Observations of actin dynamics in living cells using fluorescence microscopy have been foundational in the exploration of the mechanisms underlying cell migration. We used CRISPR/Cas9 gene editing to generate neutrophil‐like HL‐60 cell lines expressing GFP‐β‐actin from the endogenous locus (ACTB). In light of many previous reports outlining functional deficiencies of labeled actin, we anticipated that HL‐60 cells would only tolerate a monoallelic edit, as biallelic edited cells would produce no normal β‐actin. Surprisingly, we recovered viable monoallelic GFP‐β‐actin cells as well as biallelic edited GFP‐β‐actin cells, in which one copy of the ACTB gene is silenced and the other contains the GFP tag. Furthermore, the edited cells migrate with similar speeds and persistence as unmodified cells in a variety of motility assays, and have nearly normal cell shapes. These results might partially be explained by our observation that GFP‐β‐actin incorporates into the F‐actin network in biallelic edited cells at similar efficiencies as normal β‐actin in unedited cells. Additionally, the edited cells significantly upregulate γ‐actin, perhaps helping to compensate for the loss of normal β‐actin. Interestingly, biallelic edited cells have only modest changes in global gene expression relative to the monoallelic line, as measured by RNA sequencing. While monoallelic edited cells downregulate expression of the tagged allele and are thus only weakly fluorescent, biallelic edited cells are quite bright and well‐suited for live cell microscopy. The nondisruptive phenotype and direct interpretability of this fluorescent tagging approach make it a promising tool for studying actin dynamics in these rapidly migrating and highly phagocytic cells.

中文翻译：

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仅表达GFP标记的β-肌动蛋白的嗜中性粒细胞样HL-60细胞表现出几乎正常的运动性。

使用荧光显微镜观察活细胞中肌动蛋白的动力学一直是探索细胞迁移机制的基础。我们使用CRISPR / Cas9基因编辑来生成内源性位点（ACTB）的表达GFP-β-肌动蛋白的嗜中性粒细胞样HL-60细胞系。根据以前的许多报道，概述了标记肌动蛋白的功能缺陷，我们预计HL-60细胞只能耐受单等位基因编辑，因为双等位基因编辑的细胞不会产生正常的β-肌动蛋白。出乎意料的是，我们回收了可行的单等位基因GFP-β-肌动蛋白细胞以及双等位基因编辑的GFP-β-肌动蛋白细胞，其中ACTB基因的一个拷贝被沉默，而另一个拷贝则包含GFP标签。此外，在各种运动性测定中，编辑后的细胞以与未修饰的细胞相似的速度和持久性迁移，并具有接近正常的细胞形状 我们的观察结果可能部分解释了这些结果，即GFP-β-肌动蛋白以与未编辑细胞中正常β-肌动蛋白相似的效率掺入双等位基因编辑的细胞中的F-肌动蛋白网络中。此外，编辑后的细胞可显着上调γ-肌动蛋白，也许有助于补偿正常β-肌动蛋白的损失。有趣的是，如通过RNA测序所测量的，双等位基因编辑的细胞相对于单等位基因系在整体基因表达中仅具有适度的变化。虽然单等位基因编辑的细胞下调了标记的等位基因的表达，因此只有微弱的荧光，但双等位基因编辑的细胞却非常明亮，非常适合活细胞显微镜检查。