ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells.
ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells.
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DOI：
10.1186/s12864-023-09377-3
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发表时间：
2023-05-29
期刊：
影响因子：
4.4
通讯作者：
中科院分区：
文献类型：
--
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CRISPR技术的最新进展使我们能够在不同物种和细胞系中进行基因敲入。CRISPR介导的敲入需要供体DNA作为同源定向修复(HDR)的模板。对于短序列的敲入或碱基替换，单链DNA供体经常与各种其他形式的HDR供体一起使用，例如线性dsDNA。然而，部分由于长单链DNA制备的复杂性，目前尚不清楚单链DNA是否是将长转基因如荧光报告基因插入人类细胞的最佳HDR供体类型。在本研究中，我们建立了一种基于核酸酶的简单的高产率和高纯度长单链DNA的制备方法，并综合比较了单链DNA供体和双链DNA供体与90个碱基的同源臂在人二倍体RPE1和HCT116细胞中长转基因标记内源基因的性能。流式细胞术的定量结果显示，用单链DNA供体进行内源荧光标记的效率低于用双链DNA。通过使用长阅读扩增子测序和分类框架分析敲入结果，无论供体类型如何，都检测到了各种误整合事件。重要的是，单链DNA供体的精确插入率低于双链DNA供体。此外，在使用没有同源臂的供体进行的非靶向整合分析中，ssDNA和dsDNA相对容易发生非同源整合。这些结果表明，在人RPE1和HCT116细胞中，作为具有相对较短的同源臂的长HDR供体，ssDNA并不优于dsDNA。网上版载有补充材料，可在10.1186/s12864-023-09377-3查阅。
Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells. In this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration. These results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells. The online version contains supplementary material available at 10.1186/s12864-023-09377-3.
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