GFP to BFP Conversion: A Versatile Assay for the Quantification of CRISPR/Cas9-mediated Genome Editing.
GFP to BFP Conversion: A Versatile Assay for the Quantification of CRISPR/Cas9-mediated Genome Editing.
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DOI：
10.1038/mtna.2016.48
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发表时间：
2016-07-12
期刊：
影响因子：
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通讯作者：
Vadolas J
中科院分区：
文献类型：
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作者：
Glaser A;McColl B;Vadolas J
格拉泽等人。 2.额外的沉默突变（201C>G）。根据我们的预期，在 K562-50 和 HEK293T-EGFP 细胞中使用 ssODN2 实现了最高 HDR 频率（分别为 5.8% 和 23.3%，图 2a）。 ssODN2 的正义和反义构型之间没有观察到显着差异（图 1d）。该测定通过对 K562-50 细胞中用 gRNA1 和 ssODN2 编辑后的 GFP+、BFP+ 和非荧光群体生长的克隆进行测序来验证（图 2b）。总之，我们证明 GFP 到 BFP 的转换是量化 HDR 和 NHEJ 的可靠且简单的方法。 Arpino 等人证明了 GFP 发色团区域对单个氨基酸缺失的高敏感性。支持我们的假设，即甚至+ 3 和− 3 插入/缺失都可以被检测为荧光损失。 13 我们已将其应用于 GFP 到 BFP 转换的 HDR 模板的优化，并通过测序验证了该策略。虽然我们使用 EGFP+ 细胞作为靶标，但 wt GFP 也可能是代替 EGFP 的靶标。该策略可用于高通量筛选，以识别增强 HDR 频率的条件（图 2c）。为了解决依赖于所使用的供体模板而独特影响基因组编辑率的机制差异，可以轻松地调整筛选以使用不同的模板类型（例如，dsDNA、腺相关病毒）。丰富的 EGFP 表达细胞系和动物模型允许应用该策略在各种原代细胞和转化细胞中优化 HDR，从而建立体内基因修复策略。
Glaser et al. 2 additional silent mutation (201C> G). In accordance with our expectations, the highest HDR frequency was achieved with ssODN2 in both K562-50 and HEK293T-EGFP cells (5.8% and 23.3%, respectively, Figure 2a). No significant difference was observed between sense and antisense configuration of ssODN2 (Figure 1d). The assay was validated through sequencing of clones grown from the GFP+, BFP+, and nonfluorescent populations after editing with gRNA1 and ssODN2 in K562-50 cells (Figure 2b). In summary, we demonstrate that GFP to BFP conversion is a reliable and simple method for the quantification of HDR and NHEJ. The high sensitivity of the GFP chromophore region to single amino acid deletions demonstrated by Arpino et al. supports our hypothesis that even+ 3 and− 3 insertions/deletions can be detected as loss of fluorescence. 13 We have applied this to the optimization of a HDR template for GFP to BFP conversion and verified the strategy through sequencing. While we used EGFP+ cells as targets, wt GFP may also be a target in place of EGFP. This strategy could be used in a high-throughput screen to identify conditions that enhance HDR frequency (Figure 2c). In order to address mechanistic differences that uniquely affect genome-editing rates dependent on the donor template used, the screen can easily be adapted to use a different template type (eg, dsDNA, adenoassociated virus). The abundance of EGFP-expressing cell lines and animal models permit the application of this strategy for optimization of HDR in a wide range of primary and transformed cells for the establishment of in vivo gene repair strategies.