Abstract
Genetic control aims to reduce the ability of insect pest populations to cause harm via the release of modified insects. One strategy is to bias the reproductive sex ratio towards males so that a population decreases in size or is eliminated altogether due to a lack of females. We have shown previously that sex ratio distortion can be generated synthetically in the main human malaria vector Anopheles gambiae, by selectively destroying the X-chromosome during spermatogenesis, through the activity of a naturally-occurring endonuclease that targets a repetitive rDNA sequence highly-conserved in a wide range of organisms. Here we describe a CRISPR-Cas9 sex distortion system that targets ribosomal sequences restricted to the member species of the Anopheles gambiae complex. Expression of Cas9 during spermatogenesis resulted in RNA-guided shredding of the X-chromosome during male meiosis and produced extreme male bias among progeny in the absence of any significant reduction in fertility. The flexibility of CRISPR-Cas9 combined with the availability of genomic data for a range of insects renders this strategy broadly applicable for the species-specific control of any pest or vector species with an XY sex-determination system by targeting sequences exclusive to the female sex chromosome.
Highlights
PiggyBac transformation vector to allow transposon-mediated insertion into the genome
Male germline expression of CRISPR-Cas[9] does not appear to suffer markedly from problems related to undesired endonuclease stability and carry-over into the fertilized embryo that had complicated previous attempts to establish sex distortion based on the I-PpoI endonuclease[2]. This is confirmed by another set of experiments in which we find that maternally deposited Cas[9] in combination with zygotic expression of the gRNA from the CRISPRSD locus is able to induce embryo lethality but we find no evidence for an effect of paternal carryover of either the gRNA or Cas[9] protein on embryo viability (Supplementary Table S2, Supplementary Fig. S4)
For five consecutive generations transgenic males showed consistent levels of male-biased sex ratios in their offspring (Supplementary Table S2). In those rare females that had inherited an X-chromosome from a CRISPRSD father most of the repeats remained susceptible to cleavage, indicating that the vast majority of the ribosomal DNA (rDNA) copies retain the intact target sequence (Supplementary Fig. S5)
Summary
PiggyBac transformation vector to allow transposon-mediated insertion into the genome. Residual somatic expression of the CRISPRSD transgene may explain reduced fertility in strain N, probably due to position effects associated with the insertion site (Supplementary Fig. S3). All CRISPRSD strains showed no difference in the average number of eggs laid per mated female when compared to the wild-type control (Fig. 2, Supplementary Table S2).
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