CRISPR Mutants of Three Y Chromosome Genes Suggest Gradual Evolution of Fertility Functions inDrosophila melanogaster

Abstract

21Gene-poor, repeat-rich regions of the genome are poorly understood and have been understudied 22 due to technical challenges and the misconception that they are degenerating "junk". Yet 23 multiple lines of evidence indicate these regions may be an important source of variation that 24 could drive adaptation and species divergence, particularly through regulation of fertility. The 25 ~40 Mb Y chromosome of Drosophila melanogaster contains only 16 known protein-coding 26 genes and is highly repetitive and entirely heterochromatic. Most of the genes originated from 27 duplication of autosomal genes and have reduced nonsynonymous substitution rates, suggesting 28 functional constraint. We devised a genetic strategy for recovering and retaining stocks with 29 sterile Y-linked mutations and combined it with CRISPR to create mutants with deletions that 30 disrupt three Y-linked genes. Two genes, PRY and FDY, had no previously identified functions. 31We found that PRY mutant males are sub-fertile, but FDY mutant males had no detectable 32 fertility defects. FDY, the newest known gene on the Y chromosome, may have fertility effects 33 that are conditional or too subtle to detect. The third gene, CCY, had been predicted but never 34 formally shown to be required for male fertility. CRISPR-targeting and RNAi of CCY caused 35 male sterility. Surprisingly, however, our CCY mutants were sterile even in the presence of an 36 extra wild-type Y chromosome, suggesting that perturbation of the Y chromosome can lead to 37 dominant sterility. Our approach provides an important step toward understanding the complex 38 functions of the Y chromosome and parsing which functions are accomplished by genes versus 39 repeat elements. 40 41 melanogaster the ~40 Mb Y chromosome is entirely composed of constitutive heterochromatin 46 (Heitz 1933), densely populated by repetitive sequences (Lohe et al. 1993;Hoskins et al.