Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and ‘cleaner’ knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

中文翻译：

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使用多个物种的细菌重组因子增强的双刻痕，可以有效地降低基因组编辑的风险。

位点特异性DNA双链断裂已用于通过同源依赖性或非依赖性途径产生敲入。然而，低效率和伴随的负面影响，例如不期望的插入缺失或致瘤潜力仍然是有问题的。在这项研究中，我们提出了一种名为NEO的增强型降低风险的基因组编辑策略，该策略使用位点特异性反式或顺式四个细菌重组因子（RecOFAR）促进了双切口形成。与目前可用的方法相比，NEO获得了更高的敲入（KI）种系传递频率（效率从零提高到了10％，平均8个基因座提高了5倍），并且“清除”了较长的敲入DNA片段（最大5.5 kb）进入斑马鱼，小鼠和大鼠的各种基因组区域。此外，NEO在猴胚胎中产生高达50％的敲入，在非分裂原代人外周血淋巴细胞（hPBLC）中产生20％的相对整合效率。值得注意的是，NEO可以有效抑制目标上和目标外插入缺失。NEO也可用于有效，精确地引入低风险的非限制性点突变。因此，通过在效率与安全性和质量之间取得平衡，最近开发的体内基因编辑策略。