Safe Harbor Targeted CRISPR-Cas9 Tools for Molecular-Genetic Imaging of Cells in Living Subjects
Safe Harbor Targeted CRISPR-Cas9 Tools for Molecular-Genetic Imaging of Cells in Living Subjects
复制标题
DOI：
10.1089/crispr.2018.0030
复制
发表时间：
2018-12-01
期刊：
影响因子：
3.7
通讯作者：
Ronald, John A.
中科院分区：
文献类型：
--
作者：
Dubois, Veronica P.;Zotova, Darya;Ronald, John A.
表达成像报告基因的细胞的非侵入性分子遗传成像是纵向监测临床前模型和患者中癌细胞的生物分布和活力以及基于细胞的治疗的宝贵方法。然而，用报告基因标记细胞通常依赖于使用将报告基因随机整合到基因组中的基因转移方法，这可能导致不希望的和严重的有害影响。为了克服这一点，我们开发了CRISPR-Cas9工具来编辑腺相关病毒位点1（AAVS 1）安全港的细胞，其中具有编码抗生素抗性基因和用于生物发光（BLI）和荧光成像的报告基因的大型供体构建体（类似于6.3内切酶）。HEK 293 T细胞用在一个质粒中编码Cas9内切核酸酶和靶向AAVS 1的指导RNA的双质粒系统以及编码嘌呤霉素抗性基因、tdTomato和侧翼为AAVS 1同源臂的萤火虫荧光素酶的供体质粒转染。分离嘌呤霉素抗性克隆细胞，并通过PCR和PCR产物测序确认AAVS 1整合。体外BLI信号与细胞数量良好相关（R-2 = 0.9988; p< 0.05），并且在多次传代中稳定。将工程化细胞（2.5 × 10（6））注射到裸鼠的左后胁腹，并在第0、7、14、21和28天进行体内BLI。BLI信号从第0天至第7天呈下降趋势，但由于细胞生长，到第28天显著增加（p< 0.05）。这描述了第一个用于AAVS 1整合大基因构建体的CRISPR-Cas9系统，用于体内细胞的分子遗传成像。随着进一步的发展，包括提高编辑效率，使用临床相关的报告基因，以及在培养中容易扩增的其他细胞群中进行评估（例如，由于CRISPR-Cas9报告基因系统可以用于多种体内细胞追踪研究（例如，永生化细胞或T细胞），因此该CRISPR-Cas9报告基因系统可以广泛应用于多种体内细胞追踪研究。
Noninvasive molecular-genetic imaging of cells expressing imaging reporter genes is an invaluable approach for longitudinal monitoring of the biodistribution and viability of cancer cells and cell-based therapies in preclinical models and patients. However, labeling cells with reporter genes often relies on using gene transfer methods that randomly integrate the reporter genes into the genome, which may cause unwanted and serious detrimental effects. To overcome this, we have developed CRISPR-Cas9 tools to edit cells at the adeno-associated virus site 1 (AAVS1) safe harbour with a large donor construct (similar to 6.3 kilobases) encoding an antibiotic resistance gene and reporter genes for bioluminescence (BLI) and fluorescence imaging. HEK293T cells were transfected with a dual plasmid system encoding the Cas9 endonuclease and an AAVS1-targeted guide RNA in one plasmid, and a donor plasmid encoding a puromycin resistance gene, tdTomato and firefly luciferase flanked by AAVS1 homology arms. Puromycin-resistant clonal cells were isolated and AAVS1 integration was confirmed via PCR and sequencing of the PCR product. In vitro BLI signal correlated well to cell number (R-2 = 0.9988; p< 0.05) and was stable over multiple passages. Engineered cells (2.5 x 10(6)) were injected into the left hind flank of nude mice and in vivo BLI was performed on days 0, 7, 14, 21, and 28. BLI signal trended down from day 0 to day 7, but significantly increased by day 28 due to cell growth (p< 0.05). This describes the first CRISPR-Cas9 system for AAVS1 integration of large gene constructs for molecular-genetic imaging of cells in vivo. With further development, including improving editing efficiency, use of clinically relevant reporters, and evaluation in other cell populations that can be readily expanded in culture (e.g., immortalized cells or T cells), this CRISPR-Cas9 reporter gene system could be broadly applied to a number of in vivo cell tracking studies.