Project Summary/Abstract Genome integrity is essential to life. Considerable efforts are made to maintain the stability of genomes. Yet genomes also undergo constant changes, often random and small in scale, providing mechanisms for evolution and adaptation. In contrast, programmed DNA elimination is a dramatic form of genome change with large amounts of DNA, ranging from 0.5 to 95% of the genome, eliminated during development. DNA elimination is highly selective and reproducible and is an integral part of biology for diverse organisms, including single-cell ciliates, a variety of multicellular organisms across animal phyla and some plants. The broad phylogenetic distribution suggests DNA elimination has evolved independently and has important biological functions. A common theme for metazoan DNA elimination is the removal of both germline-expressed genes and repetitive sequences. This suggests that a possible function of DNA elimination in metazoa is to permanently silence certain germline sequences potentially harmful to somatic cells. Despite progress in genomics and cytology, functional and mechanistic studies of metazoan DNA elimination are limited, largely due to the lack of genetic and functional tools. Recently, we built upon and extended a genomic observation and established a genetic and functional model for DNA elimination in the free-living nematode Oscheius tipulae, a member of the Rhabditidae family, which includes Caenorhabditis elegans. We show that DNA elimination in O. tipulae occurs during 8-16 cell embryos. We identified and characterized a conserved sequence (Sequence For Elimination, SFE) motif associated with the DNA break sites and demonstrated its direct role in DNA elimination. DNA breaks occur within the motif, followed by end resection and telomere healing. Additional breaks occur simultaneously in the eliminated DNA, perhaps serving as a fail-safe mechanism for DNA elimination. We revealed the abundance and variations of this motif in many wild isolates of O. tipulae from around the world. In this proposal, we will (1) study the functions of DNA elimination in O. tipulae by characterizing the fail-to-eliminate phenotypes from CRISPR edited SFE mutants. We will use RNA-seq, ChIP-seq and small RNA sequencing to determine the changes of RNA expression and silencing mechanisms in these mutants. We will also (2) study the molecular mechanisms of O. tipulae DNA elimination by investigating the sequence and genomic position required for SFEs using CRISPR, as well as proteins that interact with SFEs using in vitro biochemistry, bioinformatic predictions, and genetics. We will further (3) study the variations of DNA elimination by building telomere-to-telomere genomes for divergent strains of O. tipulae, identifying SFEs, and carrying out comparative genomics. Overall, this proposal will use our established genetic model in the free-living nematode O. tipulae to examine the functions, mechanisms, and variations of DNA eliminat...