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.

通过短单链寡脱氧核糖核苷酸（ssODNs）的转染，可以将小的基因组改变高精度地引入哺乳动物细胞。ssODN在DNA复制过程中整合到基因组中，但是产生的异源双链体易于通过DNA错配修复（MMR）进行检测，从而无法进行有效的基因修饰。先前我们已经证明，当ssODN中的错配核苷酸为锁定核酸（LNA）时，可以避免MMR的抑制作用。在这里，我们揭示了LNA修饰的ssODNs（LMOs）并没有整合为哺乳动物细胞中的完整实体，但是在目标杂交之前和之后均被严重截短。我们发现，LMO 5'臂中的其他单个突变（未经LNA修饰）对靶定靶点产生了负面影响，并激活了MMR途径。相反，3'-臂中的其他突变不会影响靶向效率，也不受MMR的影响。更惊人的是，在3'臂中的同源性对于有效的靶向作用很大程度上是必需的，这暗示了对3'末端的广泛修饰。我们提出了一种针对包括LMO降解在内的LMO指导的哺乳动物细胞基因修饰的改进模型。