Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting , , or with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites.

中文翻译：

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配对 CRISPR-Cas 切口酶在原代人类细胞中的靶向和脱靶效应

CRISPR-Cas 核酸酶的不良脱靶效应仍然是基因组编辑中的一个挑战。虽然 Cas9 切口酶的使用已被证明可以最大限度地减少脱靶诱变，但它们在治疗性基因组编辑中的使用因缺乏功效而受到阻碍。为了克服这一限制，我们和其他人开发了基于双切口酶的策略来产生交错的 DNA 双链断裂，从而高效地介导基因破坏或基因校正。然而，配对单链切口对基因组完整性的影响在很大程度上仍未得到探索。在这里，我们开发了一种新的 CAST-seq 流程，即双 CAST，来表征由成对 CRISPR-Cas9 切口酶在源自大疱性表皮松解症患者的原代角质形成细胞的三个不同位点诱导的染色体畸变。虽然靶向 、 、 或 Cas9 核酸酶引起了先前未描述的染色体重排，但在配对切口酶编辑后没有检测到染色体易位。虽然双切口策略在所有三个位点的靶位点周围的 10 kb 区域内诱导大量缺失/倒位，与核酸酶类似，但染色体靶标畸变在性质上有所不同，并且包括高比例的插入。综上所述，我们的数据表明，双切口酶方法将高效编辑与大大减少脱靶效应结合起来，但仍然在靶位点留下大量染色体畸变。