Research Unit Apoptosis in Hematopoietic Stem Cells, German Research Center for Environmental Health (HMGU), Helmholtz Zentrum München, Marchioninistraße 25, 81377, Munich, Germany.
German Cancer Consortium (DKTK), Partner Site Munich, Munich, Germany.
Sci Rep. 2021 Jun 16;11(1):12649. doi: 10.1038/s41598-021-91760-9.
CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.
CRISPR/Cas9 代表了一种用于确定蛋白质功能的有价值的工具，但技术障碍限制了其在具有挑战性的环境中的使用，例如无法在体外生长的原代白血病细胞和源自这些细胞的异种移植物（PDX）。为了富集 CRISPR/Cas9 编辑的细胞，我们改进了双报告系统，并将感兴趣基因（GOI）的基因组靶序列克隆到一个无框荧光蛋白的上游，该荧光蛋白仅在成功基因编辑时表达。为了减少 PDX 白血病生长所需的体内传代数，将 17 个 GOI 的靶序列串联克隆，两侧是改进的接头，并通过慢病毒转导 PDX 细胞进行稳定表达。该报告器富集了稀缺的、成功编辑的 PDX 细胞，高达 80%。使用该报告器，我们表明 SRC 家族激酶 LYN 的 KO 增加了 PDX 细胞对长春新碱的反应，即使在杂合 KO 时也是如此，表明杂合不足。总之，我们的报告系统能够在技术上具有挑战性的环境中富集 KO 细胞，并将基因编辑扩展到高度与患者相关的模型系统。
