5′-Modifications improve potency and efficacy of DNA donors for precision genome editing

• Published in: eLife Vol. 10
• Main Authors: Ghanta, Krishna S, Chen, Zexiang, Mir, Aamir, Dokshin, Gregoriy A, Krishnamurthy, Pranathi M, Yoon, Yeonsoo, Gallant, Judith, Xu, Ping, Zhang, Xiao-Ou, Ozturk, Ahmet Rasit, Shin, Masahiro, Idrizi, Feston, Liu, Pengpeng, Gneid, Hassan, Edraki, Alireza, Lawson, Nathan D, Rivera-Pérez, Jaime A, Sontheimer, Erik J, Watts, Jonathan K, Mello, Craig C
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 19.10.2021; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: Analysis; Animal models; Animals; Biochemistry and Chemical Biology; Caenorhabditis elegans - genetics; Cell cycle; Cell division; Cells; chemical modifications; CRISPR; DNA; DNA - genetics; DNA Repair; DNA, Single-Stranded - genetics; Efficiency; Flow cytometry; Gene Editing - methods; Gene loci; Genetics and Genomics; Genome editing; Genomes; Genomics; HDR; Health aspects; HEK293 Cells; Homology; Humans; K562 Cells; Localization; Mello, Craig; Mice - genetics; modified donors; Nuclease; Ovaries; Peptides; Physiological aspects; Proteins; Single-stranded DNA; Toxicity; Triethylene glycol; Zebrafish - genetics; CRISPR; genome editing; C. elegans; mouse; genetics; chemical biology; modified donors; HDR; biochemistry; chemical modifications; genomics; zebrafish; human
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.72216

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.