Gerlach Max, Kraft Theresia, Brenner Bernhard, Petersen Björn, Niemann Heiner, Montag Judith
Institute for Molecular and Cell Physiology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Hoeltystrasse 10, Mariensee, 31535 Neustadt, Germany.
Genes (Basel). 2018 Jun 13;9(6):296. doi: 10.3390/genes9060296.
During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the -gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene (). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9- and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9- provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.
在CRISPR/Cas9介导的基因组编辑过程中，会引入位点特异性双链断裂，这些断裂可通过非同源末端连接（NHEJ）进行非特异性修复，或通过同源定向修复（HDR）进行序列依赖性修复。基于NHEJ的基因敲除生成已被广泛应用，而基于HDR的特定突变敲入仍然是一个瓶颈。特别是在对于遗传性人类疾病的细胞培养和动物模型生成至关重要的原代细胞系中，敲入效率不足，需要显著提高。在此，我们测试了两种不同的方法来提高猪胎儿成纤维细胞中特定点突变敲入基因的频率。在基因组编辑过程中，我们添加了一种NHEJ的小分子抑制剂SCR7（5,6-双（（E）-亚苄基氨基）-2-巯基嘧啶-4-醇），并筛选细胞培养物中的点突变。然而，这种方法并没有提高敲入率。在另一种方法中，我们将人源化Cas9（hCas9）与Geminin基因的N端肽融合。该融合蛋白在以NHEJ为主导的细胞周期阶段会被降解，这应该会提高HDR率。通过使用hCas9和点突变特异性实时PCR筛选，我们发现基因组编辑的细胞培养物增加了两倍。hCas9介导HDR的增加为在猪成纤维细胞培养物中富集特定敲入以用于体细胞克隆方法提供了一种有前景的方法。
