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 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.
Highlights
The ability to generate gene modifications is of great importance to a wide variety of research fields in molecular biology
We have shown before that short single-stranded DNA molecules (LMOs) are very useful to introduce and study genetic variants that may predispose patients to cancer
We have demonstrated the applicability of this technology in mammalian cells by setting up screens that enable the classification of Lynch syndrome-associated variants of uncertain clinical significance in MSH2, MSH6 and MLH1 [9,10,11]
Summary
The ability to generate gene modifications is of great importance to a wide variety of research fields in molecular biology. The ability to generate precise gene modifications at endogenous loci with the resolution of single nucleotides enables the study of specific protein residues. Various strategies have been developed to edit the genome with single-stranded repair templates in combination with site-specific nucleases such as Zinc-finger nucleases [1], TAL-effector nucleases (TALENs) [2] or CRISPR/Cas9 [3]. Besides use as repair-template in combination with a site-specific DNA double stranded break (DSB), ssODNs with a centrally positioned mutation are used to generate subtle gene modifications in the absence of DSBs. Targeting chromosomal DNA during replication has proven to be highly effective for multiplex genome engineering in simple prokaryotic and eukaryotic model organisms like Escherichia coli [4,5,6] and Saccharomyces cerevisiae [7,8]. We have demonstrated the applicability of this technology in mammalian cells by setting up screens that enable the classification of Lynch syndrome-associated variants of uncertain clinical significance in MSH2, MSH6 and MLH1 [9,10,11]
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