Max-Delbrück-Centrum für Molekulare Medizin, 13125, Berlin, Germany.
Present Address: BioNTech Cell & Gene Therapies GmbH, Mainz, Germany.
BMC Biotechnol. 2020 Oct 23;20(1):57. doi: 10.1186/s12896-020-00650-x.
Precise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathway and depends on the presence of repair templates its efficiency is often limited and demands optimized methodology.
For the enhancement of HDR we redirect the DSB repair pathway choice by targeting the Ubiquitin mark for damaged chromatin at Histone H2A-K15. We used fusions of the Ubiquitin binding domain (UBD) of Rad18 or RNF169 with BRCA1 to promote HDR initiation and UBD fusions with DNA binding domains to attract donor templates and facilitate HDR processing. Using a traffic light reporter system in human HEK293 cells we found that the coexpression of both types of UBD fusion proteins promotes HDR, reduces NHEJ and shifts the HDR/NHEJ balance up to 6-fold. The HDR enhancing effect of UBD fusion proteins was confirmed at multiple endogenous loci.
Our findings provide a novel efficient approach to promote precise gene editing in human cells.
精确的基因修饰是 CRISPR-Cas9 介导的哺乳动物细胞基因编辑的首选产物，但需要通过同源定向修复 (HDR) 修复诱导的双链断裂 (DSB)。由于 HDR 与普遍存在的非同源末端连接 (NHEJ) 途径竞争，并依赖于修复模板的存在，其效率通常受到限制，需要优化方法。
为了增强 HDR，我们通过靶向组蛋白 H2A-K15 上受损染色质的泛素标记来引导 DSB 修复途径的选择。我们使用 Rad18 或 RNF169 的泛素结合结构域 (UBD) 与 BRCA1 的融合来促进 HDR 的起始，以及 UBD 与 DNA 结合结构域的融合来吸引供体模板并促进 HDR 处理。在人 HEK293 细胞中使用红绿灯报告系统，我们发现两种类型的 UBD 融合蛋白的共表达均促进 HDR，减少 NHEJ，并将 HDR/NHEJ 平衡提高 6 倍。在多个内源性基因座上证实了 UBD 融合蛋白的 HDR 增强作用。
我们的发现为促进人类细胞中精确的基因编辑提供了一种新颖有效的方法。
