Center for Genome Engineering, Institute for Basic Science, Daejeon, Republic of Korea.
Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
Nat Commun. 2023 Mar 30;14(1):1786. doi: 10.1038/s41467-023-37507-8.
Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s), which are created by replacing key catalytic amino-acid residues in one of the two nuclease domains of S. pyogenesis Cas9 (SpCas9), produce nicks or single-strand breaks. Two SpCas9 variants, namely, nCas9 (D10A) and nCas9 (H840A), which cleave target (guide RNA-pairing) and non-target DNA strands, respectively, are widely used for various purposes, including paired nicking, homology-directed repair, base editing, and prime editing. In an effort to define the off-target nicks caused by these nickases, we perform Digenome-seq, a method based on whole genome sequencing of genomic DNA treated with a nuclease or nickase of interest, and find that nCas9 (H840A) but not nCas9 (D10A) can cleave both strands, producing unwanted DSBs, albeit less efficiently than wild-type Cas9. To inactivate the HNH nuclease domain further, we incorporate additional mutations into nCas9 (H840A). Double-mutant nCas9 (H840A + N863A) does not exhibit the DSB-inducing behavior in vitro and, either alone or in fusion with the M-MLV reverse transcriptase (prime editor, PE2 or PE3), induces a lower frequency of unwanted indels, compared to nCas9 (H840A), caused by error-prone repair of DSBs. When incorporated into prime editor and used with engineered pegRNAs (ePE3), we find that the nCas9 variant (H840A + N854A) dramatically increases the frequency of correct edits, but not unwanted indels, yielding the highest purity of editing outcomes compared to nCas9 (H840A).
与产生 DNA 双链断裂 (DSBs) 的 CRISPR-Cas9 核酸酶不同,酿脓链球菌 Cas9 (SpCas9) 的两个核酸酶结构域之一中的关键催化氨基酸残基被替换后产生的 Cas9 切口酶 (nCas9s) 会产生切口或单链断裂。两种 SpCas9 变体,即分别切割靶标 (向导 RNA 配对) 和非靶标 DNA 链的 nCas9 (D10A) 和 nCas9 (H840A),被广泛用于各种目的,包括配对切口、同源定向修复、碱基编辑和先导编辑。为了确定这些切口酶引起的脱靶切口,我们进行了 Digenome-seq,这是一种基于感兴趣的核酸酶或切口酶处理的基因组 DNA 的全基因组测序的方法,发现 nCas9 (H840A) 但不是 nCas9 (D10A) 可以切割两条链,产生不需要的 DSBs,尽管效率低于野生型 Cas9。为了进一步使 HNH 核酸酶结构域失活,我们将额外的突变引入 nCas9 (H840A)。双突变体 nCas9 (H840A + N863A) 既不在体外表现出 DSB 诱导行为,也没有在与 M-MLV 逆转录酶 (prime editor,PE2 或 PE3) 融合时,与 nCas9 (H840A) 相比,由于 DSB 易错修复而导致不需要的插入缺失的频率更低。当整合到 prime editor 中并与工程化 pegRNA (ePE3) 一起使用时,我们发现 nCas9 变体 (H840A + N854A) 显著增加了正确编辑的频率,但不会增加不需要的插入缺失,与 nCas9 (H840A) 相比,编辑结果的纯度更高。
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