Wyss Institute for Biologically Inspired Engineering, Harvard University, Department of Genetics, Harvard Medical School, Harvard University, Boston, MA 02115, USA.
Nucleic Acids Res. 2011 Sep 1;39(16):7336-47. doi: 10.1093/nar/gkr183. Epub 2011 May 23.
Genome engineering using single-stranded oligonucleotides is an efficient method for generating small chromosomal and episomal modifications in a variety of host organisms. The efficiency of this allelic replacement strategy is highly dependent on avoidance of the endogenous mismatch repair (MMR) machinery. However, global MMR inactivation generally results in significant accumulation of undesired background mutations. Here, we present a novel strategy using oligos containing chemically modified bases (2'-Fluoro-Uridine, 5-Methyl-deoxyCytidine, 2,6-Diaminopurine or Iso-deoxyGuanosine) in place of the standard T, C, A or G to avoid mismatch detection and repair, which we tested in Escherichia coli. This strategy increases transient allelic-replacement efficiencies by up to 20-fold, while maintaining a 100-fold lower background mutation level. We further show that the mismatched bases between the full length oligo and the chromosome are often not incorporated at the target site, probably due to nuclease activity at the 5' and 3' termini of the oligo. These results further elucidate the mechanism of oligo-mediated allelic replacement (OMAR) and enable improved methodologies for efficient, large-scale engineering of genomes.
利用单链寡核苷酸进行基因组工程是在多种宿主生物中生成小染色体和附加体修饰的有效方法。这种等位基因替换策略的效率高度依赖于避免内源性错配修复（MMR）机制。然而，全局 MMR 失活通常会导致不期望的背景突变的显著积累。在这里，我们提出了一种使用含有化学修饰碱基（2'-氟尿嘧啶、5-甲基脱氧胞嘧啶、2,6-二氨基嘌呤或异脱氧鸟嘌呤）的寡核苷酸替代标准的 T、C、A 或 G 来避免错配检测和修复的新策略，我们在大肠杆菌中对此进行了测试。该策略将瞬时等位基因替换效率提高了多达 20 倍，同时保持了低 100 倍的背景突变水平。我们进一步表明，全长寡核苷酸和染色体之间的错配碱基通常不会在靶位点掺入，可能是由于寡核苷酸 5' 和 3' 末端的核酸酶活性。这些结果进一步阐明了寡核苷酸介导的等位基因替换（OMAR）的机制，并为高效、大规模基因组工程提供了改进的方法。
Zotero 插件
