Extensive trimming of short single-stranded DNA oligonucleotides during replication-coupled gene editing in mammalian cells

• Published in: PLoS genetics Vol. 16; no. 10; p. e1009041
• Main Authors: van Ravesteyn, Thomas W., Arranz Dols, Marcos, Pieters, Wietske, Dekker, Marleen, te Riele, Hein
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 29.10.2020; Public Library of Science (PLoS)
• Subjects: Analysis; Animals; Biology; Biology and life sciences; Cancer; Cell Line; Cells; Control; Deoxyribonucleic acid; DNA; DNA biosynthesis; DNA Mismatch Repair - genetics; DNA repair; DNA replication; DNA Replication - genetics; DNA, Single-Stranded - genetics; Efficiency; Gene Editing; Gene Targeting; Genetic aspects; Genome editing; Genomes; Homology; Humans; Hybridization; Immunology; Mammalian cells; Mammals - genetics; Mismatch repair; Mutation; Mutation - genetics; Oligonucleotides; Oligonucleotides - genetics; Physical sciences; Replication; Research and Analysis Methods; Single-stranded DNA; Stem cells; Transfection; Netherlands
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1009041

Through transfection of short single-stranded oligodeoxyribonucleotides (ssODNs) small genomic alterations can be introduced into mammalian cells with high precision. ssODNs integrate into the genome during DNA replication, but the resulting heteroduplex is prone to detection by DNA mismatch repair (MMR), which prevents effective gene modification. We have previously demonstrated that the suppressive action of MMR can be avoided when the mismatching nucleotide in the ssODN is a locked nucleic acid (LNA). Here, we reveal that LNA-modified ssODNs (LMOs) are not integrated as intact entities in mammalian cells, but are severely truncated before and after target hybridization. We found that single additional (non-LNA-modified) mutations in the 5'-arm of LMOs influenced targeting efficiencies negatively and activated the MMR pathway. In contrast, additional mutations in the 3'-arm did not affect targeting efficiencies and were not subject to MMR. Even more strikingly, homology in the 3'-arm was largely dispensable for effective targeting, suggestive for extensive 3'-end trimming. We propose a refined model for LMO-directed gene modification in mammalian cells that includes LMO degradation.