Targeted editing of pericentromeric satellite DNA alters sensitivity to meiotic drive

Eukaryotic genomes are abundant in satellite DNA (satDNA): large blocks of tandemly-repeated sequences that accumulate in heterochromatic genome regions. SatDNAs are dynamic in their genomic location and abundance across species. Some satDNAs overlap essential genome regions such as centromeres and telomeres, but even pericentromeric satDNA can have effects on phenotypes, raising questions about their functional significance. However, it remains unclear whether these effects depend on satDNA sequence, copy number, higher-order structural organization, or genomic context. The highly repetitive nature of satDNA arrays has long hindered detailed genomic and genetic analyses. Recent advances in long-read sequencing now facilitate both the detailed characterization of satDNA structure and the development of more targeted approaches to genetic analysis. Here we present a sequential CRISPR/Cas9-based strategy to make mutations in satDNA arrays and demonstrate its utility using an autosomal pericentromeric satDNA in <i>Drosophila melanogaster</i> called <i>Responder (Rsp)</i>. <i>Rsp</i> is the target of a sperm-killing male meiotic driver, <i>Segregation Distorter</i> (<i>SD</i>), where sensitivity to sperm killing positively correlates with <i>Rsp</i> copy number. Using our CRISPR/Cas9 approach, we generated an allelic series of <i>Rsp</i> deletion and expansion variants in two genetic backgrounds and examined their effects on spermatogenesis. Our approach produced precise satDNA variants efficiently, with minimal detectable off-target effects. The resulting mutations affect sensitivity to <i>SD</i> that scale with <i>Rsp</i> copy number. This work establishes a new framework for experimentally dissecting satDNA function and provides insights into the evolutionary and functional roles of satDNA in genome organization.