A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models.
A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models.
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DOI：
10.1038/s41598-021-91760-9
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发表时间：
2021-06-16
影响因子：
4.6
通讯作者：
Jeremias I
中科院分区：
文献类型：
--
作者：
Liu WH;Völse K;Senft D;Jeremias I
CRISPR/Cas9是确定蛋白质功能的一种有价值的工具，但技术障碍限制了其在具有挑战性的环境中的应用，例如无法在体外生长的细胞，如原发性白血病细胞及其衍生的异种移植物（PDX）。为了丰富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只有在基因编辑成功后才能表达的框外荧光色素上游。为了减少PDX白血病生长所需的体内传代轮次，我们连续克隆了17个GOI靶点，在其两侧添加了一个改进的连接体，然后用慢病毒转导PDX细胞以稳定表达。报道者富集了高达80%的稀缺的、成功基因编辑的PDX细胞。使用报告基因，我们发现src家族激酶LYN的KO增加了B前体细胞ALL的PDX细胞对Vincristine的反应，即使是杂合KO，也表明单倍功能不全。总之，我们的报告系统能够在具有技术挑战性的环境中富集KO细胞，并将基因编辑的使用扩展到与患者高度相关的模型系统。
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.
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通讯作者：
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