and it would be interesting to model whether these alternatives would be expected to have the same impact as an X-shredder in the context of a double drive.…”

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

“…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).…”

“…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.…”

Adequacy and sufficiency evaluation of existing EFSA guidelines for the molecular characterisation, environmental risk assessment and post‐market environmental monitoring of genetically modified insects containing engineered gene drives

“…Nonetheless, future efforts to exploit randomly generated Y-chromosome docking strains (Bernardini et al, 2014) or even generate new site-specific docking strains on the Y-chromosome using CRISPR-Cas9 (Buchman and Akbari, 2019) may improve functionality of Y-chromosome nucleases and thus the development of engineered X-shredders (Raban et al, 2020). Moreover, X-shredder systems may be adaptable to other organisms (Bernardini et al, 2019;Fasulo et al, 2020), though the development of such technology has been hampered by the inability to express transgenes, under the control of meiotic promoters, from the sexual chromosomes (Bernardini et al, 2019).…”