Resolution of structural variation in diverse mouse genomes reveals chromatin remodeling due to transposable elements.

Diverse inbred mouse strains are important biomedical research models, yet genome characterization of many strains is fundamentally lacking in comparison with humans. In particular, catalogs of structural variants (SVs) (variants ≥ 50 bp) are incomplete, limiting the discovery of causative alleles for phenotypic variation. Here, we resolve genome-wide SVs in 20 genetically distinct inbred mice with long-read sequencing. We report 413,758 site-specific SVs affecting 13% (356 Mbp) of the mouse reference assembly, including 510 previously unannotated coding variants. We substantially improve the Mus musculus transposable element (TE) callset, and we find that TEs comprise 39% of SVs and account for 75% of altered bases. We further utilize this callset to investigate how TE heterogeneity affects mouse embryonic stem cells and find multiple TE classes that influence chromatin accessibility. Our work provides a comprehensive analysis of SVs found in diverse mouse genomes and illustrates the role of TEs in epigenetic differences.

Transposable element-mediated rearrangements are prevalent in human genomes.

Transposable elements constitute about half of human genomes, and their role in generating human variation through retrotransposition is broadly studied and appreciated. Structural variants mediated by transposons, which we call transposable element-mediated rearrangements (TEMRs), are less well studied, and the mechanisms leading to their formation as well as their broader impact on human diversity are poorly understood. Here, we identify 493 unique TEMRs across the genomes of three individuals. While homology directed repair is the dominant driver of TEMRs, our sequence-resolved TEMR resource allows us to identify complex inversion breakpoints, triplications or other high copy number polymorphisms, and additional complexities. TEMRs are enriched in genic loci and can create potentially important risk alleles such as a deletion in TRIM65, a known cancer biomarker and therapeutic target. These findings expand our understanding of this important class of structural variation, the mechanisms responsible for their formation, and establish them as an important driver of human diversity.

SvAnna: efficient and accurate pathogenicity prediction of coding and regulatory structural variants in long-read genome sequencing.

Structural variants (SVs) are implicated in the etiology of Mendelian diseases but have been systematically underascertained owing to sequencing technology limitations. Long-read sequencing enables comprehensive detection of SVs, but approaches for prioritization of candidate SVs are needed. Structural variant Annotation and analysis (SvAnna) assesses all classes of SVs and their intersection with transcripts and regulatory sequences, relating predicted effects on gene function with clinical phenotype data. SvAnna places 87% of deleterious SVs in the top ten ranks. The interpretable prioritizations offered by SvAnna will facilitate the widespread adoption of long-read sequencing in diagnostic genomics. SvAnna is available at https://github.com/TheJacksonLaboratory/SvAnn a .

Structural variant identification and characterization.

Structural variant (SV) differences between human genomes can cause germline and mosaic disease as well as inter-individual variation. De-regulation of accurate DNA repair and genomic surveillance mechanisms results in a large number of SVs in cancer. Analysis of the DNA sequences at SV breakpoints can help identify pathways of mutagenesis and regions of the genome that are more susceptible to rearrangement. Large-scale SV analyses have been enabled by high-throughput genome-level sequencing on humans in the past decade. These studies have shed light on the mechanisms and prevalence of complex genomic rearrangements. Recent advancements in both sequencing and other mapping technologies as well as calling algorithms for detection of genomic rearrangements have helped propel SV detection into population-scale studies, and have begun to elucidate previously inaccessible regions of the genome. Here, we discuss the genomic organization of simple and complex SVs, the molecular mechanisms of their formation, and various ways to detect them. We also introduce methods for characterizing SVs and their consequences on human genomes.

RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and Integration.

CRISPR-based technologies have become central to genome engineering. However, CRISPR-based editing strategies are dependent on the repair of DNA breaks via endogenous DNA repair mechanisms, which increases susceptibility to unwanted mutations. Here we complement Cas9 with a recombinase's functionality by fusing a hyperactive mutant resolvase from transposon Tn3, a member of serine recombinases, to a catalytically inactive Cas9, which we term integrase Cas9 (iCas9). We demonstrate iCas9 targets DNA deletion and integration. First, we validate iCas9's function in Saccharomyces cerevisiae using a genome-integrated reporter. Cooperative targeting by CRISPR RNAs at spacings of 22 or 40 bp enables iCas9-mediated recombination. Next, iCas9's ability to target DNA deletion and integration in human HEK293 cells is demonstrated using dual GFP-mCherry fluorescent reporter plasmid systems. Finally, we show that iCas9 is capable of targeting integration into a genomic reporter locus. We envision targeting and design concepts of iCas9 will contribute to genome engineering and synthetic biology.