Genome editing via the homology‐directed repair (HDR) pathway in somatic plant cells is very inefficient compared with error‐prone repair by nonhomologous end joining (NHEJ). Here, we increased HDR‐based genome editing efficiency approximately threefold compared with a Cas9‐based single‐replicon system via the use of de novo multi‐replicon systems equipped with CRISPR/LbCpf1 in tomato and obtained replicon‐free but stable HDR alleles. The efficiency of CRISPR/LbCpf1‐based HDR was significantly modulated by physical culture conditions such as temperature and light. Ten days of incubation at 31 °C under a light/dark cycle after Agrobacterium‐mediated transformation resulted in the best performance among the tested conditions. Furthermore, we developed our single‐replicon system into a multi‐replicon system that effectively increased HDR efficiency. Although this approach is still challenging, we showed the feasibility of HDR‐based genome editing of a salt‐tolerant SlHKT1;2 allele without genomic integration of antibiotic markers or any phenotypic selection. Self‐pollinated offspring plants carrying the HKT1;2 HDR allele showed stable inheritance and germination tolerance in the presence of 100 mm NaCl. Our work may pave the way for transgene‐free editing of alleles of interest in asexually and sexually reproducing plants.

中文翻译：

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使用 CRISPR/Cpf1 双病毒复制子在番茄中进行高效同源定向修复


与通过非同源末端连接（NHEJ）进行的容易出错的修复相比，在植物体细胞中通过同源定向修复（HDR）途径进行基因组编辑的效率非常低。在这里，我们通过在番茄中使用配备 CRISPR/LbCpf1 的从头多复制子系统，将基于 HDR 的基因组编辑效率比基于 Cas9 的单复制子系统提高了大约三倍，并获得了无复制子但稳定的 HDR 等位基因。基于 CRISPR/LbCpf1 的 HDR 的效率受到温度和光照等物理培养条件的显着调节。农杆菌介导的转化后，在 31°C 的光/暗循环下孵育 10 天，在测试条件下获得了最佳性能。此外，我们将单复制子系统发展为多复制子系统，有效提高了 HDR 效率。尽管这种方法仍然具有挑战性，但我们展示了基于 HDR 的耐盐 SlHKT1;2 等位基因基因组编辑的可行性，无需抗生素标记的基因组整合或任何表型选择。携带 HKT1;2 HDR 等位基因的自花授粉后代植物在 100 m m NaCl 存在下表现出稳定的遗传和发芽耐受性。我们的工作可能为无性繁殖和有性繁殖植物中感兴趣的等位基因的非转基因编辑铺平道路。