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

ABSTRACT: 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 n

SUBMITTER: van Ravesteyn TW 

PROVIDER: S-EPMC7595315 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature