Deleuze Virginie, Garcia Leonor, Rouaisnel Betty, Salma Mohammad, Kinoo Alexia, Andrieu-Soler Charlotte, Soler Eric
IGMM, University Montpellier, CNRS, Montpellier, France.
Laboratory of Excellence GR-Ex, Université de Paris, Paris, France.
iScience. 2023 Aug 15;26(9):107641. doi: 10.1016/j.isci.2023.107641. eCollection 2023 Sep 15.
Targeted genome editing holds great promise in biology. However, efficient genome modification, including gene knock-in (KI), remains an unattained goal in multiple cell types and loci due to poor transfection efficiencies and low target genes expression, impeding the positive selection of recombined cells. Here, we describe a genome editing approach to achieve efficient gene targeting using hard to transfect erythroid cell lines. We demonstrate robust fluorescent protein KI efficiency in low expressed transcription factor (TF) genes (e.g., or ). We further show the ability to target two independent loci in individual cells, exemplified by MYB-GFP and NuMA-Cherry double KI, allowing multicolor labeling of regulatory factors at physiological endogenous levels. Our KI tagging approach allowed us to perform genome-wide TF analysis at increased signal-to-noise ratios, and highlighted previously unidentified MYB target genes and pathways. Overall, we establish a versatile CRISPR-Cas9-based platform, offering attractive opportunities for the dissection of the erythroid differentiation process.
靶向基因组编辑在生物学领域具有巨大潜力。然而，由于转染效率低下和靶基因表达水平低，包括基因敲入（KI）在内的高效基因组修饰在多种细胞类型和基因座中仍然是一个未实现的目标，这阻碍了重组细胞的阳性选择。在这里，我们描述了一种基因组编辑方法，用于使用难以转染的红细胞系实现高效的基因靶向。我们展示了在低表达转录因子（TF）基因（例如 或 ）中强大的荧光蛋白敲入效率。我们进一步展示了在单个细胞中靶向两个独立基因座的能力，以MYB-GFP和NuMA-Cherry双敲入为例，允许在生理内源性水平对调节因子进行多色标记。我们的敲入标记方法使我们能够以更高的信噪比进行全基因组TF分析，并突出显示了以前未鉴定的MYB靶基因和途径。总体而言，我们建立了一个通用的基于CRISPR-Cas9的平台，为剖析红细胞分化过程提供了有吸引力的机会。
