1Department of Pathology, University of Washington, Seattle, Washington.
2Department of Genome Sciences, University of Washington, Seattle, Washington.
Hum Gene Ther. 2019 Jul;30(7):814-828. doi: 10.1089/hum.2018.169. Epub 2019 Mar 28.
This study identified 35 new sites for targeted transgene insertion that have the potential to serve as new human genomic "safe harbor" sites (SHS). SHS potential for these 35 sites, located on 16 chromosomes, including both arms of the human X chromosome, and for the existing human SHS , , and was assessed using eight different desirable, widely accepted criteria for SHS verifiable with human genomic data. Three representative newly identified sites on human chromosomes 2 and 4 were then experimentally validated by and cleavage-sensitivity tests, and analyzed for population-level and cell line-specific sequence variants that might confound site targeting. The highly ranked site on chromosome 4 (SHS231) was further characterized by targeted homology-dependent and -independent transgene insertion and expression in different human cell lines. The structure and fidelity of transgene insertions at this site were confirmed, together with analyses that demonstrated stable expression and function of transgene-encoded proteins, including fluorescent protein markers, selectable marker cassettes, and Cas9 protein variants. SHS-integrated transgene-encoded Cas9 proteins were shown to be capable of introducing a large (17 kb) gRNA-specified deletion in the fusion oncogene in human rhabdomyosarcoma cells and as a Cas9-VPR fusion protein to upregulate expression of the muscle-specific transcription factor in human rhabdomyosarcoma cells. An engineering "toolkit" was developed to enable easy use of the most extensively characterized of these new human sites, SHS231, located on the proximal long arm of chromosome 4. The target sites identified here have the potential to serve as additional human SHS to enable basic and clinical gene editing and genome-engineering applications.
本研究确定了 35 个新的靶向转基因插入位点，这些位点有可能成为新的人类基因组“安全港”（SHS）。使用八项不同的、可通过人类基因组数据验证的 SHS 理想标准，评估了这 35 个位于 16 条染色体（包括人类 X 染色体的两条臂）上的位点以及现有的人类 SHS 、 和 的 SHS 潜力。然后，通过 和 切割敏感性测试，对位于人类染色体 2 和 4 上的三个具有代表性的新鉴定位点进行了实验验证，并分析了可能影响靶点定位的人群水平和细胞系特异性序列变异。染色体 4 上排名较高的位点（SHS231）进一步通过靶向同源依赖性和非依赖性转基因插入和在不同的人类细胞系中表达进行了表征。该位点的转基因插入结构和保真度得到了确认，同时还进行了分析，证明了转基因编码蛋白的稳定表达和功能，包括荧光蛋白标记物、可选择标记盒和 Cas9 蛋白变体。在人横纹肌肉瘤细胞中，SHS 整合的转基因编码 Cas9 蛋白能够引入 17kb 的 gRNA 特异性融合癌基因缺失，Cas9-VPR 融合蛋白能够上调人横纹肌肉瘤细胞中肌肉特异性转录因子的表达。开发了一个工程“工具包”，以方便使用这些新的人类位点中最广泛表征的位点 SHS231，该位点位于染色体 4 的近端长臂上。这里确定的靶位点有可能成为额外的人类 SHS，以实现基础和临床基因编辑和基因组工程应用。
