Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

Simple 1,3-diphenylureas are capable of transporting carboxylic acid-based drugs across phospholipid bilayers without affecting the permeability of other physiological anions such as chloride.

20 21 2Nuclease-directed genome editing is a powerful tool for investigating physiology and has 22 great promise as a therapeutic approach that directly addresses the underlying genetic basis 23 of disease. In its most precise form, genome editing can use cellular homology-directed repair 24 (HDR) pathways to insert information from an exogenously supplied DNA repair template 25 (donor) directly into a targeted genomic location. Unfortunately, particularly for long 26 insertions, toxicity and delivery considerations associated with repair template DNA can 27 limit the number of donor molecules available to the HDR machinery, thus limiting HDR 28 efficacy. Here, we explore modifications to both double-stranded and single-stranded repair 29 template DNAs and describe simple 5′ end modifications that consistently and dramatically 30 increase donor potency and HDR efficacy across cell types and species. 31In the nematode worm C. elegans, efficient genome editing can be achieved by direct 32 injection of editing enzyme guide-RNA ribonucleoprotein (RNP) complexes into the syncytial 33 ovary 1 . Such injections afford simultaneous access of the editing machinery to hundreds of meiotic 34 germ nuclei within a common cytoplasm (Supplementary Fig. 1a). In the worm germline, high 35 rates of HDR are readily achieved using short (under ~200 nucleotide [nt]), single-stranded 36 oligodeoxynucleotide (ssODN) donor templates that permit insertions of up to ~150 nt in length 2, 37 3 4 . However, HDR is less efficient by 1-2 orders of magnitude when longer, double-stranded DNA 38 (dsDNA) templates are used as donors 4 . 39Longer repair templates are likely at a disadvantage for multiple reasons. First, toxicity 40 associated with high concentrations of DNA limits the safe injectable amount of a ~1kb dsDNA 41 donor to roughly ~10-fold fewer molecules than is commonly used for a 200 nt ssODN donor 2-5 . 42Second, long dsDNA donor molecules may not readily transit across the nuclear envelope into the 43 post-mitotic germ-nuclei, further reducing the effective concentration at the site of repair. We 44 3 hypothesized that the disparity in availability of ssODN and dsDNA donor molecules inside germ 45 nuclei could account for the differences in observed HDR efficiencies. To increase potency of long 46 dsDNA donors we set out to attach an SV40 peptide containing the core nuclear localization signal 47 (NLS) to the donor molecule, reasoning that the modification might promote nuclear uptake and 48 retention. Previous studies using mammalian cell cultures demonstrated that the addition of an 49 NLS enhances nuclear uptake of plasmid DNA following transfection 6 . To attach an NLS to a 50 long donor DNA, we first conjugated 15-nucleotide 2′-O-methyl (2′OMe) RNA adapters via a tri-51 or tetraethylene glycol (TEG) linkage to the 5′ ends of two target-locus specific synthetic ~20-52 nucleotide DNA oligonucleotides. The DNA sequences in these molecules serve as PCR primers 53 to amplify the donor from plasmid containing the homology arms and g...

An increasing number of people are infected with antibiotic-resistant bacteria each year, sometimes with fatal consequences. In this manuscript, we report a novel urea-functionalized crown ether that can bind to...