Investigating the potential of X chromosome shredding for mouse genetic biocontrol

Abstract CRISPR-Cas9 technology has facilitated development of strategies that can potentially provide more humane and effective methods to control invasive vertebrate species, such as mice. One promising strategy is X chromosome shredding which aims to bias offspring towards males, resulting in a gradual and unsustainable decline of females. This method has been explored in insects with encouraging results. Here, we investigated this strategy in Mus musculus by targeting repeat DNA sequences on the X chromosome with the aim of inducing sufficient DNA damage to specifically eliminate X chromosome-bearing sperm during gametogenesis. We tested three different guide RNAs (gRNAs) targeting different repeats on the X chromosome, together with three male germline-specific promoters for inducing Cas9 expression at different stages of spermatogenesis. A modest bias towards mature Y-bearing sperm was detected in some transgenic males, although this did not translate into significant male-biasing of offspring. Instead, cleavage of the X chromosome during meiosis typically resulted in a spermatogenic block, manifest as small testes volume, empty tubules, low sperm concentration, and sub/infertility. Our study highlights the importance of controlling the timing of CRISPR-Cas9 activity during mammalian spermatogenesis and the sensitivity of spermatocytes to X chromosome disruption.