CRISPR- Cas9 has revolutionized genetic engineering. However, the inability to control double-strand break (DSB) repair has severely limited both therapeutic and academic applications. Many attempts have been made to control DSB repair choice. However, particularly in the case of larger edits, none have been able to bypass the rate-limiting step of homologous recombination (HR): long-range 5′ end resection. Here, we describe a novel set of Cas9 fusions, Cas9-HRs, designed to bypass the rate-limiting step of HR repair by simultaneously coupling initial and long-range end resection. Here, we demonstrate that Cas9-HRs can increase the rate of homology directed repair (HDR) by 2- to 2.5-fold and decrease p53 mediated cellular toxicity by two- to fourfold compared to Cas9 and are functional in multiple mammalian cell lines with minimal apparent editing site bias. These properties should make Cas9-HRs an attractive option for applications demanding increased HDR rates for long inserts and/or reduced p53 pathway activation.

中文翻译：

---

一组新的 Cas9 融合蛋白刺激同源重组：Cas9-HRs

CRISPR-Cas9 彻底改变了基因工程。然而，无法控制双链断裂 (DSB) 修复严重限制了治疗和学术应用。已经进行了许多尝试来控制 DSB 维修选择。然而，特别是在较大编辑的情况下，没有人能够绕过同源重组 (HR) 的限速步骤：远程 5' 末端切除。在这里，我们描述了一组新的 Cas9 融合，Cas9-HR，旨在通过同时耦合初始和远程末端切除来绕过 HR 修复的限速步骤。在这里，我们证明 Cas9-HR 可以将同源定向修复 (HDR) 的速率提高 2 到 2。与 Cas9 相比，p53 介导的细胞毒性增加 5 倍并将其降低 2 到 4 倍，并且在多种哺乳动物细胞系中发挥作用，具有最小的明显编辑位点偏差。这些特性应该使 Cas9-HRs 成为需要增加长插入和/或减少 p53 通路激活的 HDR 速率的应用的有吸引力的选择。