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Co-introduction of targeted nucleases and DNA/RNA templates encoding new genomic sequence is the basis for rapid, effective, and iterable gene editing workflows for therapeutic, agricultural, and basic science applications. Extensive optimization of reagent delivery and nuclease activity have improved genome editing workflows, but comparatively few efforts have been made to alter the gene editing activity of template molecules. Here, we report template DNA modified with interstrand crosslinks (ICLs) – xHDRTs - increases editing frequencies in Cas9-directed gene editing workflows by up to five-fold. xHDRTs increase gene editing frequencies independent of DNA template topology, amount of sequence added, or cell type. Gene editing using xHDRTs requires the DNA repair kinase, ATR, and partially requires Fanconi Anemia proteins, including FANCA, but is independent of other ICL-repair pathways. Covalent modification of donor DNA thus presents a compelling opportunity to improve nonviral gene editing workflows.

The AAA-ATPases Pex1 and Pex6 are required for the formation and maintenance of peroxisomes, membrane-bound organelles that harbor enzymes for specialized metabolism. Together, Pex1 and Pex6 form a heterohexameric AAA-ATPase capable of unfolding substrate proteins via processive threading through a central pore. Here, we review the proposed roles for Pex1/Pex6 in peroxisome biogenesis and degradation, discussing how the unfolding of potential substrates contributes to peroxisome homeostasis. We also consider how advances in cryo-EM, computational structure prediction, and mechanisms of related ATPases are improving our understanding of how Pex1/Pex6 converts ATP hydrolysis into mechanical force. Since mutations in PEX1 and PEX6 cause the majority of known cases of peroxisome biogenesis disorders such as Zellweger syndrome, insights into Pex1/Pex6 structure and function are important for understanding peroxisomes in human health and disease.