Correction of a CD55 mutation to quantify the efficiency of targeted knock-in via flow cytometry
Correction of a CD55 mutation to quantify the efficiency of targeted knock-in via flow cytometry
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DOI：
10.1007/s11033-022-07422-0
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发表时间：
2022-04-14
影响因子：
2.8
通讯作者：
Konishi, Hiroyuki
中科院分区：
文献类型：
--
作者：
Rahman, Md. Lutfur;Hyodo, Toshinori;Konishi, Hiroyuki
背景由CRISPR/Cas9系统辅助的靶向敲入是一项先进的技术，在包括医学和农业科学在内的各个研究领域都有很好的应用前景。然而，提高靶向敲入的效率、精确度和特异性是促进这项技术实际应用的先决条件。为了提高靶向敲入的效率，有必要建立一个分子系统，允许以简单的程序灵敏地监测靶向敲入事件。方法与结果我们建立了一种检测靶向敲入完成CD55基因校正的方法，命名为CD55校正实验。为了创建本实验中使用的报告克隆，我们首先引入了覆盖CD55外显子2-5的7.7kb杂合性缺失，然后在人类细胞系中剩余的CD55等位基因的外显子4中加入了截断突变。将在细胞膜上丢失CD55蛋白的报告克隆与携带野生型CD55外显子4的供体质粒一起，用Cas9构建载体，然后对细胞进行荧光标记的CD55抗体染色，并用流式细胞仪分析CD55阳性细胞。这些程序允许高通量、定量地检测发生在内源性人类基因中的目标基因校正事件。结论本研究证实了CD55校正分析在灵敏地定量靶向敲入效率方面的作用。当与PIGA校正分析一起使用时，CD55校正分析将有助于准确确定靶向敲入的效率，排除可能由细胞系特定和位点特定因素引起的实验偏差。
Background Targeted knock-in assisted by the CRISPR/Cas9 system is an advanced technology with promising applications in various research fields including medical and agricultural sciences. However, improvements in the efficiency, precision, and specificity of targeted knock-in are prerequisites to facilitate the practical application of this technology. To improve the efficiency of targeted knock-in, it is necessary to have a molecular system that allows sensitive monitoring of targeted knock-in events with simple procedures. Methods and results We developed an assay, named CD55 correction assay, with which to monitor CD55 gene correction accomplished by targeted knock-in. To create the reporter clones used in this assay, we initially introduced a 7.7-kb heterozygous deletion covering CD55 exons 2-5, and then incorporated a truncating mutation within exon 4 of the remaining CD55 allele in human cell lines. The resultant reporter clones that lost the CD55 protein on the cell membrane were next transfected with Cas9 constructs along with a donor plasmid carrying wild-type CD55 exon 4. The cells were subsequently stained with fluorescence-labeled CD55 antibody and analyzed by flow cytometry to detect CD55-positive cells. These procedures allow high-throughput, quantitative detection of targeted gene correction events occurring in an endogenous human gene. Conclusions The current study demonstrated the utility of the CD55 correction assay to sensitively quantify the efficiency of targeted knock-in. When used with the PIGA correction assay, the CD55 correction assay will help accurately determine the efficiency of targeted knock-in, precluding possible experimental biases caused by cell line-specific and locus-specific factors.