Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan.
Department of Neurology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan.
Cell Rep. 2020 Jan 28;30(4):1195-1207.e7. doi: 10.1016/j.celrep.2019.12.064.
Targeted knockin mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knockin. Here, we demonstrate that tandem paired nicking, a method for targeted knockin involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knockin largely equivalent to that of the Cas9-nuclease-based approach. Tandem paired nicking seems to accomplish targeted knockin by DNA recombination analogous to Holliday's model and creates intended genomic changes without introducing additional nucleotide changes, such as silent mutations. Targeted knockin through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.
双链 DNA 切割介导的靶向敲入伴随着在靶和脱靶位点的不必要的插入和缺失（indels）。切口介导的方法几乎不会产生 indels，但靶向敲入的效率降低。在这里，我们证明了串联配对的切口，这是一种涉及两个 Cas9 切口酶的靶向敲入方法，它在供体 DNA 的同源区域和同一链上的基因组中创建切口，在编辑的基因组位点几乎不会产生 indels，同时允许靶向敲入的效率与 Cas9-核酸酶为基础的方法基本相当。串联配对的切口似乎通过类似于霍利迪模型的 DNA 重组来实现靶向敲入，并在不引入额外核苷酸变化（如沉默突变）的情况下产生预期的基因组变化。通过串联配对的切口进行靶向敲入既不会引发明显的 p53 激活，也不会优先发生在 p53 抑制的细胞中。串联配对切口的这些特性证明了其在精确基因组工程中的实用性。
