Department of Pharmacology, Yonsei University College of Medicine, Seoul, South Korea.
Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, South Korea.
Nucleic Acids Res. 2018 Jul 6;46(12):e71. doi: 10.1093/nar/gky222.
The use of paired Cas9 nickases instead of Cas9 nuclease drastically reduces off-target effects. Because both nickases must function for a nickase pair to make a double-strand break, the efficiency of paired nickases can intuitively be expected to be lower than that of either corresponding nuclease alone. Here, we carefully compared the gene-disrupting efficiency of Cas9 paired nickases with that of nucleases. Interestingly, the T7E1 assay and deep sequencing showed that on-target efficiency of paired D10A Cas9 nickases was frequently comparable, but sometimes higher than that of either corresponding nucleases in mammalian cells. As the underlying mechanism, we found that the HNH domain, which is preserved in the D10A Cas9 nickase, has higher activity than the RuvC domain in mammalian cells. In this study, we showed: (i) the in vivo cleavage efficiency of the HNH domain of Cas9 in mammalian cells is higher than that of the RuvC domain, (ii) paired Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. We envision that our findings which were overlooked in previous reports will serve as a new potential guideline for tool selection for CRISPR-Cas9-mediated gene disruption, facilitating efficient and precise genome editing.
使用成对的 Cas9 切口酶而非 Cas9 核酸酶可显著降低脱靶效应。由于双链断裂需要两个切口酶同时作用，因此可以直观地预期成对切口酶的效率会低于相应的单个核酸酶。在这里，我们仔细比较了 Cas9 成对切口酶与核酸酶的基因破坏效率。有趣的是，T7E1 测定和深度测序表明，在哺乳动物细胞中，成对的 D10A Cas9 切口酶的靶标效率通常相当，但有时高于相应的单个核酸酶。作为潜在的机制，我们发现，在哺乳动物细胞中，D10A Cas9 切口酶中保留的 HNH 结构域比 RuvC 结构域具有更高的活性。在这项研究中，我们表明：（i）Cas9 的 HNH 结构域在哺乳动物细胞中的体内切割效率高于 RuvC 结构域，（ii）成对的 Cas9 切口酶在基因破坏方面有时比单个核酸酶更有效。我们设想，我们的发现将成为 CRISPR-Cas9 介导的基因破坏工具选择的新潜在指南，有助于高效、精确的基因组编辑。
