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bioRxiv · 10.64898/2025.12.22.695236

Increasing the effective gene drive homing rate by targeting the haploinsufficient spermatogenesis gene KLHL10

Peng, C. L.·Kamau, W. S.·Freeman, J.·Hill, Z. J.·Esvelt, K. M.
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Abstract

CRISPR-based gene drives represent a powerful new technology for limiting disease transmission and controlling invasive populations. These systems rely on homology-directed repair (HDR) to drive a genetic element through a population. However, mammals tend to favor non-homologous end joining (NHEJ), which generates mutations that halt further drive propagation. Here, we describe the experimental characterization of a gene drive system targeting the haploinsufficient spermatogenesis gene KLHL10 in the laboratory mouse. Using a newly designed coding sequence cassette we introduce downstream guide RNAs within the gene, ensuring that sperm undergoing NHEJ are selectively removed from the population. As a proof of principle, we demonstrate that targeting KLHL10 with constitutively expressed LbCas12a results in strong selection against frameshift-containing sperm, validating the core purification mechanism required for this drive strategy. Unexpectedly, we also observed that female offspring lacked most frameshift mutations, suggesting a previously unrecognized role for KLHL10 in oogenesis or early embryonic development.

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Peng, C. L., Kamau, W. S., Freeman, J., Hill, Z. J., Esvelt, K. M.. 2025-12-26. Increasing the effective gene drive homing rate by targeting the haploinsufficient spermatogenesis gene KLHL10. https://doi.org/10.64898/2025.12.22.695236

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