Dissecting Fertility Functions of a Chromosome Genes with CRISPR.

Gene-poor, repeat-rich regions of the genome are poorly understood and have been understudied due to technical challenges and the misconception that they are degenerating "junk." Yet multiple lines of evidence indicate these regions may be an important source of variation that could drive adaptation and species divergence, particularly through regulation of fertility. The ∼40 Mb chromosome of contains only 16 known protein-coding genes, and is highly repetitive and entirely heterochromatic. Most of the genes originated from duplication of autosomal genes and have reduced nonsynonymous substitution rates, suggesting functional constraint. We devised a genetic strategy for recovering and retaining stocks with sterile -linked mutations and combined it with CRISPR to create mutants with deletions that disrupt three -linked genes. Two genes, and , had no previously identified functions. We found that mutant males are subfertile, but mutant males had no detectable fertility defects. , the newest known gene on the chromosome, may have fertility effects that are conditional or too subtle to detect. The third gene, , had been predicted but never formally shown to be required for male fertility. CRISPR targeting and RNA interference of caused male sterility. Surprisingly, however, our mutants were sterile even in the presence of an extra wild-type chromosome, suggesting that perturbation of the chromosome can lead to dominant sterility. Our approach provides an important step toward understanding the complex functions of the chromosome and parsing which functions are accomplished by genes repeat elements.