Abstract：
Replication-coupled gene editing using locked nucleic acid-modified single-stranded DNA oligonucleotides (LMOs) can genetically engineer mammalian cells with high precision at single nucleotide resolution. Based on this method, oligonucleotide-directed mutation screening (ODMS) was developed to determine whether variants of uncertain clinical significance of DNA mismatch repair (MMR) genes can cause Lynch syndrome. In ODMS, the appearance of 6-thioguanine-resistant colonies upon introduction of the variant is indicative for defective MMR and hence pathogenicity. Whereas mouse embryonic stem cells (mESCs) hemizygous for MMR genes were used previously, we now show that ODMS can also be applied in wild-type mESCs carrying two functional alleles of each MMR gene. 6-Thioguanine resistance can result from two possible events: first, the mutation is present in only one allele, which is indicative for dominant-negative activity of the variant; and second, both alleles contain the planned modification, which is indicative for a regular loss-of-function variant. Thus, ODMS in wild-type mESCs can discriminate fully disruptive and dominant-negative MMR variants. The feasibility of biallelic targeting suggests that the efficiency of LMO-mediated gene targeting at a nonselectable locus may be enriched in cells that had undergone a simultaneous selectable LMO targeting event. This turned out to be the case and provided a protocol to improve recovery of LMO-mediated gene modification events.
摘要：
使用锁核酸修饰的单链寡脱氧核糖核苷酸（LMO）的复制偶联基因编辑可以以单核苷酸分辨率高精度地基因工程化哺乳动物细胞。基于这种方法，我们开发了寡核苷酸指导的突变筛查(ODMS),以确定DNA错配修复(MMR)基因的临床意义不确定的变异是否可导致Lynch综合征.在ODMS中，在引入变体后6-硫代鸟嘌呤(6TG)抗性菌落的出现表明MMR缺陷和因此的致病性。尽管以前使用小鼠胚胎干细胞（mESCs）半合子用于DNA错配修复（MMR）基因，我们现在表明ODMS也可以应用于携带每个MMR基因的两个功能等位基因的野生型mESC。6TG电阻可以由两个可能的事件引起：第一，突变只存在于一个等位基因中,这表明变体的显性负活性；第二，两个等位基因都包含计划的修饰，这表明有规律的功能丧失变体。因此，野生型mESC中的ODMS可以区分完全破坏性和显性阴性MMR变体。双等位基因靶向的可行性表明,在非选择性基因座处LMO介导的基因靶向的效率可以在已经经历了同时选择性LMO靶向事件的细胞中富集。事实证明是这种情况，并提供了改善LMO介导的基因修饰事件恢复的方案。
