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

English version

Autoři: Thomas W. van Ravesteyn ^aff001; Marcos Arranz Dols ^aff001; Wietske Pieters ^aff001; Marleen Dekker ^aff001; Hein te Riele ^aff001
Působiště autorů: Division of Tumor Biology and Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands ^aff001
Vyšlo v časopise: Extensive trimming of short single-stranded DNA oligonucleotides during replication-coupled gene editing in mammalian cells. PLoS Genet 16(10): e32767. doi:10.1371/journal.pgen.1009041
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1009041

Souhrn

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.

Klíčová slova:

Cloning – Dideoxy DNA sequencing – DNA – DNA annealing – DNA replication – Mammalian genomics – Nucleotides – Transfection

Zdroje

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